Elucidating the Function of Penetratin and a Static Magnetic Field in Cellular Uptake of Magnetic Nanoparticles

PubMed Central

Chaudhary, Suman; Smith, Carol Anne; del Pino, Pablo; de la Fuente, Jesus M.; Mullin, Margaret; Hursthouse, Andrew; Stirling, David; Berry, Catherine C.

2013-01-01

Nanotechnology plays an increasingly important role in the biomedical arena. In particular, magnetic nanoparticles (mNPs) have become important tools in molecular diagnostics, in vivo imaging and improved treatment of disease, with the ultimate aim of producing a more theranostic approach. Due to their small sizes, the nanoparticles can cross most of the biological barriers such as the blood vessels and the blood brain barrier, thus providing ubiquitous access to most tissues. In all biomedical applications maximum nanoparticle uptake into cells is required. Two promising methods employed to this end include functionalization of mNPs with cell-penetrating peptides to promote efficient translocation of cargo into the cell and the use of external magnetic fields for enhanced delivery. This study aimed to compare the effect of both penetratin and a static magnetic field with regards to the cellular uptake of 200 nm magnetic NPs and determine the route of uptake by both methods. Results demonstrated that both techniques increased particle uptake, with penetratin proving more cell specific. Clathrin- medicated endocytosis appeared to be responsible for uptake as shown via PCR and western blot, with Pitstop 2 (known to selectively block clathrin formation) blocking particle uptake. Interestingly, it was further shown that a magnetic field was able to reverse or overcome the blocking, suggesting an alternative route of uptake. PMID:24275948

Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis

PubMed Central

Phuc, Le Thi Minh; Taniguchi, Akiyoshi

2017-01-01

The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF–EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications. PMID:28629179

Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis.

PubMed

Phuc, Le Thi Minh; Taniguchi, Akiyoshi

2017-06-19

The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF-EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications.

Novel guanidine-containing molecular transporters based on lactose scaffolds: lipophilicity effect on the intracellular organellar selectivity.

PubMed

Biswas, Goutam; Jeon, Ock-Youm; Lee, Woo Sirl; Kim, Dong-Chan; Kim, Kyong-Tai; Lee, Suho; Chang, Sunghoe; Chung, Sung-Kee

2008-01-01

We have synthesized two lactose-based molecular transporters, each containing seven guanidine residues attached to the lactose scaffold through omega-aminocarboxylate linker chains of two different lengths, and have examined their cellular uptakes and intracellular and organellar localizations in HeLa cells, as well as their tissue distributions in mice. Both molecular transporters showed higher cellular uptake efficiencies than Arg8, and wide tissue distributions including the brain. Mitochondrial localization is of special interest because of its potential relevance to "mitochondrial diseases". Interestingly, it has been found that the intracellular localization sites of the G7 molecular transporters-namely either mitochondria or lysosomes and endocytic vesicles-are largely determined by the linker chain lengths, or their associated lipophilicities.

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

PubMed

Navrátilová, Jarmila; Karasová, Martina; Kohutková Lánová, Martina; Jiráková, Ludmila; Budková, Zuzana; Pacherník, Jiří; Šmarda, Jan; Beneš, Petr

2017-09-01

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Cell uptake survey of pegylated nanographene oxide.

PubMed

Vila, M; Portolés, M T; Marques, P A A P; Feito, M J; Matesanz, M C; Ramírez-Santillán, C; Gonçalves, G; Cruz, S M A; Nieto, A; Vallet-Regi, M

2012-11-23

Graphene and more specifically, nanographene oxide (GO) has been proposed as a highly efficient antitumoral therapy agent. Nevertheless, its cell uptake kinetics, its influence in different types of cells and the possibility of controlling cellular internalization timing, is still a field that remains unexplored. Herein, different cell types have been cultured in vitro for several incubation periods in the presence of 0.075 mg ml(-1) pegylated GO solutions. GO uptake kinetics revealed differences in the agent's uptake amount and speed as a function of the type of cell involved. Osteoblast-like cells GO uptake is higher and faster without resulting in greater cell membrane damage. Moreover, the dependence on the commonly used PEG nature (number of branches) also influences the viability and cell uptake speed. These facts play an important role in the future definition of timing parameters and selective cell uptake control in order to achieve an effective therapy.

Carbon "Quantum" Dots for Fluorescence Labeling of Cells.

PubMed

Liu, Jia-Hui; Cao, Li; LeCroy, Gregory E; Wang, Ping; Meziani, Mohammed J; Dong, Yiyang; Liu, Yuanfang; Luo, Pengju G; Sun, Ya-Ping

2015-09-02

The specifically synthesized and selected carbon dots of relatively high fluorescence quantum yields were evaluated in their fluorescence labeling of cells. For the cancer cell lines, the cellular uptake of the carbon dots was generally efficient, resulting in the labeling of the cells with bright fluorescence emissions for both one- and two-photon excitations from predominantly the cell membrane and cytoplasm. In the exploration on labeling the live stem cells, the cellular uptake of the carbon dots was relatively less efficient, though fluorescence emissions could still be adequately detected in the labeled cells, with the emissions again predominantly from the cell membrane and cytoplasm. This combined with the observed more efficient internalization of the same carbon dots by the fixed stem cells might suggest some significant selectivity of the stem cells toward surface functionalities of the carbon dots. The needs and possible strategies for more systematic and comparative studies on the fluorescence labeling of different cells, including especially live stem cells, by carbon dots as a new class of brightly fluorescent probes are discussed.

In vitro cellular uptake of evodiamine and rutaecarpine using a microemulsion

PubMed Central

Zhang, Yong-Tai; Huang, Zhe-Bin; Zhang, Su-Juan; Zhao, Ji-Hui; Wang, Zhi; Liu, Ying; Feng, Nian-Ping

2012-01-01

Objective To investigate the cellular uptake of evodiamine and rutaecarpine in a microemulsion in comparison with aqueous suspensions and tinctures. Materials and methods A microemulsion was prepared using the dropwise addition method. Mouse skin fibroblasts were cultured in vitro to investigate the optimal conditions for evodiamine and rutaecarpine uptake with different drug concentrations and administration times. Under optimal conditions, the cellular uptake of microemulsified drugs was assayed and compared to tinctures and aqueous suspensions. Rhodamine B labeling and laser scanning confocal microscopy (LSCM) were used to explore the distribution of fluorochrome transferred with the microemulsion in fibroblasts. Cellular morphology was also investigated, using optical microscopy to evaluate microemulsion-induced cellular toxicity. Results The maximum cellular drug uptake amounts were obtained with a 20% concentration (v/v) of microemulsion and an 8 hour administration time. Drug uptake by mouse skin fibroblasts was lowest when the drugs were loaded in microemulsion. After incubation with rhodamine B-labeled microemulsion for 8 hours, the highest fluorescence intensity was achieved, and the fluorochrome was primarily distributed in the cytochylema. No obvious cellular morphologic changes were observed with the administration of either the microemulsion or the aqueous suspension; for the tincture group, however, massive cellular necrocytosis was observed. Conclusion The lower cellular uptake with microemulsion may be due to the fact that most of the drug loaded in the microemulsion vehicle was transported via the intercellular space, while a small quantity of free drug (released from the vehicle) was ingested through transmembrane transport. Mouse skin fibroblasts rarely endocytosed evodiamine and rutaecarpine with a microemulsion as the vehicle. The microemulsion had no obvious effect on cellular morphology, suggesting there is little or no cellular toxicity associated with the administration of microemulsion on mouse skin fibroblasts. PMID:22679361

In vitro cellular uptake of evodiamine and rutaecarpine using a microemulsion.

PubMed

Zhang, Yong-Tai; Huang, Zhe-Bin; Zhang, Su-Juan; Zhao, Ji-Hui; Wang, Zhi; Liu, Ying; Feng, Nian-Ping

2012-01-01

To investigate the cellular uptake of evodiamine and rutaecarpine in a microemulsion in comparison with aqueous suspensions and tinctures. A microemulsion was prepared using the dropwise addition method. Mouse skin fibroblasts were cultured in vitro to investigate the optimal conditions for evodiamine and rutaecarpine uptake with different drug concentrations and administration times. Under optimal conditions, the cellular uptake of microemulsified drugs was assayed and compared to tinctures and aqueous suspensions. Rhodamine B labeling and laser scanning confocal microscopy (LSCM) were used to explore the distribution of fluorochrome transferred with the microemulsion in fibroblasts. Cellular morphology was also investigated, using optical microscopy to evaluate microemulsion-induced cellular toxicity. The maximum cellular drug uptake amounts were obtained with a 20% concentration (v/v) of microemulsion and an 8 hour administration time. Drug uptake by mouse skin fibroblasts was lowest when the drugs were loaded in microemulsion. After incubation with rhodamine B-labeled microemulsion for 8 hours, the highest fluorescence intensity was achieved, and the fluorochrome was primarily distributed in the cytochylema. No obvious cellular morphologic changes were observed with the administration of either the microemulsion or the aqueous suspension; for the tincture group, however, massive cellular necrocytosis was observed. The lower cellular uptake with microemulsion may be due to the fact that most of the drug loaded in the microemulsion vehicle was transported via the intercellular space, while a small quantity of free drug (released from the vehicle) was ingested through transmembrane transport. Mouse skin fibroblasts rarely endocytosed evodiamine and rutaecarpine with a microemulsion as the vehicle. The microemulsion had no obvious effect on cellular morphology, suggesting there is little or no cellular toxicity associated with the administration of microemulsion on mouse skin fibroblasts.

Cell Type-Specific Modulation of Cobalamin Uptake by Bovine Serum

PubMed Central

Zhao, Hua; Ruberu, Kalani; Li, Hongyun; Garner, Brett

2016-01-01

Tracking cellular 57Co-labelled cobalamin (57Co-Cbl) uptake is a well-established method for studying Cbl homeostasis. Previous studies established that bovine serum is not generally permissive for cellular Cbl uptake when used as a supplement in cell culture medium, whereas supplementation with human serum promotes cellular Cbl uptake. The underlying reasons for these differences are not fully defined. In the current study we address this question. We extend earlier observations by showing that fetal calf serum inhibits cellular 57Co-Cbl uptake by HT1080 cells (a fibrosarcoma-derived fibroblast cell line). Furthermore, we discovered that a simple heat-treatment protocol (95°C for 10 min) ameliorates this inhibitory activity for HT1080 cell 57Co-Cbl uptake. We provide evidence that the very high level of haptocorrin in bovine serum (as compared to human serum) is responsible for this inhibitory activity. We suggest that bovine haptocorrin competes with cell-derived transcobalamin for Cbl binding, and that cellular Cbl uptake may be minimised in the presence of large amounts of bovine haptocorrin that are present under routine in vitro cell culture conditions. In experiments conducted with AG01518 cells (a neonatal foreskin-derived fibroblast cell line), overall cellular 57Co-Cbl uptake was 86% lower than for HT1080 cells, cellular TC production was below levels detectable by western blotting, and heat treatment of fetal calf serum resulted in only a modest increase in cellular 57Co-Cbl uptake. We recommend a careful assessment of cell culture protocols should be conducted in order to determine the potential benefits that heat-treated bovine serum may provide for in vitro studies of mammalian cell lines. PMID:27893837

Enhanced cellular uptake of maleimide-modified liposomes via thiol-mediated transport

PubMed Central

Li, Tianshu; Takeoka, Shinji

2014-01-01

With a small amount of maleimide modification on the liposome surface, enhanced cellular uptake of liposomes and drug-delivery efficiency can be obtained both in vitro and in vivo. Herein, we describe the mechanisms underlying this enhanced cellular uptake. Suppression of the cellular uptake of maleimide-modified liposomes (M-GGLG, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate [GGLG]/cholesterol/poly(ethylene glycol) – 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [PEG5000-DSPE]/maleimide [M]-PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03) caused by temperature block and addition of serum was alleviated compared with that of liposomes without maleimide modification (GGLG liposomes, composed of GGLG/cholesterol/PEG5000-DSPE/PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03). When 0.01 nM N-ethylmaleimide was used to pre-block cellular thiols, the cellular uptake of M-GGLG liposomes was decreased to approximately 70% in HeLa, HCC1954, MDA-MB-468, and COS-7 cell lines. Moreover, inhibition of a thiol-related reductase such as protein disulfide isomerase resulted in a 15%–45% inhibition of the cellular uptake of M-GGLG liposomes, whereas GGLG liposomes were not influenced. Further, single and mixed inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis did not efficiently inhibit the cellular uptake of M-GGLG liposomes. Using confocal microscopy, we verified that M-GGLG liposomes were localized partially in lysosomes after inhibition of the mentioned conventional endocytic pathways. Therefore, it was hypothesized that the mechanisms underlying the enhanced cellular uptake of liposomes by maleimide modification was thiol-mediated membrane trafficking, including endocytosis and energy-independent transport. PMID:24940060

Enhanced cellular uptake of maleimide-modified liposomes via thiol-mediated transport.

PubMed

Li, Tianshu; Takeoka, Shinji

2014-01-01

With a small amount of maleimide modification on the liposome surface, enhanced cellular uptake of liposomes and drug-delivery efficiency can be obtained both in vitro and in vivo. Herein, we describe the mechanisms underlying this enhanced cellular uptake. Suppression of the cellular uptake of maleimide-modified liposomes (M-GGLG, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate [GGLG]/cholesterol/poly(ethylene glycol) - 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [PEG₅₀₀₀-DSPE]/maleimide [M]-PEG₅₀₀₀-Glu2C18 at a molar ratio of 5:5:0.03:0.03) caused by temperature block and addition of serum was alleviated compared with that of liposomes without maleimide modification (GGLG liposomes, composed of GGLG/cholesterol/PEG₅₀₀₀-DSPE/PEG₅₀₀₀-Glu2C₁₈ at a molar ratio of 5:5:0.03:0.03). When 0.01 nM N-ethylmaleimide was used to pre-block cellular thiols, the cellular uptake of M-GGLG liposomes was decreased to approximately 70% in HeLa, HCC1954, MDA-MB-468, and COS-7 cell lines. Moreover, inhibition of a thiol-related reductase such as protein disulfide isomerase resulted in a 15%-45% inhibition of the cellular uptake of M-GGLG liposomes, whereas GGLG liposomes were not influenced. Further, single and mixed inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis did not efficiently inhibit the cellular uptake of M-GGLG liposomes. Using confocal microscopy, we verified that M-GGLG liposomes were localized partially in lysosomes after inhibition of the mentioned conventional endocytic pathways. Therefore, it was hypothesized that the mechanisms underlying the enhanced cellular uptake of liposomes by maleimide modification was thiol-mediated membrane trafficking, including endocytosis and energy-independent transport.

Measurement of cellular copper levels in Bacillus megaterium during exponential growth and sporulation.

PubMed

Krueger, W B; Kolodziej, B J

1976-01-01

Both atomic absorption spectrophotometry (AAS) and neutron activation analysis have been utilized to determine cellular Cu levels in Bacillus megaterium ATCC 19213. Both methods were selected for their sensitivity to detection of nanogram quantities of Cu. Data from both methods demonstrated identical patterms of Cu uptake during exponenetial growth and sporulation. Late exponential phase cells contained less Cu than postexponential t2 cells while t5 cells contained amounts equivalent to exponential cells. The t11 phase-bright forespore containing cells had a higher Cu content than those of earlier time periods, and the free spores had the highest Cu content. Analysis of the culture medium by AAS corroborated these data by showing concomitant Cu uptake during exponential growth and into t2 postexponential phase of sporulation. From t2 to t4, Cu egressed from the cells followed by a secondary uptake during the maturation of phase-dark forespores into phase-bright forespores (t6--t9).

Apoptosis selectively induced in BEL-7402 cells by folic acid-modified magnetic nanoparticles combined with 100 Hz magnetic field

PubMed Central

Wen, Jian; Jiang, Shulian; Chen, Zhiqiang; Zhao, Wei; Yi, Yongxiang; Yang, Ruili; Chen, Baoan

2014-01-01

Objective To explore the effect of folic acid-modified magnetic nanoparticles (FA-MNPs) combined with a 100 Hz extremely low-frequency electromagnetic field (ELF-EMF) on the apoptosis of liver cancer BEL-7402 cells. Materials and methods MNPs (20 nm) were prepared by coprecipitation, and then folic acid was coated onto MNPs to prepare FA-MNPs. BEL-7402 cells and HL7702 cells were selected as liver cancer cells and normal liver cells, respectively. The ELF-EMF was generated from a solenoid coil. Cellular uptake of NPs was determined by inductively coupled plasma atomic emission spectroscopy. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to evaluate cell inhibition. Apoptosis was analyzed by flow cytometry. Statistical analyses were performed using two-way analysis of variance. Results FA-MNPs combined with a 100 Hz magnetic field significantly inhibited cell proliferation and induced higher apoptosis compared to either the ELF-EMF alone or FA-MNPs alone. FA-MNPs showed a better apoptosis effect and higher iron uptake in BEL-7402 cells compared to in HL7702 cells. On the basis of the ELF-EMF, higher doses of FA-MNPs brought higher apoptosis and higher iron uptake in either BEL-7402 cells or HL7702 cells. Conclusion These results suggest that FA-MNPs may induce apoptosis in a cellular iron uptake-dependent manner when combined with an ELF-EMF in BEL-7402 cells. PMID:24790442

Contribution of electrostatics to the binding of pancreatic-type ribonucleases to membranes.

PubMed

Sundlass, Nadia K; Eller, Chelcie H; Cui, Qiang; Raines, Ronald T

2013-09-17

Pancreatic-type ribonucleases show clinical promise as chemotherapeutic agents but are limited in efficacy by the inefficiency of their uptake by human cells. Cellular uptake can be increased by the addition of positive charges to the surface of ribonucleases, either by site-directed mutagenesis or by chemical modification. This observation has led to the hypothesis that ribonuclease uptake by cells depends on electrostatics. Here, we use a combination of experimental and computational methods to ascertain the contribution of electrostatics to the cellular uptake of ribonucleases. We focus on three homologous ribonucleases: Onconase (frog), ribonuclease A (cow), and ribonuclease 1 (human). Our results support the hypothesis that electrostatics are necessary for the cellular uptake of Onconase. In contrast, specific interactions with cell-surface components likely contribute more to the cellular uptake of ribonuclease A and ribonuclease 1 than do electrostatics. These findings provide insight for the design of new cytotoxic ribonucleases.

Enhanced and Selective Antiproliferative Activity of Methotrexate-Functionalized-Nanocapsules to Human Breast Cancer Cells (MCF-7).

PubMed

de Oliveira, Catiúscia P; Büttenbender, Sabrina L; Prado, Willian A; Beckenkamp, Aline; Asbahr, Ana C; Buffon, Andréia; Guterres, Silvia S; Pohlmann, Adriana R

2018-01-04

Methotrexate is a folic acid antagonist and its incorporation into nanoformulations is a promising strategy to increase the drug antiproliferative effect on human breast cancer cells by overexpressing folate receptors. To evaluate the efficiency and selectivity of nanoformulations containing methotrexate and its diethyl ester derivative, using two mechanisms of drug incorporation (encapsulation and surface functionalization) in the in vitro cellular uptake and antiproliferative activity in non-tumoral immortalized human keratinocytes (HaCaT) and in human breast carcinoma cells (MCF-7). Methotrexate and its diethyl ester derivative were incorporated into multiwall lipid-core nanocapsules with hydrodynamic diameters lower than 160 nm and higher drug incorporation efficiency. The nanoformulations were applied to semiconfluent HaCaT or MCF-7 cells. After 24 h, the nanocapsules were internalized into HaCaT and MCF-7 cells; however, no significant difference was observed between the nanoformulations in HaCaT (low expression of folate receptors), while they showed significantly higher cellular uptakes than the blank-nanoformulation in MCF-7, which was the highest uptakes observed for the drug functionalized-nanocapsules. No antiproliferative activity was observed in HaCaT culture, whereas drug-containing nanoformulations showed antiproliferative activity against MCF-7 cells. The effect was higher for drug-surface functionalized nanocapsules. In conclusion, methotrexate-functionalized-nanocapsules showed enhanced and selective antiproliferative activity to human breast cancer cells (MCF-7) being promising products for further in vivo pre-clinical evaluations.

Cationic Phosphorus Dendrimer Enhances Photodynamic Activity of Rose Bengal against Basal Cell Carcinoma Cell Lines.

PubMed

Dabrzalska, Monika; Janaszewska, Anna; Zablocka, Maria; Mignani, Serge; Majoral, Jean Pierre; Klajnert-Maculewicz, Barbara

2017-05-01

In the last couple of decades, photodynamic therapy emerged as a useful tool in the treatment of basal cell carcinoma. However, it still meets limitations due to unfavorable properties of photosensitizers such as poor solubility or lack of selectivity. Dendrimers, polymers widely studied in biomedical field, may play a role as photosensitizer carriers and improve the efficacy of photodynamic treatment. Here, we describe the evaluation of an electrostatic complex of cationic phosphorus dendrimer and rose bengal in such aspects as singlet oxygen production, cellular uptake, and phototoxicity against three basal cell carcinoma cell lines. Rose bengal-cationic dendrimer complex in molar ratio 5:1 was compared to free rose bengal. Obtained results showed that the singlet oxygen production in aqueous medium was significantly higher for the complex than for free rose bengal. The cellular uptake of the complex was 2-7-fold higher compared to a free photosensitizer. Importantly, rose bengal, rose bengal-dendrimer complex, and dendrimer itself showed no dark toxicity against all three cell lines. Moreover, we observed that phototoxicity of the complex was remarkably enhanced presumably due to high cellular uptake. On the basis of the obtained results, we conclude that rose bengal-cationic dendrimer complex has a potential in photodynamic treatment of basal cell carcinoma.

Novel mucus-penetrating liposomes as a potential oral drug delivery system: preparation, in vitro characterization, and enhanced cellular uptake

PubMed Central

Li, Xiuying; Chen, Dan; Le, Chaoyi; Zhu, Chunliu; Gan, Yong; Hovgaard, Lars; Yang, Mingshi

2011-01-01

Background The aim of this study was to investigate the intestinal mucus-penetrating properties and intestinal cellular uptake of two types of liposomes modified by Pluronic F127 (PF127). Methods The two types of liposomes, ie, PF127-inlaid liposomes and PF127-adsorbed liposomes, were prepared by a thin-film hydration method followed by extrusion, in which coumarin 6 was loaded as a fluorescence marker. A modified Franz diffusion cell mounted with the intestinal mucus of rats was used to study the diffusion characteristics of the two types of PF127 liposomes. Cell uptake studies were conducted in Caco-2 cells and analyzed using confocal laser scanning microcopy as well as flow cytometry. Results The diffusion efficiency of the two types of PF127-modified liposomes through intestinal rat mucus was 5–7-fold higher than that of unmodified liposomes. Compared with unmodified liposomes, PF127-inlaid liposomes showed significantly higher cellular uptake of courmarin 6. PF127-adsorbed liposomes showed a lower cellular uptake. Moreover, and interestingly, the two types of PF127-modified liposomes showed different cellular uptake mechanisms in Caco-2 cells. Conclusion PF127-inlaid liposomes with improved intestinal mucus-penetrating ability and enhanced cellular uptake might be a potential carrier candidate for oral drug delivery. PMID:22163166

Differential polymer structure tunes mechanism of cellular uptake and transfection routes of poly(β-amino ester) polyplexes in human breast cancer cells.

PubMed

Kim, Jayoung; Sunshine, Joel C; Green, Jordan J

2014-01-15

Successful gene delivery with nonviral particles has several barriers, including cellular uptake, endosomal escape, and nuclear transport. Understanding the mechanisms behind these steps is critical to enhancing the effectiveness of gene delivery. Polyplexes formed with poly(β-amino ester)s (PBAEs) have been shown to effectively transfer DNA to various cell types, but the mechanism of their cellular uptake has not been identified. This is the first study to evaluate the uptake mechanism of PBAE polyplexes and the dependence of cellular uptake on the end group and molecular weight of the polymer. We synthesized three different analogues of PBAEs with the same base polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (B4S4) but with small changes in the end group or molecular weight. We quantified the uptake and transfection efficiencies of the pDNA polyplexes formulated from these polymers in hard-to-transfect triple negative human breast cancer cells (MDA-MB 231). All polymers formed positively charged (10-17 mV) nanoparticles of ∼200 nm in size. Cellular internalization of all three formulations was inhibited the most (60-90% decrease in cellular uptake) by blocking caveolae-mediated endocytosis. Greater inhibition was shown with polymers that had a 1-(3-aminopropyl)-4-methylpiperazine end group (E7) than the others with a 2-(3-aminopropylamino)-ethanol end group (E6) or higher molecular weight. However, caveolae-mediated endocytosis was generally not as efficient as clathrin-mediated endocytosis in leading to transfection. These findings indicate that PBAE polyplexes can be used to transfect triple negative human breast cancer cells and that small changes to the same base polymer can modulate their cellular uptake and transfection routes.

Lactose-modified DNA tile nanostructures as drug carriers.

PubMed

Akkus Sut, Pinar; Tunc, Cansu Umran; Culha, Mustafa

2016-09-01

DNA hybridization allows the preparation of nanoscale DNA structures with desired shape and size. DNA structures using simple base pairing can be used for the delivery of drug molecules into the cells. Since DNA carries multiple negative charges, their cellular uptake efficiency is low. Thus, the modification of the DNA structures with molecules that may enhance the cellular internalization may be an option. The objective of this study is to construct DNA-based nanocarrier system and to investigate the cellular uptake of DNA tile with/without lactose modification. Doxorubicin was intercalated to DNA tile and cellular uptake of drug-loaded DNA-based carrier with/without lactose modification was investigated in vitro. HeLa, BT-474, and MDA-MB-231 cancer cells were used for cellular uptake studies and cytotoxicity assays. Using fluorescence spectroscopy, flow cytometry, and confocal microscopy, cellular uptake behavior of DNA tile was investigated. The cytotoxicity of DNA tile structures was determined with WST-1 assay. The results show that modification with lactose effectively increases the intracellular uptake of doxorubicin loaded DNA tile structure by cancer cells compared with the unmodified DNA tile. The findings of this study suggest that DNA-based nanostructures modified with carbohydrates can be used as suitable multifunctional nanocarriers with simple chemical modifications.

Cellular Stress Response to Engineered Nanoparticles: Effect of Size, Surface Coating, and Cellular Uptake

EPA Science Inventory

CELLULAR STRESS RESPONSE TO ENGINEERED NANOPARTICLES: EFFECT OF SIZE, SURFACE COATING, AND CELLULAR UPTAKE RY Prasad 1, JK McGee2, MG Killius1 D Ackerman2, CF Blackman2 DM DeMarini2 , SO Simmons2 1 Student Services Contractor, US EPA, RTP, NC 2 US EPA, RTP, NC The num...

Selective Cytotoxicity of Rhodium Metalloinsertors in Mismatch Repair-Deficient Cells†

PubMed Central

Ernst, Russell J.; Komor, Alexis C.; Barton, Jacqueline K.

2011-01-01

Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. Rhodium metalloinsertors bind to DNA mismatches with high affinity and specificity and represent a promising strategy to target mismatches in cells. Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands in cells deficient in MMR versus those that are MMR-proficient. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents. PMID:22103240

The Role of Hydrophobicity in the Cellular Uptake of Negatively Charged Macromolecules.

PubMed

Abou Matar, Tamara; Karam, Pierre

2018-02-01

It is generally accepted that positively charged molecules are the gold standard to by-pass the negatively charged cell membrane. Here, it is shown that cellular uptake is also possible for polymers with negatively charged side chains and hydrophobic backbones. Specifically, poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene], a conjugated polyelectrolyte with sulfonate, as water-soluble functional groups, is shown to accumulate in the intracellular region. When the polymer hydrophobic backbone is dissolved using polyvinylpyrrolidone, an amphiphilic macromolecule, the cellular uptake is dramatically reduced. The report sheds light on the fine balance between negatively charged side groups and the hydrophobicity of polymers to either enhance or reduce cellular uptake. As a result, these findings will have important ramifications on the future design of targeted cellular delivery nanocarriers for imaging and therapeutic applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

pHLIP®-Mediated Delivery of PEGylated Liposomes to Cancer Cells

PubMed Central

Yao, Lan; Daniels, Jennifer; Wijesinghe, Dayanjali; Andreev, Oleg A.; Reshetnyak, Yana K.

2013-01-01

We develop a method for pH-dependent fusion between liposomes and cellular membranes using pHLIP® (pH Low Insertion Peptide), which inserts into lipid bilayer of membrane only at low pH. Previously we establish the molecular mechanism of peptide action and show that pHLIP can target acidic diseased tissue. Here we investigate how coating of PEGylated liposomes with pHLIP might affect liposomal uptake by cells. The presence of pHLIP on the surface of PEGylated-liposomes enhanced membrane fusion and lipid exchange in a pH dependent fashion, leading to increase of cellular uptake and payload release, and inhibition of cell proliferation by liposomes containing ceramide. A novel type of pH-sensitive, “fusogenic” pHLIP-liposomes was developed, which could be used to selectively deliver various diagnostic and therapeutic agents to acidic diseased cells. PMID:23416366

Catecholamine transport in isolated lung parenchyma of pig

PubMed Central

Goldie, Roy G.; Paterson, James W.

1982-01-01

1 Lung parenchyma strips of the pig incubated at 37°C with [3H]-(-)-noradrenaline ([3H]-NA) or [3H]-(±)-isoprenaline ([3H]-Iso), accumulated radioactivity via saturable, high affinity uptake processes. Apparent saturation constants (Km) for [3H]-NA and [3H]-Iso were 1.34 × 10-6 M and 1.63 × 10-6 M respectively, while apparent transport maxima (Vmax) were 4.86 and 1.63 × 10-9 mol min-1 g-1 respectively. 2 Cellular accumulation of radioactivity from radiolabelled catecholamines was greatly reduced by lowering the temperature to 7°C, pretreatment with ouabain (100 μM), phentolamine (15 μM) or phenoxybenzamine (80 μM). However, accumulation of radioactivity derived from (3H]-NA was inhibited selectively by cocaine (10 μM) and desipramine (1 μM), while normetanephrine (80 μM) and 3-O-methylisoprenaline (50 μM) caused much greater reductions in cellular radioactivity from [3H]-Iso than from (3H]-NA. Taken together with information from kinetic studies, the results indicate that these amines are transported by separate uptake processes. 3 Cocaine (50 μM) which selectively reduced [3H]-NA transport, had no significant effect on the sensitivity (EC50) of isolated parenchyma lung strips of the pig to the contractile effects of cumulative concentrations of NA. The catechol-O-methyl transferase (COMT) inhibitor, U-0521 (60 μM), also failed to alter the potency of NA, while normetanephrine (80 μM) caused a 2 fold decrease in potency. 4 Phentolamine (15 μM), which reduced the cellular accumulation of radioactivity derived from [3H]-Iso by 64%, caused a small potentiation of Iso-induced relaxations of porcine lung strips. Normetanephrine (80 μM) and 3-O-methylisoprenaline (50 μM), which also depressed the accumulation of cellular radioactivity from [3H]-Iso by > 50%, caused rightward shifts in Iso concentration-effect curves as a result of β-adrenoceptor blockade. In sharp contrast, cortisol (80 μM) and U-0521 (60 μM), which caused smaller reductions in the cellular accumulation of radioactivity derived from [3H]-Iso, both caused an approximately 9 fold potentiation of responses to Iso in isolated lung strips. 5 The results indicate that the major sites of uptake and metabolism of NA in porcine parenchyma strip are remote from α-adrenoceptors mediating NA-induced contraction. Similarly, some major sites of uptake of Iso are remote from β-adrenoceptors mediating Iso-induced relaxation. However, β-adrenoceptors are apparently in close proximity to a compartment containing COMT activity. PMID:7104521

Positive Newborn Screen for Methylmalonic Aciduria Identifies the First Mutation in TCblR/CD320, the Gene for Cellular Uptake of Transcobalamin-bound Vitamin B12

PubMed Central

Quadros, Edward V.; Lai, Shao-Chiang; Nakayama, Yasumi; Sequeira, Jeffrey M.; Hannibal, Luciana; Wang, Sihe; Jacobsen, Donald W.; Fedosov, Sergey; Wright, Erica; Gallagher, Renata C.; Anastasio, Natascia; Watkins, David; Rosenblatt, David S.

2010-01-01

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-directed mutagenesis fully restored TCblR function. PMID:20524213

Preferential tumor cellular uptake and retention of indocyanine green for in vivo tumor imaging.

PubMed

Onda, Nobuhiko; Kimura, Masayuki; Yoshida, Toshinori; Shibutani, Makoto

2016-08-01

Indocyanine green (ICG) is a fluorescent agent approved for clinical applications by the Food and Drug Administration and European Medicines Agency. This study examined the mechanism of tumor imaging using intravenously administered ICG. The in vivo kinetics of intravenously administered ICG were determined in tumor xenografts using microscopic approaches that enabled both spatio-temporal and high-magnification analyses. The mechanism of ICG-based tumor imaging was examined at the cellular level in six phenotypically different human colon cancer cell lines exhibiting different grades of epithelioid organization. ICG fluorescence imaging detected xenograft tumors, even those < 1 mm in size, based on their preferential cellular uptake and retention of the dye following its rapid tissue-non-specific delivery, in contrast to its rapid clearance by normal tissue. Live-cell imaging revealed that cellular ICG uptake is temperature-dependent and occurs after ICG binding to the cellular membrane, a pattern suggesting endocytic uptake as the mechanism. Cellular ICG uptake correlated inversely with the formation of tight junctions. Intracellular ICG was entrapped in the membrane traffic system, resulting in its slow turnover and prolonged retention by tumor cells. Our results suggest that tumor-specific imaging by ICG involves non-specific delivery of the dye to tissues followed by preferential tumor cellular uptake and retention. The tumor cell-preference of ICG is driven by passive tumor cell-targeting, the inherent ability of ICG to bind to cell membranes, and the high endocytic activity of tumor cells in association with the disruption of their tight junctions. © 2016 UICC.

Combinatorial approaches to evaluate nanodiamond uptake and induced cellular fate

NASA Astrophysics Data System (ADS)

Eldawud, Reem; Reitzig, Manuela; Opitz, Jörg; Rojansakul, Yon; Jiang, Wenjuan; Nangia, Shikha; Zoica Dinu, Cerasela

2016-02-01

Nanodiamonds (NDs) are an emerging class of engineered nanomaterials that hold great promise for the next generation of bionanotechnological products to be used for drug and gene delivery, or for bio-imaging and biosensing. Previous studies have shown that upon their cellular uptake, NDs exhibit high biocompatibility in various in vitro and in vivo set-ups. Herein we hypothesized that the increased NDs biocompatibility is a result of minimum membrane perturbations and their reduced ability to induce disruption or damage during cellular translocation. Using multi-scale combinatorial approaches that simulate ND-membrane interactions, we correlated NDs real-time cellular uptake and kinetics with the ND-induced membrane fluctuations to derive energy requirements for the uptake to occur. Our discrete and real-time analyses showed that the majority of NDs internalization occurs within 2 h of cellular exposure, however, with no effects on cellular viability, proliferation or cellular behavior. Furthermore, our simulation analyses using coarse-grained models identified key changes in the energy profile, membrane deformation and recovery time, all functions of the average ND or ND-based agglomerate size. Understanding the mechanisms responsible for ND-cell membrane interactions could possibly advance their implementation in various biomedical applications.

Combinatorial approaches to evaluate nanodiamond uptake and induced cellular fate

PubMed Central

Eldawud, Reem; Reitzig, Manuela; Opitz, Jörg; Rojansakul, Yon; Jiang, Wenjuan; Nangia, Shikha; Dinu, Cerasela Zoica

2016-01-01

Nanodiamonds (NDs) are an emerging class of engineered nanomaterials that hold great promise for the next generation of bionanotechnological products to be used for drug and gene delivery, or for bio-imaging and biosensing. Previous studies have shown that upon their cellular uptake, NDs exhibit high biocompatibility in various in vitro and in vivo set-ups. Herein we hypothesized that the increased NDs biocompatibility is a result of minimum membrane perturbations and their reduced ability to induce disruption or damage during cellular translocation. Using multi-scale combinatorial approaches that simulate ND-membrane interactions, we correlated NDs real-time cellular uptake and kinetics with the ND-induced membrane fluctuations to derive energy requirements for the uptake to occur. Our discrete and real-time analyses showed that the majority of NDs internalization occurs within 2 h of cellular exposure, however, with no effects on cellular viability, proliferation or cellular behavior. Furthermore, our simulation analyses using coarse-grained models identified key changes in the energy profile, membrane deformation and recovery time, all functions of the average ND or ND-based agglomerate size. Understanding the mechanisms responsible for ND-cell membrane interactions could possibly advance their implementation in various biomedical applications. PMID:26820775

Enantioselective cellular localisation of europium(iii) coordination complexes† †Electronic supplementary information (ESI) available: Complex synthesis and characterisation, selected cell images showing correspondence with commercial lysosomal or mitochondrial stains and spectral imaging profiles. See DOI: 10.1039/c7sc04422d

PubMed Central

Frawley, Andrew T.; Linford, Holly V.; Starck, Matthieu; Pal, Robert

2017-01-01

The selective mitochondrial localisation of the Λ enantiomer of three different emissive europium(iii) complexes in NIH 3T3 and MCF7 cells contrasts with the behaviour of the Δ enantiomer, for which a predominant lysosomal localisation was observed by confocal microscopy. In each case, cell uptake occurs via macropinocytosis. PMID:29675151

The significance of transferrin receptors in oncology: the development of functional nano-based drug delivery systems.

PubMed

Tortorella, Stephanie; Karagiannis, Tom C

2014-01-01

Anticancer therapeutic research aims to improve clinical management of the disease through the development of strategies that involve currently-relevant treatment options and targeted delivery. Tumour-specific and -targeted delivery of compounds to the site of malignancy allows for enhanced cellular uptake, increased therapeutic benefit with high intratumoural drug concentrations, and decreased systemic exposure. Due to the upregulation of transferrin receptor expression in a wide variety of cancers, its function and its highly efficient recycling pathway, strategies involving the selective targeting of the receptor are well documented. Direct conjugation and immunotoxin studies using the transferrin peptide or anti-transferrin receptor antibodies as the targeting moiety have established the capacity to enhance cellular uptake, cross the blood brain barrier, limit systemic toxicity and reverse multi-drug resistance. Limitations in direct conjugation, including the difficulty in linking an adequate amount of therapeutic compound to the ligand or antibody have identified the requirement to develop novel delivery methods. The application of nanoparticulate theory in the development of functional drug delivery systems has proven to be most promising, with the ability to selectively modify size-dependent properties and surface chemistry. The transferrin modification on a range of nanoparticle formulations enhances selective cellular uptake through transferrin-mediated processes, and increases therapeutic benefit through the ability to encapsulate high concentrations of relevant drug to the tumour site. Although ineffective in crossing the blood brain barrier in its free form, chemotherapeutic compounds including doxorubicin, may be loaded into transferrin-conjugated nanocarriers and impart cytotoxic effects in glioma cells in vitro and in vivo. Additionally, transferrin-targeted nanoparticles may be used in selective diagnostic applications with enhanced selectivity and sensitivity. Four transferrin-modified nano-based drug delivery systems are currently in early phases of human clinical trials. Despite the collective promise, inconsistencies in some studies have exposed some limitations in current formulations and the difficulty in translating preliminary studies into clinically-relevant therapeutic options. The main objective of this review is to investigate the development of transferrin targeted nano-based drug delivery systems in order to establish the use of transferrin as a cancer-targeted moiety, and to ultimately evaluate the progression of cancer therapeutic strategies for future research.

Selective Intracellular Delivery of Ganglioside GM3-Binding Peptide through Caveolae/Raft-Mediated Endocytosis.

PubMed

Matsubara, Teruhiko; Otani, Ryohei; Yamashita, Miki; Maeno, Haruka; Nodono, Hanae; Sato, Toshinori

2017-02-13

Glycosphingolipids are major components of the membrane raft, and several kinds of viruses and bacterial toxins are known to bind to glycosphingolipids in the membrane raft. Since the viral genes and pathogenic proteins that are taken into cells are directly delivered to their target organelles, caveolae/raft-mediated endocytosis represents a promising pathway for specific delivery. In the present study, we demonstrated the ability of an artificial pentadecapeptide, which binds to ganglioside GM3, to deliver protein into cells by caveolae/raft-mediated endocytosis. The cellular uptake of a biotinylated GM3-binding peptide (GM3BP)-avidin complex into HeLa cells was observed, and the cellular uptake of this complex was inhibited by an incubation with sialic acid or endocytic inhibitors such as methyl-ß-cyclodextrin, and also by an incubation at 4 °C. These results indicate that the GM3BP-avidin complex bind to GM3 in membrane raft, and are taken into cell through caveolae/raft-mediated endocytosis. The GM3BP-avidin complex was transported into cells and localized around the nucleus more slowly than a human immunodeficiency virus type 1 TAT peptide. Furthermore, the uptake of a green fluorescent protein (GFP) linked with GM3BP into HeLa cells was similar to that of the GM3BP-avidin complex, and the localization of the GM3BP-GFP fusion protein was markedly different with that of the TAT-GFP fusion protein. The uptake and trafficking of GM3BP were distinguished from conventional cell-penetrating peptides. GM3BP has potential as a novel peptide for the selective delivery of therapeutic proteins and materials into cells in addition to being a cell-penetrating peptide.

Cellular uptake and retention measurements of alkylphosphocholines in the SK-BR-3 breast cancer and Molt-4 leukemia cell line using capillary gas chromatography.

PubMed

Brochez, V; Van Heuverswyn, D; Diniz, J A; De Potter, C R; Van den Eeckhout, E G

1999-05-01

The determination of cellular content of octadecylphosphocholine (D-19391) and hexadecylphosphocholine (HePC, D-18506), two anticancer agents of the alkylphosphocholine group, using capillary gas chromatography is described. The compounds' cytotoxicity was first determined by the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium] assay, being indicative for the concentration used in the uptake and retention measurements. D-19391 was added to the SK-BR-3 breast cancer cell line and HePC to the Molt-4 leukemia cell line in concentrations of, respectively, 18.6 and 15.0 microM, during a 36-h incubation period at 37 degrees C, 5% CO2. HePC uptake in the leukemia cells was followed by a 24-h reversibility test in drug-free medium. Subsequently, sample clean-up was performed on a weak cation-exchange column. For the quantitative analysis, HePC was used as internal standard for the D-19391 measurements and vice versa. Derivatization of the samples with trimethylsilylbromide was followed by capillary gas chromatographic analysis. From these data we conclude that our uptake results are quite similar with those of a previous study of HePC cellular uptake in the more resistant Caco-2T colon cancer cell line. Without having investigated the mechanism that underlies the cellular uptake results obtained, our study points to no direct correlation between the compounds' cellular uptake and their cytotoxic effects.

[Effects of sub-micro emulsion composition on cellular disposition of incorporated lipophilic drug].

PubMed

Sun, Xiao-Yi; Xiang, Zhi-Qiang; Wu, Shuo; Lv, Yuan-Yuan; Liang, Wen-Quan

2013-09-01

To investigate the effects of sub-micro emulsion composition on cellular uptake and disposition of incorporated lipophilic drug. Sub-micro emulsions containing 10 % oil, 1.2 % lecithin and 2.25 % glycerol were prepared, and the fluorescent agent coumarin 6 was used as a model drug. The effects of oil types, co-surfactants and cationic lipid on uptake and elimination kinetics of 6-coumarin in HeLa cells were studied. The uptake mechanism of sub-micro emulsions was further investigated. Oil type and Tweens had no influence on the cellular uptake. Modifications of surfactants with Span series increased the cellular influx, among which Span 20 with hydrophilic-lipophilic balance (HLB) value of 8.6 was the best enhancer. The intracellular drug level reached up to (46.09 ± 1.98)ng/μg protein which had significant difference with control group [(38.54 ± 0.34)ng/μg protein]. The positively charged emulsions significantly increased the uptake rate constant and elimination rate constant which were 4 times and 1.5 times of those in anionic groups, respectively. The uptake enhancement was also observed in cationic emulsions, cellular concentrations at plateau were (42.73 ± 0.84)ng/μg protein, which was about 3 times of that in anionic emulsions [(15.71 ± 0.74)ng/μg protein], when extracellular drug concentration kept at 100 ng/ml. Cationic emulsions delivered the payload mainly by direct drug transfer to contacted cells, while the negative ones depended on both drug passive diffusion and clathrin-mediated endocytosis of drug containing oil droplets which accounted for 20% of the intracellular drug. Interfacial characteristic of sub-micro emulsions such as co-surfactants HLB as well as zeta potentials can influence lipophilic drug both in cellular uptake and elimination.

Inhibitory effect of a toxic peptide isolated from a waterbloom of Microcystis sp. (Cyanobacteria) on iron uptake by rabbit reticulocytes.

PubMed

Rojas, M; Nuñez, M T; Zambrano, F

1990-01-01

The effect of a soluble toxin purified from the algae bloom of a eutrophic lake dominated by Microcystis on the receptor-mediated endocytosis of ferro-transferrin in rabbit reticulocytes was studied. The toxin was a very effective inhibitor of cell iron uptake. Kinetic studies using 125I, 59Fe-labeled transferrin indicated that the step of ferrotransferrin internalization was selectively inhibited by the toxin while the surface receptor-binding capacity, the externalization of previously internalized transferrin, and the cellular ATP levels were not affected. These findings indicate that the reduction of iron uptake caused by the toxin is due to inhibition of the internalization of surface-located transferrin-transferrin receptor complexes, perhaps due to a disruption of cytoskeleton integrity.

Discovery of novel, high potent, ABC type PTP1B inhibitors with TCPTP selectivity and cellular activity.

PubMed

Liu, Peihong; Du, Yongli; Song, Lianhua; Shen, Jingkang; Li, Qunyi

2016-08-08

Protein tyrosine phosphatase 1B (PTP1B) as a key negative regulator of both insulin and leptin receptor pathways has been an attractive therapeutic target for the treatment of type 2 diabetes mellitus (T2DM) and obesity. With the goal of enhancing potency and selectivity of the PTP1B inhibitors, a series of methyl salicylate derivatives as ABC type PTP1B inhibitors (P1-P7) were discovered. More importantly, compound P6 exhibited high potent inhibitory activity (IC50 = 50 nM) for PTP1B with 15-fold selectivity over T-cell PTPase (TCPTP). Further studies on cellular activities revealed that compound P6 could enhance insulin-mediated insulin receptor β (IRβ) phosphorylation and insulin-stimulated glucose uptake. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Fluorescence-encoded gold nanoparticles: library design and modulation of cellular uptake into dendritic cells.

PubMed

Rodriguez-Lorenzo, Laura; Fytianos, Kleanthis; Blank, Fabian; von Garnier, Christophe; Rothen-Rutishauser, Barbara; Petri-Fink, Alke

2014-04-09

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo- and hetero-functional fluorescence-encoded gold nanoparticles (Au-NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)-based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV-Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence-activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero-functionalized Au-NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Correlation of Emulsion Structure with Cellular Uptake Behavior of Encapsulated Bioactive Nutrients: Influence of Droplet Size and Interfacial Structure.

PubMed

Lu, Wei; Kelly, Alan L; Maguire, Pierce; Zhang, Hongzhou; Stanton, Catherine; Miao, Song

2016-11-16

In this study, an in vitro Caco-2 cell culture assay was employed to evaluate the correlation between emulsion structure and cellular uptake of encapsulated β-carotene. After 4 h of incubation, an emulsion stabilized with whey protein isolate showed the highest intracellular accumulation of β-carotene (1.06 μg), followed by that stabilized with sodium caseinate (0.60 μg) and Tween 80 (0.20 μg), which are 13-, 7.5-, and 2.5-fold higher than that of free β-carotene (0.08 μg), respectively. Emulsions with small droplet size (239 ± 5 nm) showed a higher cellular uptake of β-carotene (1.56 μg) than emulsiond with large droplet size (489 ± 9 nm) (0.93 μg) (p < 0.01). The results suggested that delivery in an emulsion significantly improved the cellular uptake of β-carotene and thus potentially its bioavailability; uptake was closely correlated with the interfacial composition and droplet size of emulsions. The findings support the potential for achieving optimal controlled and targeted delivery of bioactive nutrients by structuring emulsions.

Incorporation of a selective sigma-2 receptor ligand enhances uptake of liposomes by multiple cancer cells

PubMed Central

Zhang, Yifei; Huang, Yixian; Zhang, Peng; Gao, Xiang; Gibbs, Robert B; Li, Song

2012-01-01

Background: The sigma-2 receptor is an attractive target for tumor imaging and targeted therapy because it is overexpressed in multiple types of solid tumors, including prostate cancer, breast cancer, and lung cancer. SV119 is a synthetic small molecule that binds to sigma-2 receptors with high affinity and specificity. This study investigates the utility of SV119 in mediating the selective targeting of liposomal vectors in various types of cancer cells. Methods: SV119 was covalently linked with polyethylene glycol-dioleyl amido aspartic acid conjugate (PEG-DOA) to generate a novel functional lipid, SV119-PEG-DOA. This lipid was utilized for the preparation of targeted liposomes to enhance their uptake by cancer cells. Liposomes with various SV119 densities (0, 1, 3, and 5 mole%) were prepared and their cellular uptake was investigated in several tumor cell lines. In addition, doxorubicin (DOX) was loaded into the targeted and unmodified liposomes, and the cytotoxic effect on the DU-145 cells was evaluated by MTT assay. Results: Liposomes with or without SV119-PEG-DOA both have a mean diameter of approximately 90 nm and a neutral charge. The incorporation of SV119-PEG-DOA significantly increased the cellular uptake of liposomes by the DU-145, PC-3, A549, 201T, and MCF-7 tumor cells, which was shown by fluorescence microscopy and the quantitative measurement of fluorescence intensity. In contrast, the incorporation of SV119 did not increase the uptake of liposomes by the normal BEAS-2B cells. In a time course study, the uptake of SV119 liposomes by DU-145 cells was also significantly higher at each time point compared to the unmodified liposomes. Furthermore, the DOX-loaded SV119 liposomes showed significantly higher cytotoxicity to DU-145 cells compared to the DOX-loaded unmodified liposomes. Conclusion: SV119 liposomes were developed for targeted drug delivery to cancer cells. The targeting efficiency and specificity of SV119 liposomes to cancer cells was demonstrated in vitro. The results of this study suggest that SV119-modified liposomes might be a promising drug carrier for tumor-targeted delivery. PMID:22927761

Cholesteryl oleate-loaded cationic solid lipid nanoparticles as carriers for efficient gene-silencing therapy

PubMed Central

Suñé-Pou, Marc; Prieto-Sánchez, Silvia; El Yousfi, Younes; Boyero-Corral, Sofía; Nardi-Ricart, Anna; Nofrerias-Roig, Isaac; Pérez-Lozano, Pilar; García-Montoya, Encarna; Miñarro-Carmona, Montserrat; Ticó, Josep Ramón; Suñé-Negre, Josep Mª; Hernández-Munain, Cristina; Suñé, Carlos

2018-01-01

Background Cationic solid lipid nanoparticles (SLNs) have been given considerable attention for therapeutic nucleic acid delivery owing to their advantages over viral and other nanoparticle delivery systems. However, poor delivery efficiency and complex formulations hinder the clinical translation of SLNs. Aim The aim of this study was to formulate and characterize SLNs incorporating the cholesterol derivative cholesteryl oleate to produce SLN–nucleic acid complexes with reduced cytotoxicity and more efficient cellular uptake. Methods Five cholesteryl oleate-containing formulations were prepared. Laser diffraction and laser Doppler microelectrophoresis were used to evaluate particle size and zeta potential, respectively. Nanoparticle morphology was analyzed using electron microscopy. Cytotoxicity and cellular uptake of lipoplexes were evaluated using flow cytometry and fluorescence microscopy. The gene inhibition capacity of the lipoplexes was assessed using siRNAs to block constitutive luciferase expression. Results We obtained nanoparticles with a mean diameter of approximately 150–200 nm in size and zeta potential values of 25–40 mV. SLN formulations with intermediate concentrations of cholesteryl oleate exhibited good stability and spherical structures with no aggregation. No cell toxicity of any reference SLN was observed. Finally, cellular uptake experiments with DNA-and RNA-SLNs were performed to select one reference with superior transient transfection efficiency that significantly decreased gene activity upon siRNA complexation. Conclusion The results indicate that cholesteryl oleate-loaded SLNs are a safe and effective platform for nonviral nucleic acid delivery. PMID:29881274

Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity.

PubMed

Othman, Basmah A; Greenwood, Christina; Abuelela, Ayman F; Bharath, Anil A; Chen, Shu; Theodorou, Ioannis; Douglas, Trevor; Uchida, Maskai; Ryan, Mary; Merzaban, Jasmeen S; Porter, Alexandra E

2016-06-01

ZnO nanoparticles (NPs) are reported to show a high degree of cancer cell selectivity with potential use in cancer imaging and therapy. Questions remain about the mode by which the ZnO NPs cause cell death, whether they exert an intra- or extracellular effect, and the resistance among different cancer cell types to ZnO NP exposure. The present study quantifies the variability between the cellular toxicity, dynamics of cellular uptake, and dissolution of bare and RGD (Arg-Gly-Asp)-targeted ZnO NPs by MDA-MB-231 cells. Compared to bare ZnO NPs, RGD-targeting of the ZnO NPs to integrin αvβ3 receptors expressed on MDA-MB-231 cells appears to increase the toxicity of the ZnO NPs to breast cancer cells at lower doses. Confocal microscopy of live MDA-MB-231 cells confirms uptake of both classes of ZnO NPs with a commensurate rise in intracellular Zn(2+) concentration prior to cell death. The response of the cells within the population to intracellular Zn(2+) is highly heterogeneous. In addition, the results emphasize the utility of dynamic and quantitative imaging in understanding cell uptake and processing of targeted therapeutic ZnO NPs at the cellular level by heterogeneous cancer cell populations, which can be crucial for the development of optimized treatment strategies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modification of meta-iodobenzylguanidine uptake in neuroblastoma cells by elevated temperature.

PubMed Central

Armour, A.; Mairs, R. J.; Gaze, M. N.; Wheldon, T. E.

1994-01-01

Successful imaging or treatment of neuroblastoma with 131I-meta-iodobenzylguanidine (131I-mIBG) depends on the selectivity of active (type 1) uptake of mIBG in neuroblastoma cells relative to passive (type 2) uptake present in most normal tissues. This study investigates the effects of moderately elevated temperature (39-41 degrees C) on the cellular uptake of 131I-mIBG in two neuroblastoma cell lines [SK-N-BE(2c) and IMR-32] and in a non-neuronal (ovarian carcinoma) cell line (A2780). In SK-N-BE(2c), a cell line with high active uptake capacity, the specific (type 1) uptake was reduced by 75% (P < 0.001) at 39 degrees C. Both IMR-32 and A2780 have a low capacity for accumulation of mIBG by active uptake. These cell lines demonstrated a statistically significant increase in accumulation at 39 degrees C, mainly as a result of increased non-specific transport. At 41 degrees C uptake of 131I-mIBG was reduced in all cell lines. Thus, the active component of mIBG uptake is more vulnerable to increased temperature than the passive component. It seems probable that moderately increased temperature will have an unfavourable effect on the therapeutic differential for targeted radiotherapy of neuroblastoma using radiolabelled mIBG. PMID:8080728

Bioaccessibility and Cellular Uptake of β-Carotene Encapsulated in Model O/W Emulsions: Influence of Initial Droplet Size and Emulsifiers

PubMed Central

Kelly, Alan L.

2017-01-01

The effects of the initial emulsion structure (droplet size and emulsifier) on the properties of β-carotene-loaded emulsions and the bioavailability of β-carotene after passing through simulated gastrointestinal tract (GIT) digestion were investigated. Exposure to GIT significantly changed the droplet size, surface charge and composition of all emulsions, and these changes were dependent on their initial droplet size and the emulsifiers used. Whey protein isolate (WPI)-stabilized emulsion showed the highest β-carotene bioaccessibility, while sodium caseinate (SCN)-stabilized emulsion showed the highest cellular uptake of β-carotene. The bioavailability of emulsion-encapsulated β-carotene based on the results of bioaccessibility and cellular uptake showed the same order with the results of cellular uptake being SCN > TW80 > WPI. An inconsistency between the results of bioaccessibility and bioavailability was observed, indicating that the cellular uptake assay is necessary for a reliable evaluation of the bioavailability of emulsion-encapsulated compounds. The findings in this study contribute to a better understanding of the correlation between emulsion structure and the digestive fate of emulsion-encapsulated nutrients, which make it possible to achieve controlled or potential targeted delivery of nutrients by designing the structure of emulsion-based carriers. PMID:28930195

Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group.

PubMed

Petrova, Natalya S; Chernikov, Ivan V; Meschaninova, Mariya I; Dovydenko, Iiya S; Venyaminova, Aliya G; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

2012-03-01

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5'-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

Spectroscopic studies of anthracyclines: Structural characterization and in vitro tracking

NASA Astrophysics Data System (ADS)

Szafraniec, Ewelina; Majzner, Katarzyna; Farhane, Zeineb; Byrne, Hugh J.; Lukawska, Malgorzata; Oszczapowicz, Irena; Chlopicki, Stefan; Baranska, Malgorzata

2016-12-01

A broad spectroscopic characterization, using ultraviolet-visible (UV-vis) and Fourier transform infrared absorption as well as Raman scattering, of two commonly used anthracyclines antibiotics (DOX) daunorubicin (DNR), their epimers (EDOX, EDNR) and ten selected analogs is presented. The paper serves as a comprehensive spectral library of UV-vis, IR and Raman spectra of anthracyclines in the solid state and in solution. The particular advantage of Raman spectroscopy for the measurement and analysis of individual antibiotics is demonstrated. Raman spectroscopy can be used to monitor the in vitro uptake and distribution of the drug in cells, using both 488 nm and 785 nm as source wavelengths, with submicrometer spatial resolution, although the cellular accumulation of the drug is different in each case. The high information content of Raman spectra allows studies of the drug-cell interactions, and so the method seems very suitable for monitoring drug uptake and mechanisms of interaction with cellular compartments at the subcellular level.

Interaction of carbon nanohorns with plants: Uptake and biological effects

DOE PAGES

Lahiani, Mohamed H.; Chen, Jihua; Irin, Fahmida; ...

2014-10-07

Single-Walled Carbon Nanohorns (SWCNHs) are a unique carbon-based nanomaterial with promising application in different fields including, medicine, genetic engineering and horticulture. Here, we investigated the biological response of six crop species (barley, corn, rice, soybean, switchgrass, tomato) and tobacco cell culture to the exposure of SWCNHs. We found that SWCNHs can activate seed germination of selected crops and enhance growth of different organs of corn, tomato, rice and soybean. At cellular level, growth of tobacco cells was increased in response to exposure of SWCNHs (78% increase compared to control). Uptake of SWCNHs by exposed crops and tobacco cells was confirmedmore » by transmission electron microscopy (TEM) and quantified by microwave induced heating (MIH) technique. At genetic level, SWCNHs were able to affect expression of a number of tomato genes that are involved in stress responses, cellular responses and metabolic processes. Our conclusion is that SWCNHs can be used as plant growth regulators and have the potential for plant-related applications.« less

Substrate Profile and Metal-ion Selectivity of Human Divalent Metal-ion Transporter-1*

PubMed Central

Illing, Anthony C.; Shawki, Ali; Cunningham, Christopher L.; Mackenzie, Bryan

2012-01-01

Divalent metal-ion transporter-1 (DMT1) is a H+-coupled metal-ion transporter that plays essential roles in iron homeostasis. DMT1 exhibits reactivity (based on evoked currents) with a broad range of metal ions; however, direct measurement of transport is lacking for many of its potential substrates. We performed a comprehensive substrate-profile analysis for human DMT1 expressed in RNA-injected Xenopus oocytes by using radiotracer assays and the continuous measurement of transport by fluorescence with the metal-sensitive PhenGreen SK fluorophore. We provide validation for the use of PhenGreen SK fluorescence quenching as a reporter of cellular metal-ion uptake. We determined metal-ion selectivity under fixed conditions using the voltage clamp. Radiotracer and continuous measurement of transport by fluorescence assays revealed that DMT1 mediates the transport of several metal ions that were ranked in selectivity by using the ratio Imax/K0.5 (determined from evoked currents at −70 mV): Cd2+ > Fe2+ > Co2+, Mn2+ ≫ Zn2+, Ni2+, VO2+. DMT1 expression did not stimulate the transport of Cr2+, Cr3+, Cu+, Cu2+, Fe3+, Ga3+, Hg2+, or VO+. 55Fe2+ transport was competitively inhibited by Co2+ and Mn2+. Zn2+ only weakly inhibited 55Fe2+ transport. Our data reveal that DMT1 selects Fe2+ over its other physiological substrates and provides a basis for predicting the contribution of DMT1 to intestinal, nasal, and pulmonary absorption of metal ions and their cellular uptake in other tissues. Whereas DMT1 is a likely route of entry for the toxic heavy metal cadmium, and may serve the metabolism of cobalt, manganese, and vanadium, we predict that DMT1 should contribute little if at all to the absorption or uptake of zinc. The conclusion in previous reports that copper is a substrate of DMT1 is not supported. PMID:22736759

Effect of arginine methylation on the RNA recognition and cellular uptake of Tat-derived peptides.

PubMed

Li, Jhe-Hao; Chiu, Wen-Chieh; Yao, Yun-Chiao; Cheng, Richard P

2015-05-01

Arginine (Arg) methylation is a common post-translational modification that regulates gene expression and viral infection. The HIV-1 Tat protein is an essential regulatory protein for HIV proliferation, and is methylated in the cell. The basic region (residues 47-57) of the Tat protein contains six Arg residues, and is responsible for two biological functions: RNA recognition and cellular uptake. In this study, we explore the effect of three different methylation states at each Arg residue in Tat-derived peptides on the two biological functions. The Tat-derived peptides were synthesized by solid phase peptide synthesis. TAR RNA binding of the peptides was assessed by electrophoresis mobility shift assays. The cellular uptake of the peptides into Jurkat cells was determined by flow cytometry. Our results showed that RNA recognition was affected by both methylation state and position. In particular, asymmetric dimethylation at position 53 decreased TAR RNA binding affinity significantly, but unexpectedly less so upon asymmetric dimethylation at position 52. The RNA binding affinity even slightly increased upon methylation at some of the flanking Arg residues. Upon Arg methylation, the cellular uptake of Tat-derived peptides mostly decreased. Interestingly, cellular uptake of Tat-derived peptides with a single asymmetrically dimethylated Arg residue was similar to the native all Arg peptide (at 120 μM). Based on our results, TAR RNA binding apparently required both guanidinium terminal NH groups on Arg53, whereas cellular uptake apparently required guanidinium terminal NH₂ groups instead. These results should provide insight into how nature uses arginine methylation to regulate different biological functions, and should be useful for the development of functional molecules with methylated arginines. Copyright © 2015. Published by Elsevier Ltd.

(18)F-FBPA as a tumor specific tracer of L-type amino acid transporter 1 (LAT1): PET evaluation in tumor and inflammation compared to (18)F-FDG and (11)C-methionine.

PubMed

Watabe, Tadashi; Hatazawa, Jun

2015-01-01

(18)F-FDG-PET is used worldwide for oncology patients. However, we sometimes encounter false positive cases of (18)F-FDG PET, such as moderate uptake in the inflammatory lesion, because (18)F-FDG accumulates not only in the cancer cells but also in the inflammatory cells (macrophage, granulation tissue, etc). To overcome this limitation of (18)F-FDG, we started to use (4-borono-2- [(18)F]fluoro-L-phenylalanine) (18)F-FBPA, an artificial amino acid tracer which is focusing attention as a tumor specific PET tracer. Physiological accumulation of (18)F-FBPA is limited in the kidney and urinary tract in humans, which enable preferable evaluation of uptake in the abdominal organs compared to (11)C-methionine ((11)C-MET). The purpose of this study was to evaluate (18)F-FBPA as a tumor specific tracer by in vitro cellular uptake analysis focusing on the selectivity of L-type amino acid transporter 1 (LAT1), which is specifically expressed in tumor cells, and in vivo PET analysis in rat xenograft and inflammation models compared to (18)F-FDG and (11)C-methionine. Uptake inhibition and efflux experiments were performed in HEK293-LAT1 and LAT2 cells using cold BPA, cold (18)F-FBPA, and hot (18)F-FBPA to evaluate LAT affinity and transport capacity. Position emission tomography studies were performed in rat xenograft model of C6 glioma 2 weeks after the implantation (n=9, body weight=197±10.5g) and subcutaneous inflammation model 4 days after the injection of turpentine oil (n=9, body weight=197±14.4g). Uptake on static PET images were compared among (18)F-FBPA at 60-70min post injection, (18)F-FDG at 60-70min, and (11)C-MET at 20-30min in the tumors and the inflammatory lesions by maximum standardized uptake value (SUVmax). Cellular uptake analysis showed no significant difference in inhibitory effect and efflux of LAT1 between cold (18)F-FBPA and cold BPA, suggesting the same affinity and transport capacity via LAT1. Uptake of (18)F-FBPA via LAT1 was superior to LAT2 by the concentration dependent uptake analysis. Position emission tomography analysis using SUVmax showed significantly higher accumulation of (18)F-FDG in the tumor and the inflammatory lesions (7.19±2.11 and 4.66±0.63, respectively) compared to (18)F-FBPA (3.23±0.40 and 1.86±0.19, respectively) and (11)C-MET (3.39±0.43 and 1.63±0.11, respectively) (P<0.01 by Tukey test). No significant difference was observed between (18)F-FBPA and (11)C-MET. (18)F-FBPA showed high selectivity of LAT1 by in vitro cellular uptake analysis, suggesting the potential as a tumor-specific substrate. In vivo PET analysis showed significantly lower uptake of (18)F-FBPA and (11)C-MET in the inflammatory lesions compared to (18)F-FDG, suggesting comparable utility of (18)F-FBPA PET to (11)C-MET PET in differentiating between the tumor and the inflammation.

Cellular uptake of titanium and vanadium from addition of salts or fretting corrosion in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maurer, A.M.; Merritt, K.; Brown, S.A.

1994-02-01

The use of titanium and titanium-6% aluminum-4% vanadium alloy for dental and orthopedic implants has increased in the last decade. The implants are presumed to be compatible because oseointegration, bony apposition, and cell attachment are known. However, the cellular association of titanium and vanadium have remained unknown. This study examined the uptake of salts or fretting corrosion products. Titanium was not observed to be toxic to the cells. Vanadium was toxic at levels greater than 10[mu]g/mL. The percentage of cellular association of titanium was shown to be about 10 times that of vanadium. The percentage of cellular association of eithermore » element was greater from fretting corrosion than from the addition of salts. The presence of vanadium did not affect the cellular uptake of titanium. The presence of titanium decreased the cell association of vanadium.« less

Selective electrodiffusion of zinc ions in a Zrt-, Irt-like protein, ZIPB

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, W.; Fu, D.; Chai, J.

2010-12-10

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstratedmore » that ZIPB is selective for two group 12 transition metal ions, Zn{sup 2+} and Cd{sup 2+}, whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.« less

Non-toxic fluorescent phosphonium probes to detect mitochondrial potential.

PubMed

Šarić, Ana; Crnolatac, Ivo; Bouillaud, Frédéric; Sobočanec, Sandra; Mikecin, Ana-Matea; Mačak Šafranko, Željka; Delgeorgiev, Todor; Piantanida, Ivo; Balog, Tihomir; Petit, Patrice X

2017-03-22

We evaluated our phosphonium-based fluorescent probes for selective staining of mitochondria. Currently used probes for monitoring mitochondrial membrane potential show varying degrees of interference with cell metabolism, photo-induced damage and probe binding. Here presented probes are characterised by highly efficient cellular uptake and specific accumulation in mitochondria. Fluorescent detection of the probes was accomplished using flow cytometry and confocal microscopy imaging of yeast and mammalian cells. Toxicity analysis (impedimetry-xCELLigence for the cellular proliferation and Seahorse technology for respiratory properties) confirms that these dyes exhibit no-toxicity on mitochondrial or cellular functioning even for long time incubation. The excellent chemical and photophysical stability of the dyes makes them promising leads toward improved fluorescent probes. Therefore, the probes described here offer to circumvent the problems associated with existing-probe's limitations.

Non-toxic fluorescent phosphonium probes to detect mitochondrial potential

NASA Astrophysics Data System (ADS)

Šarić, Ana; Crnolatac, Ivo; Bouillaud, Frédéric; Sobočanec, Sandra; Mikecin, Ana-Matea; Mačak Šafranko, Željka; Delgeorgiev, Todor; Piantanida, Ivo; Balog, Tihomir; Petit, Patrice X.

2017-03-01

We evaluated our phosphonium-based fluorescent probes for selective staining of mitochondria. Currently used probes for monitoring mitochondrial membrane potential show varying degrees of interference with cell metabolism, photo-induced damage and probe binding. Here presented probes are characterised by highly efficient cellular uptake and specific accumulation in mitochondria. Fluorescent detection of the probes was accomplished using flow cytometry and confocal microscopy imaging of yeast and mammalian cells. Toxicity analysis (impedimetry—xCELLigence for the cellular proliferation and Seahorse technology for respiratory properties) confirms that these dyes exhibit no-toxicity on mitochondrial or cellular functioning even for long time incubation. The excellent chemical and photophysical stability of the dyes makes them promising leads toward improved fluorescent probes. Therefore, the probes described here offer to circumvent the problems associated with existing-probe’s limitations.

Target-specific cellular uptake of PLGA nanoparticles coated with poly(L-lysine)-poly(ethylene glycol)-folate conjugate.

PubMed

Kim, Sun Hwa; Jeong, Ji Hoon; Chun, Ki Woo; Park, Tae Gwan

2005-09-13

Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with anionic surface charge were surface coated with cationic di-block copolymer, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL) conjugate, for enhancing their site-specific intracellular delivery against folate receptor overexpressing cancer cells. The PLGA nanoparticles coated with the conjugate were characterized in terms of size, surface charge, and change in surface composition by XPS. By employing the flow cytometry method and confocal image analysis, the extent of cellular uptake was comparatively evaluated under various conditions. PLL-PEG-FOL coated PLGA nanoparticles demonstrated far greater extent of cellular uptake to KB cells, suggesting that they were mainly taken up by folate receptor-mediated endocytosis. The enhanced cellular uptake was also observed even in the presence of serum proteins, possibly due to the densely seeded PEG chains. The PLL-PEG-FOL coated PLGA nanoparticles could be potentially applied for cancer cell targeted delivery of various therapeutic agents.

Selenium uptake through cystine transporter mediated by glutathione conjugation.

PubMed

Tobe, Takao; Ueda, Koji; Aoki, Akira; Okamoto, Yoshinori; Kojima, Nakao; Jinno, Hideto

2017-01-01

Selenium (Se) is an essential trace element and is regarded as a protective agent against cancer. In particular, antioxidant effects of selenoenzymes contribute to cancer prevention. Se can also produce reactive oxygen species and, thereby, exert cancer-selective cytotoxicity. Selenodiglutathione (SDG) is a primary Se metabolite conjugated to two glutathione (GSH) moieties. SDG increases intracellular Se accumulation and is more toxic than selenous acid (H 2 SeO 3 ), but the mechanisms for importing Se compounds into cells are not fully understood. Here, we propose a novel mechanism for importing Se, in the form of SDG. Cellular intake of Se compounds was assessed based on Se accumulation, as detected by ICP-MS. SDG incorporation was decreased in the presence of thiols (GSH, cysteine or their oxidized forms, GSSG and cystine), whereas H 2 SeO 3 uptake was increased by addition of GSH or cysteine. Cellular SDG uptake was decreased by pretreatment with specific inhibitors against gamma-glutamyl transpeptidase (GGT) or the cystine/glutamate antiporter (system x c - ). Furthermore, siRNA against xCT, which is the light chain component of system x c - , significantly decreased SDG incorporation. These data suggest an involvement of SDG in Se incorporation, with SDG processed at the cell surface by GGT, leading to formation of selenodicysteine which, in turn, is likely to be imported via xCT. Because GGT and xCT are highly expressed in cancer cells, these mechanisms mediated by the cystine transporter might underlie the cancer-selective toxicity of Se. In addition, the system described in our study appears to represent a physiological transport mechanism for the essential element Se.

Impact of silica nanoparticle surface chemistry on protein corona formation and consequential interactions with biological cells.

PubMed

Kurtz-Chalot, Andréa; Villiers, Christian; Pourchez, Jérémie; Boudard, Delphine; Martini, Matteo; Marche, Patrice N; Cottier, Michèle; Forest, Valérie

2017-06-01

Nanoparticles (NP) physico-chemical features greatly influence NP/cell interactions. NP surface functionalization is often used to improve NP biocompatibility or to enhance cellular uptake. But in biological media, the formation of a protein corona adds a level of complexity. The aim of this study was to investigate in vitro the influence of NP surface functionalization on their cellular uptake and the biological response induced. 50nm fluorescent silica NP were functionalized either with amine or carboxylic groups, in presence or in absence of polyethylene glycol (PEG). NP were incubated with macrophages, cellular uptake and cellular response were assessed in terms of cytotoxicity, pro-inflammatory response and oxidative stress. The NP protein corona was also characterized by protein mass spectroscopy. Results showed that NP uptake was enhanced in absence of PEG, while NP adsorption at the cell membrane was fostered by an initial positively charged NP surface. NP toxicity was not correlated with NP uptake. NP surface functionalization also influenced the formation of the protein corona as the profile of protein binding differed among the NP types. Copyright © 2017 Elsevier B.V. All rights reserved.

Cellular delivery of PEGylated PLGA nanoparticles.

PubMed

Pamujula, Sarala; Hazari, Sidhartha; Bolden, Gevoni; Graves, Richard A; Chinta, Dakshinamurthy Devanga; Dash, Srikanta; Kishore, Vimal; Mandal, Tarun K

2012-01-01

The objective of this study was to investigate the efficiency of uptake of PEGylated polylactide-co-gycolide (PLGA) nanoparticles by breast cancer cells. Nanoparticles of PLGA containing various amounts of polyethylene glycol (PEG, 5%-15%) were prepared using a double emulsion solvent evaporation method. The nanoparticles were loaded with coumarin-6 (C6) as a fluorescence marker. The particles were characterized for surface morphology, particle size, zeta potential, and for cellular uptake by 4T1 murine breast cancer cells. Irrespective of the amount of PEG, all formulations yielded smooth spherical particles. However, a comparison of the particle size of various formulations showed bimodal distribution of particles. Each formulation was later passed through a 1.2 µm filter to obtain target size particles (114-335 nm) with zeta potentials ranging from -2.8 mV to -26.2 mV. While PLGA-PEG di-block (15% PEG) formulation showed significantly higher 4T1 cellular uptake than all other formulations, there was no statistical difference in cellular uptake among PLGA, PLGA-PEG-PLGA tri-block (10% PEG), PLGA-PEG di-block (5% PEG) and PLGA-PEG di-block (10% PEG) nanoparticles. These preliminary findings indicated that the nanoparticle formulation prepared with 15% PEGylated PLGA showed maximum cellular uptake due to it having the smallest particle size and lowest zeta potential. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

Recent Advances in Chemical Modification of Peptide Nucleic Acids

PubMed Central

Rozners, Eriks

2012-01-01

Peptide nucleic acid (PNA) has become an extremely powerful tool in chemistry and biology. Although PNA recognizes single-stranded nucleic acids with exceptionally high affinity and sequence selectivity, there is considerable ongoing effort to further improve properties of PNA for both fundamental science and practical applications. The present paper discusses selected recent studies that improve on cellular uptake and binding of PNA to double-stranded DNA and RNA. The focus is on chemical modifications of PNA's backbone and heterocyclic nucleobases. The paper selects representative recent studies and does not attempt to provide comprehensive coverage of the broad and vibrant field of PNA modification. PMID:22991652

The extracellular microenvironment explains variations in passive drug transport across different airway epithelial cell types.

PubMed

Min, Kyoung Ah; Talattof, Arjang; Tsume, Yasuhiro; Stringer, Kathleen A; Yu, Jing-Yu; Lim, Dong Hyun; Rosania, Gus R

2013-08-01

We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types. Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types. Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in the extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells. Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types.

The Extracellular Microenvironment Explains Variations in Passive Drug Transport across Different Airway Epithelial Cell Types

PubMed Central

Min, Kyoung Ah; Talattof, Arjang; Tsume, Yasuhiro; Stringer, Kathleen A.; Yu, Jing-yu; Lim, Dong Hyun; Rosania, Gus R.

2013-01-01

Purpose We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types. Methods Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types. Results Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells. Conclusion Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types. PMID:23708857

Quantifying the cellular uptake of semiconductor quantum dot nanoparticles by analytical electron microscopy.

PubMed

Hondow, Nicole; Brown, M Rowan; Starborg, Tobias; Monteith, Alexander G; Brydson, Rik; Summers, Huw D; Rees, Paul; Brown, Andy

2016-02-01

Semiconductor quantum dot nanoparticles are in demand as optical biomarkers yet the cellular uptake process is not fully understood; quantification of numbers and the fate of internalized particles are still to be achieved. We have focussed on the characterization of cellular uptake of quantum dots using a combination of analytical electron microscopies because of the spatial resolution available to examine uptake at the nanoparticle level, using both imaging to locate particles and spectroscopy to confirm identity. In this study, commercially available quantum dots, CdSe/ZnS core/shell particles coated in peptides to target cellular uptake by endocytosis, have been investigated in terms of the agglomeration state in typical cell culture media, the traverse of particle agglomerates across U-2 OS cell membranes during endocytosis, the merging of endosomal vesicles during incubation of cells and in the correlation of imaging flow cytometry and transmission electron microscopy to measure the final nanoparticle dose internalized by the U-2 OS cells. We show that a combination of analytical transmission electron microscopy and serial block face scanning electron microscopy can provide a comprehensive description of the internalization of an initial exposure dose of nanoparticles by an endocytically active cell population and how the internalized, membrane bound nanoparticle load is processed by the cells. We present a stochastic model of an endosome merging process and show that this provides a data-driven modelling framework for the prediction of cellular uptake of engineered nanoparticles in general. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

The effect of sedimentation and diffusion on cellular uptake of gold nanoparticles

PubMed Central

Cho, Eun Chul; Zhang, Qiang; Xia, Younan

2011-01-01

In vitro experiments typically measure the uptake of nanoparticles by exposing cells at the bottom of a culture plate to a suspension of nanoparticles, which is assumed to be well-dispersed. However, nanoparticles can sediment and this means the concentration of particles on the cell surface and those actually taken up by the cells may be higher than the initial bulk concentration. Here we use upright and inverted cell culture configurations to show that cellular uptake of gold nanoparticles depends on the sedimentation and diffusion velocities of the nanoparticles and is independent of size, shape, density, surface coating and initial concentration of the nanoparticles. Generally more nanoparticles are taken up in the upright configuration than the inverted one and nanoparticles that sediment faster showed greater differences in uptake between the two configurations. Our results suggest that cellular uptake of nanoparticles is sensitive to the way cells are positioned and sedimentation need to be considered when performing in vitro studies for large and heavy nanoparticles. PMID:21516092

Y-shaped Folic Acid-Conjugated PEG-PCL Copolymeric Micelles for Delivery of Curcumin.

PubMed

Feng, Runliang; Zhu, Wenxia; Chu, Wei; Teng, Fangfang; Meng, Ning; Deng, Peizong; Song, Zhimei

2017-01-01

Curcumin is a natural hydrophobic product showing anticancer activity. Many studies show its potential use in the field of cancer treatment due to its safety and efficiency. However, its application is limited due to its low water-solubility and poor selective delivery to cancer. A Y-shaped folic acid-modified poly (ethylene glycol)-b-poly (ε-caprolactone)2 copolymer was prepared to improve curcumin solubility and realize its selective delivery to cancer. The copolymer was synthesized through selective acylation reaction of folic acid with α- monoamino poly(ethylene glycol)-b-poly(ε-caprolactone)2. Curcumin was encapsulated into the copolymeric micelles with 93.71% of encapsulation efficiency and 11.94 % of loading capacity. The results from confocal microscopy and cellular uptake tests showed that folic acid-modified copolymeric micelles could improve cellular uptake of curcumin in Hela and HepG2 cells compared with folic acid-unmodified micelles. In vitro cytotoxicity assay showed that folic acid-modified micelles improved anticancer activity against Hela and HepG2 cells in comparison to folic acidunmodified micelles. Meanwhile, both drug-loaded micelles demonstrated higher activity against Hela cell lines than HepG2. The research results suggested that the folic acid-modified Y-shaped copolymeric micelles should be used to enhance hydrophobic anticancer drugs' solubility and their specific delivery to folic acid receptors-overexpressed cancer. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Chemical probes to potently and selectively inhibit endocannabinoid cellular reuptake.

PubMed

Chicca, Andrea; Nicolussi, Simon; Bartholomäus, Ruben; Blunder, Martina; Aparisi Rey, Alejandro; Petrucci, Vanessa; Reynoso-Moreno, Ines Del Carmen; Viveros-Paredes, Juan Manuel; Dalghi Gens, Marianela; Lutz, Beat; Schiöth, Helgi B; Soeberdt, Michael; Abels, Christoph; Charles, Roch-Philippe; Altmann, Karl-Heinz; Gertsch, Jürg

2017-06-20

The extracellular effects of the endocannabinoids anandamide and 2-arachidonoyl glycerol are terminated by enzymatic hydrolysis after crossing cellular membranes by facilitated diffusion. The lack of potent and selective inhibitors for endocannabinoid transport has prevented the molecular characterization of this process, thus hindering its biochemical investigation and pharmacological exploitation. Here, we report the design, chemical synthesis, and biological profiling of natural product-derived N -substituted 2,4-dodecadienamides as a selective endocannabinoid uptake inhibitor. The highly potent (IC 50 = 10 nM) inhibitor N -(3,4-dimethoxyphenyl)ethyl amide (WOBE437) exerted pronounced cannabinoid receptor-dependent anxiolytic, antiinflammatory, and analgesic effects in mice by increasing endocannabinoid levels. A tailored WOBE437-derived diazirine-containing photoaffinity probe (RX-055) irreversibly blocked membrane transport of both endocannabinoids, providing mechanistic insights into this complex process. Moreover, RX-055 exerted site-specific anxiolytic effects on in situ photoactivation in the brain. This study describes suitable inhibitors to target endocannabinoid membrane trafficking and uncovers an alternative endocannabinoid pharmacology.

Reciprocal Regulation of Endocytosis and Metabolism

PubMed Central

Antonescu, Costin N.; McGraw, Timothy E.; Klip, Amira

2014-01-01

The cellular uptake of many nutrients and micronutrients governs both their cellular availability and their systemic homeostasis. The cellular rate of nutrient or ion uptake (e.g., glucose, Fe3+, K+) or efflux (e.g., Na+) is governed by a complement of membrane transporters and receptors that show dynamic localization at both the plasma membrane and defined intracellular membrane compartments. Regulation of the rate and mechanism of endocytosis controls the amounts of these proteins on the cell surface, which in many cases determines nutrient uptake or secretion. Moreover, the metabolic action of diverse hormones is initiated upon binding to surface receptors that then undergo regulated endocytosis and show distinct signaling patterns once internalized. Here, we examine how the endocytosis of nutrient transporters and carriers as well as signaling receptors governs cellular metabolism and thereby systemic (whole-body) metabolite homeostasis. PMID:24984778

Lanthanoplatins: emissive Eu(iii) and Tb(iii) complexes staining nucleoli targeted through Pt-DNA crosslinking.

PubMed

Singh, Khushbu; Singh, Swati; Srivastava, Payal; Sivakumar, Sri; Patra, Ashis K

2017-06-01

Two highly luminescent water-soluble heterometallic LnPt 2 complexes, [{cis-PtCl(NH 3 ) 2 } 2 Ln(L)(H 2 O)](NO 3 ) 2 (Ln = Eu (1), Tb (2)), have been designed for their selective nucleoli staining through formation of Pt-DNA crosslinks. The complexes showed significant cellular uptake and distinctive nucleoli localization through intrinsic emission from Eu III or Tb III observed through confocal fluorescence microscopy.

The laforin-malin complex negatively regulates glycogen synthesis by modulating cellular glucose uptake via glucose transporters.

PubMed

Singh, Pankaj Kumar; Singh, Sweta; Ganesh, Subramaniam

2012-02-01

Lafora disease (LD), an inherited and fatal neurodegenerative disorder, is characterized by increased cellular glycogen content and the formation of abnormally branched glycogen inclusions, called Lafora bodies, in the affected tissues, including neurons. Therefore, laforin phosphatase and malin ubiquitin E3 ligase, the two proteins that are defective in LD, are thought to regulate glycogen synthesis through an unknown mechanism, the defects in which are likely to underlie some of the symptoms of LD. We show here that laforin's subcellular localization is dependent on the cellular glycogen content and that the stability of laforin is determined by the cellular ATP level, the activity of 5'-AMP-activated protein kinase, and the affinity of malin toward laforin. By using cell and animal models, we further show that the laforin-malin complex regulates cellular glucose uptake by modulating the subcellular localization of glucose transporters; loss of malin or laforin resulted in an increased abundance of glucose transporters in the plasma membrane and therefore excessive glucose uptake. Loss of laforin or malin, however, did not affect glycogen catabolism. Thus, the excessive cellular glucose level appears to be the primary trigger for the abnormally higher levels of cellular glycogen seen in LD.

Cellular uptake and metabolism of curcuminoids in monocytes/macrophages: regulatory effects on lipid accumulation

USDA-ARS?s Scientific Manuscript database

We previously showed that curcumin (CUR) may increase lipid accumulation in cultured THP-1 monocytes/macrophages, but tetrahydrocurcumin (THC), an in vivo metabolite of CUR, had no such effect. In the present study, we have hypothesized that different cellular uptake and/or metabolism of CUR and THC...

Endocytotic uptake of HPMA-based polymers by different cancer cells: impact of extracellular acidosis and hypoxia

PubMed Central

Gündel, Daniel; Allmeroth, Mareli; Reime, Sarah; Zentel, Rudolf; Thews, Oliver

2017-01-01

Background Polymeric nanoparticles allow to selectively transport chemotherapeutic drugs to the tumor tissue. These nanocarriers have to be taken up into the cells to release the drug. In addition, tumors often show pathological metabolic characteristics (hypoxia and acidosis) which might affect the polymer endocytosis. Materials and methods Six different N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer structures (homopolymer as well as random and block copolymers with lauryl methacrylate containing hydrophobic side chains) varying in molecular weight and size were analyzed in two different tumor models. The cellular uptake of fluorescence-labeled polymers was measured under hypoxic (pO2 ≈1.5 mmHg) and acidic (pH 6.6) conditions. By using specific inhibitors, different endocytotic routes (macropinocytosis and clathrin-mediated, dynamin-dependent, cholesterol-dependent endocytosis) were analyzed separately. Results The current results revealed that the polymer uptake depends on the molecular structure, molecular weight and tumor line used. In AT1 cells, the uptake of random copolymer was five times stronger than the homopolymer, whereas in Walker-256 cells, the uptake of all polymers was much stronger, but this was independent of the molecular structure and size. Acidosis increased the uptake of random copolymer in AT1 cells but reduced the intracellular accumulation of homopolymer and block copolymer. Hypoxia reduced the uptake of all polymers in Walker-256 cells. Hydrophilic polymers (homopolymer and block copolymer) were taken up by all endocytotic routes studied, whereas the more lipophilic random copolymer seemed to be taken up preferentially by cholesterol- and dynamin-dependent endocytosis. Conclusion The study indicates that numerous parameters of the polymer (structure, size) and of the tumor (perfusion, vascular permeability, pH, pO2) modulate drug delivery, which makes it difficult to select the appropriate polymer for the individual patient. PMID:28831253

Endocytotic uptake of HPMA-based polymers by different cancer cells: impact of extracellular acidosis and hypoxia.

PubMed

Gündel, Daniel; Allmeroth, Mareli; Reime, Sarah; Zentel, Rudolf; Thews, Oliver

2017-01-01

Polymeric nanoparticles allow to selectively transport chemotherapeutic drugs to the tumor tissue. These nanocarriers have to be taken up into the cells to release the drug. In addition, tumors often show pathological metabolic characteristics (hypoxia and acidosis) which might affect the polymer endocytosis. Six different N -(2-hydroxypropyl)methacrylamide (HPMA)-based polymer structures (homopolymer as well as random and block copolymers with lauryl methacrylate containing hydrophobic side chains) varying in molecular weight and size were analyzed in two different tumor models. The cellular uptake of fluorescence-labeled polymers was measured under hypoxic (pO 2 ≈1.5 mmHg) and acidic (pH 6.6) conditions. By using specific inhibitors, different endocytotic routes (macropinocytosis and clathrin-mediated, dynamin-dependent, cholesterol-dependent endocytosis) were analyzed separately. The current results revealed that the polymer uptake depends on the molecular structure, molecular weight and tumor line used. In AT1 cells, the uptake of random copolymer was five times stronger than the homopolymer, whereas in Walker-256 cells, the uptake of all polymers was much stronger, but this was independent of the molecular structure and size. Acidosis increased the uptake of random copolymer in AT1 cells but reduced the intracellular accumulation of homopolymer and block copolymer. Hypoxia reduced the uptake of all polymers in Walker-256 cells. Hydrophilic polymers (homopolymer and block copolymer) were taken up by all endocytotic routes studied, whereas the more lipophilic random copolymer seemed to be taken up preferentially by cholesterol- and dynamin-dependent endocytosis. The study indicates that numerous parameters of the polymer (structure, size) and of the tumor (perfusion, vascular permeability, pH, pO 2 ) modulate drug delivery, which makes it difficult to select the appropriate polymer for the individual patient.

Rational Design of Cancer-Targeted Benzoselenadiazole by RGD Peptide Functionalization for Cancer Theranostics.

PubMed

Yang, Liye; Li, Wenying; Huang, Yanyu; Zhou, Yangliang; Chen, Tianfeng

2015-09-01

A cancer-targeted conjugate of the selenadiazole derivative BSeC (benzo[1,2,5] selenadiazole-5-carboxylic acid) with RGD peptide as targeting molecule and PEI (polyethylenimine) as a linker is rationally designed and synthesized in the present study. The results show that RGD-PEI-BSeC forms nanoparticles in aqueous solution with a core-shell nanostructure and high stability under physiological conditions. This rational design effectively enhances the selective cellular uptake and cellular retention of BSeC in human glioma cells, and increases its selectivity between cancer and normal cells. The nanoparticles enter the cells through receptor-mediated endocytosis via clathrin-mediated and nystatin-dependent lipid raft-mediated pathways. Internalized nanoparticles trigger glioma cell apoptosis by activation of ROS-mediated p53 phosphorylation. Therefore, this study provides a strategy for the rational design of selenium-containing cancer-targeted theranostics. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulation of Acetate Utilization by Monocarboxylate Transporter 1 (MCT1) in Hepatocellular Carcinoma (HCC).

PubMed

Jeon, Jeong Yong; Lee, Misu; Whang, Sang Hyun; Kim, Jung-Whan; Cho, Arthur; Yun, Mijin

2018-01-19

Altered energy metabolism is a biochemical fingerprint of cancer cells. Hepatocellular carcinoma (HCC) shows reciprocal [18F]fluorodeoxyglucose (FDG) and [11C]acetate uptake, as revealed by positron emission tomography/computed tomography (PET/CT). Previous studies have focused on the role of FDG uptake in cancer cells. In this study, we evaluated the mechanism and roles of [11C]acetate uptake in human HCCs and cell lines. The expression of monocarboxylate transporters (MCTs) was assessed to determine the transporters of [11C]acetate uptake in HCC cell lines and human HCCs with different [11C]acetate uptake. Using two representative cell lines with widely different [11C]acetate uptake (HepG2 for high uptake and Hep3B for low uptake), changes in [11C]acetate uptake were measured after treatment with an MCT1 inhibitor or MCT1-targeted siRNA. To verify the roles of MCT1 in cells, oxygen consumption rate and the amount of lipid synthesis were measured. HepG2 cells with high [11C]acetate uptake showed higher MCT1 expression than other HCC cell lines with low [11C]acetate uptake. MCT1 expression was elevated in human HCCs with high [11C]acetate uptake compared to those with low [11C]acetate uptake. After blocking MCT1 with AR-C155858 or MCT1 knockdown, [11C]acetate uptake in HepG2 cells was significantly reduced. Additionally, inhibition of MCT1 suppressed mitochondrial oxidative phosphorylation, lipid synthesis, and cellular proliferation in HCC cells with high [11C]acetate uptake. MCT1 may be a new therapeutic target for acetate-dependent HCCs with high [11C]acetate uptake, which can be selected by [11C]acetate PET/CT imaging in clinical practice.

Acute changes in cellular zinc alters zinc uptake rates prior to zinc transporter gene expression in Jurkat cells.

PubMed

Holland, Tai C; Killilea, David W; Shenvi, Swapna V; King, Janet C

2015-12-01

A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells.

Cellular transport of l-arginine determines renal medullary blood flow in control rats, but not in diabetic rats despite enhanced cellular uptake capacity.

PubMed

Persson, Patrik; Fasching, Angelica; Teerlink, Tom; Hansell, Peter; Palm, Fredrik

2017-02-01

Diabetes mellitus is associated with decreased nitric oxide bioavailability thereby affecting renal blood flow regulation. Previous reports have demonstrated that cellular uptake of l-arginine is rate limiting for nitric oxide production and that plasma l-arginine concentration is decreased in diabetes. We therefore investigated whether regional renal blood flow regulation is affected by cellular l-arginine uptake in streptozotocin-induced diabetic rats. Rats were anesthetized with thiobutabarbital, and the left kidney was exposed. Total, cortical, and medullary renal blood flow was investigated before and after renal artery infusion of increasing doses of either l-homoarginine to inhibit cellular uptake of l-arginine or N ω -nitro- l-arginine methyl ester (l-NAME) to inhibit nitric oxide synthase. l-Homoarginine infusion did not affect total or cortical blood flow in any of the groups, but caused a dose-dependent reduction in medullary blood flow. l-NAME decreased total, cortical and medullary blood flow in both groups. However, the reductions in medullary blood flow in response to both l-homoarginine and l-NAME were more pronounced in the control groups compared with the diabetic groups. Isolated cortical tubular cells displayed similar l-arginine uptake capacity whereas medullary tubular cells isolated from diabetic rats had increased l-arginine uptake capacity. Diabetics had reduced l-arginine concentrations in plasma and medullary tissue but increased l-arginine concentration in cortical tissue. In conclusion, the reduced l-arginine availability in plasma and medullary tissue in diabetes results in reduced nitric oxide-mediated regulation of renal medullary hemodynamics. Cortical blood flow regulation displays less dependency on extracellular l-arginine and the upregulated cortical tissue l-arginine may protect cortical hemodynamics in diabetes. Copyright © 2017 the American Physiological Society.

Biomechanics and Thermodynamics of Nanoparticle Interactions with Plasma and Endosomal Membrane Lipids in Cellular Uptake and Endosomal Escape

PubMed Central

2015-01-01

To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(d,l-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In conclusion, biomechanical interactions with membrane lipids are involved in cellular uptake and endosomal escape of NPs. Biophysical interaction studies could help us better understand the role of membrane lipids in cellular uptake and intracellular trafficking of NPs. PMID:24911361

Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Levesque, Martine; Martineau, Corine; Jumarie, Catherine

Since bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation, alterations in osteoblast proliferation and differentiation may disturb the equilibrium of bone remodeling. Exposure to cadmium (Cd) has been associated with the alteration of bone metabolism and the development of osteoporosis. Because little information is available about the direct effects of Cd on osteoblastic cells, we have characterized in vitro the cellular accumulation and cytotoxicity of Cd in human osteoblastic cells. Incubation of osteoblast-like MG-63 cells with increasing concentrations of Cd in serum-free culture medium reduced cell viability in a time- and concentration-dependentmore » manner, suggesting that Cd accumulates in osteoblasts. Consequently, an uptake time-course could be characterized for the cellular accumulation of {sup 109}Cd in serum-free culture medium. In order to characterize the mechanisms of Cd uptake, experiments have been conducted under well-defined metal speciation conditions in chloride and nitrate transport media. The results revealed a preferential uptake of Cd{sup 2+} species. The cellular accumulation and cytotoxicity of Cd increased in the absence of extracellular calcium (Ca), suggesting that Cd may enter the cells in part through Ca channels. However, neither the cellular accumulation nor the cytotoxicity of Cd was modified by voltage-dependent Ca channel (VDCC) modulators or potassium-induced depolarization. Moreover, exposure conditions activating or inhibiting capacitative Ca entry (CCE) failed to modify the cellular accumulation and cytotoxicity of Cd, which excludes the involvement of canonical transient receptor potential (TRPC) channels. The cellular accumulation and cytotoxicity of Cd were reduced by 2-APB, a known inhibitor of the Mg and Ca channel TRPM7 and were increased in the absence of extracellular magnesium (Mg). The inhibition of Cd uptake by Mg and Ca was not additive, suggesting that each ion inhibits the same uptake mechanism. Our results indicate that Cd uptake in human osteoblastic cells occurs, at least in part, through Ca- and Mg-inhibitable transport mechanisms, which may involve channels of the TRPM family. The effect of Cd on bone metabolism may be enhanced under low Ca and/or Mg levels.« less

Increase in Dye:Dendrimer Ratio Decreases Cellular Uptake of Neutral Dendrimers in RAW Cells.

PubMed

Vaidyanathan, Sriram; Kaushik, Milan; Dougherty, Casey; Rattan, Rahul; Goonewardena, Sascha N; Banaszak Holl, Mark M; Monano, Janet; DiMaggio, Stassi

2016-09-12

Neutral generation 3 poly(amidoamine) dendrimers were labeled with Oregon Green 488 (G3-OG n ) to obtain materials with controlled fluorophore:dendrimer ratios (n = 1-2), a mixture containing mostly 3 dyes per dendrimer, a mixture containing primarily 4 or more dyes per dendrimer ( n = 4+), and a stochastic mixture ( n = 4 avg ). The UV absorbance of the dye conjugates increased linearly as n increased and the fluorescence emission decreased linearly as n increased. Cellular uptake was studied in RAW cells and HEK 293A cells as a function of the fluorophore:dendrimer ratio (n). The cellular uptake of G3-OG n ( n = 3, 4+, 4 avg ) into RAW cells was significantly lower than G3-OG n ( n = 1, 2). The uptake of G3-OG n ( n = 3, 4+, 4 avg ) into HEK 293A cells was not significantly different from G3-OG 1 . Thus, the fluorophore:dendrimer ratio was observed to change the extent of uptake in the macrophage uptake mechanism but not in the HEK 293A cell. This difference in endocytosis indicates the presence of a pathway in the macrophage that is sensitive to hydrophobicity of the particle.

Coating barium titanate nanoparticles with polyethylenimine improves cellular uptake and allows for coupled imaging and gene delivery

PubMed Central

Dempsey, Christopher; Lee, Isac; Cowan, Katie; Suh, Junghae

2015-01-01

Barium titanate nanoparticles (BT NP) belong to a class of second harmonic generating (SHG) nanoprobes that have recently demonstrated promise in biological imaging. Unfortunately, BT NPs display low cellular uptake efficiencies, which may be a problem if cellular internalization is desired or required for a particular application. To overcome this issue, while concomitantly developing a particle platform that can also deliver nucleic acids into cells, we coated the BT NPs with the cationic polymer polyethylenimine (PEI) – one of the most effective nonviral gene delivery agents. Coating of BT with PEI yielded complexes with positive zeta potentials and resulted in an 8-fold increase in cellular uptake of the BT NPs. Importantly, we were able to achieve high levels of gene delivery with the BT-PEI/DNA complexes, supporting further efforts to generate BT platforms for coupled imaging and gene therapy. PMID:23973999

Mechanism for the Cellular Uptake of Targeted Gold Nanorods of Defined Aspect Ratios.

PubMed

Yang, Hongrong; Chen, Zhong; Zhang, Lei; Yung, Wing-Yin; Leung, Ken Cham-Fai; Chan, Ho Yin Edwin; Choi, Chung Hang Jonathan

2016-10-01

Biomedical applications of non-spherical nanoparticles such as photothermal therapy and molecular imaging require their efficient intracellular delivery, yet reported details on their interactions with the cell remain inconsistent. Here, the effects of nanoparticle geometry and receptor targeting on the cellular uptake and intracellular trafficking are systematically explored by using C166 (mouse endothelial) cells and gold nanoparticles of four different aspect ratios (ARs) from 1 to 7. When coated with poly(ethylene glycol) strands, the cellular uptake of untargeted nanoparticles monotonically decreases with AR. Next, gold nanoparticles are functionalized with DNA oligonucleotides to target Class A scavenger receptors expressed by C166 cells. Intriguingly, cellular uptake is maximized at a particular AR: shorter nanorods (AR = 2) enter C166 cells more than nanospheres (AR = 1) and longer nanorods (AR = 4 or 7). Strikingly, long targeted nanorods align to the cell membrane in a near-parallel manner followed by rotating by ≈90° to enter the cell via a caveolae-mediated pathway. Upon cellular entry, targeted nanorods of all ARs predominantly traffic to the late endosome without progressing to the lysosome. The studies yield important materials design rules for drug delivery carriers based on targeted, anisotropic nanoparticles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Complexing Methylene Blue with Phosphorus Dendrimers to Increase Photodynamic Activity.

PubMed

Dabrzalska, Monika; Janaszewska, Anna; Zablocka, Maria; Mignani, Serge; Majoral, Jean Pierre; Klajnert-Maculewicz, Barbara

2017-02-23

The efficiency of photodynamic therapy is limited mainly due to low selectivity, unfavorable biodistribution of photosensitizers, and long-lasting skin sensitivity to light. However, drug delivery systems based on nanoparticles may overcome the limitations mentioned above. Among others, dendrimers are particularly attractive as carriers, because of their globular architecture and high loading capacity. The goal of the study was to check whether an anionic phosphorus dendrimer is suitable as a carrier of a photosensitizer-methylene blue (MB). As a biological model, basal cell carcinoma cell lines were used. We checked the influence of the MB complexation on its singlet oxygen production ability using a commercial fluorescence probe. Next, cellular uptake, phototoxicity, reactive oxygen species (ROS) generation, and cell death were investigated. The MB-anionic dendrimer complex (MB-1an) was found to generate less singlet oxygen; however, the complex showed higher cellular uptake and phototoxicity against basal cell carcinoma cell lines, which was accompanied with enhanced ROS production. Owing to the obtained results, we conclude that the photodynamic activity of MB complexed with an anionic dendrimer is higher than free MB against basal cell carcinoma cell lines.

Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism

NASA Technical Reports Server (NTRS)

Attieh, Z. K.; Mukhopadhyay, C. K.; Seshadri, V.; Tripoulas, N. A.; Fox, P. L.

1999-01-01

The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

[Cellular uptake of TPS-L-carnitine synthesised as transporter-based renal targeting prodrug].

PubMed

Li, Li; Zhu, Di; Sun, Xun

2012-11-01

To synthesize transporter-based renal targeting prodrug TPS-L-Carnitine and to determine its cellular uptake in vitro. Triptolide (TP) was conjugated with L-carnitine using succinate as the linker to form TPS-L-Carnitine, which could be specifically recognized by OCTN2, a cationic transporter with high affinity to L-Carnitine and is highly expressed on the apical membrane of renal proximal tubule cells. Cellular uptake assays of the prodrug and its parent drug were performed on HK-2 cells, a human proximal tubule cell line, in different temperature, concentration and in the presence of competitive inhibitors. TPS-L-Carnitine was taken up into HK-2 cells in a saturable and temperature- and concentration-dependent manner. The uptake process could be inhibited by the competitive inhibitors. The uptake of TPS-L-Carnitine was significantly higher than that of TP at 37 degrees C in the same drug concentration. TPS-L-Carnitine was taken through endocytosis mediated by transporter. TPS-L-Carnitine provides a good renal targeting property and lays the foundation for further studies in vivo.

Polymeric nanocomposites loaded with fluoridated hydroxyapatite Ln3+ (Ln = Eu or Tb)/iron oxide for magnetic targeted cellular imaging

PubMed Central

Pan, Jie; Liu, Wei-Jiao; Hua, Chao; Wang, Li-Li; Wan, Dong; Gong, Jun-Bo

2015-01-01

Objective To fabricate polymeric nanocomposites with excellent photoluminescence, magnetic properties, and stability in aqueous solutions, in order to improve specificity and sensitivity of cellular imaging under a magnetic field. Methods Fluoridated Ln3+-doped HAP (Ln3+-HAP) NPs and iron oxides (IOs) can be encapsulated with biocompatible polymers via a modified solvent exaction/evaporation technique to prepare polymeric nanocomposites with fluoridated Ln3+-HAP/iron oxide. The nanocomposites were characterized for surface morphology, fluorescence spectra, magnetic properties and in vitro cytotoxicity. Magnetic targeted cellular imaging of such nanocomposites was also evaluated with confocal laser scanning microscope using A549 cells with or without magnetic field. Results The fabricated nanocomposites showed good stability and excellent luminescent properties, as well as low in vitro cytotoxicity, indicating that the nanocomposites are suitable for biological applications. Nanocomposites under magnetic field achieved much higher cellular uptake via an energy-dependent pathway than those without magnetic field. Conclusion The nanocomposites fabricated in this study will be a promising tool for magnetic targeted cellular imaging with improved specificity and enhanced selection. PMID:26487962

Enantioselective cellular uptake of chiral semiconductor nanocrystals

NASA Astrophysics Data System (ADS)

Martynenko, I. V.; Kuznetsova, V. A.; Litvinov, I. K.; Orlova, A. O.; Maslov, V. G.; Fedorov, A. V.; Dubavik, A.; Purcell-Milton, F.; Gun'ko, Yu K.; Baranov, A. V.

2016-02-01

The influence of the chirality of semiconductor nanocrystals, CdSe/ZnS quantum dots (QDs) capped with L- and D-cysteine, on the efficiency of their uptake by living Ehrlich Ascite carcinoma cells is studied by spectral- and time-resolved fluorescence microspectroscopy. We report an evident enantioselective process where cellular uptake of the L-Cys QDs is almost twice as effective as that of the D-Cys QDs. This finding paves the way for the creation of novel approaches to control the biological properties and behavior of nanomaterials in living cells.

Multifunctional gold nanorods for selective plasmonic photothermal therapy in pancreatic cancer cells using ultra-short pulse near-infrared laser irradiation.

PubMed

Patino, Tania; Mahajan, Ujjwal; Palankar, Raghavendra; Medvedev, Nikolay; Walowski, Jakob; Münzenberg, Markus; Mayerle, Julia; Delcea, Mihaela

2015-03-12

Gold nanorods (AuNRs) have attracted considerable attention in plasmonic photothermal therapy for cancer treatment by exploiting their selective and localized heating effect due to their unique photophysical properties. Here we describe a strategy to design a novel multifunctional platform based on AuNRs to: (i) specifically target the adenocarcinoma MUC-1 marker through the use of the EPPT-1 peptide, (ii) enhance cellular uptake through a myristoylated polyarginine peptide (MPAP) and (iii) selectively induce cell death by ultra-short near infrared laser pulses. We used a biotin-avidin based approach to conjugate EPPT-1 and MPAP to AuNRs. Dual-peptide (EPPT-1+MPAP) labelled AuNRs showed a significantly higher uptake by pancreatic ductal adenocarcinoma cells when compared to their single peptide or avidin conjugated counterparts. In addition, we selectively induced cell death by ultra-short near infrared laser pulses in small target volumes (∼1 μm3), through the creation of plasmonic nanobubbles that lead to the destruction of a local cell environment. Our approach opens new avenues for conjugation of multiple ligands on AuNRs targeting cancer cells and tumors and it is relevant for plasmonic photothermal therapy.

Uptake of gentamicin by separated, viable renal tubules from rabbits.

PubMed

Barza, M; Murray, T; Hamburger, R J

1980-04-01

The proximal renal tubules have a marked affinity for gentamicin; they also are the major site of nephrotoxicity caused by this drug. The uptake of radiolabeled gentamicin in separated, viable renal tubules prepared by enzymatic digestion of rabbit kidneys was studied. The preparations showed rapid initial uptake of gentamicin followed by continued slower uptake. Accumulation was not affected by pH, but was significantly inhibited by ouabain, dinitrophenol, anoxia, and hypothermia in the absence of evident cellular damage. At gentamicin concentrations of greater than 50 microgram/ml in the medium, there was competition for drug uptake. Gentamicin efflux in tubules that were taken from a medium containing antibiotic and placed into antibiotic-free fluid was slow and incomplete. From these data it appears that gentamicin uptake by separated renal tubules occurs by a process that requires metabolic energy; thereafter, the drug resides in a poorly exchangeable cellular pool.

Bupivacaine-induced cellular entry of QX-314 and its contribution to differential nerve block

PubMed Central

Brenneis, C; Kistner, K; Puopolo, M; Jo, S; Roberson, DP; Sisignano, M; Segal, D; Cobos, EJ; Wainger, BJ; Labocha, S; Ferreirós, N; Hehn, C; Tran, J; Geisslinger, G; Reeh, PW; Bean, BP; Woolf, C J

2014-01-01

Background and Purpose: Selective nociceptor fibre block is achieved by introducing the cell membrane impermeant sodium channel blocker lidocaine N-ethyl bromide (QX-314) through transient receptor potential V1 (TRPV1) channels into nociceptors. We screened local anaesthetics for their capacity to activate TRP channels, and characterized the nerve block obtained by combination with QX-314. Experimental Approach: We investigated TRP channel activation in dorsal root ganglion (DRG) neurons by calcium imaging and patch-clamp recordings, and cellular QX-314 uptake by MS. To characterize nerve block, compound action potential (CAP) recordings from isolated nerves and behavioural responses were analysed. Key Results: Of the 12 compounds tested, bupivacaine was the most potent activator of ruthenium red-sensitive calcium entry in DRG neurons and activated heterologously expressed TRPA1 channels. QX-314 permeated through TRPA1 channels and accumulated intracellularly after activation of these channels. Upon sciatic injections, QX-314 markedly prolonged bupivacaine's nociceptive block and also extended (to a lesser degree) its motor block. Bupivacaine's blockade of C-, but not A-fibre, CAPs in sciatic nerves was extended by co-application of QX-314. Surprisingly, however, this action was the same in wild-type, TRPA1-knockout and TRPV1/TRPA1-double knockout mice, suggesting a TRP-channel independent entry pathway. Consistent with this, high doses of bupivacaine promoted a non-selective, cellular uptake of QX-314. Conclusions and Implications: Bupivacaine, combined with QX-314, produced a long-lasting sensory nerve block. This did not require QX-314 permeation through TRPA1, although bupivacaine activated these channels. Regardless of entry pathway, the greatly extended duration of block produced by QX-314 and bupivacaine may be clinically useful. PMID:24117225

Effect of molecular characteristics on cellular uptake, subcellular localization, and phototoxicity of Zn(II) N-alkylpyridylporphyrins.

PubMed

Ezzeddine, Rima; Al-Banaw, Anwar; Tovmasyan, Artak; Craik, James D; Batinic-Haberle, Ines; Benov, Ludmil T

2013-12-20

Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para > meta > ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs.

Effect of Molecular Characteristics on Cellular Uptake, Subcellular Localization, and Phototoxicity of Zn(II) N-Alkylpyridylporphyrins*

PubMed Central

Ezzeddine, Rima; Al-Banaw, Anwar; Tovmasyan, Artak; Craik, James D.; Batinic-Haberle, Ines; Benov, Ludmil T.

2013-01-01

Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para > meta > ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs. PMID:24214973

Gracilaria lemaneiformis polysaccharide as integrin-targeting surface decorator of selenium nanoparticles to achieve enhanced anticancer efficacy.

PubMed

Jiang, Wenting; Fu, Yuanting; Yang, Fang; Yang, Yufeng; Liu, Ting; Zheng, Wenjie; Zeng, Lilan; Chen, Tianfeng

2014-08-27

The poor permeability of glioma parenchyma represents a major limit for antiglioblastoma drug delivery. Gracilaria lemaneiformis polysaccharide (GLP), which has a high binding affinity to αvβ3 integrin overexpressed in glioma cells, was employed in the present study to functionalize selenium nanoparticles (SeNPs) to achieve antiglioblastoma efficacy. GLP-SeNPs showed satisfactory size distribution, high stability, and selectivity between cancer and normal cells. In U87 glioma cell membrane, which has a high integrin expression level, GLP-SeNPs exhibited significantly higher cellular uptake than unmodified SeNPs. As expected, U87 cells exhibited a greater uptake of GLP-SeNPs than C6 cells with low integrin expression level. Furthermore, the internalization of GLP-SeNPs was inhibited by cyclo-(Arg-Gly-Asp-Phe-Lys) peptides, suggesting that cellular uptake into U87 cells and C6 cells occurred via αvβ3 integrin-mediated endocytosis. For U87 cells, the cytotoxicity of SeNPs decorated by GLP was enhanced significantly because of the induction of various apoptosis signaling pathways. Internalized GLP-SeNPs triggered intracellular reactive oxygen species downregulation. Therefore, p53, MAPKs, and AKT pathways were activated to advance cell apoptosis. These findings suggest that surface decoration of nanomaterials with GLP could be an efficient strategy for design and preparation of glioblastoma targeting nanodrugs.

Determination of cellular injury and death thresholds following exposure to high voltage 10ns electrical pulses

NASA Astrophysics Data System (ADS)

Ibey, Bennett L.; Roth, Caleb C.; Bernhard, Joshua A.; Pakhomov, Andrei G.; Wilmink, Gerald J.; Pakhomova, Olga

2011-03-01

Intense, nanosecond-duration electric pulses (nsEP) have been introduced as a novel modality to alter cellular function, with a mechanism of action qualitatively different from micro- and millisecond duration pulses used in electroporation. In this study, we determined the thresholds for plasma membrane injury (within 15 minutes) and cell death (at 24 hours) for 4 different cell types (CHO-K1, HeLa, Jurkat and U937). Plasma membrane injury was measured by flow cytometry using two fluorescent dyes, namely Annexin V-FITC, which binds to phosphatidylserine (PS) upon its externalization (subtle membrane injury), and propidium iodide (PI), which is typically impermeable to the cell, but enters when large pores are formed in the plasma membrane. In all cell types, 10-ns pulses caused phosphatidylserine (PS) externalization at low doses (<150kV/cm and 100 pulses for each cell type) and no PI uptake. Jurkat and U937 cell lines showed substantial cell death without uptake of PI (15 minutes post exposure) suggesting either delayed permeabilization due to swelling, or damage to intracellular components. In CHO-K1 and HeLa cell lines, PI uptake occurred at low doses relative to that necessary to cause cell death suggesting a necrotic death similar to longer pulse exposures. These findings suggest that nanosecond pulses may be beneficial in applications that require selective elimination of specific cell types.

The Role of Transporters in the Toxicity of Nucleoside and Nucleotide Analogs

PubMed Central

Koczor, Christopher A; Torres, Rebecca A

2013-01-01

Introduction Two families of nucleoside analogs have been developed to treat viral infections and cancer, but these compounds can cause tissue and cell-specific toxicity related to their uptake and subcellular activity which are dictated by host enzymes and transporters. Cellular uptake of these compounds requires nucleoside transporters that share functional similarities but differ in substrate specificity. Tissue-specific cellular expression of these transporters enables nucleoside analogs to produce their tissue specific toxic effects, a limiting factor in the treatment of retroviruses and cancer. Areas Covered This review discusses the families of nucleoside transporters and how they mediate cellular uptake of nucleoside analogs. Specific focus is placed on examples of known cases of transporter-mediated cellular toxicity and classification of the toxicities resulting. Efflux transporters are also explored as a contributor to analog toxicity and cell-specific effects. Expert Opinion Efforts to modulate transporter uptake/clearance remain long-term goals of oncologists and virologists. Accordingly, subcellular approaches that either increase or decrease intracellular nucleoside analog concentrations are eagerly sought and include transporter inhibitors and targeting transporter expression. However, additional understanding of nucleoside transporter kinetics, tissue expression, and genetic polymorphisms are required to design better molecules and better therapies. PMID:22509856

Surface decoration by Spirulina polysaccharide enhances the cellular uptake and anticancer efficacy of selenium nanoparticles.

PubMed

Yang, Fang; Tang, Quanming; Zhong, Xueyun; Bai, Yan; Chen, Tianfeng; Zhang, Yibo; Li, Yinghua; Zheng, Wenjie

2012-01-01

A simple and solution-phase method for functionalization of selenium nanoparticles (SeNPs) with Spirulina polysaccharides (SPS) has been developed in the present study. The cellular uptake and anticancer activity of SPS-SeNPs were also evaluated. Monodisperse and homogeneous spherical SPS-SeNPs with diameters ranging from 20 nm to 50 nm were achieved under optimized conditions, which were stable in the solution phase for at least 3 months. SPS surface decoration significantly enhanced the cellular uptake and cytotoxicity of SeNPs toward several human cancer cell lines. A375 human melanoma cells were found extremely susceptible to SPS-SeNPs with half maximal (50%) inhibitory concentration value of 7.94 μM. Investigation of the underlying mechanisms revealed that SPS-SeNPs inhibited cancer cell growth through induction of apoptosis, as evidenced by an increase in sub-G(1) cell population, deoxyribonucleic acid fragmentation, chromatin condensation, and phosphatidylserine translocation. Results suggest that the strategy to use SPS as a surface decorator could be an effective way to enhance the cellular uptake and anticancer efficacy of nanomaterials. SPS-SeNPs may be a potential candidate for further evaluation as a chemopreventive and chemotherapeutic agent against human cancers.

Surface decoration by Spirulina polysaccharide enhances the cellular uptake and anticancer efficacy of selenium nanoparticles

PubMed Central

Yang, Fang; Tang, Quanming; Zhong, Xueyun; Bai, Yan; Chen, Tianfeng; Zhang, Yibo; Li, Yinghua; Zheng, Wenjie

2012-01-01

A simple and solution-phase method for functionalization of selenium nanoparticles (SeNPs) with Spirulina polysaccharides (SPS) has been developed in the present study. The cellular uptake and anticancer activity of SPS-SeNPs were also evaluated. Monodisperse and homogeneous spherical SPS-SeNPs with diameters ranging from 20 nm to 50 nm were achieved under optimized conditions, which were stable in the solution phase for at least 3 months. SPS surface decoration significantly enhanced the cellular uptake and cytotoxicity of SeNPs toward several human cancer cell lines. A375 human melanoma cells were found extremely susceptible to SPS-SeNPs with half maximal (50%) inhibitory concentration value of 7.94 μM. Investigation of the underlying mechanisms revealed that SPS-SeNPs inhibited cancer cell growth through induction of apoptosis, as evidenced by an increase in sub-G1 cell population, deoxyribonucleic acid fragmentation, chromatin condensation, and phosphatidylserine translocation. Results suggest that the strategy to use SPS as a surface decorator could be an effective way to enhance the cellular uptake and anticancer efficacy of nanomaterials. SPS-SeNPs may be a potential candidate for further evaluation as a chemopreventive and chemotherapeutic agent against human cancers. PMID:22359460

Selective killing of hepatocellular carcinoma HepG2 cells by three-dimensional nanographene nanoparticles based on triptycene

NASA Astrophysics Data System (ADS)

Xiong, Xiaoqin; Gan, Lu; Liu, Ying; Zhang, Chun; Yong, Tuying; Wang, Ziyi; Xu, Huibi; Yang, Xiangliang

2015-03-01

Carbon-based materials have been widely used in the biomedical fields including drug delivery and cancer therapies. In this paper, a recently synthesized three-dimensional nanographene (NG) based on triptycene self-assembles into nanoparticles which selectively kill human hepatocellular carcinoma HepG2 cells as compared to human normal liver HL7702 cells. Obvious differences in cellular accumulation, the endocytic pathway and intracellular trafficking of NG nanoparticles are observed in HepG2 cells and HL7702 cells. Further studies reveal that NG nanoparticles significantly increase the levels of reactive oxygen species (ROS) in HepG2 cells, but not in HL7702 cells. NG nanoparticle-induced ROS result in apoptosis induction and the decrease in mitochondrial membrane potential in HepG2 cells. Moreover, IKK/nuclear factor-κB (NF-κB) signaling is found to be activated by NG nanoparticle-induced ROS and serves to antagonize NG nanoparticle-induced apoptosis in HepG2 cells. Our studies show that the distinct behaviors of cellular uptake and ROS-mediated cytotoxicity are responsible for the selective killing of HepG2 cells. This study provides a foundation for understanding the mechanism of selective induction of apoptosis in cancer cells by NG nanoparticles and designing more effective chemotherapeutical agents.Carbon-based materials have been widely used in the biomedical fields including drug delivery and cancer therapies. In this paper, a recently synthesized three-dimensional nanographene (NG) based on triptycene self-assembles into nanoparticles which selectively kill human hepatocellular carcinoma HepG2 cells as compared to human normal liver HL7702 cells. Obvious differences in cellular accumulation, the endocytic pathway and intracellular trafficking of NG nanoparticles are observed in HepG2 cells and HL7702 cells. Further studies reveal that NG nanoparticles significantly increase the levels of reactive oxygen species (ROS) in HepG2 cells, but not in HL7702 cells. NG nanoparticle-induced ROS result in apoptosis induction and the decrease in mitochondrial membrane potential in HepG2 cells. Moreover, IKK/nuclear factor-κB (NF-κB) signaling is found to be activated by NG nanoparticle-induced ROS and serves to antagonize NG nanoparticle-induced apoptosis in HepG2 cells. Our studies show that the distinct behaviors of cellular uptake and ROS-mediated cytotoxicity are responsible for the selective killing of HepG2 cells. This study provides a foundation for understanding the mechanism of selective induction of apoptosis in cancer cells by NG nanoparticles and designing more effective chemotherapeutical agents. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr07248k

Cellular uptake and transport of zein nanoparticles: effects of sodium caseinate.

PubMed

Luo, Yangchao; Teng, Zi; Wang, Thomas T Y; Wang, Qin

2013-08-07

Cellular evaluation of zein nanoparticles has not been studied systematically due to their poor redispersibility. Caseinate (CAS)-stabilized zein nanoparticles have been recently developed with better redispersibility in salt solutions. In this study, zein-CAS nanoparticles were prepared with different zein/CAS mass ratios. The prepared nanoparticles demonstrated good stabilities to maintain particle size (120-140 nm) in cell culture medium and HBSS buffer at 37 °C. The nanoparticles showed no cytotoxicity for Caco-2 cells for 72 h. CAS not only significantly enhanced cell uptake of zein nanoparticles in a concentration- and time-dependent manner but also remarkably improved epithelial transport through Caco-2 cell monolayer. The cell uptake of zein-CAS nanoparticles indicated an energy-dependent endocytosis process as evidenced by cell uptake under blocking conditions, that is, 4 °C, sodium azide, and colchicine. Fluorescent microscopy clearly showed the internalization of zein-CAS nanoparticles. This study may shed some light on the cellular evaluations of hydrophobic protein nanoparticles.

Towards increased selectivity of drug delivery to cancer cells: development of a LDL-based nanodelivery system for hydrophobic photosensitizers

NASA Astrophysics Data System (ADS)

Buzova, Diana; Huntosova, Veronika; Kasak, Peter; Petrovajova, Dana; Joniova, Jaroslava; Dzurova, Lenka; Nadova, Zuzana; Sureau, Franck; Midkovsky, Pavol; Jancura, Daniel

2012-10-01

Low-density lipoproteins (LDL), a natural in vivo carrier of cholesterol in the vascular system, play a key role in the delivery of hydrophobic photosensitizers (pts) to tumor cells in photodynamic therapy (PDT) of cancer. To make this delivery system even more efficient, we have constructed a nano-delivery system by coating of LDL surface by polyethylene glycol (PEG) and dextran. Fluorescence spectroscopy and confocal fluorescence imaging were used to characterize redistribution of hypericin (Hyp), a natural potent pts, loaded in LDL/PEG and LDL/dextran complexes to free LDL molecules as well as to monitor cellular uptake of Hyp by U87-MG cells. It was shown than the redistribution process of Hyp between LDL molecules is significantly suppressed by dextran coating of LDL surface. On the other hand, PEG does not significantly influence this process. The modification of LDL molecules by the polymers does not inhibit their recognition by cellular LDL receptors. U-87 MG cellular uptake of Hyp loaded in LDL/PEG and LDL/dextran complexes appears to be similar to that one observed for Hyp transported by unmodified LDL particles. It is proposed that by polymers modified LDL molecules could be used as a basis for construction of a drug transport system for targeted delivery of hydrophobic drugs to cancer cells expressing high level of LDL receptors.

Enhancement of cellular uptake and cytotoxicity of curcumin-loaded PLGA nanoparticles by conjugation with anti-P-glycoprotein in drug resistance cancer cells.

PubMed

Punfa, Wanisa; Yodkeeree, Supachai; Pitchakarn, Pornsiri; Ampasavate, Chadarat; Limtrakul, Pornngarm

2012-06-01

To compare the anti-cancer activity and cellular uptake of curcumin (Cur) delivered by targeted and non-targeted drug delivery systems in multidrug-resistant cervical cancer cells. Cur was entrapped into poly (DL-lactide-co-glycolide) (PLGA) nanoparticles (Cur-NPs) in the presence of modified-pluronic F127 stabilizer using nano-precipitation technique. On the surface of Cur-NPs, the carboxy-terminal of modified pluronic F127 was conjugated to the amino-terminal of anti-P-glycoprotein (P-gp) (Cur-NPs-APgp). The physical properties of the Cur-NPs, including particle size, zeta potential, particle morphology and Cur release kinetics, were investigated. Cellular uptake and specificity of the Cur-NPs and Cur-NPs-APgp were detected in cervical cancer cell lines KB-V1 (higher expression of P-gp) and KB-3-1 (lower expression of P-gp) using fluorescence microscope and flow cytometry, respectively. Cytotoxicity of the Cur-NPs and Cur-NPs-APgp was determined using MTT assay. The particle size of Cur-NPs and Cur-NPs-APgp was 127 and 132 nm, respectively. The entrapment efficiency and actual loading of Cur-NPs-APgp (60% and 5 μg Cur/mg NP) were lower than those of Cur-NPs (99% and 7 μg Cur/mg NP). The specific binding of Cur-NPs-APgp to KB-V1 cells was significantly higher than that to KB-3-1 cells. Cellular uptake of Cur-NPs-APgp into KB-V1 cells was higher, as compared to KB-3-1 cells. However, the cellular uptake of Cur-NPs and Cur-NPs-IgG did not differ between the two types of cells. Besides, the cytotoxicity of Cur-NPs-APgp in KB-V1 cells was higher than those of Cur and Cur-NPs. The results demonstrate that Cur-NPs-APgp targeted to P-gp on the cell surface membrane of KB-V1 cells, thus enhancing the cellular uptake and cytotoxicity of Cur.

Vacuolar amino acid transporter Avt5p is responsible for lithium uptake in Schizosaccharomyces pombe.

PubMed

Iwaki, Tomoko; Sekito, Takayuki; Kakinuma, Yoshimi

2010-01-01

The fission yeast Schizosaccharomyces pombe was sensitive to salinity; cell growth was stopped by 0.5 M NaCl and by 10 mM LiCl. The avt5+ gene encodes a vacuolar transporter with a broad specificity for amino acids. We found that the avt5Delta mutant became highly tolerant of Li+ and Na+ in growth. Concanamycin A-sensitive Li+ uptake as well as cellular Li+ content was lower in the avt5 mutant, suggesting a role of Avt5p in cellular uptake of toxic Li+.

Photoluminescent diamond nanoparticles for cell labeling: study of the uptake mechanism in mammalian cells.

PubMed

Faklaris, Orestis; Joshi, Vandana; Irinopoulou, Theano; Tauc, Patrick; Sennour, Mohamed; Girard, Hugues; Gesset, Céline; Arnault, Jean-Charles; Thorel, Alain; Boudou, Jean-Paul; Curmi, Patrick A; Treussart, François

2009-12-22

Diamond nanoparticles (nanodiamonds) have been recently proposed as new labels for cellular imaging. For small nanodiamonds (size <40 nm), resonant laser scattering and Raman scattering cross sections are too small to allow single nanoparticle observation. Nanodiamonds can, however, be rendered photoluminescent with a perfect photostability at room temperature. Such a remarkable property allows easier single-particle tracking over long time scales. In this work, we use photoluminescent nanodiamonds of size <50 nm for intracellular labeling and investigate the mechanism of their uptake by living cells. By blocking selectively different uptake processes, we show that nanodiamonds enter cells mainly by endocytosis, and converging data indicate that it is clathrin-mediated. We also examine nanodiamond intracellular localization in endocytic vesicles using immunofluorescence and transmission electron microscopy. We find a high degree of colocalization between vesicles and the biggest nanoparticles or aggregates, while the smallest particles appear free in the cytosol. Our results pave the way for the use of photoluminescent nanodiamonds in targeted intracellular labeling or biomolecule delivery.

Target Nanoparticles for Therapy - SANS and DLS of Drug Carrier Liposomes and Polymer Nanoparticles

NASA Astrophysics Data System (ADS)

Nawroth, T.; Johnson, R.; Krebs, L.; Khoshakhlagh, P.; Langguth, P.; Hellmann, N.; Goerigk, G.; Boesecke, P.; Bravin, A.; Le Duc, G.; Szekely, N.; Schweins, R.

2016-09-01

T arget Nano-Pharmaceutics shall improve therapy and diagnosis of severe diseases, e.g. cancer, by individual targeting of drug-loaded nano-pharmaceuticals towards cancer cells, and drug uptake receptors in other diseases. Specific ligands, proteins or cofactors, which are recognized by the diseased cells or cells of food and drug uptake, are bound to the nanoparticle surface, and thus capable of directing the drug carriers. The strategy has two branches: a) for parenteral cancer medicine a ligand set (2-5 different, surface-linked) are selected according to the biopsy analysis of the patient tissue e.g. from tumor.; b) in the oral drug delivery part the drug transport is enforced by excipients/ detergents in combination with targeting materials for cellular receptors resulting in an induced drug uptake. Both targeting nanomaterials are characterized by a combination of SANS + DLS and SAXS or ASAXS in a feedback process during development by synthesis, nanoparticle assembly and formulation.

Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements.

PubMed

Wolfe, Kelly L; Liu, Rui Hai

2007-10-31

A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds to prevent the formation of DCF by 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds. The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay, and the results were expressed in micromoles of quercetin equivalents per 100 micromol of phytochemical or micromoles of quercetin equivalents per 100 g of fresh fruit. Quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin among the pure compounds tested. Among the selected fruits tested, blueberry had the highest CAA value, followed by cranberry > apple = red grape > green grape. The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells.

Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis.

PubMed

Nakase, Ikuhiko; Noguchi, Kosuke; Fujii, Ikuo; Futaki, Shiroh

2016-10-17

Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained.

Reduction in clonogenic survival of sodium-iodide symporter (NIS)-positive cells following intracellular uptake of (99m)Tc versus (188)Re.

PubMed

Freudenberg, Robert; Wendisch, Maria; Runge, Roswitha; Wunderlich, Gerd; Kotzerke, Jörg

2012-12-01

Cellular radionuclide uptake increases the heterogeneity of absorbed dose to biological structures. Dose increase depends on uptake yield and emission characteristics of radioisotopes. We used an in vitro model to compare the impact of cellular uptake of (188)Re-perrhenate and (99m)Tc-pertechnetate on cellular survival. Rat thyroid PC Cl3 cells in culture were incubated with (188)Re or (99m)Tc in the presence or absence of perchlorate for 1 hour. Clonogenic cell survival was measured by colony formation. In addition, intracellular radionuclide uptake was quantified. Dose effect curves were established for (188)Re and (99m)Tc for various extra- and intracellular distributions of the radioactivity. In the presence of perchlorate, no uptake of radionuclides was detected and (188)Re reduced cell survival more efficiently than (99m)Tc. A(37), the activity that is necessary to yield 37% cell survival was 14 MBq/ml for (188)Re and 480 MBq/ml for (99m)Tc. In the absence of perchlorate, both radionuclides showed similar uptakes; however, A(37) was reduced by 30% for the beta-emitter and by 95% for (99m)Tc. The dose D(37) that yields 37% cell survival was between 2.3 and 2.8 Gy for both radionuclides. Uptake of (188)Re and (99m)Tc decreased cell survival. Intracellular (99m)Tc yielded a dose increase that was higher compared to (188)Re due to emitted Auger and internal conversion-electrons. Up to 5 Gy there was no difference in radiotoxicity of (188)Re and (99m)Tc. At doses higher than 5 Gy intracellular (99m)Tc became less radiotoxic than (188)Re, probably due to a non-uniform lognormal radionuclide uptake.

Multifunctional gold nanorods for selective plasmonic photothermal therapy in pancreatic cancer cells using ultra-short pulse near-infrared laser irradiation

NASA Astrophysics Data System (ADS)

Patino, Tania; Mahajan, Ujjwal; Palankar, Raghavendra; Medvedev, Nikolay; Walowski, Jakob; Münzenberg, Markus; Mayerle, Julia; Delcea, Mihaela

2015-03-01

Gold nanorods (AuNRs) have attracted considerable attention in plasmonic photothermal therapy for cancer treatment by exploiting their selective and localized heating effect due to their unique photophysical properties. Here we describe a strategy to design a novel multifunctional platform based on AuNRs to: (i) specifically target the adenocarcinoma MUC-1 marker through the use of the EPPT-1 peptide, (ii) enhance cellular uptake through a myristoylated polyarginine peptide (MPAP) and (iii) selectively induce cell death by ultra-short near infrared laser pulses. We used a biotin-avidin based approach to conjugate EPPT-1 and MPAP to AuNRs. Dual-peptide (EPPT-1 + MPAP) labelled AuNRs showed a significantly higher uptake by pancreatic ductal adenocarcinoma cells when compared to their single peptide or avidin conjugated counterparts. In addition, we selectively induced cell death by ultra-short near infrared laser pulses in small target volumes (~1 μm3), through the creation of plasmonic nanobubbles that lead to the destruction of a local cell environment. Our approach opens new avenues for conjugation of multiple ligands on AuNRs targeting cancer cells and tumors and it is relevant for plasmonic photothermal therapy.Gold nanorods (AuNRs) have attracted considerable attention in plasmonic photothermal therapy for cancer treatment by exploiting their selective and localized heating effect due to their unique photophysical properties. Here we describe a strategy to design a novel multifunctional platform based on AuNRs to: (i) specifically target the adenocarcinoma MUC-1 marker through the use of the EPPT-1 peptide, (ii) enhance cellular uptake through a myristoylated polyarginine peptide (MPAP) and (iii) selectively induce cell death by ultra-short near infrared laser pulses. We used a biotin-avidin based approach to conjugate EPPT-1 and MPAP to AuNRs. Dual-peptide (EPPT-1 + MPAP) labelled AuNRs showed a significantly higher uptake by pancreatic ductal adenocarcinoma cells when compared to their single peptide or avidin conjugated counterparts. In addition, we selectively induced cell death by ultra-short near infrared laser pulses in small target volumes (~1 μm3), through the creation of plasmonic nanobubbles that lead to the destruction of a local cell environment. Our approach opens new avenues for conjugation of multiple ligands on AuNRs targeting cancer cells and tumors and it is relevant for plasmonic photothermal therapy. Electronic supplementary information (ESI) available: Size distribution histograms of the hydrodynamic diameter and ζ-potential of functionalized and CTAB stabilized AuNRs. Characterization of TR-AV functionalized AuNRs after 48 h at 4 °C. The ζ-potential of TR-AV functionalized AuNRs and CTAB stabilized AuNRs. The ζ-potential of peptide-conjugated AuNRs in water. See DOI: 10.1039/c5nr00114e

Increased cellular uptake of peptide-modified PEGylated gold nanoparticles.

PubMed

He, Bo; Yang, Dan; Qin, Mengmeng; Zhang, Yuan; He, Bing; Dai, Wenbing; Wang, Xueqing; Zhang, Qiang; Zhang, Hua; Yin, Changcheng

2017-12-09

Gold nanoparticles are promising drug delivery vehicles for nucleic acids, small molecules, and proteins, allowing various modifications on the particle surface. However, the instability and low bioavailability of gold nanoparticles compromise their clinical application. Here, we functionalized gold nanoparticles with CPP fragments (CALNNPFVYLI, CALRRRRRRRR) through sulfhydryl PEG to increase their stability and bioavailability. The resulting gold nanoparticles were characterized with transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-visible spectrometry and X-ray photoelectron spectroscopy (XPS), and the stability in biological solutions was evaluated. Comparing to PEGylated gold nanoparticles, CPP (CALNNPFVYLI, CALRRRRRRRR)-modified gold nanoparticles showed 46 folds increase in cellular uptake in A549 and B16 cell lines, as evidenced by the inductively coupled plasma atomic emission spectroscopy (ICP-AES). The interactions between gold nanoparticles and liposomes indicated CPP-modified gold nanoparticles bind to cell membrane more effectively than PEGylated gold nanoparticles. Surface plasmon resonance (SPR) was used to measure interactions between nanoparticles and the membrane. TEM and uptake inhibitor experiments indicated that the cellular entry of gold nanoparticles was mediated by clathrin and macropinocytosis. Other energy independent endocytosis pathways were also identified. Our work revealed a new strategy to modify gold nanoparticles with CPP and illustrated the cellular uptake pathway of CPP-modified gold nanoparticles. Copyright © 2017 Elsevier Inc. All rights reserved.

Radiolabeled probes for imaging Alzheimer’s plaques

NASA Astrophysics Data System (ADS)

Kulkarni, P. V.; Arora, V.; Roney, A. C.; White, C.; Bennett, M.; Antich, P. P.; Bonte, F. J.

2005-12-01

Alzheimer's disease (AD) is a debilitating disease characterized by the presence of extra-cellular plaques and intra-cellular neurofibrillary tangles (NFTs) in the brain. The major protein component of these plaques is beta amyloid peptide (Aβ), a 40-42 amino acid peptide cleaved from amyloid precursor protein (APP) by β-secretase and a putative γ-secretase. We radioiodinated quinoline derivatives (clioquinol and oxine) and evaluated them as potential amyloid imaging agents based on their ability to cross the blood brain barrier (BBB) and on their selectivity to metal binding sites on amyloid plaques. The uptake of theses tracers in the brains of normal swiss-webster mice was rapid and so was the clearance. Selectivity was demonstrated by higher binding to AD brain homogenates compared to normal brain. Autoradiographic studies demonstrated the localization of the tracers in the plaque regions of the AD brain sections as well as in liver tissue with amyloidosis. Further optimization and evaluations would likely lead to development of these molecules as AD plaque imaging agents.

Genotoxic capacity of Cd/Se semiconductor quantum dots with differing surface chemistries

PubMed Central

Manshian, Bella B.; Soenen, Stefaan J.; Brown, Andy; Hondow, Nicole; Wills, John; Jenkins, Gareth J. S.; Doak, Shareen H.

2016-01-01

Quantum dots (QD) have unique electronic and optical properties promoting biotechnological advances. However, our understanding of the toxicological structure–activity relationships remains limited. This study aimed to determine the biological impact of varying nanomaterial surface chemistry by assessing the interaction of QD with either a negative (carboxyl), neutral (hexadecylamine; HDA) or positive (amine) polymer coating with human lymphoblastoid TK6 cells. Following QD physico-chemical characterisation, cellular uptake was quantified by optical and electron microscopy. Cytotoxicity was evaluated and genotoxicity was characterised using the micronucleus assay (gross chromosomal damage) and the HPRT forward mutation assay (point mutagenicity). Cellular damage mechanisms were also explored, focusing on oxidative stress and mitochondrial damage. Cell uptake, cytotoxicity and genotoxicity were found to be dependent on QD surface chemistry. Carboxyl-QD demonstrated the smallest agglomerate size and greatest cellular uptake, which correlated with a dose dependent increase in cytotoxicity and genotoxicity. Amine-QD induced minimal cellular damage, while HDA-QD promoted substantial induction of cell death and genotoxicity. However, HDA-QD were not internalised by the cells and the damage they caused was most likely due to free cadmium release caused by QD dissolution. Oxidative stress and induced mitochondrial reactive oxygen species were only partially associated with cytotoxicity and genotoxicity induced by the QD, hence were not the only mechanisms of importance. Colloidal stability, nanoparticle (NP) surface chemistry, cellular uptake levels and the intrinsic characteristics of the NPs are therefore critical parameters impacting genotoxicity induced by QD. PMID:26275419

Intracellular delivery of etoposide loaded biodegradable nanoparticles: cytotoxicity and cellular uptake studies.

PubMed

Yadav, Khushwant S; Jacob, Sheeba; Sachdeva, Geetanjali; Sawant, Krutika K

2011-08-01

The preferred delivery systems for anticancer drugs would be the one which would have selective and effective destruction of cancer cells. In the present study etoposide (ETO) loaded nanoparticles (NP) were prepared using PLGA (ETO-PLGA NP), PLGA-MPEG block copolymer (ETO-PLGA-MPEG NP) and PLGA-Pluronic copolymer (ETO-PLGA-PLU NP) and they were evaluated for cytotoxicity and cellular uptake studies using two cancer cell lines, L1210 and DU145. The IC50 values for L1210 cells were 18.0, 6.2, 4.8 and 5.4 microM and for DU145 cells the IC50 values were 98.4, 75.1, 60.1 and 71.3 microM for ETO, ETO-PLGA NP, ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP respectively. The increased cytotoxicities were attributed to increased uptake of the NPs by the cells. Moreover the ETO loaded PLGA-MPEG NP and PLGA-Pluronic NP showed a sustained cytotoxic effect till 5 days on both the cell lines. Results of the long term cytotoxicity study concluded that the drug loaded PLGA nanoparticulate formulations were efficient in decreasing the viability of the L1210 cells over a period of three days, whereas the pure drug exerted its maximum efficiency on the day one itself. Z-stack confocal images of NPs showed fluorescence activity in each section of DU 145 and L1210 cells indicating that the nanoparticles were internalized by the cells. The study concluded that ETO loaded PLGA NPs had higher cytotoxicity compared with that of the free drug and ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP had higher cell uptake efficiency compared with that of ETO-PLGA NP. The developed PLGA based NPs shows promise to be used for cancer therapy.

Zwitterion-Coated Iron Oxide Nanoparticles: Surface Chemistry and Intracellular Uptake by Hepatocarcinoma (HepG2) Cells.

PubMed

Mondini, Sara; Leonzino, Marianna; Drago, Carmelo; Ferretti, Anna M; Usseglio, Sandro; Maggioni, Daniela; Tornese, Paolo; Chini, Bice; Ponti, Alessandro

2015-07-07

Nanoparticles (NPs) have received much attention in recent years for their diverse potential biomedical applications. However, the synthesis of NPs with desired biodistribution and pharmacokinetics is still a major challenge, with NP size and surface chemistry being the main factors determining the behavior of NPs in vivo. Here we report on the surface chemistry and in vitro cellular uptake of magnetic iron oxide NPs coated with zwitterionic dopamine sulfonate (ZDS). ZDS-coated NPs were compared to similar iron oxide NPs coated with PEG-like 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (MEEA) to investigate how surface chemistry affects their in vitro behavior. ZDS-coated NPs had a very dense coating, guaranteeing high colloidal stability in several aqueous media and negligible interaction with proteins. Treatment of HepG2 cells with increasing doses (2.5-100 μg Fe/mL) of ZDS-coated iron oxide NPs had no effect on cell viability and resulted in a low, dose-dependent NP uptake, inferior than most reported data for the internalization of iron oxide NPs by HepG2 cells. MEEA-coated NPs were scarcely stable and formed micrometer-sized aggregates in aqueous media. They decreased cell viability for dose ≥50 μg Fe/mL, and were more efficiently internalized than ZDS-coated NPs. In conclusion, our data indicate that the ZDS layer prevented both aggregation and sedimentation of iron oxide NPs and formed a biocompatible coating that did not display any biocorona effect. The very low cellular uptake of ZDS-coated iron NPs can be useful to achieve highly selective targeting upon specific functionalization.

Preparation and In Vitro Photodynamic Activity of Glucosylated Zinc(II) Phthalocyanines as Underlying Targeting Photosensitizers.

PubMed

Liu, Jian-Yong; Wang, Chen; Zhu, Chun-Hui; Zhang, Zhi-Hong; Xue, Jin-Ping

2017-05-19

Two novel glucosylated zinc(ІІ) phthalocyanines 7a-7b, as well as the acetyl-protected counterparts 6a-6b, have been synthesized by the Cu(I)-catalyzed 1,3-dipolar cycloaddition between the propargylated phthalocyanine and azide-substituted glucoses. All of these phthalocyanines were characterized with various spectroscopic methods and studied for their photo-physical, photo-chemical, and photo-biological properties. With glucose as the targeting unit, phthalocyanines 7a-7b exhibit a specific affinity to MCF-7 breast cancer cells over human embryonic lung fibroblast (HELF) cells, showing higher cellular uptake. Upon illumination, both photosensitizers show high cytotoxicity with IC 50 as low as 0.032 µM toward MCF-7 cells, which are attributed to their high cellular uptake and low aggregation tendency in the biological media, promoting the generation of intracellular reactive oxygen species (ROS). Confocal laser fluorescence microscopic studies have also revealed that they have high and selective affinities to the lysosomes, but not the mitochondria, of MCF-7 cells. The results show that these two glucosylated zinc(II) phthalocyanines are potential anticancer agents for targeting photodynamic therapy.

Diselenolane-mediated cellular uptake.

PubMed

Chuard, Nicolas; Poblador-Bahamonde, Amalia I; Zong, Lili; Bartolami, Eline; Hildebrandt, Jana; Weigand, Wolfgang; Sakai, Naomi; Matile, Stefan

2018-02-21

The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to selenium. We report that according to results with fluorescent model substrates, cellular uptake with 1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This 1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-β-cyclodextrin, wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum. Selenophilicity, the extreme CSeSeC dihedral angle of 0° and the high but different acidity of primary and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved selenosulfides.

Factors Influencing the Diversity of Iron Uptake Systems in Aquatic Microorganisms

PubMed Central

Desai, Dhwani K.; Desai, Falguni D.; LaRoche, Julie

2012-01-01

Iron (Fe) is an essential micronutrient for many processes in all living cells. Dissolved Fe (dFe) concentrations in the ocean are of the order of a few nM, and Fe is often a factor limiting primary production. Bioavailability of Fe in aquatic environments is believed to be primarily controlled through chelation by Fe-binding ligands. Marine microbes have evolved different mechanisms to cope with the scarcity of bioavailable dFe. Gradients in dFe concentrations and diversity of the Fe-ligand pool from coastal to open ocean waters have presumably imposed selection pressures that should be reflected in the genomes of microbial communities inhabiting the pelagic realm. We applied a hidden Markov model (HMM)-based search for proteins related to cellular iron metabolism, and in particular those involved in Fe uptake mechanisms in 164 microbial genomes belonging to diverse taxa and occupying different aquatic niches. A multivariate statistical approach demonstrated that in phototrophic organisms, there is a clear influence of the ecological niche on the diversity of Fe uptake systems. Extending the analyses to the metagenome database from the Global Ocean Sampling expedition, we demonstrated that the Fe uptake and homeostasis mechanisms differed significantly across marine niches defined by temperatures and dFe concentrations, and that this difference was linked to the distribution of microbial taxa in these niches. Using the dN/dS ratios (which signify the rate of non-synonymous mutations) of the nucleotide sequences, we identified that genes encoding for TonB, Ferritin, Ferric reductase, IdiA, ZupT, and Fe2+ transport proteins FeoA and FeoB were evolving at a faster rate (positive selection pressure) while genes encoding ferrisiderophore, heme and Vitamin B12 uptake systems, siderophore biosynthesis, and IsiA and IsiB were under purifying selection pressure (evolving slowly). PMID:23087680

Albumin-based nanoparticles as methylprednisolone carriers for targeted delivery towards the neonatal Fc receptor in glomerular podocytes

PubMed Central

Wu, Lin; Chen, Mingyu; Mao, Huijuan; Wang, Ningning; Zhang, Bo; Zhao, Xiufen; Qian, Jun; Xing, Changying

2017-01-01

Glucocorticoids (GCs) are commonly used in the treatment of nephrotic syndrome. However, high doses and long periods of GC therapy can result in severe side effects. The present study aimed to selectively deliver albumin-methylprednisolone (MP) nanoparticles towards glomerular podocytes, which highly express the specific neonatal Fc receptor (FcRn) of albumin. Bovine serum albumin (BSA) was labeled with a fluorescent dye and linked with modified MP via an amide bond. The outcome nanoparticle named BSA633-MP showed a uniform size with a diameter of approximately 10 nm and contained 12 drug molecules on average. The nanoconjugates were found to be stable at pH 7.4 and acid-sensitive at pH 4.0, with approximately 72% release of the MP drug after 48 h of incubation. The nanoparticle demonstrated a 36-fold uptake in receptor-specific cellular delivery in the FcRn-expressing human podocytes compared to the uptake in the non-FcRn-expressing control cells. Co-localization further confirmed that uptake of the nanoconjugates involved receptor-mediated endocytosis followed by lysosome associated transportation. In vitro cellular experiments indicated that the BSA633-MP ameliorated puromycin aminonucleoside-induced podocyte apoptosis. Moreover, in vivo fluorescence molecular imaging showed that BSA633-MP was mainly accumulated in the liver and kidney after intravenous dosing for 24 h. Collectively, this study may provide an approach for the effective and safe therapy of nephrotic syndrome. PMID:28259932

Efficient delivery of cell impermeable phosphopeptides by a cyclic peptide amphiphile containing tryptophan and arginine.

PubMed

Nasrolahi Shirazi, Amir; Tiwari, Rakesh Kumar; Oh, Donghoon; Banerjee, Antara; Yadav, Arpita; Parang, Keykavous

2013-05-06

Phosphopeptides are valuable reagent probes for studying protein-protein and protein-ligand interactions. The cellular delivery of phosphopeptides is challenging because of the presence of the negatively charged phosphate group. The cellular uptake of a number of fluorescent-labeled phosphopeptides, including F'-GpYLPQTV, F'-NEpYTARQ, F'-AEEEIYGEFEAKKKK, F'-PEpYLGLD, F'-pYVNVQN-NH2, and F'-GpYEEI (F' = fluorescein), was evaluated in the presence or absence of a [WR]4, a cyclic peptide containing alternative arginine (R) and tryptophan (W) residues, in human leukemia cells (CCRF-CEM) after 2 h incubation using flow cytometry. [WR]4 improved significantly the cellular uptake of all phosphopeptides. PEpYLGLD is a sequence that mimics the pTyr1246 of ErbB2 that is responsible for binding to the Chk SH2 domain. The cellular uptake of F'-PEpYLGLD was enhanced dramatically by 27-fold in the presence of [WR]4 and was found to be time-dependent. Confocal microscopy of a mixture of F'-PEpYLGLD and [WR]4 in live cells exhibited intracellular localization and significantly higher cellular uptake compared to that of F'-PEpYLGLD alone. Transmission electron microscopy (TEM) and isothermal calorimetry (ITC) were used to study the interaction of PEpYLGLD and [WR]4. TEM results showed that the mixture of PEpYLGLD and [WR]4 formed noncircular nanosized structures with width and height of 125 and 60 nm, respectively. ITC binding studies confirmed the interaction between [WR]4 and PEpYLGLD. The binding isotherm curves, derived from sequential binding models, showed an exothermic interaction driven by entropy. These studies suggest that amphiphilic peptide [WR]4 can be used as a cellular delivery tool of cell-impermeable negatively charged phosphopeptides.

Anticancer efficacy of the metabolic blocker 3-bromopyruvate: specific molecular targeting.

PubMed

Ganapathy-Kanniappan, Shanmugasundaram; Kunjithapatham, Rani; Geschwind, Jean-Francois

2013-01-01

The anticancer efficacy of the pyruvate analog 3-bromopyruvate has been demonstrated in multiple tumor models. The chief principle underlying the antitumor effects of 3-bromopyruvate is its ability to effectively target the energy metabolism of cancer cells. Biochemically, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been identified as the primary target of 3-bromopyruvate. Its inhibition results in the depletion of intracellular ATP, causing cell death. Several reports have also demonstrated that in addition to GAPDH inhibition, the induction of cellular stress also contributes to 3-bromopyruvate treatment-dependent apoptosis. Furthermore, recent evidence shows that 3-bromopyruvate is taken up selectively by tumor cells via the monocarboxylate transporters (MCTs) that are frequently overexpressed in cancer cells (for the export of lactate produced during aerobic glycolysis). The preferential uptake of 3-bromopyruvate via MCTs facilitates selective targeting of tumor cells while leaving healthy and non-malignant tissue untouched. Taken together, the specificity of molecular (GAPDH) targeting and selective uptake by tumor cells, underscore the potential of 3-bromopyruvate as a potent and promising anticancer agent. In this review, we highlight the mechanistic characteristics of 3-bromopyruvate and discuss its potential for translation into the clinic.

Ceramic nanoparticles: Recompense, cellular uptake and toxicity concerns.

PubMed

Singh, Deependra; Singh, Satpal; Sahu, Jageshwari; Srivastava, Shikha; Singh, Manju Rawat

2016-01-01

Over the past few years, nanoparticles and their role in drug delivery have been the centre of attraction as new drug delivery systems. Various forms of nanosystems have been designed, such as nanoclays, scaffolds and nanotubes, having numerous applications in areas such as drug loading, target cell uptake, bioassay and imaging. The present study discusses various types of nanoparticles, with special emphasis on ceramic nanocarriers. Ceramic materials have high mechanical strength, good body response and low or non-existing biodegradability. In this article, the various aspects concerning ceramic nanoparticles, such as their advantages over other systems, their cellular uptake and toxicity concerns are discussed in detail.

Designing Superoxide-Generating Quantum Dots for Selective Light-Activated Nanotherapy

NASA Astrophysics Data System (ADS)

Goodman, Samuel M.; Levy, Max; Li, Fei-Fei; Ding, Yuchen; Courtney, Colleen M.; Chowdhury, Partha P.; Erbse, Annette; Chatterjee, Anushree; Nagpal, Prashant

2018-03-01

The rapid emergence of superbugs or multi-drug resistant (MDR) organisms has prompted a search for novel antibiotics, beyond traditional small-molecule therapies. Nanotherapeutics are being investigated as alternatives, and recently superoxide-generating quantum dots (QDs) have been shown as important candidates for selective light-activated therapy and potentiating existing antibiotics against MDR superbugs. Their therapeutic action is selective, can be tailored by simply changing their quantum-confined conduction-valence bands and their alignment with different redox half-reactions, and hence their ability to generate specific radical species in biological media. Here, we show the design of superoxide-generating QDs using optimal QD material and size well matched to superoxide redox potential, charged ligands to modulate their uptake in cells and selective redox interventions, and core/shell structures to improve their stability for therapeutic action. We show that cadmium telluride (CdTe) QDs with conduction band position at -0.5V with respect to Normal Hydrogen Electron (NHE) and visible 2.4 eV bandgap generate a large flux of selective superoxide radicals, thereby demonstrating the most effective light-activated therapy. Although the positively charged QDs demonstrate large cellular uptake, they bind indiscriminately to cell surfaces and cause non-selective cell death, while negatively charged and zwitterionic QD ligands reduce the uptake and allow selective therapeutic action via interaction with redox species. The stability of designed QDs in biologically-relevant media increases with the formation of core-shell QD structures, but an appropriate design of core-shell structures is needed to minimize any reduction in charge injection efficiency to adsorbed oxygen molecules (to form superoxide) and maintain similar quantitative generation of tailored redox species, as measured using electron paramagnetic resonance (EPR) spectroscopy and electrochemical impedance spectroscopy. Using these findings, we demonstrate the rational design of QDs as selective therapeutic kills more than 99% of priority class I pathogens, thus providing an effective therapy against MDR superbugs.

Designing Superoxide-Generating Quantum Dots for Selective Light-Activated Nanotherapy.

PubMed

Goodman, Samuel M; Levy, Max; Li, Fei-Fei; Ding, Yuchen; Courtney, Colleen M; Chowdhury, Partha P; Erbse, Annette; Chatterjee, Anushree; Nagpal, Prashant

2018-01-01

The rapid emergence of superbugs, or multi-drug resistant (MDR) organisms, has prompted a search for novel antibiotics, beyond traditional small-molecule therapies. Nanotherapeutics are being investigated as alternatives, and recently superoxide-generating quantum dots (QDs) have been shown as important candidates for selective light-activated therapy, while also potentiating existing antibiotics against MDR superbugs. Their therapeutic action is selective, can be tailored by simply changing their quantum-confined conduction-valence band (CB-VB) positions and alignment with different redox half-reactions-and hence their ability to generate specific radical species in biological media. Here, we show the design of superoxide-generating QDs using optimal QD material and size well-matched to superoxide redox potential, charged ligands to modulate their uptake in cells and selective redox interventions, and core/shell structures to improve their stability for therapeutic action. We show that cadmium telluride (CdTe) QDs with conduction band (CB) position at -0.5 V with respect to Normal Hydrogen Electron (NHE) and visible 2.4 eV bandgap generate a large flux of selective superoxide radicals, thereby demonstrating the effective light-activated therapy. Although the positively charged QDs demonstrate large cellular uptake, they bind indiscriminately to cell surfaces and cause non-selective cell death, while negatively charged and zwitterionic QD ligands reduce the uptake and allow selective therapeutic action via interaction with redox species. The stability of designed QDs in biologically-relevant media increases with the formation of core-shell QD structures, but an appropriate design of core-shell structures is needed to minimize any reduction in charge injection efficiency to adsorbed oxygen molecules (to form superoxide) and maintain similar quantitative generation of tailored redox species, as measured using electron paramagnetic resonance (EPR) spectroscopy and electrochemical impedance spectroscopy (EIS). Using these findings, we demonstrate the rational design of QDs as selective therapeutic to kill more than 99% of a priority class I pathogen, thus providing an effective therapy against MDR superbugs.

A MARCH6 and IDOL E3 Ubiquitin Ligase Circuit Uncouples Cholesterol Synthesis from Lipoprotein Uptake in Hepatocytes

PubMed Central

Loregger, Anke; Cook, Emma Claire Laura; Nelson, Jessica Kristin; Moeton, Martina; Sharpe, Laura Jane; Engberg, Susanna; Karimova, Madina; Lambert, Gilles; Brown, Andrew John

2015-01-01

Cholesterol synthesis and lipoprotein uptake are tightly coordinated to ensure that the cellular level of cholesterol is adequately maintained. Hepatic dysregulation of these processes is associated with pathological conditions, most notably cardiovascular disease. Using a genetic approach, we have recently identified the E3 ubiquitin ligase MARCH6 as a regulator of cholesterol biosynthesis, owing to its ability to promote degradation of the rate-limiting enzymes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and squalene epoxidase (SQLE). Here, we present evidence for MARCH6 playing a multifaceted role in the control of cholesterol homeostasis in hepatocytes. We identify MARCH6 as an endogenous inhibitor of the sterol regulatory element binding protein (SREBP) transcriptional program. Accordingly, loss of MARCH6 increases expression of SREBP-regulated genes involved in cholesterol biosynthesis and lipoprotein uptake. Unexpectedly, this is associated with a decrease in cellular lipoprotein uptake, induced by enhanced lysosomal degradation of the low-density lipoprotein receptor (LDLR). Finally, we provide evidence that induction of the E3 ubiquitin ligase IDOL represents the molecular mechanism underlying this MARCH6-induced phenotype. Our study thus highlights a MARCH6-dependent mechanism to direct cellular cholesterol accretion that relies on uncoupling of cholesterol synthesis from lipoprotein uptake. PMID:26527619

Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures.

PubMed

Fiorentino, Ilaria; Gualtieri, Roberto; Barbato, Vincenza; Mollo, Valentina; Braun, Sabrina; Angrisani, Alberto; Turano, Mimmo; Furia, Maria; Netti, Paolo A; Guarnieri, Daniela; Fusco, Sabato; Talevi, Riccardo

2015-01-15

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Variable phosphorus uptake rates and allocation across microbial groups in the oligotrophic Gulf of Mexico.

PubMed

Popendorf, Kimberly J; Duhamel, Solange

2015-10-01

Microbial uptake of dissolved phosphorus (P) is an important lever in controlling both microbial production and the fate and cycling of marine P. We investigated the relative role of heterotrophic bacteria and phytoplankton in P cycling by measuring the P uptake rates of individual microbial groups (heterotrophic bacteria and the phytoplankton groups Synechococcus, Prochlorococcus and picoeukaryotic phytoplankton) in the P-depleted Gulf of Mexico. Phosphorus uptake rates were measured using incubations with radiolabelled phosphate and adenosine triphosphate coupled with cell sorting flow cytometry. We found that heterotrophic bacteria were the dominant consumers of P on both a biomass basis and a population basis. Biovolume normalized heterotrophic bacteria P uptake rate per cell (amol P μm(-3) h(-1)) was roughly an order of magnitude greater than phytoplankton uptake rates, and heterotrophic bacteria were responsible for generally greater than 50% of total picoplankton P uptake. We hypothesized that this variation in uptake rates reflects variation in cellular P allocation strategies, and found that, indeed, the fraction of cellular P uptake utilized for phospholipid production was significantly higher in heterotrophic bacteria compared with cyanobacterial phytoplankton. These findings indicate that heterotrophic bacteria have a uniquely P-oriented physiology and play a dominant role in cycling dissolved P. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kahl, S.B.

Although significant progress toward the application of boronated LDL for site selective boron drug delivery has been made during the past year, the loss of our animal and cell culture testing site at WSU has seriously hampered progress on this project. Results are reported of one in vivo biodistribution study in B16BL6-bearing mice and a series of in vitro studies that demonstrate very substantial uptake of boronated LDL in certain cell lines. The influence of 17{alpha}-ethinyl estradiol (EE) on cellular uptake has also been probed. Most significantly LDL loaded with HC was demonstrated to be taken up with exceptional ariditymore » in several lines. Significant progress has also been made in the development of a rapid infrared analysis of boron in boronated LDL solution, which may be useful for clinical BNCT studies in Finland.« less

Tempo-spatially resolved cellular dynamics of human immunodeficiency virus transacting activator of transcription (Tat) peptide-modified nanocargos in living cells

NASA Astrophysics Data System (ADS)

Wei, Lin; Yang, Qiaoyu; Xiao, Lehui

2014-08-01

Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs could not actively target the cell nuclei, which is contrary to previous observations based on fixed cell results. More importantly, the inheritance of TGNPs to the daughter cells through mitosis was found to be the major route to metabolize TGNPs by HeLa cells. These understandings on the cellular uptake mechanism and intracellular fate of nanocargos in living cells would provide deep insight on how to improve and controllably manipulate their translocation efficiency for targeted drug delivery.Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs could not actively target the cell nuclei, which is contrary to previous observations based on fixed cell results. More importantly, the inheritance of TGNPs to the daughter cells through mitosis was found to be the major route to metabolize TGNPs by HeLa cells. These understandings on the cellular uptake mechanism and intracellular fate of nanocargos in living cells would provide deep insight on how to improve and controllably manipulate their translocation efficiency for targeted drug delivery. Electronic supplementary information (ESI) available: Experimental section and additional supporting results as noted in the text. See DOI: 10.1039/c4nr02732a

Functions of Intracellular Retinoid Binding-Proteins.

PubMed

Napoli, Joseph L

Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

PubMed Central

Sohel, Md. Mahmodul Hasan; Hoelker, Michael; Noferesti, Sina Seifi; Salilew-Wondim, Dessie; Tholen, Ernst; Looft, Christian; Rings, Franca; Uddin, Muhammad Jasim; Spencer, Thomas E.; Schellander, Karl; Tesfaye, Dawit

2013-01-01

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment. PMID:24223816

Citrate- and Succinate-Modified Carbonate Apatite Nanoparticles with Loaded Doxorubicin Exhibit Potent Anticancer Activity against Breast Cancer Cells

PubMed Central

Mehbuba Hossain, Sultana; Chowdhury, Ezharul Hoque

2018-01-01

Biodegradable inorganic apatite-based particle complex is popular for its pH-sensitivity at the endosomal acidic environment to facilitate drug release following cellular uptake. Despite being a powerful anticancer drug, doxorubicin shows severe off-target effects and therefore would need a carrier for the highest effectiveness. We aimed to chemically modify carbonate apatite (CA) with Krebs cycle intermediates, such as citrate and succinate in order to control the growth of the resultant particles to more efficiently carry and transport the anticancer drug into the cancer cells. Citrate- or succinate-modified CA particles were synthesized with different concentrations of sodium citrate or sodium succinate, respectively, in the absence or presence of doxorubicin. The drug loading efficiency of the particles and their cellular uptake were observed by quantifying fluorescence intensity. The average diameter and surface charge of the particles were determined using Zetasizer. Cell viability was assessed by MTT assay. Citrate-modified carbonate apatite (CMCA) exhibited the highest (31.38%) binding affinity for doxorubicin and promoted rapid cellular uptake of the drug, leading to the half-maximal inhibitory concentration 1000 times less than that of the free drug in MCF-7 cells. Hence, CMCA nanoparticles with greater surface area enhance cytotoxicity in different breast cancer cells by enabling higher loading and more efficient cellular uptake of the drug. PMID:29534497

Cd(2+) Triggered the FRET "ON": A New Molecular Switch for the Ratiometric Detection of Cd(2+) with Live-Cell Imaging and Bound X-ray Structure.

PubMed

Aich, Krishnendu; Goswami, Shyamaprosad; Das, Sangita; Mukhopadhyay, Chitrangada Das; Quah, Ching Kheng; Fun, Hoong-Kun

2015-08-03

On the basis of the Förster resonance energy transfer mechanism between rhodamine and quinoline-benzothiazole conjugated dyad, a new colorimetric as well as fluorescence ratiometric probe was synthesized for the selective detection of Cd(2+). The complex formation of the probe with Cd(2+) was confirmed through Cd(2+)-bound single-crystal structure. Capability of the probe as imaging agent to detect the cellular uptake of Cd(2+) was demonstrated here using living RAW cells.

Engineering the lipid layer of lipid-PLGA hybrid nanoparticles for enhanced in vitro cellular uptake and improved stability.

PubMed

Hu, Yun; Hoerle, Reece; Ehrich, Marion; Zhang, Chenming

2015-12-01

Lipid-polymer hybrid nanoparticles (NPs), consisting of a polymeric core and a lipid shell, have been intensively examined as delivery systems for cancer drugs, imaging agents, and vaccines. For applications in vaccine particularly, the hybrid NPs need to be able to protect the enclosed antigens during circulation, easily be up-taken by dendritic cells, and possess good stability for prolonged storage. However, the influence of lipid composition on the performance of hybrid NPs has not been well studied. In this study, we demonstrate that higher concentrations of cholesterol in the lipid layer enable slower and more controlled antigen release from lipid-poly(lactide-co-glycolide) acid (lipid-PLGA) NPs in human serum and phosphate buffered saline (PBS). Higher concentrations of cholesterol also promoted in vitro cellular uptake of hybrid NPs, improved the stability of the lipid layer, and protected the integrity of the hybrid structure during long-term storage. However, stabilized hybrid structures of high cholesterol content tended to fuse with each other during storage, resulting in significant size increase and lowered cellular uptake. Additional experiments demonstrated that PEGylation of NPs could effectively minimize fusion-caused size increase after long term storage, leading to improved cellular uptake, although excessive PEGylation will not be beneficial and led to reduced improvement. This paper reports the engineering of the lipid layer that encloses a polymeric nanoparticle, which can be used as a carrier for drug and vaccine molecules for targeted delivery. We demonstrated that the concentration of cholesterol is critical for the stability and uptake of the hybrid nanoparticles by dendritic cells, a targeted cell for the delivery of immune effector molecules. However, we found that hybrid nanoparticles with high cholesterol concentration tend to fuse during storage resulting in larger particles with decreased cellular uptake. This problem is subsequently solved by PEGylating the hybrid nanoparticles. With increased research and clinical applications of lipid-polymer hybrid nanoparticles in drug and vaccine delivery, this work will significantly impact the design of the hybrid nanoparticles for minimized molecule release during circulation and increased bioavailability of the target molecules. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Mathematical Modeling and Experimental Validation of Nanoemulsion-Based Drug Transport across Cellular Barriers.

PubMed

Kadakia, Ekta; Shah, Lipa; Amiji, Mansoor M

2017-07-01

Nanoemulsions have shown potential in delivering drug across epithelial and endothelial cell barriers, which express efflux transporters. However, their transport mechanisms are not entirely understood. Our goal was to investigate the cellular permeability of nanoemulsion-encapsulated drugs and apply mathematical modeling to elucidate transport mechanisms and sensitive nanoemulsion attributes. Transport studies were performed in Caco-2 cells, using fish oil nanoemulsions and a model substrate, rhodamine-123. Permeability data was modeled using a semi-mechanistic approach, capturing the following cellular processes: endocytotic uptake of the nanoemulsion, release of rhodamine-123 from the nanoemulsion, efflux and passive permeability of rhodamine-123 in aqueous solution. Nanoemulsions not only improved the permeability of rhodamine-123, but were also less sensitive to efflux transporters. The model captured bidirectional permeability results and identified sensitive processes, such as the release of the nanoemulsion-encapsulated drug and cellular uptake of the nanoemulsion. Mathematical description of cellular processes, improved our understanding of transport mechanisms, such as nanoemulsions don't inhibit efflux to improve drug permeability. Instead, their endocytotic uptake, results in higher intracellular drug concentrations, thereby increasing the concentration gradient and transcellular permeability across biological barriers. Modeling results indicated optimizing nanoemulsion attributes like the droplet size and intracellular drug release rate, may further improve drug permeability.

Expression of a monocarboxylate transporter in reticular cells of the mouse lymph node and its involvement in the uptake of exogenous particles.

PubMed

Zheng, Miao; Ishiguro-Oonuma, Toshina; Iwanaga, Toshihiko

2014-01-01

The monocarboxylate transporter (MCT)-1 plays an important role in the transfer of monocarboxylate metabolites such as lactate, ketone bodies, and acetic acid. The present study revealed the selective localization of MCT1 in reticular cells of the murine lymph node. An intense MCT1 immunoreactivity was found in the reticular cells forming a cellular network together with sinus-lining cells in the medullary sinuses and in cells covering the inside of subcapsular sinuses.Electron-microscopically, MCT1 was localized along the plasma membrane of the reticular cells.The medullary reticular cells vigorously ingested carboxylate-modified latex particles, but any reticular cells within the cortical lymphoid follicles and medullary cords neither expressed MCT1 nor incorporated latex particles. MCT1-immunoreactive reticular cells also expressed LYVE-1,which is a hyaluronan receptor abundant in both the lymphatic endothelium and hepatic sinusoidal epithelium. The selective localization of MCT1 and LYVE-1 suggests a high level of activity for lymphoid reticular cells in the uptake of carboxylate-modified and hyaluronate waste substances circulating in the body.

Differential Roles of Cysteine Residues in Cellular Trafficking, Dimerization, and Function of the HDL Receptor, SR-BI *

PubMed Central

Hu, Jie; Zhang, Zhonghua; Shen, Wen-Jun; Nomoto, Ann; Azhar, Salman

2011-01-01

The scavenger receptor, class B, type I (SR-BI) binds high-density lipoprotein (HDL) and mediates selective delivery of cholesteryl esters (CEs) to the liver and steroidogenic cells of the adrenal and gonads. Although it is clear that the large extracellular domain (ECD) of SR-BI binds HDL, the role of ECD in the selective HDL-CE transport remains poorly understood. In this study, we used a combination of mutational and chemical approaches to systematically evaluate the contribution of cysteine residues, especially six cysteine residues of ECD, in SR-BI-mediated selective HDL-CE uptake, intracellular trafficking and SR-BI dimerization. Pretreatment of SR-BI overexpressing COS-7 cells with disulfide (S-S) bond reducing agent, β-mercaptoethanol (100 mM) or dithiothreitol (DTT) (10 mM) modestly, but significantly impaired the SR-BI mediated selective HDL-CE uptake. Treatment of SR-BI overexpressing COS-7 cells with the optimum doses of membrane permeant alkyl methanethiosulfonate (MTS) reagents, positively charged MTSEA or neutral MMTS that specifically react with the free sulfhydryl group of cysteine reduced the SR-BI-mediated selective HDL-CE uptake, indicating that certain intracellular free cysteine residues may also be critically involved in the selective cholesterol transport process. In contrast, use of membrane impermeant MTS reagent, positively charged MTSET and negatively charged MTSES showed no such effect. Next, the importance of eight cysteine residues in SR-BI expression, cell surface expression, dimer formation and selective HDL-derived CE transport was evaluated. These cysteine residues were replaced either singly or in pairs with serine and the mutant SR-BIs expressed in either COS-7 or CHO cells. Four mutations, C280S, C321S, C323S or C334S of the ECD, either singly or in various pair combinations, resulted in significant decreases in SR-BI (HDL) binding activity, selective-CE uptake, and trafficking to cell surface. Surprisingly, we found that mutation of the two remaining cysteine residues, C251 and C384 of the ECD, had no effect on either SR-BI expression or function. Other cysteine mutations and substitutions were also without any effect. Western blot data indicated that single and double mutants of C280, C321, C323 and C334 residues strongly favor dimer formation. However, they are rendered non-functional presumably due to mutation-induced formation of aberrant disulfide linkages resulting in inhibition of optimal HDL binding and, thus, selective HDL-CE uptake. These results provide novel insights about the functional role of four cysteine residues, C280, C321, C323 and C334 of SR-BI ECD domain in SR-BI expression and trafficking to cell surface, its dimerization, and associated selective CE transport function. PMID:22097902

Assessing the Role of Dissolved Organic Phosphate on Rates of Microbial Phosphorus Cycling

NASA Astrophysics Data System (ADS)

Gonzalez, A. C.; Popendorf, K. J.; Duhamel, S.

2016-02-01

Phosphorus (P) is an element crucial to life, and it is limiting in many parts of the ocean. In oligotrophic environments, the dissolved P pool is cycled rapidly through the activity of microbes, with turnover times of several hours or less. The overarching aim of this study was to assess the flux of P from picoplankton to the dissolved pool and the role this plays in fueling rapid P cycling. To determine if specific microbial groups are responsible for significant return of P to the dissolved pool during cell lifetime, we compared the rate of cellular P turnover (cell-Pτ, the rate of cellular P uptake divided by cellular P content) to the rate of cellular biomass turnover (cellτ). High rates of P return to the dissolved pool during cell lifetime (high cell-Pτ/cellτ) indicate significant P regeneration, fueling more rapid turnover of the dissolved P pool. We hypothesized that cell-Pτ/cellτ varies widely across picoplankton groups. One factor influencing this variation may be each microbial group's relative uptake of dissolved organic phosphorus (DOP) versus dissolved inorganic phosphorus (DIP). As extracellular hydrolysis is necessary for P incorporation from DOP, this process may return more P to the dissolved pool than DIP incorporation. This leads to the question: does a picoplankton's relative uptake of DOP (versus DIP) affect the rate at which it returns phosphorus to the dissolved pool? To address this question, we compared the rate of cellular P turnover based on uptake of DOP and uptake DIP using cultured representatives of three environmentally significant picoplankton groups: Prochlorococcus, Synechococcus, and heterotrophic bacteria. These different picoplankton groups are known to take up different ratios of DOP to DIP, and may in turn make significantly different contributions to the regeneration and cycling phosphorus. These findings have implications towards our understanding of the timeframes of biogeochemical cycling of phosphorus in the ocean.

In vitro and in vivo evaluations of a hydrophilic 64Cu-bis(thiosemicarbazonato)-glucose conjugate for hypoxia imaging.

PubMed

Bayly, Simon R; King, Robert C; Honess, Davina J; Barnard, Peter J; Betts, Helen M; Holland, Jason P; Hueting, Rebekka; Bonnitcha, Paul D; Dilworth, Jonathan R; Aigbirhio, Franklin I; Christlieb, Martin

2008-11-01

A water-soluble glucose conjugate of the hypoxia tracer 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) was synthesized and radiolabeled (64Cu-ATSE/A-G). Here we report our initial biological experiments with 64Cu-ATSE/A-G and compare the results with those obtained for 64Cu-ATSM and 18F-FDG. The uptake of 64Cu-ATSE/A-G and 64Cu-ATSM into HeLa cells in vitro was investigated at a range of dissolved oxygen concentrations representing normoxia, hypoxia, and anoxia. Small-animal PET with 64Cu-ATSE/A-G was performed in male BDIX rats implanted with P22 syngeneic carcinosarcomas. Images of 64Cu-ATSM and 18F-FDG were obtained in the same model for comparison. 64CuATSE/A-G showed oxygen concentration-dependent uptake in vitro and, under anoxic conditions, showed slightly lower levels of cellular uptake than 64Cu-ATSM; uptake levels under hypoxic conditions were also lower. Whereas the normoxic uptake of 64Cu-ATSM increased linearly over time, 64Cu-ATSE/A-G uptake remained at low levels over the entire time course. In the PET study, 64CuATSE/A-G showed good tumor uptake and a biodistribution pattern substantially different from that of each of the controls. In marked contrast to the findings for 64Cu-ATSM, renal clearance and accumulation in the bladder were observed. 64Cu-ATSE/A-G did not display the characteristic brain and heart uptake of 18F-FDG. The in vitro cell uptake studies demonstrated that 64Cu-ATSE/A-G retained hypoxia selectivity and had improved characteristics when compared with 64Cu-ATSM. The in vivo PET results indicated a difference in the excretion pathways, with a shift from primarily hepatointestinal for 64Cu-ATSM to partially renal with 64Cu-ATSE/A-G. This finding is consistent with the hydrophilic nature of the glucose conjugate. A comparison with 18F-FDG PET results revealed that 64Cu-ATSE/A-G was not a surrogate for glucose metabolism. We have demonstrated that our method for the modification of Cu-bis(thiosemicarbazonato) complexes allows their biodistribution to be modified without negating their hypoxia selectivity or tumor uptake properties.

Mitotic trafficking of silicon microparticles†

PubMed Central

Serda, Rita E.; Ferrati, Silvia; Godin, Biana; Tasciotti, Ennio; Liu, XueWu

2010-01-01

Multistage carriers were recently introduced by our laboratory, with the concurrent objectives of co-localized delivery of multiple therapeutic agents, the “theranostic” integration of bioactive moieties with imaging contrast, and the selective, potentially personalized bypassing of the multiplicity of biological barriers that adversely impact biodistribution of vascularly injected particulates. Mesoporous (“nanoporous”) silicon microparticles were selected as primary carriers in multi-stage devices, with targets including vascular endothelia at pathological lesions. The objective of this study was to evaluate biocompatibility of mesoporous silicon microparticles with endothelial cells using in vitro assays with an emphasis on microparticle compatibility with mitotic events. We observed that vascular endothelial cells, following internalization of silicon microparticles, maintain cellular integrity, as demonstrated by cellular morphology, viability and intact mitotic trafficking of vesicles bearing silicon microparticles. The presence of gold or iron oxide nanoparticles within the porous matrix did not alter the cellular uptake of particles or the viability of endothelial cells subsequent to engulfment of microparticles. Endothelial cells maintained basal levels of IL-6 and IL-8 release in the presence of silicon microparticles. This is the first study that demonstrates polarized, ordered partitioning of endosomes based on tracking microparticles. The finding that mitotic sorting of endosomes is unencumbered by the presence of nanoporous silicon microparticles advocates the use of silicon microparticles for biomedical applications. PMID:20644846

On Guanidinium and Cellular Uptake

PubMed Central

2015-01-01

Guanidinium-rich scaffolds facilitate cellular translocation and delivery of bioactive cargos through biological barriers. Although impressive uptake has been demonstrated for nonoligomeric and nonpept(o)idic guanidinylated scaffolds in cell cultures and animal models, the fundamental understanding of these processes is lacking. Charge pairing and hydrogen bonding with cell surface counterparts have been proposed, but their exact role remains putative. The impact of the number and spatial relationships of the guanidinium groups on delivery and organelle/organ localization is yet to be established. PMID:25019333

Surface Chemistry Manipulation of Gold Nanorods Displays High Cellular Uptake In Vitro While Preserving Optical Properties for Bio-Imaging and Photo-Thermal Applications

DTIC Science & Technology

2016-03-28

PROPERTIES FOR BIO -IMAGING AND PHOTO-THERMAL APPLICATIONS ANTHONY B. POLITO III, Maj, USAF, BSC, PhD, MT(ASCP)SBB March 2016 Final Report for March...HIGH CELLULAR UPTAKE IN VITRO WHILE PRESERVING OPTICAL PROPERTIES FOR BIO -IMAGING AND PHOTO-THERMAL APPLICATIONS. 5a. CONTRACT NUMBER 5b...These findings identify MTAB-TA GNRs as prime candidates for use in nano-based bio -imaging and photo-thermal applications. 15. SUBJECT TERMS

Size-dependent cellular uptake mechanism and cytotoxicity toward calcium oxalate on Vero cells

NASA Astrophysics Data System (ADS)

Sun, Xin-Yuan; Gan, Qiong-Zhi; Ouyang, Jian-Ming

2017-02-01

Urinary crystals with various sizes are present in healthy individuals and patients with kidney stone; however, the cellular uptake mechanism of calcium oxalate of various sizes has not been elucidated. This study aims to compare the internalization of nano-/micron-sized (50 nm, 100 nm, and 1 μm) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals in African green monkey renal epithelial (Vero) cells. The internalization and adhesion of COM and COD crystals to Vero cells were enhanced with decreasing crystal size. Cell death rate was positively related to the amount of adhered and internalized crystals and exhibited higher correlation with internalization than that with adhesion. Vero cells mainly internalized nano-sized COM and COD crystals through clathrin-mediated pathways as well as micron-sized crystals through macropinocytosis. The internalized COM and COD crystals were distributed in the lysosomes and destroyed lysosomal integrity to some extent. The results of this study indicated that the size of crystal affected cellular uptake mechanism, and may provide an enlightenment for finding potential inhibitors of crystal uptake, thereby decreasing cell injury and the occurrence of kidney stones.

The minute virus of mice exploits different endocytic pathways for cellular uptake

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcin, Pierre O.; Panté, Nelly, E-mail: pante@zoology.ubc.ca

The minute virus of mice, prototype strain (MVMp), is a non-enveloped, single-stranded DNA virus of the family Parvoviridae. Unlike other parvoviruses, the mechanism of cellular uptake of MVMp has not been studied in detail. We analyzed MVMp endocytosis in mouse LA9 fibroblasts and a tumor cell line derived from epithelial–mesenchymal transition through polyomavirus middle T antigen transformation in transgenic mice. By a combination of immunofluorescence and electron microscopy, we found that MVMp endocytosis occurs at the leading edge of migrating cells in proximity to focal adhesion sites. By using drug inhibitors of various endocytic pathways together with immunofluorescence microscopy andmore » flow cytometry analysis, we discovered that MVMp can use a number of endocytic pathways, depending on the host cell type. At least three different mechanisms were identified: clathrin-, caveolin-, and clathrin-independent carrier-mediated endocytosis, with the latter occurring in transformed cells but not in LA9 fibroblasts. - Highlights: • MVMp uptake takes place at the leading edge of migrating cells. • MVMp exploits a variety of endocytic pathways. • MVMp could use clathrin- and caveolin-mediated endocytosis. • MVMp could also use clathrin-independent carriers for cellular uptake.« less

Mechanisms of the ultrasound-mediated intracellular delivery of liposomes and dextrans.

PubMed

Afadzi, Mercy; Strand, Sabina P; Nilssen, Esben A; Måsøy, Svein-Erik; Johansen, Tonni F; Hansen, Rune; Angelsen, Bjørn A; de L Davies, Catharina

2013-01-01

The mechanism involved in the ultrasoundenhanced intracellular delivery of fluorescein-isothiocyanate (FITC)-dextran (molecular weight 4 to 2000 kDa) and liposomes containing doxorubicin (Dox) was studied using HeLa cells and an ultrasound transducer at 300 kHz, varying the acoustic power. The cellular uptake and cell viability were measured using flow cytometry and confocal microscopy. The role of endocytosis was investigated by inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis. Microbubbles were found to be required during ultrasound treatment to obtain enhanced cellular uptake. The percentage of cells internalizing Dox and dextran increased with increasing mechanical index. Confocal images and flow cytometric analysis indicated that the liposomes were disrupted extracellularly and that released Dox was taken up by the cells. The percentage of cells internalizing dextran was independent of the molecular weight of dextrans, but the amount of the small 4-kDa dextran molecules internalized per cell was higher than for the other dextrans. The inhibition of endocytosis during ultrasound exposure resulted in a significant decrease in cellular uptake of dextrans. Therefore, the improved uptake of Dox and dextrans may be a result of both sonoporation and endocytosis.

Development of therapeutic Au-methylene blue nanoparticles for targeted photodynamic therapy of cervical cancer cells.

PubMed

Yu, Jiashing; Hsu, Che-Hao; Huang, Chih-Chia; Chang, Po-Yang

2015-01-14

Photodynamic therapy (PDT) involves the cellular uptake of a photosensitizer (PS) combined with oxygen molecules and light at a specific wavelength to be able to trigger cancer cell death via the apoptosis pathway, which is less harmful and has less inflammatory side effect than necrosis. However, the traditional PDT treatment has two main deficiencies: the dark toxicity of the PS and the poor selectivity of the cellular uptake of PS between the target cells and normal tissues. In this work, methylene blue (MB), a known effective PS, combined with Au nanoparticles (NPs) was prepared using an intermolecular interaction between a polystyrene-alt-maleic acid (PSMA) layer on the Au NPs and MB. The Au@polymer/MB NPs produced a high quantum yield of singlet oxygen molecules, over 50% as much as that of free MB, when they were excited by a dark red light source at 660 nm, but without significant dark toxicity. Furthermore, transferrin (Tf) was conjugated on the Au@polymer/MB NPs via an EDC/NHS reaction to enhance the selectivity to HeLa cells compared to 3T3 fibroblasts. With a hand-held single laser treatment (32 mW/cm) for 4 min, the new Au@polymer/MB-Tf NPs showed a 2-fold enhancement of PDT efficiency toward HeLa cells over the use of free MB at 4 times dosage. Cellular staining examinations showed that the HeLa cells reacted with Au@polymer/MB-Tf NPs and the 660 nm light excitation triggered PDT, which caused the cells to undergo apoptosis ("programmed" cell death). We propose that applying this therapeutic Au@polymer/MB-Tf nanoagent is facile and safe for delivery and cancer cell targeting to simultaneously minimize side effects and accomplish a significant enhancement in photodynamic therapeutic efficiency toward next-generation nanomedicine development.

Synergistic effect of pH-responsive folate-functionalized poloxamer 407-TPGS-mixed micelles on targeted delivery of anticancer drugs.

PubMed

Butt, Adeel Masood; Mohd Amin, Mohd Cairul Iqbal; Katas, Haliza

2015-01-01

Doxorubicin (DOX), an anthracycline anticancer antibiotic, is used for treating various types of cancers. However, its use is associated with toxicity to normal cells and development of resistance due to overexpression of drug efflux pumps. Poloxamer 407 (P407) and vitamin E TPGS (D-α-tocopheryl polyethylene glycol succinate, TPGS) are widely used polymers as drug delivery carriers and excipients for enhancing the drug retention times and stability. TPGS reduces multidrug resistance, induces apoptosis, and shows selective anticancer activity against tumor cells. Keeping in view the problems, we designed a mixed micelle system encapsulating DOX comprising TPGS for its selective anticancer activity and P407 conjugated with folic acid (FA) for folate-mediated receptor targeting to cancer cells. FA-functionalized P407 was prepared by carbodiimide crosslinker chemistry. P407-TPGS/FA-P407-TPGS-mixed micelles were prepared by thin-film hydration method. Cytotoxicity of blank micelles, DOX, and DOX-loaded micelles was determined by alamarBlue(®) assay. The size of micelles was less than 200 nm with encapsulation efficiency of 85% and 73% for P407-TPGS and FA-P407-TPGS micelles, respectively. Intracellular trafficking study using nile red-loaded micelles indicated improved drug uptake and perinuclear drug localization. The micelles show minimal toxicity to normal human cell line WRL-68, enhanced cellular uptake of DOX, reduced drug efflux, increased DOX-DNA binding in SKOV3 and DOX-resistant SKOV3 human ovarian carcinoma cell lines, and enhanced in vitro cytotoxicity as compared to free DOX. FA-P407-TPGS-DOX micelles show potential as a targeted nano-drug delivery system for DOX due to their multiple synergistic factors of selective anticancer activity, inhibition of multidrug resistance, and folate-mediated selective uptake.

Synergistic effect of pH-responsive folate-functionalized poloxamer 407-TPGS-mixed micelles on targeted delivery of anticancer drugs

PubMed Central

Butt, Adeel Masood; Mohd Amin, Mohd Cairul Iqbal; Katas, Haliza

2015-01-01

Background Doxorubicin (DOX), an anthracycline anticancer antibiotic, is used for treating various types of cancers. However, its use is associated with toxicity to normal cells and development of resistance due to overexpression of drug efflux pumps. Poloxamer 407 (P407) and vitamin E TPGS (D-α-tocopheryl polyethylene glycol succinate, TPGS) are widely used polymers as drug delivery carriers and excipients for enhancing the drug retention times and stability. TPGS reduces multidrug resistance, induces apoptosis, and shows selective anticancer activity against tumor cells. Keeping in view the problems, we designed a mixed micelle system encapsulating DOX comprising TPGS for its selective anticancer activity and P407 conjugated with folic acid (FA) for folate-mediated receptor targeting to cancer cells. Methods FA-functionalized P407 was prepared by carbodiimide crosslinker chemistry. P407-TPGS/FA-P407-TPGS-mixed micelles were prepared by thin-film hydration method. Cytotoxicity of blank micelles, DOX, and DOX-loaded micelles was determined by alamarBlue® assay. Results The size of micelles was less than 200 nm with encapsulation efficiency of 85% and 73% for P407-TPGS and FA-P407-TPGS micelles, respectively. Intracellular trafficking study using nile red-loaded micelles indicated improved drug uptake and perinuclear drug localization. The micelles show minimal toxicity to normal human cell line WRL-68, enhanced cellular uptake of DOX, reduced drug efflux, increased DOX–DNA binding in SKOV3 and DOX-resistant SKOV3 human ovarian carcinoma cell lines, and enhanced in vitro cytotoxicity as compared to free DOX. Conclusion FA-P407-TPGS-DOX micelles show potential as a targeted nano-drug delivery system for DOX due to their multiple synergistic factors of selective anticancer activity, inhibition of multidrug resistance, and folate-mediated selective uptake. PMID:25709451

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin.

PubMed

Rink, Jonathan S; Yang, Shuo; Cen, Osman; Taxter, Tim; McMahon, Kaylin M; Misener, Sol; Behdad, Amir; Longnecker, Richard; Gordon, Leo I; Thaxton, C Shad

2017-11-06

Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy, while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer.

Olanzapine and aripiprazole differentially affect glucose uptake and energy metabolism in human mononuclear blood cells.

PubMed

Stapel, Britta; Kotsiari, Alexandra; Scherr, Michaela; Hilfiker-Kleiner, Denise; Bleich, Stefan; Frieling, Helge; Kahl, Kai G

2017-05-01

The use of antipsychotics carries the risk of metabolic side effects, such as weight gain and new onset type-2 diabetes mellitus. The mechanisms of the observed metabolic alterations are not fully understood. We compared the effects of two atypical antipsychotics, one known to favor weight gain (olanzapine), the other not (aripiprazole), on glucose metabolism. Primary human peripheral blood mononuclear cells (PBMC) were isolated and stimulated with olanzapine or aripiprazole for 72 h. Cellular glucose uptake was analyzed in vitro by 18F-FDG uptake. Further measurements comprised mRNA expression of glucose transporter (GLUT) 1 and 3, GLUT1 protein expression, DNA methylation of GLUT1 promoter region, and proteins involved in downstream glucometabolic processes. We observed a 2-fold increase in glucose uptake after stimulation with aripiprazole. In contrast, olanzapine stimulation decreased glucose uptake by 40%, accompanied by downregulation of the cellular energy sensor AMP activated protein kinase (AMPK). GLUT1 protein expression increased, GLUT1 mRNA expression decreased, and GLUT1 promoter was hypermethylated with both antipsychotics. Pyruvat-dehydrogenase (PDH) complex activity decreased with olanzapine only. Our findings suggest that the atypical antipsychotics olanzapine and aripiprazole differentially affect energy metabolism in PBMC. The observed decrease in glucose uptake in olanzapine stimulated PBMC, accompanied by decreased PDH point to a worsening in cellular energy metabolism not compensated by AMKP upregulation. In contrast, aripiprazole stimulation lead to increased glucose uptake, while not affecting PDH complex expression. The observed differences may be involved in the different metabolic profiles observed in aripiprazole and olanzapine treated patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

Diffusion and cellular uptake of drugs in live cells studied with surface-enhanced Raman scattering probes

NASA Astrophysics Data System (ADS)

Bálint, Štefan; Rao, Satish; Sánchez, Mónica Marro; Huntošová, Veronika; Miškovský, Pavol; Petrov, Dmitri

2010-03-01

An understanding of the mechanisms of drug diffusion and uptake through cellular membranes is critical for elucidating drug action and in the development of effective drug delivery systems. We study these processes for emodin, a potential anticancer drug, in live cancer cells using surface-enhanced Raman scattering. Micrometer-sized silica beads covered by nanosized silver colloids are passively embedded into the cell and used as sensors of the drug. We demonstrate that the technique offers distinct advantages: the possibility to study the kinetics of drug diffusion through the cellular membrane toward specific cell organelles, the detection of lower drug concentrations compared to fluorescence techniques, and less damage imparted on the cell.

Wheat germ agglutinin-conjugated PLGA nanoparticles for enhanced intracellular delivery of paclitaxel to colon cancer cells.

PubMed

Wang, Chunxia; Ho, Paul C; Lim, Lee Yong

2010-11-15

The purpose of this study was to investigate the potentiation of the anticancer activity and enhanced cellular retention of paclitaxel-loaded PLGA nanoparticles after surface conjugation with wheat germ agglutinin (WGA) against colon cancer cells. Glycosylation patterns of representative colon cancer cells confirmed the higher expression levels of WGA-binding glycoproteins in the Caco-2 and HT-29 cells, than in the CCD-18Co cells. Cellular uptake and in vitro cytotoxicity of WNP (final formulation) against colon cell lines was evaluated alongside control formulations. Confocal microscopy and quantitative analysis of intracellular paclitaxel were used to monitor the endocytosis and retention of nanoparticles inside the cells. WNP showed enhanced anti-proliferative activity against Caco-2 and HT-29 cells compared to corresponding nanoparticles without WGA conjugation (PNP). The greater efficacy of WNP was associated with higher cellular uptake and sustained intracellular retention of paclitaxel, which in turn was attributed to the over-expression of N-acetyl-D-glucosamine-containing glycoprotein on the colon cell membrane. WNP also demonstrated increased intracellular retention in the Caco-2 (30% of uptake) and HT-29 (40% of uptake) cells, following post-uptake incubation with fresh medium, compared to the unconjugated PNP nanoparticles (18% in Caco-2) and (27% in HT-29), respectively. Cellular trafficking study of WNP showed endocytosed WNP could successful escape from the endo-lysosome compartment and release into the cytosol with increasing incubation time. It may be concluded that WNP has the potential to be applied as a targeted delivery platform for paclitaxel in the treatment of colon cancer. Copyright © 2010 Elsevier B.V. All rights reserved.

Transport of selenium across the plasma membrane of primary hepatocytes and enterocytes of rainbow trout.

PubMed

Misra, Sougat; Kwong, Raymond W M; Niyogi, Som

2012-05-01

Transport of essential solutes across biological membranes is one of the fundamental characteristics of living cells. Although selenium is an essential micronutrient, little is known about the cellular mechanisms of chemical species-specific selenium transport in fish. We report here the kinetic and pharmacological transport characteristics of selenite and its thiol (glutathione and l-cysteine) derivatives in primary cultures of hepatocytes and isolated enterocytes of rainbow trout. Findings from the current study suggest an apparent low-affinity linear transport system for selenite in both cell types. However, we recorded high-affinity Hill kinetics (K(d)=3.61±0.28 μmol l(-1)) in enterocytes exposed to selenite in the presence of glutathione. The uptake of selenite in the presence of thiols was severalfold higher than uptake of selenite alone (at equimolar concentration) in both hepatocytes and enterocytes. Cellular accumulation of selenium was found to be energy independent. Interestingly, we observed a decrease in selenite transport with increasing pH, whereas selenite uptake increased with increasing pH in the presence glutathione in both cell types. The cellular uptake of selenite demonstrated a pronounced competitive interaction with a structurally similar compound, sulfite. The uptake of selenite as well as its thiol derivatives was found to be sensitive to the anion transport blocker DIDS, irrespective of the cell type. Inorganic mercury (Hg(2+)) elicited an inhibition of selenite transport in both cell types, but augmented the transport of reduced forms of selenite in hepatocytes. Based on the substrate choice and comparable pharmacological properties, we advocate that multiple anion transport systems are probably involved in the cellular transport of selenite in fish.

Smart Nanoparticles Undergo Phase Transition for Enhanced Cellular Uptake and Subsequent Intracellular Drug Release in a Tumor Microenvironment.

PubMed

Ye, Guihua; Jiang, Yajun; Yang, Xiaoying; Hu, Hongxiang; Wang, Beibei; Sun, Lu; Yang, Victor C; Sun, Duxin; Gao, Wei

2018-01-10

Inefficient cellular uptake and intracellular drug release at the tumor site are two major obstacles limiting the antitumor efficacy of nanoparticle delivery systems. To overcome both problems, we designed a smart nanoparticle that undergoes phase transition in a tumor microenvironment (TME). The smart nanoparticle is generated using a lipid-polypetide hybrid nanoparticle, which comprises a PEGylated lipid monolayer shell and a pH-sensitive hydrophobic poly-l-histidine core and is loaded with the antitumor drug doxorubicin (DOX). The smart nanoparticle undergoes a two-step phase transition at two different pH values in the TME: (i) At the TME (pH e : 7.0-6.5), the smart nanoparticle swells, and its surface potential turns from negative to neutral, facilitating the cellular uptake; (ii) After internalization, at the acid endolysosome (pH endo : 6.5-4.5), the smart nanoparticle dissociates and induces endolysosome escape to release DOX into the cytoplasm. In addition, a tumor-penetrating peptide iNRG was modified on the surface of the smart nanoparticle as a tumor target moiety. The in vitro studies demonstrated that the iNGR-modified smart nanoparticles promoted cellular uptake in the acidic environment (pH 6.8). The in vivo studies showed that the iNGR-modified smart nanoparticles exerted more potent antitumor efficacy against late-stage aggressive breast carcinoma than free DOX. These data suggest that the smart nanoparticles may serve as a promising delivery system for sequential uptake and intracellular drug release of antitumor agents. The easy preparation of these smart nanoparticles may also have advantages in the future manufacture for clinical trials and clinical use.

Carbon nanotubes' surface chemistry determines their potency as vaccine nanocarriers in vitro and in vivo

PubMed Central

Hassan, Hatem A.F.M.; Smyth, Lesley; Rubio, Noelia; Ratnasothy, Kulachelvy; Wang, Julie T.-W.; Bansal, Sukhvinder S.; Summers, Huw D.; Diebold, Sandra S.; Lombardi, Giovanna; Al-Jamal, Khuloud T.

2016-01-01

Carbon nanotubes (CNTs) have shown marked capabilities in enhancing antigen delivery to antigen presenting cells. However, proper understanding of how altering the physical properties of CNTs may influence antigen uptake by antigen presenting cells, such as dendritic cells (DCs), has not been established yet. We hypothesized that altering the physical properties of multi-walled CNTs (MWNTs)-antigen conjugates, e.g. length and surface charge, can affect the internalization of MWNT-antigen by DCs, hence the induced immune response potency. For this purpose, pristine MWNTs (p-MWNTs) were exposed to various chemical reactions to modify their physical properties then conjugated to ovalbumin (OVA), a model antigen. The yielded MWNTs-OVA conjugates were long MWNT-OVA (~ 386 nm), bearing net positive charge (5.8 mV), or short MWNTs-OVA (~ 122 nm) of increasing negative charges (− 23.4, − 35.8 or − 39 mV). Compared to the short MWNTs-OVA bearing high negative charges, short MWNT-OVA with the lowest negative charge demonstrated better cellular uptake and OVA-specific immune response both in vitro and in vivo. However, long positively-charged MWNT-OVA showed limited cellular uptake and OVA specific immune response in contrast to short MWNT-OVA displaying the least negative charge. We suggest that reduction in charge negativity of MWNT-antigen conjugate enhances cellular uptake and thus the elicited immune response intensity. Nevertheless, length of MWNT-antigen conjugate might also affect the cellular uptake and immune response potency; highlighting the importance of physical properties as a consideration in designing a MWNT-based vaccine delivery system. PMID:26802552

Increased cellular uptake of lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles due to surface modification with folic acid.

PubMed

Feuser, Paulo Emilio; Arévalo, Juan Marcelo Carpio; Junior, Enio Lima; Rossi, Gustavo Rodrigues; da Silva Trindade, Edvaldo; Rocha, Maria Eliane Merlin; Jacques, Amanda Virtuoso; Ricci-Júnior, Eduardo; Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H Hermes

2016-12-01

Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid were synthesized by miniemulsion polymerization in just one step. In vitro biocompatibility and cytotoxicity assays on L929 (murine fibroblast), human red blood, and HeLa (uterine colon cancer) cells were performed. The effect of folic acid at the nanoparticles surface was evaluated through cellular uptake assays in HeLa cells. Results showed that the presence of folic acid did not affect substantially the polymer particle size (~120 nm), the superparamagnetic behavior, the encapsulation efficiency of lauryl gallate (~87 %), the Zeta potential (~38 mV) of the polymeric nanoparticles or the release profile of lauryl gallate. The release profile of lauryl gallate from superparamagnetic poly(methyl methacrylate) nanoparticles presented an initial burst effect (0-1 h) followed by a slow and sustained release, indicating a biphasic release system. Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles with folic acid did not present cytotoxicity effects on L929 and human red blood cells. However, free lauryl gallate presented significant cytotoxic effects on L929 and human red blood cells at all tested concentrations. The presence of folic acid increased the cytotoxicity of lauryl gallate loaded in nanoparticles on HeLa cells due to a higher cellular uptake when HeLa cells were incubated at 37 °C. On the other hand, when the nanoparticles were incubated at low temperature (4 °C) cellular uptake was not observed, suggesting that the uptake occurred by folate receptor mediated energy-dependent endocytosis. Based on presented results our work suggests that this carrier system can be an excellent alternative in targeted drug delivery by folate receptor.

Impact of protein pre-coating on the protein corona composition and nanoparticle cellular uptake.

PubMed

Mirshafiee, Vahid; Kim, Raehyun; Park, Soyun; Mahmoudi, Morteza; Kraft, Mary L

2016-01-01

Nanoparticles (NPs) are functionalized with targeting ligands to enable selectively delivering drugs to desired locations in the body. When these functionalized NPs enter the blood stream, plasma proteins bind to their surfaces, forming a protein corona that affects NP uptake and targeting efficiency. To address this problem, new strategies for directing the formation of a protein corona that has targeting capabilities are emerging. Here, we have investigated the feasibility of directing corona composition to promote targeted NP uptake by specific types of cells. We used the well-characterized process of opsonin-induced phagocytosis by macrophages as a simplified model of corona-mediated NP uptake by a desired cell type. We demonstrate that pre-coating silica NPs with gamma-globulins (γ-globulins) produced a protein corona that was enriched with opsonins, such as immunoglobulins. Although immunoglobulins are ligands that bind to receptors on macrophages and elicit phagocytois, the opsonin-rich protein corona did not increase NP uptake by macrophage RAW 264.7 cells. Immunolabeling experiments indicated that the binding of opsonins to their target cell surface receptors was impeded by other proteins in the corona. Thus, corona-mediated NP targeting strategies must optimize both the recruitment of the desired plasma proteins as well as their accessibility and orientation in the corona layer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Approaching the cellular processes involved in the positive effect of glycosaminoglycans on Fe uptake to Caco-2 cells

USDA-ARS?s Scientific Manuscript database

This study constitutes an approach to understand the enhancing effect of glycosaminoglycans (GAGs) on Fe uptake to Caco-2 cells. The high-sulfated GAGs fraction was isolated and purified from cooked haddock. An in vitro digestion/Caco-2 cell culture model was used to monitor Fe uptake (cell ferritin...

Luminescent single-walled carbon nanotube-sensitized europium nanoprobes for cellular imaging

PubMed Central

Avti, Pramod K; Sitharaman, Balaji

2012-01-01

Lanthanoid-based optical probes with excitation wavelengths in the ultra-violet (UV) range (300–325 nm) have been widely developed as imaging probes. Efficient cellular imaging requires that lanthanoid optical probes be excited at visible wavelengths, to avoid UV damage to cells. The efficacy of europium-catalyzed single-walled carbon nanotubes (Eu-SWCNTs), as visible nanoprobes for cellular imaging, is reported in this study. Confocal fluorescence microscopy images of breast cancer cells (SK-BR-3 and MCF-7) and normal cells (NIH 3T3), treated with Eu-SWCNT at 0.2 μg/mL concentration, showed bright red luminescence after excitation at 365 nm and 458 nm wavelengths. Cell viability analysis showed no cytotoxic effects after the incubation of cells with Eu-SWCNTs at this concentration. Eu-SWCNT uptake is via the endocytosis mechanism. Labeling efficiency, defined as the percentage of incubated cells that uptake Eu-SWCNT, was 95%–100% for all cell types. The average cellular uptake concentration was 6.68 ng Eu per cell. Intracellular localization was further corroborated by transmission electron microscopy and Raman microscopy. The results indicate that Eu-SWCNT shows potential as a novel cellular imaging probe, wherein SWCNT sensitizes Eu3+ ions to allow excitation at visible wavelengths, and stable time-resolved red emission. The ability to functionalize biomolecules on the exterior surface of Eu-SWCNT makes it an excellent candidate for targeted cellular imaging. PMID:22619533

Non-specific cellular uptake of surface-functionalized quantum dots

NASA Astrophysics Data System (ADS)

Kelf, T. A.; Sreenivasan, V. K. A.; Sun, J.; Kim, E. J.; Goldys, E. M.; Zvyagin, A. V.

2010-07-01

We report a systematic empirical study of nanoparticle internalization into cells via non-specific pathways. The nanoparticles were comprised of commercial quantum dots (QDs) that were highly visible under a fluorescence confocal microscope. Surface-modified QDs with basic biologically significant moieties, e.g. carboxyl, amino, and streptavidin, were used, in combination with surface derivatization with polyethylene glycol (PEG) for a range of immortalized cell lines. Internalization rates were derived from image analysis and a detailed discussion about the effect of nanoparticle size, charge and surface groups is presented. We find that PEG derivatization dramatically suppresses the non-specific uptake while PEG-free carboxyl and amine functional groups promote QD internalization. These uptake variations displayed a remarkable consistency across different cell types. The reported results are important for experiments concerned with cellular uptake of surface-functionalized nanomaterials, both when non-specific internalization is undesirable and when it is intended for material to be internalized as efficiently as possible.

Simulation of tissue activity curves of 64Cu-ATSM for sub-target volume delineation in radiotherapy

NASA Astrophysics Data System (ADS)

Dalah, E.; Bradley, D.; Nisbet, A.

2010-02-01

There is much interest in positron emission tomography (PET) for measurements of regional tracer concentration in hypoxic tumour-bearing tissue, focusing on the need for accurate radiotherapy treatment planning. Generally, relevant data are taken over multiple time frames in the form of tissue activity curves (TACs), thus providing an indication of vasculature structure and geometry. This is a potential key in providing information on cellular perfusion and limited diffusion. A number of theoretical studies have attempted to describe tracer uptake in tissue cells in an effort to understand such complicated behaviour of cellular uptake and the mechanism of washout. More recently, a novel computerized reaction diffusion equation method was developed by Kelly and Brady (2006 A model to simulate tumour oxygenation and dynamic [18F]-FMISO PET data Phys. Med. Biol. 51 5859-73), where they managed to simulate the realistic dynamic TACs of 18F-FMISO. The model was developed over a multi-step process. Here we present a refinement to the work of Kelly and Brady, such that the model allows simulation of a realistic tissue activity curve (TAC) of any hypoxia selective PET tracer, in a single step process. In this work we show particular interest in simulating the TAC of perhaps the most promising hypoxia selective tracer, 64Cu-ATSM. In addition, we demonstrate its potential role in tumour sub-volume delineation for radiotherapy treatment planning. Simulation results have demonstrated the significant high contrast of imaging using ATSM, with a tumour to blood ratio ranging from 2.24 to 4.1.

The epidermal growth factor receptor (EGFR) promotes uptake of influenza A viruses (IAV) into host cells.

PubMed

Eierhoff, Thorsten; Hrincius, Eike R; Rescher, Ursula; Ludwig, Stephan; Ehrhardt, Christina

2010-09-09

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake.

The Epidermal Growth Factor Receptor (EGFR) Promotes Uptake of Influenza A Viruses (IAV) into Host Cells

PubMed Central

Eierhoff, Thorsten; Hrincius, Eike R.; Rescher, Ursula; Ludwig, Stephan; Ehrhardt, Christina

2010-01-01

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake. PMID:20844577

Single-cell analysis of radiotracers' uptake by fluorescence microscopy: direct and droplet approach

NASA Astrophysics Data System (ADS)

Gallina, M. E.; Kim, T. J.; Vasquez, J.; Tuerkcan, S.; Abbyad, P.; Pratx, G.

2017-02-01

Radionuclides are used for sensitive and specific detection of small molecules in vivo and in vitro. Recently, radioluminescence microscopy extended their use to single-cell studies. Here we propose a new single-cell radioisotopic assay that improves throughput while adding sorting capabilities. The new method uses fluorescence-based sensor for revealing single-cell interactions with radioactive molecular markers. This study focuses on comparing two different experimental approaches. Several probes were tested and Dihydrorhodamine 123 was selected as the best compromise between sensitivity, brightness and stability. The sensor was incorporated either directly within the cell cytoplasm (direct approach), or it was coencapsulated with radiolabeled single-cells in oil-dispersed water droplets (droplet approach). Both approaches successfully activated the fluorescence signal following cellular uptake of 18F-fluorodeoxyglucose (FDG) and external Xrays exposure. The direct approach offered single-cell resolution and longtime stability ( > 20 hours), moreover it could discriminate FDG uptake at labelling concentration as low as 300 μCi/ml. In cells incubated with Dihydrorhodamine 123 after exposure to high radiation doses (8-16 Gy), the fluorescence signal was found to increase with the depletion of ROS quenchers. On the other side, the droplet approach required higher labelling concentrations (1.00 mCi/ml), and, at the current state of art, three cells per droplet are necessary to produce a fluorescent signal. This approach, however, is independent on cellular oxidative stress and, with further improvements, will be more suitable for studying heterogeneous populations. We anticipate this technology to pave the way for the analysis of single-cell interactions with radiomarkers by radiofluorogenic-activated single-cell sorting.

CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

PubMed Central

Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

2016-01-01

Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

Synthesis, characterisation, and in vitro cellular uptake kinetics of nanoprecipitated poly(2-methacryloyloxyethyl phosphorylcholine)- b-poly(2-(diisopropylamino)ethyl methacrylate) (MPC-DPA) polymeric nanoparticle micelles for nanomedicine applications

NASA Astrophysics Data System (ADS)

Salvage, Jonathan P.; Smith, Tia; Lu, Tao; Sanghera, Amendeep; Standen, Guy; Tang, Yiqing; Lewis, Andrew L.

2016-10-01

Nanoscience offers the potential for great advances in medical technology and therapies in the form of nanomedicine. As such, developing controllable, predictable, and effective, nanoparticle-based therapeutic systems remains a significant challenge. Many polymer-based nanoparticle systems have been reported to date, but few harness materials with accepted biocompatibility. Phosphorylcholine (PC) based biomimetic materials have a long history of successful translation into effective commercial medical technologies. This study investigated the synthesis, characterisation, nanoprecipitation, and in vitro cellular uptake kinetics of PC-based polymeric nanoparticle micelles (PNM) formed by the biocompatible and pH responsive block copolymer poly(2-methacryloyloxyethyl phosphorylcholine)- b-poly(2-(diisopropylamino)ethyl methacrylate) (MPC-DPA). Atom transfer radical polymerisation (ATRP), and gel permeation chromatography (GPC) were used to synthesise and characterise the well-defined MPC100-DPA100 polymer, revealing organic GPC, using evaporative light scatter detection, to be more accurate than aqueous GPC for this application. Subsequent nanoprecipitation investigations utilising photon correlation spectroscopy (PCS) revealed PNM size increased with polymer concentration, and conferred Cryo-stability. PNM diameters ranged from circa 64-69 nm, and increased upon hydrophobic compound loading, circa 65-71 nm, with loading efficiencies of circa 60 % achieved, whilst remaining monodisperse. In vitro studies demonstrated that the PNM were of low cellular toxicity, with colony formation and MTT assays, utilising V79 and 3T3 cells, yielding comparable results. Investigation of the in vitro cellular uptake kinetics revealed rapid, 1 h, cellular uptake of MPC100-DPA100 PNM delivered fluorescent probes, with fluorescence persistence for 48 h. This paper presents the first report of these novel findings, which highlight the potential of the system for nanomedicine application development.

Preparation of poly(β-L-malic acid)-based charge-conversional nanoconjugates for tumor-specific uptake and cellular delivery.

PubMed

Zhou, Qing; Yang, Tiehong; Qiao, Youbei; Guo, Songyan; Zhu, Lin; Wu, Hong

2015-01-01

In this study, a multifunctional poly(β-L-malic acid)-based nanoconjugate with a pH-dependent charge conversional characteristic was developed for tumor-specific drug delivery. The short branched polyethylenimine-modified poly(β-L-malic acid) (PEPM) was first synthesized. Then, the fragment HAb18 F(ab')2 and 2,3-dimethylmaleic anhydride were covalently attached to the PEPM to form the nanoconjugate, HDPEPM. In this nanoconjugate, the 2,3-dimethylmaleic anhydride, the shielding group, could shield the positive charge of the conjugate at pH 7.4, while it was selectively hydrolyzed in the tumor extracellular space (pH 6.8) to expose the previously-shielded positive charge. To study the anticancer activity, the anticancer drug, doxorubicin, was covalently attached to the nanoconjugate. The doxorubicin-loaded HDPEPM nanoconjugate was able to efficiently undergo a quick charge conversion from -11.62 mV to 9.04 mV in response to the tumor extracellular pH. The electrostatic interaction between the positively charged HDPEPM nanoconjugates and the negatively charged cell membrane significantly enhanced their cellular uptake, resulting in the enhanced anticancer activity. Also, the tumor targetability of the nanoconjugates could be further improved via the fragment HAb18 F(ab')2 ligand-receptor-mediated tumor cell-specific endocytosis.

Quantification of endocytosis using a folate functionalized silica hollow nanoshell platform

PubMed Central

Sandoval, Sergio; Mendez, Natalie; Alfaro, Jesus G.; Yang, Jian; Aschemeyer, Sharraya; Liberman, Alex; Trogler, William C.; Kummel, Andrew C.

2015-01-01

Abstract. A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N-hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900  μg, respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization. PMID:26315280

Multi-walled carbon nanotubes (NM401) induce ROS-mediated HPRT mutations in Chinese hamster lung fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rubio, Laura; El Yamani, Naouale; Kazimirova, Alena

Although there is an important set of data showing potential genotoxic effects of nanomaterials (NMs) at the DNA (comet assay) and chromosome (micronucleus test) levels, few studies have been conducted to analyze their potential mutagenic effects at gene level. We have determined the ability of multi-walled carbon nanotubes (MWCNT, NM401), to induce mutations in the HPRT gene in Chinese hamster lung (V79) fibroblasts. NM401, characterized in the EU NanoGenotox project, were further studied within the EU Framework Programme Seven (FP7) project NANoREG. From the proliferation assay data we selected a dose-range of 0.12 to 12 µg/cm{sup 2} At these rangemore » we have been able to observe significant cellular uptake of MWCNT by using transmission electron microscopy (TEM), as well as a concentration-dependent induction of intracellular reactive oxygen species. In addition, a clear concentration-dependent increase in the induction of HPRT mutations was also observed. Data support a potential genotoxic/ carcinogenic risk associated with MWCNT exposure. - Highlights: • MWCNT were tested in V79 cells. • Cellular uptake of MWCNT was detected using TEM. • Intracellular ROS induction was observed after MWCNT exposure. • MWCNT induced a concentration-dependent increase of HPRT mutations.« less

Membrane Microdomain Structures of Liposomes and Their Contribution to the Cellular Uptake Efficiency into HeLa Cells.

PubMed

Onuki, Yoshinori; Obata, Yasuko; Kawano, Kumi; Sano, Hiromu; Matsumoto, Reina; Hayashi, Yoshihiro; Takayama, Kozo

2016-02-01

The purpose of this study is to obtain a comprehensive relationship between membrane microdomain structures of liposomes and their cellular uptake efficiency. Model liposomes consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol (Ch) were prepared with various lipid compositions. To detect distinct membrane microdomains in the liposomes, fluorescence-quenching assays were performed at temperatures ranging from 25 to 60 °C using 1,6-diphenyl-1,3,5-hexatriene-labeled liposomes and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl. From the data analysis using the response surface method, we gained a better understanding of the conditions for forming distinct domains (Lo, Ld, and gel phase membranes) as a function of lipid composition. We further performed self-organizing maps (SOM) clustering to simplify the complicated behavior of the domain formation to obtain its essence. As a result, DPPC/DOPC/Ch liposomes in any lipid composition were integrated into five distinct clusters in terms of similarity of the domain structure. In addition, the findings from synchrotron small-angle X-ray scattering analysis offered further insight into the domain structures. As a last phase of this study, an in vitro cellular uptake study using HeLa cells was conducted using SOM clusters' liposomes with/without PEGylation. As a consequence of this study, higher cellular uptake was observed from liposomes having Ch-rich ordered domains.

Exploring the cellular and tissue uptake of nanomaterials in a range of biological samples using multimodal nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

Johnston, Helinor J.; Mouras, Rabah; Brown, David M.; Elfick, Alistair; Stone, Vicki

2015-12-01

The uptake of nanomaterials (NMs) by cells is critical in determining their potential biological impact, whether beneficial or detrimental. Thus, investigation of NM internalization by cells is a common consideration in hazard and efficacy studies. There are currently a number of approaches that are routinely used to investigate NM-cell interactions, each of which have their own advantages and limitations. Ideally, imaging modalities used to investigate NM uptake by cells should not require the NM to be labelled (e.g. with fluorophores) to facilitate its detection. We present a multimodal imaging approach employing a combination of label-free microscopies that can be used to investigate NM-cell interactions. Coherent anti-Stokes Raman scattering microscopy was used in combination with either two-photon photoluminescence or four-wave mixing (FWM) to visualize the uptake of gold or titanium dioxide NMs respectively. Live and fixed cell imaging revealed that NMs were internalized by J774 macrophage and C3A hepatocyte cell lines (15-31 μg ml-1). Sprague Dawley rats were exposed to NMs (intratracheal instillation, 62 μg) and NMs were detected in blood and lung leucocytes, lung and liver tissue, demonstrating that NMs could translocate from the exposure site. Obtained data illustrate that multimodal nonlinear optical microscopy may help overcome current challenges in the assessment of NM cellular uptake and biodistribution. It is therefore a powerful tool that can be used to investigate unlabelled NM cellular and tissue uptake in three dimensions, requires minimal sample preparation, and is applicable to live and fixed cells.

Detection of increased 64Cu uptake by human copper transporter 1 gene overexpression using PET with 64CuCl2 in human breast cancer xenograft model.

PubMed

Kim, Kwang Il; Jang, Su Jin; Park, Ju Hui; Lee, Yong Jin; Lee, Tae Sup; Woo, Kwang Sun; Park, Hyun; Choe, Jae Gol; An, Gwang Il; Kang, Joo Hyun

2014-10-01

Copper is an essential cofactor for a variety of biochemical processes including oxidative phosphorylation, cellular antioxidant activity, and elimination of free radicals. The copper transporter 1 is known to be involved in cellular uptake of copper ions. In this study, we evaluated the utility of human copper transporter 1 (hCTR1) gene as a new reporter gene for (64)Cu PET imaging. Human breast cancer cells (MDA-MB-231) were infected with a lentiviral vector constitutively expressing the hCTR1 gene under super cytomegalovirus promoter, and positive clones (MDA-MB-231-hCTR1) were selected. The expression of hCTR1 gene in MDA-MB-231-hCTR1 cells was measured by reverse transcription polymerase chain reaction, Western blot, and (64)Cu uptake assay. To evaluate the cytotoxic effects induced by hCTR1 expression, the dose-dependent cell survival rate after treatment with cisplatin (Cis-diaminedichloroplatinum (II) [CDDP]) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion. Small-animal PET images were acquired in tumor-bearing mice from 2 to 48 h after an intravenous injection of (64)Cu. The hCTR1 gene expression in MDA-MB-231-hCTR1 cells was confirmed at the RNA and protein expression and the cellular (64)Cu uptake level. MTT assay and trypan blue dye exclusion showed that the cell viability of MDA-MB-231-hCTR1 cells decreased more rapidly than that of MDA-MB-231 cells after treatment with CDDP for 96 or 72 h, respectively. Small-animal PET imaging revealed a higher accumulation of (64)Cu in MDA-MB-231-hCTR1 tumors than in MDA-MB-231 tumors. With respect to the biodistribution data, the percentage injected dose per gram of (64)Cu in the MDA-MB-231 tumors and MDA-MB-231-hCTR1 tumors at 48 h after (64)Cu injection was 2.581 ± 0.254 and 5.373 ± 1.098, respectively. An increase in (64)Cu uptake induced by the expression of hCTR1 gene was demonstrated in vivo and in vitro, suggesting the potential use of hCTR1 gene as a new imaging reporter gene for PET with (64)CuCl2. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Chlorovirus-mediated membrane depolarization of Chlorella alters secondary active transport of solutes.

PubMed

Agarkova, Irina; Dunigan, David; Gurnon, James; Greiner, Timo; Barres, Julia; Thiel, Gerhard; Van Etten, James L

2008-12-01

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of a family of large, double-stranded DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus Chlorovirus. PBCV-1 infection results in rapid host membrane depolarization and potassium ion release. One interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (Kcv) that are considered responsible for the host membrane depolarization and, as a consequence, the efflux of potassium ions. This report examines the relationship between cellular depolarization and solute uptake. Annotation of the virus host Chlorella strain NC64A genome revealed 482 putative transporter-encoding genes; 224 are secondary active transporters. Solute uptake experiments using seven radioactive compounds revealed that virus infection alters the transport of all the solutes. However, the degree of inhibition varied depending on the solute. Experiments with nystatin, a drug known to depolarize cell membranes, produced changes in solute uptake that are similar but not identical to those that occurred during virus infection. Therefore, these studies indicate that chlorovirus infection causes a rapid and sustained depolarization of the host plasma membrane and that this depolarization leads to the inhibition of secondary active transporters that changes solute uptake.

Chlorovirus-Mediated Membrane Depolarization of Chlorella Alters Secondary Active Transport of Solutes▿

PubMed Central

Agarkova, Irina; Dunigan, David; Gurnon, James; Greiner, Timo; Barres, Julia; Thiel, Gerhard; Van Etten, James L.

2008-01-01

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of a family of large, double-stranded DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus Chlorovirus. PBCV-1 infection results in rapid host membrane depolarization and potassium ion release. One interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (Kcv) that are considered responsible for the host membrane depolarization and, as a consequence, the efflux of potassium ions. This report examines the relationship between cellular depolarization and solute uptake. Annotation of the virus host Chlorella strain NC64A genome revealed 482 putative transporter-encoding genes; 224 are secondary active transporters. Solute uptake experiments using seven radioactive compounds revealed that virus infection alters the transport of all the solutes. However, the degree of inhibition varied depending on the solute. Experiments with nystatin, a drug known to depolarize cell membranes, produced changes in solute uptake that are similar but not identical to those that occurred during virus infection. Therefore, these studies indicate that chlorovirus infection causes a rapid and sustained depolarization of the host plasma membrane and that this depolarization leads to the inhibition of secondary active transporters that changes solute uptake. PMID:18842725

Cellular cytotoxic response induced by highly purified multi-wall carbon nanotube in human lung cells.

PubMed

Tsukahara, Tamotsu; Haniu, Hisao

2011-06-01

Carbon nanotubes, a promising nanomaterial with unique characteristics, have applications in a variety of fields. The cytotoxic effects of carbon nanotubes are partially due to the induction of oxidative stress; however, the detailed mechanisms of nanotube cytotoxicity and their interaction with cells remain unclear. In this study, the authors focus on the acute toxicity of vapor-grown carbon fiber, HTT2800, which is one of the most highly purified multi-wall carbon nanotubes (MWCNT) by high-temperature thermal treatment. The authors exposed human bronchial epithelial cells (BEAS-2B) to HTT2800 and measured the cellular uptake, mitochondrial function, cellular LDH release, apoptotic signaling, reactive oxygen species (ROS) generation and pro-inflammatory cytokine release. The HTT2800-exposed cells showed cellular uptake of the carbon nanotube, increased cell death, enhanced DNA damage, and induced cytokine release. However, the exposed cells showed no obvious intracellular ROS generation. These cellular and molecular findings suggest that HTT2800 could cause a potentially adverse inflammatory response in BEAS-2B cells.

Glucagon-like peptide-1 increases myocardial glucose uptake via p38alpha MAP kinase-mediated, nitric oxide-dependent mechanisms in conscious dogs with dilated cardiomyopathy.

PubMed

Bhashyam, Siva; Fields, Anjali V; Patterson, Brandy; Testani, Jeffrey M; Chen, Li; Shen, You-Tang; Shannon, Richard P

2010-07-01

We have shown that glucagon-like peptide-1 (GLP-1[7-36] amide) stimulates myocardial glucose uptake in dilated cardiomyopathy (DCM) independent of an insulinotropic effect. The cellular mechanisms of GLP-1-induced myocardial glucose uptake are unknown. Myocardial substrates and glucoregulatory hormones were measured in conscious, chronically instrumented dogs at control (n=6), DCM (n=9) and DCM after treatment with a 48-hour infusion of GLP-1 (7-36) amide (n=9) or vehicle (n=6). GLP-1 receptors and cellular pathways implicated in myocardial glucose uptake were measured in sarcolemmal membranes harvested from the 4 groups. GLP-1 stimulated myocardial glucose uptake (DCM: 20+/-7 nmol/min/g; DCM+GLP-1: 61+/-12 nmol/min/g; P=0.001) independent of increased plasma insulin levels. The GLP-1 receptors were upregulated in the sarcolemmal membranes (control: 98+/-2 density units; DCM: 256+/-58 density units; P=0.046) and were expressed in their activated (65 kDa) form in DCM. The GLP-1-induced increases in myocardial glucose uptake did not involve adenylyl cyclase or Akt activation but was associated with marked increases in p38alpha MAP kinase activity (DCM+vehicle: 97+/-22 pmol ATP/mg/min; DCM+GLP-1: 170+/-36 pmol ATP/mg/min; P=0.051), induction of nitric oxide synthase 2 (DCM+vehicle: 151+/-13 density units; DCM+GLP-1: 306+/-12 density units; P=0.001), and GLUT-1 translocation (DCM+vehicle: 21+/-3% membrane bound; DCM+GLP-1: 39+/-3% membrane bound; P=0.005). The effects of GLP-1 on myocardial glucose uptake were blocked by pretreatment with the p38alpha MAP kinase inhibitor or the nonspecific nitric oxide synthase inhibitor nitro-l-arginine. GLP-1 stimulates myocardial glucose uptake through a non-Akt-1-dependent mechanism by activating cellular pathways that have been identified in mediating chronic hibernation and the late phase of ischemic preconditioning.

Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro

PubMed Central

Claudia, Meindl; Kristin, Öhlinger; Jennifer, Ober; Eva, Roblegg; Eleonore, Fröhlich

2017-01-01

At many portals of entry the relative uptake by phagocytes and non-phagocytic cells has a prominent effect on availability and biological action of nanoparticles (NPs). Cellular uptake can be determined for fluorescence-labeled NPs. The present study compares three methods (plate reader, flow cytometry and image analysis) in order to investigate the influence of particle size and functionalization and medium content on cellular uptake of fluorescence–labeled polystyrene particles and to study the respective method’s suitability for uptake studies. For comparison between the techniques, ratios of macrophage to alveolar epithelial cell uptakes were used. Presence of serum protein in the exposure solution decreased uptake of carboxyl-functionalized and non-functionalized particles; there was no clear effect for the amine-functionalized particles. The 200 nm non- or carboxyl-functionalized NPs were taken up preferentially by phagocytes while for amine-functionalized particles preference was lowest. The presence of the serum slightly increased the preference for these particles. In conclusion, due to the possibility of calibration, plate reader measurements might present a better option than the other techniques to (semi)quantify differences between phagocytes and non-phagocytic cells for particles with different fluorescence. In order to obtain unbiased data the fluorescent labeling has to fulfill certain requirements. PMID:28065592

Celllular Uptake and Clearance of TIO2 Nanoparticles

EPA Science Inventory

Differential rates of cellular uptake and clearance of engineered nanomaterials may influence the propensity for tissue accumulation under chronic exposure conditions. A retinal pigment epithelial cell line (ARPE-19) was used to investigate 1) if Ti02 (Degussa, P25) nanoparticles...

Live Cell Imaging of a Fluorescent Gentamicin Conjugate

PubMed Central

Escobedo, Jorge O.; Chu, Yu-Hsuan; Wang, Qi; Steyger, Peter S.; Strongin, Robert M.

2012-01-01

Understanding cellular mechanisms of ototoxic and nephrotoxic drug uptake, intracellular distribution, and molecular trafficking across cellular barrier systems aids the study of potential uptake blockers that preserve sensory and renal function during critical life-saving therapy. Herein we report the design, synthesis characterization and evaluation of a fluorescent conjugate of the aminoglycoside antibiotic gentamicin. Live cell imaging results show the potential utility of this new material. Related gentamicin conjugates studied to date quench in live kindney cells, and have been largely restricted to use in fixed (delipidated) cells. PMID:22545403

Effects of calcium on hepatocyte iron uptake from transferrin, iron-pyrophosphate and iron-ascorbate.

PubMed

Nilsen, T

1991-10-16

Calcium stimulates hepatocyte iron uptake from transferrin, ferric-iron-pyrophosphate and ferrous-iron-ascorbate. Maximal stimulation of iron uptake is observed at 1-1.5 mM of extra-cellular calcium and the effect is reversible and immediate. Neither the receptor affinity for transferrin, nor the total amounts of transferrin associated with the cells or the rate of transferrin endocytosis are significantly affected by calcium. In the presence of calcium the rate of iron uptake of non-transferrin bound iron increases abruptly at approximate 17 degrees C and 27 degrees C and as assessed by Arrhenius plots, the activation energy is reduced in a calcium dependent manner at approx. 27 degrees C. At a similar temperature, i.e., between 25 degrees C and 28 degrees C, calcium increases the rates of cellular iron uptake from transferrin in a way that is not reflected in the rate of transferrin endocytosis. By the results of this study it is concluded that calcium increases iron transport across the plasma membrane by a mechanism dependent on membrane fluidity.

Overexpression of amyloid precursor protein increases copper content in HEK293 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suazo, Miriam; Hodar, Christian; Morgan, Carlos

2009-05-15

Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer's disease. However, its physiological function remains elusive. Cu{sup 2+} binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu{sup 2+} reduction and {sup 64}Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu{sup 2+} reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu{sup 2+} ions. Moreover, wild-type cells exposed to bothmore » Cu{sup 2+} ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu{sup 2+} reductase activity and increased {sup 64}Cu uptake. We conclude that Cu{sup 2+} reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.« less

Point-particle method to compute diffusion-limited cellular uptake.

PubMed

Sozza, A; Piazza, F; Cencini, M; De Lillo, F; Boffetta, G

2018-02-01

We present an efficient point-particle approach to simulate reaction-diffusion processes of spherical absorbing particles in the diffusion-limited regime, as simple models of cellular uptake. The exact solution for a single absorber is used to calibrate the method, linking the numerical parameters to the physical particle radius and uptake rate. We study the configurations of multiple absorbers of increasing complexity to examine the performance of the method by comparing our simulations with available exact analytical or numerical results. We demonstrate the potential of the method to resolve the complex diffusive interactions, here quantified by the Sherwood number, measuring the uptake rate in terms of that of isolated absorbers. We implement the method in a pseudospectral solver that can be generalized to include fluid motion and fluid-particle interactions. As a test case of the presence of a flow, we consider the uptake rate by a particle in a linear shear flow. Overall, our method represents a powerful and flexible computational tool that can be employed to investigate many complex situations in biology, chemistry, and related sciences.

Oncogene pathway activation in mammary tumors dictates [18F]-FDG-PET uptake

PubMed Central

Alvarez, James V.; Belka, George K.; Pan, Tien-chi; Chen, Chien-Chung; Blankemeyer, Eric; Alavi, Abass; Karp, Joel; Chodosh, Lewis A.

2015-01-01

Increased glucose utilization is a hallmark of human cancer that is used to image tumors clinically. In this widely used application, glucose uptake by tumors is monitored by positron emission tomography (PET) of the labeled glucose analog F-18-2-fluoro-2-deoxyglucose (18F-FDG). Despite its widespread clinical use, the cellular and molecular mechanisms that determine FDG uptake - a tool that can monitor tumor heterogeneity - remain poorly understood. In this study, we compared FDG uptake in mammary tumors driven by the Akt1, c-MYC, HER2/neu, Wnt1 or H-Ras oncogenes in genetically engineered mice, correlating it to tumor growth, cell proliferation and levels of gene expression involved in key steps of glycolytic metabolism. We found that FDG uptake by tumors was dictated principally by the driver oncogene and was not independently associated with tumor growth or cellular proliferation. Oncogene downregulation resulted in a rapid decrease in FDG uptake, preceding effects on tumor regression, irrespective of the baseline level of uptake. FDG uptake correlated positively with expression of hexokinase-2 (HK2) and HIF-1α and associated negatively with PFK-2b expression and p-AMPK. The correlation of HK2 and FDG uptake was independent of all variables tested, including the initiating oncogene, suggesting that HK2 is an independent predictor of FDG uptake. In contrast, expression of Glut1 was correlated with FDG uptake only in tumors driven by Akt or HER2/neu. Together, these results showed that the oncogenic pathway activated within a tumor is a primary determinant of its FDG uptake, mediated by key glycolytic enzymes that provide a framework to interpret effects on this key parameter in clinical imaging. PMID:25239452

Cytotoxicity and cellular uptake of doxorubicin and its formamidine derivatives in HL60 sensitive and HL60/MX2 resistant cells.

PubMed

Kik, Krzysztof; Wasowska-Lukawska, Malgorzata; Oszczapowicz, Irena; Szmigiero, Leszek

2009-04-01

In this work a comparison was made of the cytotoxicity and cellular uptake of doxorubicin (DOX) and two of its derivatives containing a formamidino group (-N=CH-N<) at the 3' position with morpholine (DOXM) or hexamethyleneimine (DOXH) ring. All tests were performed in doxorubicin-sensitive HL60 and -resistant HL60/MX2 cells which are known for the presence of altered topoisomerase II. Cytotoxic activity of DOX toward HL60/MX2 cells was about 195 times lower when compared with the sensitive HL60 cell line. DOXM and DOXH were approximately 20 times more active in resistant cells than DOX. It was found that the uptake of DOX was lower in resistant cells by about 16%, while that of DOXM and DOXH was lower by about 36% and 19%, respectively. Thus the changes in the cellular uptake of anthracyclines are not associated with the fact that cytotoxicity of DOXM and DOXH exceed the cytotoxicity of DOX. Experiments in cell-free system containing human topoisomerase II showed that topoisomerase II is not inhibited by DOXM and DOXH. Formamidinoanthracyclines may be more useful than parent drugs in therapy against tumor cells with altered topoisomerase II activity.

Construction and evaluation of BSA-CaP nanomaterials with enhanced transgene performance via biocorona-inspired caveolae-mediated endocytosis

NASA Astrophysics Data System (ADS)

Ma, Xi-Xi; Gao, Han; Zhang, Ya-Xuan; Jia, Yi-Yang; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2018-02-01

Non-viral nanovectors have attracted much attention owing to their ability to condense genetic materials and their ease of modification. However, their poor stability, low biocompatibility and gene degradation in endosomes or lysosomes has significantly hampered their application in vivo and in the clinic. In an attempt to overcome these difficulties a series of bovine serum albumin (BSA)-calcium phosphate (CaP) nanoparticles were constructed. The CaP condenses with DNA to form nanocomplexes coated with a biomimetic corona of BSA. Such complexes may retain the inherent endocytosis profile of BSA, with improved biocompatibility. In particular the transgene performance may be enhanced by stimulating the cellular uptake pathway via caveolae-mediated endocytosis. Two methods were employed to construct and optimize the formulation of BSA-CaP nanomaterials. The optimized BSA-CaP-50-M2 nanoparticles prepared by our second method exhibited good stability, negligible cytotoxicity and enhanced transgene performance with long-term expression for 72 h in vivo even with a single dose. Determination of the cellular uptake pathway and Western blot revealed that cellular uptake of the designed BSA-CaP-50-M2 nanoparticles was mainly via caveolae-mediated endocytosis in a non-degradative pathway in which the biomimetic uptake profile of BSA was retained.

Construction and evaluation of BSA-CaP nanomaterials with enhanced transgene performance via biocorona-inspired caveolae-mediated endocytosis.

PubMed

Ma, Xi-Xi; Gao, Han; Zhang, Ya-Xuan; Jia, Yi-Yang; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2018-02-23

Non-viral nanovectors have attracted much attention owing to their ability to condense genetic materials and their ease of modification. However, their poor stability, low biocompatibility and gene degradation in endosomes or lysosomes has significantly hampered their application in vivo and in the clinic. In an attempt to overcome these difficulties a series of bovine serum albumin (BSA)-calcium phosphate (CaP) nanoparticles were constructed. The CaP condenses with DNA to form nanocomplexes coated with a biomimetic corona of BSA. Such complexes may retain the inherent endocytosis profile of BSA, with improved biocompatibility. In particular the transgene performance may be enhanced by stimulating the cellular uptake pathway via caveolae-mediated endocytosis. Two methods were employed to construct and optimize the formulation of BSA-CaP nanomaterials. The optimized BSA-CaP-50-M2 nanoparticles prepared by our second method exhibited good stability, negligible cytotoxicity and enhanced transgene performance with long-term expression for 72 h in vivo even with a single dose. Determination of the cellular uptake pathway and Western blot revealed that cellular uptake of the designed BSA-CaP-50-M2 nanoparticles was mainly via caveolae-mediated endocytosis in a non-degradative pathway in which the biomimetic uptake profile of BSA was retained.

Size of submicrometric and nanometric particles affect cellular uptake and biological activity of macrophages in vitro.

PubMed

Leclerc, L; Rima, W; Boudard, D; Pourchez, J; Forest, V; Bin, V; Mowat, P; Perriat, P; Tillement, O; Grosseau, P; Bernache-Assollant, D; Cottier, M

2012-08-01

Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles. FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150 nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively). Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed.

Bumetanide hyperpolarizes Madin-Darby canine kidney cells and enhances cellular gentamicin uptake via elevating cytosolic Ca2+ thus facilitating intermediate conductance Ca2+-activated potassium channels

PubMed Central

Wang, Tian; Yang, Yu-qin; Karasawa, Takatoshi; Wang, Qi; Phillips, Amanda; Guan, Bing-Cai; Ma, Ke-Tao; Jiang, Meiyan; Xie, Ding-Hua; Steyger, Peter S.; Jiang, Zhi-Gen

2012-01-01

Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin-Darby Canine kidney (MDCK) cells. We found that bumetanide and furosemide concentration-dependently enhanced cytoplasmic GTTR fluorescence by ~60%. This enhancement was suppressed by La3+, a non-selective cation channel (NSCC) blocker, and by K+ channel blockers Ba2+ and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl− channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTRinh-172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (Vr) ~−80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba2+ or clotrimazole, and absent in elevated [Ca2+]i, but not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA or CFTRinh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca2+. Ba2+ and clotrimazole alone depolarized cells by ~18 mV and reduced I/V slope with a net current Vr near −85 mV, and reduced GTTR uptake by ~20%. La3+ alone hyperpolarized the cells by ~−14 mV, reduced the I/V slope with a net current Vr near −10 mV, and inhibited GTTR uptake by ~50%. In the presence of La3+, bumetanide caused negligible potential or I/V change. We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating the intracellular Ca2+ and thus a facilitation of the intermediate conductance Ca2+-activated K+ channels. PMID:23109177

Bumetanide hyperpolarizes madin-darby canine kidney cells and enhances cellular gentamicin uptake by elevating cytosolic Ca(2+) thus facilitating intermediate conductance Ca(2+)--activated potassium channels.

PubMed

Wang, Tian; Yang, Yu-Qin; Karasawa, Takatoshi; Wang, Qi; Phillips, Amanda; Guan, Bing-Cai; Ma, Ke-Tao; Jiang, Meiyan; Xie, Ding-Hua; Steyger, Peter S; Jiang, Zhi-Gen

2013-04-01

Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin-Darby canine kidney (MDCK) cells. We found that bumetanide and furosemide dose-dependently enhanced cytoplasmic GTTR fluorescence by ~60 %. This enhancement was suppressed by La(3+), a non-selective cation channel (NSCC) blocker, and by K(+) channel blockers Ba(2+) and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl(-) channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTRinh-172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (V r) ~-80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba(2+) or clotrimazole, and absent in elevated [Ca(2+)]i, but was not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA, or CFTRinh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca(2+). Ba(2+) and clotrimazole alone depolarized cells by ~18 mV and reduced I/V slope with a net current V r near -85 mV, and reduced GTTR uptake by ~20 %. La(3+) alone hyperpolarized the cells by ~-14 mV, reduced the I/V slope with a net current V r near -10 mV, and inhibited GTTR uptake by ~50 %. In the presence of La(3+), bumetanide-caused negligible change in potential or I/V. We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases the cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating intracellular Ca(2+) and thus facilitating activation of the intermediate conductance Ca(2+)-activated K(+) channels.

A Chemical Biology Approach to Interrogate Quorum Sensing Regulated Behaviors at the Molecular and Cellular Level

PubMed Central

Lowery, Colin A.; Matamouros, Susana; Niessen, Sherry; Zhu, Jie; Scolnick, Jonathan A.; Mee, Jenny M.; Cravatt, Benjamin F.; Miller, Samuel I.; Kaufmann, Gunnar F.; Janda, Kim D.

2013-01-01

SUMMARY Small molecule probes have been employed extensively to explore biological systems and elucidate cellular signaling pathways. In this study, we utilize an inhibitor of bacterial communication to monitor changes in the proteome of Salmonella enterica serovar Typhimurium with the aim of discovering new processes regulated by AI-2-based quorum sensing (QS), a mechanism of bacterial intracellular communication that allows for the coordination of gene expression in a cell density-dependent manner. In S. typhimurium, this system regulates the uptake and catabolism of intracellular signals and has been implicated in pathogenesis, including the invasion of host epithelial cells. We demonstrate that our QS antagonist is capable of selectively inhibiting the expression of known QS-regulated proteins in S. typhimurium, thus attesting that QS inhibitors may be used to confirm proposed and elucidate previously unidentified QS pathways without relying on genetic manipulation. PMID:23890008

Anchored but not internalized: shape dependent endocytosis of nanodiamond

NASA Astrophysics Data System (ADS)

Zhang, Bokai; Feng, Xi; Yin, Hang; Ge, Zhenpeng; Wang, Yanhuan; Chu, Zhiqin; Raabova, Helena; Vavra, Jan; Cigler, Petr; Liu, Renbao; Wang, Yi; Li, Quan

2017-04-01

Nanoparticle-cell interactions begin with the cellular uptake of the nanoparticles, a process that eventually determines their cellular fate. In the present work, we show that the morphological features of nanodiamonds (NDs) affect both the anchoring and internalization stages of their endocytosis. While a prickly ND (with sharp edges/corners) has no trouble of anchoring onto the plasma membrane, it suffers from difficult internalization afterwards. In comparison, the internalization of a round ND (obtained by selective etching of the prickly ND) is not limited by its lower anchoring amount and presents a much higher endocytosis amount. Molecular dynamics simulation and continuum modelling results suggest that the observed difference in the anchoring of round and prickly NDs likely results from the reduced contact surface area with the cell membrane of the former, while the energy penalty associated with membrane curvature generation, which is lower for a round ND, may explain its higher probability of the subsequent internalization.

A chemical biology approach to interrogate quorum-sensing regulated behaviors at the molecular and cellular level.

PubMed

Lowery, Colin A; Matamouros, Susana; Niessen, Sherry; Zhu, Jie; Scolnick, Jonathan; Lively, Jenny M; Cravatt, Benjamin F; Miller, Samuel I; Kaufmann, Gunnar F; Janda, Kim D

2013-07-25

Small molecule probes have been used extensively to explore biologic systems and elucidate cellular signaling pathways. In this study, we use an inhibitor of bacterial communication to monitor changes in the proteome of Salmonella enterica serovar Typhimurium with the aim of discovering unrecognized processes regulated by AI-2-based quorum-sensing (QS), a mechanism of bacterial intercellular communication that allows for the coordination of gene expression in a cell density-dependent manner. In S. typhimurium, this system regulates the uptake and catabolism of intercellular signals and has been implicated in pathogenesis, including the invasion of host epithelial cells. We demonstrate that our QS antagonist is capable of selectively inhibiting the expression of known QS-regulated proteins in S. typhimurium, thus attesting that QS inhibitors may be used to confirm proposed and elucidate previously unidentified QS pathways without relying on genetic manipulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cellular uptake of modified oligonucleotides: fluorescence approach

NASA Astrophysics Data System (ADS)

Kočišová, Eva; Praus, Petr; Rosenberg, Ivan; Seksek, Olivier; Sureau, Franck; Štěpánek, Josef; Turpin, Pierre-Yves

2005-06-01

Cellular uptake and intracellular distribution of the synthetic antisense analogue of dT 15 oligonucleotide (homogenously containing 3'-O-P-CH 2-O-5' internucleotide linkages and labeled with tetramethylrhodamine dye) was studied on B16 melanoma cell line by fluorescence micro-imaging and time-resolved microspectrofluorimetry. By using amphotericin B 3-dimethylaminopropyl amide as an enhancer molecule for the uptake process, homogenous staining of the cells with rather distinct nucleoli staining was achieved after 4 h of incubation. Two spectral components of 2.7 and 1.3 ns lifetime, respectively, were resolved in the emission collected from the cell nucleus. The way of staining and the long-lived component differed from our previous experiments demonstrating complexity of the intracellular oligonucleotide distribution and in particular of the binding inside the nucleus.

Surface bioengineering of diatomite based nanovectors for efficient intracellular uptake and drug delivery

NASA Astrophysics Data System (ADS)

Terracciano, Monica; Shahbazi, Mohammad-Ali; Correia, Alexandra; Rea, Ilaria; Lamberti, Annalisa; de Stefano, Luca; Santos, Hélder A.

2015-11-01

Diatomite is a natural porous silica material of sedimentary origin. Due to its peculiar properties, it can be considered as a valid surrogate of synthetic porous silica for nano-based drug delivery. In this work, we exploit the potential of diatomite nanoparticles (DNPs) for drug delivery with the aim of developing a successful dual-biofunctionalization method by polyethylene glycol (PEG) coverage and cell-penetrating peptide (CPP) bioconjugation, to improve the physicochemical and biological properties of the particles, to enhance the intracellular uptake in cancer cells, and to increase the biocompatibility of 3-aminopropyltriethoxysilane (APT) modified-DNPs. DNPs-APT-PEG-CPP showed hemocompatibility for up to 200 μg mL-1 after 48 h of incubation with erythrocytes, with a hemolysis value of only 1.3%. The cytotoxicity of the modified-DNPs with a concentration up to 200 μg mL-1 and incubation with MCF-7 and MDA-MB-231 breast cancer cells for 24 h, demonstrated that PEGylation and CPP-bioconjugation can strongly reduce the cytotoxicity of DNPs-APT. The cellular uptake of the modified-DNPs was also evaluated using the above mentioned cancer cell lines, showing that the CPP-bioconjugation can considerably increase the DNP cellular uptake. Moreover, the dual surface modification of DNPs improved both the loading of a poorly water-soluble anticancer drug, sorafenib, with a loading degree up to 22 wt%, and also enhanced the drug release profiles in aqueous solutions. Overall, this work demonstrates that the biofunctionalization of DNPs is a promising platform for drug delivery applications in cancer therapy as a result of its enhanced stability, biocompatibility, cellular uptake, and drug release profiles.Diatomite is a natural porous silica material of sedimentary origin. Due to its peculiar properties, it can be considered as a valid surrogate of synthetic porous silica for nano-based drug delivery. In this work, we exploit the potential of diatomite nanoparticles (DNPs) for drug delivery with the aim of developing a successful dual-biofunctionalization method by polyethylene glycol (PEG) coverage and cell-penetrating peptide (CPP) bioconjugation, to improve the physicochemical and biological properties of the particles, to enhance the intracellular uptake in cancer cells, and to increase the biocompatibility of 3-aminopropyltriethoxysilane (APT) modified-DNPs. DNPs-APT-PEG-CPP showed hemocompatibility for up to 200 μg mL-1 after 48 h of incubation with erythrocytes, with a hemolysis value of only 1.3%. The cytotoxicity of the modified-DNPs with a concentration up to 200 μg mL-1 and incubation with MCF-7 and MDA-MB-231 breast cancer cells for 24 h, demonstrated that PEGylation and CPP-bioconjugation can strongly reduce the cytotoxicity of DNPs-APT. The cellular uptake of the modified-DNPs was also evaluated using the above mentioned cancer cell lines, showing that the CPP-bioconjugation can considerably increase the DNP cellular uptake. Moreover, the dual surface modification of DNPs improved both the loading of a poorly water-soluble anticancer drug, sorafenib, with a loading degree up to 22 wt%, and also enhanced the drug release profiles in aqueous solutions. Overall, this work demonstrates that the biofunctionalization of DNPs is a promising platform for drug delivery applications in cancer therapy as a result of its enhanced stability, biocompatibility, cellular uptake, and drug release profiles. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05173h

Repeatability of quantitative 18F-FLT uptake measurements in solid tumors: an individual patient data multi-center meta-analysis.

PubMed

Kramer, G M; Liu, Y; de Langen, A J; Jansma, E P; Trigonis, I; Asselin, M-C; Jackson, A; Kenny, L; Aboagye, E O; Hoekstra, O S; Boellaard, R

2018-06-01

3'-deoxy-3'-[ 18 F]fluorothymidine ( 18 F-FLT) positron emission tomography (PET) provides a non-invasive method to assess cellular proliferation and response to antitumor therapy. Quantitative 18 F-FLT uptake metrics are being used for evaluation of proliferative response in investigational setting, however multi-center repeatability needs to be established. The aim of this study was to determine the repeatability of 18 F-FLT tumor uptake metrics by re-analyzing individual patient data from previously published reports using the same tumor segmentation method and repeatability metrics across cohorts. A systematic search in PubMed, EMBASE.com and the Cochrane Library from inception-October 2016 yielded five 18 F-FLT repeatability cohorts in solid tumors. 18 F-FLT avid lesions were delineated using a 50% isocontour adapted for local background on test and retest scans. SUV max , SUV mean , SUV peak , proliferative volume and total lesion uptake (TLU) were calculated. Repeatability was assessed using the repeatability coefficient (RC = 1.96 × SD of test-retest differences), linear regression analysis, and the intra-class correlation coefficient (ICC). The impact of different lesion selection criteria was also evaluated. Images from four cohorts containing 30 patients with 52 lesions were obtained and analyzed (ten in breast cancer, nine in head and neck squamous cell carcinoma, and 33 in non-small cell lung cancer patients). A good correlation was found between test-retest data for all 18 F-FLT uptake metrics (R 2  ≥ 0.93; ICC ≥ 0.96). Best repeatability was found for SUV peak (RC: 23.1%), without significant differences in RC between different SUV metrics. Repeatability of proliferative volume (RC: 36.0%) and TLU (RC: 36.4%) was worse than SUV. Lesion selection methods based on SUV max  ≥ 4.0 improved the repeatability of volumetric metrics (RC: 26-28%), but did not affect the repeatability of SUV metrics. In multi-center studies, differences ≥ 25% in 18 F-FLT SUV metrics likely represent a true change in tumor uptake. Larger differences are required for FLT metrics comprising volume estimates when no lesion selection criteria are applied.

A polymeric nanoparticle consisting of mPEG-PLA-Toco and PLMA-COONa as a drug carrier: improvements in cellular uptake and biodistribution.

PubMed

Yi, Yilwoong; Kim, Jae Hong; Kang, Hye-Won; Oh, Hun Seung; Kim, Sung Wan; Seo, Min Hyo

2005-02-01

To evaluate a new polymeric nanoparticulate drug delivery formulation that consists of two components: i) an amphiphilic diblock copolymer having tocopherol moiety at the end of the hydrophobic block in which the hydrophobic tocopherol moiety increases stability of hydrophobic core of the nanoparticle in aqueous medium; and ii) a biodegradable copolyester having carboxylate end group that is capable of forming ionic complex with positively charged compounds such as doxorubicin. A doxourubicin-loaded polymeric nanoparticle (Dox-PNP) was prepared by solvent evaporation method. The entrapment efficiency, size distribution, and in vitro release profile at various pH conditions were characterized. In vitro cellular uptake was investigated by confocal microscopy, flow cytometry, and MTT assay using drug-sensitive and drug-resistant cell lines. Pharmacokinetics and biodistribution were evaluated in rats and tumor-bearing mice. Doxorubicin (Dox) was efficiently loaded into the PNP (higher than 95% of entrapment efficiency), and the diameter of Dox-PNP was in the range 20-25 nm with a narrow size distribution. In Vitro study showed that Dox-PNP exhibited higher cellular uptake into both human breast cancer cell (MCF-7) and human uterine cancer cell (MES-SA) than free doxorubicin solution (Free-Dox), especially into drug-resistant cells (MCF-7/ADR and MES-SA/Dx-5). In pharmacokinetics and tissue distribution study, the bioavailability of Dox-PNP calculated from the area under the blood concentration-time curve (AUC) was 69.8 times higher than that of Free-Dox in rats, and Dox-PNP exhibited 2 times higher bioavailability in tumor tissue of tumor-bearing mice. Dox-PNP exhibited enhanced cellular uptake of the drug. In the cytotoxic activity study, this improved cellular uptake was proved to be more advantageous in drug-resistant cell. Dox-PNP exhibited much higher bioavailability in blood plasma and more drug accumulation in tumor tissue than conventional doxorubicin formulation. The results of this study suggest that the PNP system is an advantageous carrier for drug delivery.

Regulation of transport processes across the tonoplast

PubMed Central

Neuhaus, H. Ekkehard; Trentmann, Oliver

2014-01-01

In plants, the vacuole builds up the cellular turgor and represents an important component in cellular responses to diverse stress stimuli. Rapid volume changes of cells, particularly of motor cells, like guard cells, are caused by variation of osmolytes and consequently of the water contents in the vacuole. Moreover, directed solute uptake into or release out of the large central vacuole allows adaptation of cytosolic metabolite levels according to the current physiological requirements and specific cellular demands. Therefore, solute passage across the vacuolar membrane, the tonoplast, has to be tightly regulated. Important principles in vacuolar transport regulation are changes of tonoplast transport protein abundances by differential expression of genes or changes of their activities, e.g., due to post-translational modification or by interacting proteins. Because vacuolar transport is in most cases driven by an electro-chemical gradient altered activities of tonoplast proton pumps significantly influence vacuolar transport capacities. Intense studies on individual tonoplast proteins but also unbiased system biological approaches have provided important insights into the regulation of vacuolar transport. This short review refers to selected examples of tonoplast proteins and their regulation, with special focus on protein phosphorylation. PMID:25309559

Cellular trafficking and anticancer activity of Garcinia mangostana extract-encapsulated polymeric nanoparticles

PubMed Central

Pan-In, Porntip; Wanichwecharungruang, Supason; Hanes, Justin; Kim, Anthony J

2014-01-01

Garcinia mangostana Linn extract (GME) is a natural product that has received considerable attention in cancer therapy, and has the potential to reduce side effects of chemotherapeutics and improve efficacy. We formulated GME-encapsulated ethyl cellulose (GME-EC) and a polymer blend of ethyl cellulose and methyl cellulose (GME-EC/MC) nanoparticles. We achieved high drug-loading and encapsulation efficiency using a solvent-displacement method with particle sizes around 250 nm. Cellular uptake and accumulation of GME was higher for GME-encapsulated nanoparticles compared to free GME. In vitro cytotoxicity analysis showed effective anticancer activity of GME-EC and GME-EC/MC nanoparticles in HeLa cells in a dose-dependent manner. GME-EC/MC nanoparticles showed approximately twofold-higher anticancer activity compared to GME-EC nanoparticles, likely due to their enhanced bioavailability. GME-encapsulated nanoparticles primarily entered HeLa cells by clathrin-mediated endocytosis and trafficked through the endolysosomal pathway. As far as we know, this is the first report on the cellular uptake and intracellular trafficking mechanism of drug-loaded cellulose-based nanoparticles. In summary, encapsulation of GME using cellulose-derivative nanoparticles – GME-EC and GME-EC/MC nanoparticles – successfully improved the bioavailability of GME in aqueous solution, enhanced cellular uptake, and displayed effective anticancer activity. PMID:25125977

Dual peptide conjugation strategy for improved cellular uptake and mitochondria targeting.

PubMed

Lin, Ran; Zhang, Pengcheng; Cheetham, Andrew G; Walston, Jeremy; Abadir, Peter; Cui, Honggang

2015-01-21

Mitochondria are critical regulators of cellular function and survival. Delivery of therapeutic and diagnostic agents into mitochondria is a challenging task in modern pharmacology because the molecule to be delivered needs to first overcome the cell membrane barrier and then be able to actively target the intracellular organelle. Current strategy of conjugating either a cell penetrating peptide (CPP) or a subcellular targeting sequence to the molecule of interest only has limited success. We report here a dual peptide conjugation strategy to achieve effective delivery of a non-membrane-penetrating dye 5-carboxyfluorescein (5-FAM) into mitochondria through the incorporation of both a mitochondrial targeting sequence (MTS) and a CPP into one conjugated molecule. Notably, circular dichroism studies reveal that the combined use of α-helix and PPII-like secondary structures has an unexpected, synergistic contribution to the internalization of the conjugate. Our results suggest that although the use of positively charged MTS peptide allows for improved targeting of mitochondria, with MTS alone it showed poor cellular uptake. With further covalent linkage of the MTS-5-FAM conjugate to a CPP sequence (R8), the dually conjugated molecule was found to show both improved cellular uptake and effective mitochondria targeting. We believe these results offer important insight into the rational design of peptide conjugates for intracellular delivery.

Bioaccessibility, Cellular Uptake, and Transport of Astaxanthin Isomers and their Antioxidative Effects in Human Intestinal Epithelial Caco-2 Cells.

PubMed

Yang, Cheng; Zhang, Hua; Liu, Ronghua; Zhu, Honghui; Zhang, Lianfu; Tsao, Rong

2017-11-29

The bioaccessibility, bioavailability, and antioxidative activities of three astaxanthin geometric isomers were investigated using an in vitro digestion model and human intestinal Caco-2 cells. This study demonstrated that the trans-cis isomerization of all-E-astaxanthin and the cis-trans isomerization of Z-astaxanthins could happen both during in vitro gastrointestinal digestion and cellular uptake processes. 13Z-Astaxanthin showed higher bioaccessibility than 9Z- and all-E-astaxanthins during in vitro digestion, and 9Z-astaxanthin exhibited higher transport efficiency than all-E- and 13Z-astaxanthins. These might explain why 13Z- and 9Z-astaxanthins are found at higher concentrations in human plasma than all-E-astaxanthin in reported studies. All three astaxanthin isomers were effective in maintaining cellular redox homeostasis as seen in the antioxidant enzyme (CAT, SOD) activities ; 9Z- and 13Z- astaxanthins exhibited a higher protective effect than all-E-astaxanthin against oxidative stress as demonstrated by the lower cellular uptake of Z-astaxanthins and lower secretion and gene expression of the pro-inflammatory cytokine IL-8 in Caco-2 cells treated with H 2 O 2 . We conclude, for the first time, that Z-astaxanthin isomers may play a more important role in preventing oxidative stress induced intestinal diseases.

Catalposide is a natural agonistic ligand of peroxisome proliferator-activated receptor-{alpha}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ji Hae; Jun, Hee-jin; Hoang, Minh-Hien

2012-06-15

Highlights: Black-Right-Pointing-Pointer Catalposide is a novel ligand for PPAR{alpha}. Black-Right-Pointing-Pointer Cell stimulated with catalposide improved fatty acid uptake, regulated target genes in fatty acid {beta}-oxidation and synthesis. Black-Right-Pointing-Pointer Catalposdie reduces hepatic triacylglycerides. Black-Right-Pointing-Pointer Theses demonstrate catalposide could ameliorate hyperlipidemia and hepatic steatosis. -- Abstract: Peroxisome proliferator-activated receptor-alpha (PPAR{alpha}) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPAR{alpha} agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPAR{alpha} agonist, identified from reporter gene assay-based activity screening withmore » approximately 900 natural plant and seaweed extracts. Results of time-resolved fluorescence resonance energy transfer analyses suggested that the compound interacted directly with the ligand-binding domain of PPAR{alpha}. Cultured hepatocytes stimulated with catalposide exhibited significantly reduced cellular triglyceride concentrations, by 21%, while cellular uptake of fatty acids was increased, by 70% (P < 0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to upregulation of fatty acid transporter protein-4 (+19% vs. the control) in cells stimulated with catalposide. Additionally, expression of genes related to fatty acid oxidation and high-density lipoprotein metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed. In conclusion, catalposide is hypolipidemic by activation of PPAR{alpha} via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.« less

Dendrimer-paclitaxel complexes for efficient treatment in ovarian cancer: study on OVCAR-3 and HEK293T cells.

PubMed

Yao, Hua; Ma, Jinqi

2018-01-01

The present paper investigates the enhancement of the therapeutic effect of Paclitaxel (a potent anticancer drug) by increasing its cellular uptake in the cancerous cells with subsequent reduction in its cytotoxic effects. To fulfill these goals the Paclitaxel (PTX)-Biotinylated PAMAM dendrimer complexes were prepared using biotinylation method. The primary parameter of Biotinylated PAMAM with a terminal HN 2 group - the degree of biotinylation - was evaluated using HABA assay. The basic integrity of the complex was studied using DSC. The Drug Loading (DL) and Drug Release (DR) parameters of Biotinylated PAMAM dendrimer-PTX complexes were also examined. Cellular uptake study was performed in OVCAR-3 and HEK293T cells using fluorescence technique. The statistical analysis was also performed to support the experimental data. The results obtained from HABA assay showed the complete biotinylation of PAMAM dendrimer. DSC study confirmed the integrity of the complex as compared with pure drug, biotinylated complex and their physical mixture. Batch 9 showed the highest DL (12.09%) and DR (70%) for 72 h as compared to different concentrations of drug and biotinylated complex. The OVCAR-3 (cancerous) cells were characterized by more intensive cellular uptake of the complexes than HEK293T (normal) cells. The obtained experimental results were supported by the statistical data. The results obtained from both experimental and statistical evaluation confirmed that the biotinylated PAMAM NH 2 dendrimer-PTX complex not only displays increased cellular uptake but has also enhanced release up to 72 h with the reduction in cytotoxicity.

Sequestering ability to Cu2+ of a new bodipy-based dye and its behavior as in vitro fluorescent sensor.

PubMed

Papalia, Teresa; Barattucci, Anna; Barreca, Davide; Bellocco, Ersilia; Bonaccorsi, Paola; Minuti, Lucio; Nicolò, Marco Sebastiano; Temperini, Andrea; Foti, Claudia

2017-02-01

A Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) derivative has been conceived and synthesized starting from l-aspartic acid, as a selective turn-off sensor of Cu 2+ ions. Its acid-base properties were determined to study the formation of metal/sensor complex species by titration of solutions each containing a different metal ion, such as Cu 2+ , Ca 2+ , Zn 2+ , Pb 2+ and Hg 2+ and different metal/sensor ratios. The speciation models allowed us to simulate the distribution of the metal/sensor complex species at the normal concentrations of the corresponding metals present in biological fluids. The distribution diagrams, obtained by varying the concentration of sensor 1, clearly indicate that sensor 1 responds selectively to Cu 2+ at micromolar concentrations, even in the presence of other more abundant metal cations Ca 2+ . Finally, we analyzed the cellular uptake of sensor 1 on human erythrocytes and its ability to chelate Cu 2+ in the cellular environment. Results indicate that it crosses the plasmatic membrane and colors the cells of a bright fluorescent red. Exposing the fluorescent cells to Cu 2+ results in a complete cellular photobleaching of the red fluorescence, indicating that sensor 1 is able to detect metal changes in the cytosolic environment. Copyright © 2016 Elsevier Inc. All rights reserved.

Hydrodynamic size-dependent cellular uptake of aqueous QDs probed by fluorescence correlation spectroscopy.

PubMed

Dong, Chaoqing; Irudayaraj, Joseph

2012-10-11

Aqueous quantum dots (QDs) directly synthesized with various thiol ligands have been investigated as imaging probes in living cells. However, the effect of the surface chemistry of these ligands on QDs' cellular uptakes and their intracellular fate remains poorly understood. In this work, four CdTe QDs were directly synthesized under aqueous conditions using four different thiols as stabilizers and their interactions with cells were investigated. Fluorescence correlation spectroscopy (FCS), X-ray photoelectron spectroscopy (XPS), and zeta potential measurements on QDs primarily show that the surface structure of these QDs is highly dependent on the thiol ligands used in the preparation of QDs' precursors, including its layer thicknesses, densities, and surface charges. Subsequently, FCS integrated with the maximum-entropy-method-based FCS (MEMFCS) was used to investigate the concentration distribution and dynamics of these QDs in living A-427 cells. Our findings indicate that QDs' surface characteristics affect cell membrane adsorption and subsequent internalization. More critically, we show that the cellular uptake of aqueous QDs is dependent on their hydrodynamic diameter and might have the potential to escape trapped environments to accumulate in the cytoplasm.

Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol

PubMed Central

Infante, Rodney Elwood; Radhakrishnan, Arun

2017-01-01

Cells employ regulated transport mechanisms to ensure that their plasma membranes (PMs) are optimally supplied with cholesterol derived from uptake of low-density lipoproteins (LDL) and synthesis. To date, all inhibitors of cholesterol transport block steps in lysosomes, limiting our understanding of post-lysosomal transport steps. Here, we establish the cholesterol-binding domain 4 of anthrolysin O (ALOD4) as a reversible inhibitor of cholesterol transport from PM to endoplasmic reticulum (ER). Using ALOD4, we: (1) deplete ER cholesterol without altering PM or overall cellular cholesterol levels; (2) demonstrate that LDL-derived cholesterol travels from lysosomes first to PM to meet cholesterol needs, and subsequently from PM to regulatory domains of ER to suppress activation of SREBPs, halting cholesterol uptake and synthesis; and (3) determine that continuous PM-to-ER cholesterol transport allows ER to constantly monitor PM cholesterol levels, and respond rapidly to small declines in cellular cholesterol by activating SREBPs, increasing cholesterol uptake and synthesis. DOI: http://dx.doi.org/10.7554/eLife.25466.001 PMID:28414269

FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eyre, Nicholas S.; Cleland, Leslie G.; Mayrhofer, Graham

2008-06-06

Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. Wemore » propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells.« less

Genome-wide assessment of the carriers involved in the cellular uptake of drugs: a model system in yeast

PubMed Central

2011-01-01

Background The uptake of drugs into cells has traditionally been considered to be predominantly via passive diffusion through the bilayer portion of the cell membrane. The recent recognition that drug uptake is mostly carrier-mediated raises the question of which drugs use which carriers. Results To answer this, we have constructed a chemical genomics platform built upon the yeast gene deletion collection, using competition experiments in batch fermenters and robotic automation of cytotoxicity screens, including protection by 'natural' substrates. Using these, we tested 26 different drugs and identified the carriers required for 18 of the drugs to gain entry into yeast cells. Conclusions As well as providing a useful platform technology, these results further substantiate the notion that the cellular uptake of pharmaceutical drugs normally occurs via carrier-mediated transport and indicates that establishing the identity and tissue distribution of such carriers should be a major consideration in the design of safe and effective drugs. PMID:22023736

Synchrotron radiation induced X-ray emission studies of the antioxidant mechanism of the organoselenium drug ebselen.

PubMed

Aitken, Jade B; Lay, Peter A; Duong, T T Hong; Aran, Roshanak; Witting, Paul K; Harris, Hugh H; Lai, Barry; Vogt, Stefan; Giles, Gregory I

2012-04-01

Synchrotron radiation induced X-ray emission (SRIXE) spectroscopy was used to map the cellular uptake of the organoselenium-based antioxidant drug ebselen using differentiated ND15 cells as a neuronal model. The cellular SRIXE spectra, acquired using a hard X-ray microprobe beam (12.8-keV), showed a large enhancement of fluorescence at the K(α) line for Se (11.2-keV) following treatment with ebselen (10 μM) at time periods from 60 to 240 min. Drug uptake was quantified and ebselen was shown to induce time-dependent changes in cellular elemental content that were characteristic of oxidative stress with the efflux of K, Cl, and Ca species. The SRIXE cellular Se distribution map revealed that ebselen was predominantly localized to a discreet region of the cell which, by comparison with the K and P elemental maps, is postulated to correspond to the endoplasmic reticulum. On the basis of these findings, it is hypothesized that a major outcome of ebselen redox catalysis is the induction of cellular stress. A mechanism of action of ebselen is proposed that involves the cell responding to drug-induced stress by increasing the expression of antioxidant genes. This hypothesis is supported by the observation that ebselen also regulated the homeostasis of the transition metals Mn, Cu, Fe, and Zn, with increases in transition metal uptake paralleling known induction times for the expression of antioxidant metalloenzymes. © SBIC 2012

The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation.

PubMed

Halamoda-Kenzaoui, Blanka; Ceridono, Mara; Urbán, Patricia; Bogni, Alessia; Ponti, Jessica; Gioria, Sabrina; Kinsner-Ovaskainen, Agnieszka

2017-06-26

Significant progress of nanotechnology, including in particular biomedical and pharmaceutical applications, has resulted in a high number of studies describing the biological effects of nanomaterials. Moreover, a determination of so-called "critical quality attributes", that is specific physicochemical properties of nanomaterials triggering the observed biological response, has been recognised as crucial for the evaluation and design of novel safe and efficacious therapeutics. In the context of in vitro studies, a thorough physicochemical characterisation of nanoparticles (NPs), also in the biological medium, is necessary to allow a correlation with a cellular response. Following this concept, we examined whether the main and frequently reported characteristics of NPs such as size and the agglomeration state can influence the level and the mechanism of NP cellular internalization. We employed fluorescently-labelled 30 and 80 nm silicon dioxide NPs, both in agglomerated and non-agglomerated form. Using flow cytometry, transmission electron microscopy, the inhibitors of endocytosis and gene silencing we determined the most probable routes of cellular uptake for each form of tested silica NPs. We observed differences in cellular uptake depending on the size and the agglomeration state of NPs. Caveolae-mediated endocytosis was implicated particularly in the internalisation of well dispersed silica NPs but with an increase of the agglomeration state of NPs a combination of endocytic pathways with a predominant role of macropinocytosis was noted. We demonstrated that the agglomeration state of NPs is an important factor influencing the level of cell uptake and the mechanism of endocytosis of silica NPs.

Single-walled carbon nanohorns decorated with semiconductor quantum dots to evaluate intracellular transport

NASA Astrophysics Data System (ADS)

Zimmermann, Kristen A.; Inglefield, David L.; Zhang, Jianfei; Dorn, Harry C.; Long, Timothy E.; Rylander, Christopher G.; Rylander, M. Nichole

2014-01-01

Single-walled carbon nanohorns (SWNHs) have great potential to enhance thermal and chemotherapeutic drug efficiencies for cancer therapies. Despite their diverse capabilities, minimal research has been conducted so far to study nanoparticle intracellular transport, which is an important step in designing efficient therapies. SWNHs, like many other carbon nanomaterials, do not have inherent fluorescence properties making intracellular transport information difficult to obtain. The goals of this project were to (1) develop a simple reaction scheme to decorate the exohedral surface of SWNHs with fluorescent quantum dots (QDs) and improve conjugate stability, and (2) evaluate SWNH-QD conjugate cellular uptake kinetics and localization in various cancer cell lines of differing origins and morphologies. In this study, SWNHs were conjugated to CdSe/ZnS core/shell QDs using a unique approach to carbodiimide chemistry. Transmission electron microscopy and electron dispersive spectroscopy verified the conjugation of SWNHs and QDs. Cellular uptake kinetics and efficiency were characterized in three malignant cell lines: U-87 MG (glioblastoma), MDA-MB-231 (breast cancer), and AY-27 (bladder transitional cell carcinoma) using flow cytometry. Cellular distribution was verified by confocal microscopy, and cytotoxicity was also evaluated using an alamarBlue assay. Results indicate that cellular uptake kinetics and efficiency are highly dependent on cell type, highlighting the significance of studying nanoparticle transport at the cellular level. Nanoparticle intracellular transport investigations may provide information to optimize treatment parameters (e.g., SWNH concentration, treatment time, etc.) depending on tumor etiology.

``Sheddable'' PEG-lipid to balance the contradiction of PEGylation between long circulation and poor uptake

NASA Astrophysics Data System (ADS)

Zhao, Caiyan; Deng, Hongzhang; Xu, Jing; Li, Shuyi; Zhong, Lin; Shao, Leihou; Wu, Yan; Liang, Xing-Jie

2016-05-01

PEGylated lipids confer longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. In order to balance the contradiction between the advantages of long circulation and the disadvantages of poor uptake of PEGylated lipids, we prepared a ``sheddable'' PEG-lipid micelle system based on the conjugation of PEG and phosphatidyl ethanolamine (DSPE) with a pH sensitive benzoic imine bond. In a physiological environment, the PEG-protected micelles were not readily taken up by the reticuloendothelial system (RES) and could be successfully delivered to tumor tissue by the EPR effect. In a tumor acidic microenvironment, the PEG chains detached from the surfaces of the micelles while the degree of linker cleavage could not cause a significant particle size change, which facilitated the carrier binding to tumor cells and improved the cellular uptake. Subsequently, the ``sheddable'' PEG-lipid micelles easily internalized into cells and the increased acidity in the lysosomes further promoted drug release. Thus, this ``sheddable'' PEG-lipid nanocarrier could be a good candidate for effective intracellular drug delivery in cancer chemotherapy.PEGylated lipids confer longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. In order to balance the contradiction between the advantages of long circulation and the disadvantages of poor uptake of PEGylated lipids, we prepared a ``sheddable'' PEG-lipid micelle system based on the conjugation of PEG and phosphatidyl ethanolamine (DSPE) with a pH sensitive benzoic imine bond. In a physiological environment, the PEG-protected micelles were not readily taken up by the reticuloendothelial system (RES) and could be successfully delivered to tumor tissue by the EPR effect. In a tumor acidic microenvironment, the PEG chains detached from the surfaces of the micelles while the degree of linker cleavage could not cause a significant particle size change, which facilitated the carrier binding to tumor cells and improved the cellular uptake. Subsequently, the ``sheddable'' PEG-lipid micelles easily internalized into cells and the increased acidity in the lysosomes further promoted drug release. Thus, this ``sheddable'' PEG-lipid nanocarrier could be a good candidate for effective intracellular drug delivery in cancer chemotherapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr02174c

Targeting mitochondria by Zn(II)N-alkylpyridylporphyrins: the impact of compound sub-mitochondrial partition on cell respiration and overall photodynamic efficacy.

PubMed

Odeh, Ahmad M; Craik, James D; Ezzeddine, Rima; Tovmasyan, Artak; Batinic-Haberle, Ines; Benov, Ludmil T

2014-01-01

Mitochondria play a key role in aerobic ATP production and redox control. They harness crucial metabolic pathways and control cell death mechanisms, properties that make these organelles essential for survival of most eukaryotic cells. Cancer cells have altered cell death pathways and typically show a shift towards anaerobic glycolysis for energy production, factors which point to mitochondria as potential culprits in cancer development. Targeting mitochondria is an attractive approach to tumor control, but design of pharmaceutical agents based on rational approaches is still not well established. The aim of this study was to investigate which structural features of specially designed Zn(II)N-alkylpyridylporphyrins would direct them to mitochondria and to particular mitochondrial targets. Since Zn(II)N-alkylpyridylporphyrins can act as highly efficient photosensitizers, their localization can be confirmed by photodamage to particular mitochondrial components. Using cultured LS174T adenocarcinoma cells, we found that subcellular distribution of Zn-porphyrins is directed by the nature of the substituents attached to the meso pyridyl nitrogens at the porphyrin ring. Increasing the length of the aliphatic chain from one carbon (methyl) to six carbons (hexyl) increased mitochondrial uptake of the compounds. Such modifications also affected sub-mitochondrial distribution of the Zn-porphyrins. The amphiphilic hexyl derivative (ZnTnHex-2-PyP) localized in the vicinity of cytochrome c oxidase complex, causing its inactivation during illumination. Photoinactivation of critical cellular targets explains the superior efficiency of the hexyl derivative in causing mitochondrial photodamage, and suppressing cellular respiration and survival. Design of potent photosensitizers and redox-active scavengers of free radicals should take into consideration not only selective organelle uptake and localization, but also selective targeting of critical macromolecular structures.

Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro.

PubMed

Claudia, Meindl; Kristin, Öhlinger; Jennifer, Ober; Eva, Roblegg; Eleonore, Fröhlich

2017-03-01

At many portals of entry the relative uptake by phagocytes and non-phagocytic cells has a prominent effect on availability and biological action of nanoparticles (NPs). Cellular uptake can be determined for fluorescence-labeled NPs. The present study compares three methods (plate reader, flow cytometry and image analysis) in order to investigate the influence of particle size and functionalization and medium content on cellular uptake of fluorescence-labeled polystyrene particles and to study the respective method́s suitability for uptake studies. For comparison between the techniques, ratios of macrophage to alveolar epithelial cell uptakes were used. Presence of serum protein in the exposure solution decreased uptake of carboxyl-functionalized and non-functionalized particles; there was no clear effect for the amine-functionalized particles. The 200nm non- or carboxyl-functionalized NPs were taken up preferentially by phagocytes while for amine-functionalized particles preference was lowest. The presence of the serum slightly increased the preference for these particles. In conclusion, due to the possibility of calibration, plate reader measurements might present a better option than the other techniques to (semi)quantify differences between phagocytes and non-phagocytic cells for particles with different fluorescence. In order to obtain unbiased data the fluorescent labeling has to fulfill certain requirements. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

C282Y-HFE Gene Variant Affects Cholesterol Metabolism in Human Neuroblastoma Cells

PubMed Central

Ali-Rahmani, Fatima; Huang, Michael A.; Schengrund, C.-L.; Connor, James R.; Lee, Sang Y.

2014-01-01

Although disruptions in the maintenance of iron and cholesterol metabolism have been implicated in several cancers, the association between variants in the HFE gene that is associated with cellular iron uptake and cholesterol metabolism has not been studied. The C282Y-HFE variant is a risk factor for different cancers, is known to affect sphingolipid metabolism, and to result in increased cellular iron uptake. The effect of this variant on cholesterol metabolism and its possible relevance to cancer phenotype was investigated using wild type (WT) and C282Y-HFE transfected human neuroblastoma SH-SY5Y cells. Expression of C282Y-HFE in SH-SY5Y cells resulted in a significant increase in total cholesterol as well as increased transcription of a number of genes involved in its metabolism compared to cells expressing WT-HFE. The marked increase in expression of NPC1L1 relative to that of most other genes, was accompanied by a significant increase in expression of NPC1, a protein that functions in cholesterol uptake by cells. Because inhibitors of cholesterol metabolism have been proposed to be beneficial for treating certain cancers, their effect on the viability of C282Y-HFE neuroblastoma cells was ascertained. C282Y-HFE cells were significantly more sensitive than WT-HFE cells to U18666A, an inhibitor of desmosterol Δ24-reductase the enzyme catalyzing the last step in cholesterol biosynthesis. This was not seen for simvastatin, ezetimibe, or a sphingosine kinase inhibitor. These studies indicate that cancers presenting in carriers of the C282Y-HFE allele might be responsive to treatment designed to selectively reduce cholesterol content in their tumor cells. PMID:24533143

C282Y-HFE gene variant affects cholesterol metabolism in human neuroblastoma cells.

PubMed

Ali-Rahmani, Fatima; Huang, Michael A; Schengrund, C-L; Connor, James R; Lee, Sang Y

2014-01-01

Although disruptions in the maintenance of iron and cholesterol metabolism have been implicated in several cancers, the association between variants in the HFE gene that is associated with cellular iron uptake and cholesterol metabolism has not been studied. The C282Y-HFE variant is a risk factor for different cancers, is known to affect sphingolipid metabolism, and to result in increased cellular iron uptake. The effect of this variant on cholesterol metabolism and its possible relevance to cancer phenotype was investigated using wild type (WT) and C282Y-HFE transfected human neuroblastoma SH-SY5Y cells. Expression of C282Y-HFE in SH-SY5Y cells resulted in a significant increase in total cholesterol as well as increased transcription of a number of genes involved in its metabolism compared to cells expressing WT-HFE. The marked increase in expression of NPC1L1 relative to that of most other genes, was accompanied by a significant increase in expression of NPC1, a protein that functions in cholesterol uptake by cells. Because inhibitors of cholesterol metabolism have been proposed to be beneficial for treating certain cancers, their effect on the viability of C282Y-HFE neuroblastoma cells was ascertained. C282Y-HFE cells were significantly more sensitive than WT-HFE cells to U18666A, an inhibitor of desmosterol Δ24-reductase the enzyme catalyzing the last step in cholesterol biosynthesis. This was not seen for simvastatin, ezetimibe, or a sphingosine kinase inhibitor. These studies indicate that cancers presenting in carriers of the C282Y-HFE allele might be responsive to treatment designed to selectively reduce cholesterol content in their tumor cells.

Preparation and evaluation of microemulsion-based transdermal delivery of total flavone of rhizoma arisaematis

PubMed Central

Shen, Li-Na; Zhang, Yong-Tai; Wang, Qin; Xu, Ling; Feng, Nian-Ping

2014-01-01

The aims of the present study were to investigate the skin permeation and cellular uptake of a microemulsion (ME) containing total flavone of rhizoma arisaematis (TFRA), and to evaluate its effects on skin structure. Pseudo-ternary phase diagrams were constructed to evaluate ME regions with various surfactants and cosurfactants. Eight formulations of oil-in-water MEs were selected as vehicles, and in vitro skin-permeation experiments were performed to optimize the ME formulation and to evaluate its permeability, in comparison to that of an aqueous suspension. Laser scanning confocal microscopy and fluorescent-activated cell sorting were used to explore the cellular uptake of rhodamine 110-labeled ME in human epidermal keratinocytes (HaCaT) and human embryonic skin fibroblasts (CCC-ESF-1). The structure of stratum corneum treated with ME was observed using a scanning electron microscope. Furthermore, skin irritation was tested to evaluate the safety of ME. ME formulated with 4% ethyl oleate (weight/weight), 18% Cremophor EL® (weight/weight), and 18% Transcutol® P, with 1% Azone to enhance permeation, showed good skin permeability. ME-associated transdermal fluxes of schaftoside and isoschaftoside, two major effective constituents of TFRA, were 3.72-fold and 5.92-fold higher, respectively, than those achieved using aqueous suspensions. In contrast, in vitro studies revealed that uptake by HaCaT and CCC-ESF-1 cells was lower with ME than with an aqueous suspension. Stratum corneum loosening and shedding was observed in nude mouse skin treated with ME, although ME produced no observable skin irritation in rabbits. These findings indicated that ME enhanced transdermal TFRA delivery effectively and showed good biocompatibility with skin tissue. PMID:25092976

Bombesin functionalized gold nanoparticles show in vitro and in vivo cancer receptor specificity.

PubMed

Chanda, Nripen; Kattumuri, Vijaya; Shukla, Ravi; Zambre, Ajit; Katti, Kavita; Upendran, Anandhi; Kulkarni, Rajesh R; Kan, Para; Fent, Genevieve M; Casteel, Stan W; Smith, C Jeffrey; Boote, Evan; Robertson, J David; Cutler, Cathy; Lever, John R; Katti, Kattesh V; Kannan, Raghuraman

2010-05-11

Development of cancer receptor-specific gold nanoparticles will allow efficient targeting/optimum retention of engineered gold nanoparticles within tumors and thus provide synergistic advantages in oncology as it relates to molecular imaging and therapy. Bombesin (BBN) peptides have demonstrated high affinity toward gastrin-releasing peptide (GRP) receptors in vivo that are overexpressed in prostate, breast, and small-cell lung carcinoma. We have synthesized a library of GRP receptor-avid nanoplatforms by conjugating gold nanoparticles (AuNPs) with BBN peptides. Cellular interactions and binding affinities (IC(50)) of AuNP-BBN conjugates toward GRP receptors on human prostate cancer cells have been investigated in detail. In vivo studies using AuNP-BBN and its radiolabeled surrogate (198)AuNP-BBN, exhibiting high binding affinity (IC(50) in microgram ranges), provide unequivocal evidence that AuNP-BBN constructs are GRP-receptor-specific showing accumulation with high selectivity in GRP-receptor-rich pancreatic acne in normal mice and also in tumors in prostate-tumor-bearing, severe combined immunodeficient mice. The i.p. mode of delivery has been found to be efficient as AuNP-BBN conjugates showed reduced RES organ uptake with concomitant increase in uptake at tumor targets. The selective uptake of this new generation of GRP-receptor-specific AuNP-BBN peptide analogs has demonstrated realistic clinical potential in molecular imaging via x-ray computed tomography techniques as the contrast numbers in prostate tumor sites are severalfold higher as compared to the pretreatment group (Hounsfield unit = 150).

Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization

PubMed Central

2015-01-01

Many of the reported arginine-rich cell-penetrating peptides (CPPs) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties. Herein, we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines, namely, R5 and R6. We further explored the effect of acylation with long chain fatty acids (i.e., octanoic acid, dodecanoic acid, and hexadecanoic acid) and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R5] and linear peptide dodecanoyl-(R5) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. Dodecanoyl-[R5] and dodecanoyl-[R6] enhanced the intracellular uptake of a fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F′-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively, as shown by flow cytometry. The cellular uptake of F′-GpYEEI in the presence of hexadecanoyl-[R5] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[R5] and dodecanoyl-[R5], respectively. Dodecanoyl-[R5] enhanced the cellular uptake of the phosphopeptide by 1.4–2.5-fold higher than the corresponding linear peptide dodecanoyl-(R5) and those of representative CPPs, such as hepta-arginine (CR7) and TAT peptide. These results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids. PMID:24978295

Vibrational imaging of glucose uptake activity in live cells and tissues by stimulated Raman scattering microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hu, Fanghao; Chen, Zhixing; Zhang, Luyuan; Shen, Yihui; Wei, Lu; Min, Wei

2016-03-01

Glucose is consumed as an energy source by virtually all living organisms, from bacteria to humans. Its uptake activity closely reflects the cellular metabolic status in various pathophysiological transformations, such as diabetes and cancer. Extensive efforts such as positron emission tomography, magnetic resonance imaging and fluorescence microscopy have been made to specifically image glucose uptake activity but all with technical limitations. Here, we report a new platform to visualize glucose uptake activity in live cells and tissues with subcellular resolution and minimal perturbation. A novel glucose analogue with a small alkyne tag (carbon-carbon triple bond) is developed to mimic natural glucose for cellular uptake, which can be imaged with high sensitivity and specificity by targeting the strong and characteristic alkyne vibration on stimulated Raman scattering (SRS) microscope to generate a quantitative three dimensional concentration map. Cancer cells with differing metabolic characteristics can be distinguished. Heterogeneous uptake patterns are observed in tumor xenograft tissues, neuronal culture and mouse brain tissues with clear cell-cell variations. Therefore, by offering the distinct advantage of optical resolution but without the undesirable influence of bulky fluorophores, our method of coupling SRS with alkyne labeled glucose will be an attractive tool to study energy demands of living systems at the single cell level.

Diselenolane-mediated cellular uptake† †Electronic supplementary information (ESI) available: Detailed procedures and results for all reported experiments. See DOI: 10.1039/c7sc05151d

PubMed Central

Chuard, Nicolas; Poblador-Bahamonde, Amalia I.; Zong, Lili; Bartolami, Eline; Hildebrandt, Jana; Weigand, Wolfgang; Sakai, Naomi

2018-01-01

The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to selenium. We report that according to results with fluorescent model substrates, cellular uptake with 1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This 1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-β-cyclodextrin, wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum. Selenophilicity, the extreme CSeSeC dihedral angle of 0° and the high but different acidity of primary and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved selenosulfides. PMID:29675232

Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells.

PubMed

Lichtenstein, Dajana; Ebmeyer, Johanna; Knappe, Patrick; Juling, Sabine; Böhmert, Linda; Selve, Sören; Niemann, Birgit; Braeuning, Albert; Thünemann, Andreas F; Lampen, Alfonso

2015-11-01

Because of the rising application of nanoparticles in food and food-related products, we investigated the influence of the digestion process on the toxicity and cellular uptake of silver nanoparticles for intestinal cells. The main food components--carbohydrates, proteins and fatty acids--were implemented in an in vitro digestion process to simulate realistic conditions. Digested and undigested silver nanoparticle suspensions were used for uptake studies in the well-established Caco-2 model. Small-angle X-ray scattering was used to estimate particle core size, size distribution and stability in cell culture medium. Particles proved to be stable and showed radii from 3.6 to 16.0 nm. Undigested particles and particles digested in the presence of food components were comparably taken up by Caco-2 cells, whereas the uptake of particles digested without food components was decreased by 60%. Overall, these findings suggest that in vivo ingested poly (acrylic acid)-coated silver nanoparticles may reach the intestine in a nanoscaled form even if enclosed in a food matrix. While appropriate for studies on the uptake into intestinal cells, the Caco-2 model might be less suited for translocation studies. Moreover, we show that nanoparticle digestion protocols lacking food components may lead to misinterpretation of uptake studies and inconclusive results.

Fatty acid uptake by isolated rat heart myocytes represents a carrier-mediated transport process.

PubMed Central

Stremmel, W

1988-01-01

The mechanism by which fatty acids enter cardiomyocytes is unclear. Therefore, the influx kinetics of [3H]oleate into isolated rat heart myocytes were examined. Cells were incubated at 37 degrees C with [3H]oleate bound to albumin in various molar ratios and the initial rate of uptake (V0) was determined as a function of the unbound oleate concentration in the medium. V0 was saturable with increasing oleate concentrations incubated (Km 78 nM; Vmax 1.9 nmol X min-1 per 10(6) cells) and temperature dependent with an optimum at 37 degrees C. Furthermore, binding of [3H]oleate to isolated plasma membranes of cardiomyocytes was saturable, revealing a KD of 42 nM, and was inhibited by heat denaturation or trypsin pretreatment of the membranes. From these membranes a single 40-kD protein with high affinity for a variety of long chain fatty acids was isolated. With a monospecific antibody to this membrane protein, binding as well as cellular influx of [3H]oleate was selectively inhibited. These data indicate that at least a portion of myocardial fatty acid uptake is mediated by a specific membrane protein. Images PMID:3343344

Screening plant derived dietary phenolic compounds for bioactivity related to cardiovascular disease.

PubMed

Croft, Kevin D; Yamashita, Yoko; O'Donoghue, Helen; Shirasaya, Daishi; Ward, Natalie C; Ashida, Hitoshi

2018-04-01

The potential health benefits of phenolic acids found in food and beverages has been suggested from a number of large population studies. However, the mechanism of how these compounds may exert biological effects is less well established. It is also now recognised that many complex polyphenols in the diet are metabolised to simple phenolic acids which can be taken up in the circulation. In this paper a number of selected phenolic compounds have been tested for their bioactivity in two cell culture models. The expression and activity of endothelial nitric oxide synthase (eNOS) in human aortic endothelial cells and the uptake of glucose in muscle cells. Our data indicate that while none of the compounds tested had a significant effect on eNOS expression or activation in endothelial cells, several of the compounds increased glucose uptake in muscle cells. These compounds also enhanced the translocation of the glucose transporter GLUT4 to the plasma membrane, which may explain the observed increase in cellular glucose uptake. These results indicate that simple cell culture models may be useful to help understand the bioactivity of phenolic compounds in relation to cardiovascular protection. Copyright © 2017 Elsevier B.V. All rights reserved.

Modifications of chemical functional groups of Pandanus amaryllifolius Roxb and its effect towards biosorption of heavy metals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abdullah, Mohd Zamri, E-mail: zamriab@petronas.com.my; Ismail, Siti Salwa

2015-07-22

The utilization of non-living biomass as an alternative biosorbent for heavy metal removal has gain a tremendous consideration through the years. Pandanus amaryllifolius Roxb or pandan leaves, which is widely used as food additives in the South East Asia region, has been selected for its viability in the said effort due to the presence of chemical functional groups on its cellular network that enables the sorption to occur. In order to elucidate the possible mechanisms participated during the heavy metal removal process, the biosorbent undergone a series of modification techniques to alter the chemical functional groups present on its constituent.more » From the outcome of the chemically-modified biosorbent being subjected to the contact with metal cations, nitrogen- and oxygen-containing groups present on the biosorbent are believed to be responsible for the metal uptake to occur through complexation mechanism. Modifying amine groups causes 14% reduction of Cu(II) uptake, whereas removing protein element increases the uptake to 26% as compared to the unmodified biosorbent. Also, scanning electron micrographs further suggested that the adsorption mechanism could perform in parallel, as attributed to the evidence of porous structure throughout the biosorbent fibrous nature.« less

Modifications of chemical functional groups of Pandanus amaryllifolius Roxb and its effect towards biosorption of heavy metals

NASA Astrophysics Data System (ADS)

Abdullah, Mohd. Zamri; Ismail, Siti Salwa

2015-07-01

The utilization of non-living biomass as an alternative biosorbent for heavy metal removal has gain a tremendous consideration through the years. Pandanus amaryllifolius Roxb or pandan leaves, which is widely used as food additives in the South East Asia region, has been selected for its viability in the said effort due to the presence of chemical functional groups on its cellular network that enables the sorption to occur. In order to elucidate the possible mechanisms participated during the heavy metal removal process, the biosorbent undergone a series of modification techniques to alter the chemical functional groups present on its constituent. From the outcome of the chemically-modified biosorbent being subjected to the contact with metal cations, nitrogen- and oxygen-containing groups present on the biosorbent are believed to be responsible for the metal uptake to occur through complexation mechanism. Modifying amine groups causes 14% reduction of Cu(II) uptake, whereas removing protein element increases the uptake to 26% as compared to the unmodified biosorbent. Also, scanning electron micrographs further suggested that the adsorption mechanism could perform in parallel, as attributed to the evidence of porous structure throughout the biosorbent fibrous nature.

Human adenovirus Ad36 and its E4orf1 gene enhance cellular glucose uptake even in the presence of inflammatory cytokines.

PubMed

Na, Ha-Na; Dubuisson, Olga; Hegde, Vijay; Nam, Jae-Hwan; Dhurandhar, Nikhil V

2016-05-01

Aging and obesity are associated with elevated pro-inflammatory cytokines such as monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)α, which are linked to insulin resistance. Anti-inflammatory agents have marginal effect in improving insulin resistance. Hence, agents are needed to improve glycemic control despite the inflammation. Ad36, a human adenovirus, increases TNFα and MCP1 mRNA in adipose tissue, yet improves glycemic control in mice. Ad36 via its E4orf1 gene, up-regulates AKT/glucose transporter (Glut)-4 signaling to enhance cellular glucose uptake. Directly test a role of Ad36, or E4orf1 in enhancing cellular glucose uptake in presence of inflammatory cytokines. Experiment 1: 3T3-L1 preadipocytes were treated with 0, 10 or 100 ng/mL lipopolysaccharides (LPS), and infected with 0 or 5 plaque forming units (PFU) of Ad36/cell. 3T3-L1 cells that stably and inducibly express E4orf1 or a null vector (pTRE-E4orf1 or pTRE-null cells), were similarly treated with LPS and then with doxycycline, to induce E4orf1. Experiment 2: 3T3L1 preadipocytes were treated with 25 nM MCP1 or 20 nM TNFα for 16 h, followed by infection with 0 or 5 PFU of Ad36/cell. Experiment 3: pTRE-E4orf1 or -null cells were similarly treated with MCP1 or TNFα followed by doxycycline to induce E4orf1. Cellular glucose uptake and cellular signaling were determined 72 h post-Ad36 infection or E4orf1-induction, in continued presence of MCP1 or TNFα. In 3T3-L1 preadipocytes, Ad36, but not E4orf1, increased MCP1 and TNFα mRNA, in presence of LPS stimulation. Ad36 or E4orf1 up-regulated AKT-phosphorylation and Glut4 and increased glucose uptake (P < 0.05) in the presence of MCP1 or TNFα. Unlike Ad36, E4orf1 does not appear to stimulate inflammatory response. Ad36 and E4orf1 both enhance cellular glucose uptake even in presence of inflammation. Further research is needed to harness this novel and beneficial property of E4orf1 to improve hyperglycemia despite chronic inflammation that is commonly present in aging and obesity. Copyright © 2014 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Combinatorics of feedback in cellular uptake and metabolism of small molecules.

PubMed

Krishna, Sandeep; Semsey, Szabolcs; Sneppen, Kim

2007-12-26

We analyze the connection between structure and function for regulatory motifs associated with cellular uptake and usage of small molecules. Based on the boolean logic of the feedback we suggest four classes: the socialist, consumer, fashion, and collector motifs. We find that the socialist motif is good for homeostasis of a useful but potentially poisonous molecule, whereas the consumer motif is optimal for nutrition molecules. Accordingly, examples of these motifs are found in, respectively, the iron homeostasis system in various organisms and in the uptake of sugar molecules in bacteria. The remaining two motifs have no obvious analogs in small molecule regulation, but we illustrate their behavior using analogies to fashion and obesity. These extreme motifs could inspire construction of synthetic systems that exhibit bistable, history-dependent states, and homeostasis of flux (rather than concentration).

The role of FDG-PET in detecting rejection after liver transplantation.

PubMed

Watson, Ashley M; Bhutiani, Neal; Philips, Prejesh; Davis, Eric G; Eng, Mary; Cannon, Robert M; Jones, Christopher M

2018-05-15

The activation and increased metabolic activity of T cells in acute cellular rejection could allow fluoro-2-deoxyglucose positron emission tomography to be utilized for detection of acute cellular rejection. The objective of this study was to evaluate the effectiveness of fluoro-2-deoxyglucose positron emission tomography in detecting acute cellular rejection in the clinical setting. Fluoro-2-deoxyglucose positron emission tomography studies were performed on 88 orthotopic liver transplant patients at 7 and 17 days postoperatively (first positron emission tomography and second positron emission tomography, respectively). Additional studies were performed if patients had suspicion of rejection and at resolution of rejection (third positron emission tomography and fourth positron emission tomography, respectively). A circular region of interest was placed over the liver for semiquantitative evaluation of fluoro-2-deoxyglucose positron emission tomography images by means of standard uptake values. Eighteen of 88 patients in our study (20.5%) had histologically proven acute cellular rejection during a 16 ± 11 day follow-up. There was no significant difference between the standard uptake values of first positron emission tomography among non-rejecters versus rejecters (2.05 ±0.46 non-rejecters versus 1.82 ± 0.40 rejecters, P = .127). Within the rejection cohort, the standard uptake values from the third positron emission tomography (rejection) were higher compared to the first positron emission tomography (baseline) (2.41 ± 0.48 third positron emission tomography versus 1.82 ± 0.41 first positron emission tomography, P < .001). Increased signal on fluoro-2-deoxyglucose positron emission tomography over baseline is associated with acute cellular rejection in liver transplant recipients. Additional prospective validation studies are essential to define the role of fluoro-2-deoxyglucose positron emission tomography scan as an early marker for acute cellular rejection. Copyright © 2018 Elsevier Inc. All rights reserved.

Rapid transcapillary exchange and unidirectional neuronal uptake of noradrenaline in the perfused rabbit heart.

PubMed Central

Mann, G E; Yudilevich, D L

1984-01-01

Capillary permeability and cellular uptake of noradrenaline by the isolated artificially perfused rabbit heart was measured using rapid (less than 30 s) single-circulation tracer-dilution techniques. In a single coronary circulation capillary extractions of L-[14C]noradrenaline and D-[3H]mannitol (extracellular reference) relative to an intravascular marker, 125I-labelled albumin, were similar and above 60%. The 'apparent' volume of distribution for tracer noradrenaline was 2.5-fold larger than that measured for D-mannitol (0.32 ml g-1) suggesting cellular uptake of the amine. Unidirectional noradrenaline uptake was estimated by directly comparing coronary sinus dilution profiles of L-[3H]noradrenaline and D-[14C]mannitol. Michaelis-Menten saturation kinetics based on a single-entry system were determined (Km = 2.8 +/- 1.5 microM, Vmax = 2.1 +/- 0.5 nmol min-1 g-1, n = 4) by perfusing hearts with varying concentrations of L-noradrenaline (1-10 microM). Various known inhibitors of noradrenaline uptake were investigated to determine whether uptake was mediated by neuronal (uptake1) and/or extraneuronal (uptake2) mechanisms. Desipramine (5 microM), imipramine (5 microM) and metaraminol (2 microM) resulted in a 66-94% inhibition of noradrenaline influx. In comparison, the steroids, 17 beta-oestradiol (1 microM) and corticosterone (10 microM), and the noradrenaline metabolite normetanephrine (5 microM) caused virtually no inhibitory effects. The beta-adrenergic antagonist propranolol (5 microM) was also relatively ineffective. These results together with the kinetic constants estimated suggest that the rapid noradrenaline uptake reflects transport into adrenergic neurones lying in the coronary interstitium. The high resolution of this paired-tracer dilution technique has permitted a 'non-invasive' study of neuronal uptake mechanisms and its application may be of clinical value. PMID:6425496

Endoplasmic Reticulum Ca2+ Handling in Excitable Cells in Health and Disease

PubMed Central

Mattson, Mark P.

2011-01-01

The endoplasmic reticulum (ER) is a morphologically and functionally diverse organelle capable of integrating multiple extracellular and internal signals and generating adaptive cellular responses. It plays fundamental roles in protein synthesis and folding and in cellular responses to metabolic and proteotoxic stress. In addition, the ER stores and releases Ca2+ in sophisticated scenarios that regulate a range of processes in excitable cells throughout the body, including muscle contraction and relaxation, endocrine regulation of metabolism, learning and memory, and cell death. One or more Ca2+ ATPases and two types of ER membrane Ca2+ channels (inositol trisphosphate and ryanodine receptors) are the major proteins involved in ER Ca2+ uptake and release, respectively. There are also direct and indirect interactions of ER Ca2+ stores with plasma membrane and mitochondrial Ca2+-regulating systems. Pharmacological agents that selectively modify ER Ca2+ release or uptake have enabled studies that revealed many different physiological roles for ER Ca2+ signaling. Several inherited diseases are caused by mutations in ER Ca2+-regulating proteins, and perturbed ER Ca2+ homeostasis is implicated in a range of acquired disorders. Preclinical investigations suggest a therapeutic potential for use of agents that target ER Ca2+ handling systems of excitable cells in disorders ranging from cardiac arrhythmias and skeletal muscle myopathies to Alzheimer disease. PMID:21737534

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

PubMed Central

Wang, Shiyu; Allen, Nickolas; Vickers, Timothy A; Revenko, Alexey S; Sun, Hong; Liang, Xue-hai; Crooke, Stanley T

2018-01-01

Abstract Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs. PMID:29514240

A cellular uptake and cytotoxicity properties study of gallic acid-loaded mesoporous silica nanoparticles on Caco-2 cells

NASA Astrophysics Data System (ADS)

Rashidi, Ladan; Vasheghani-Farahani, Ebrahim; Soleimani, Masoud; Atashi, Amir; Rostami, Khosrow; Gangi, Fariba; Fallahpour, Masoud; Tahouri, Mohammad Taher

2014-03-01

In this study, the effects of intracellular delivery of various concentrations of gallic acid (GA) as a semistable antioxidant, gallic acid-loaded mesoporous silica nanoparticles (MSNs-GA), and cellular uptake of nanoparticles into Caco-2 cells were investigated. MSNs were synthesized and loaded with GA, then characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, N2 adsorption isotherms, X-ray diffraction, and thermal gravimetric analysis. The cytotoxicity of MSNs and MSNs-GA at low and high concentrations were studied by means of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry. MSNs did not show significant toxicity in various concentrations (0-500 μg/ml) on Caco-2 cells. For MSNs-GA, cell viability was reduced as a function of incubation time and different concentrations of nanoparticles. The in vitro GA release from MSNs-GA exhibited the same antitumor properties as free GA on Caco-2 cells. Flow cytometry results confirmed those obtained using MTT assay. TEM and fluorescent microscopy confirmed the internalization of MSNs by Caco-2 cells through nonspecific cellular uptake. MSNs can easily internalize into Caco-2 cells without deleterious effects on cell viability. The cell viability of Caco-2 cells was affected during MSNs-GA uptake. MSNs could be designed as suitable nanocarriers for antioxidants delivery.

The uptake of 3H-vincristine by a mouse carcinoma during a course of fractionated radiotherapy.

PubMed

Zanelli, G D; Rota, L; Trovo, M; Grigoletto, E; Roncadin, M

1989-09-01

The variations in uptake of 3H-vincristine sulphate, given as a bolus i.v. injection, by a transplantable murine tumour during a realistic course of fractionated daily gamma-radiation of 25 x 2.0 Gy have been investigated. Maximum levels of 3H in the tumours are found when the tracer is injected 4h after irradiation and the tumours are dissected out 1 h after injection. During the course of daily irradiation the pattern of uptake varies considerably but reproducibly. There are peaks of uptake after 7, 13 and 22 fractions of 2.0 Gy when the amount of 3H in the tumours is as much as three times that found in non-irradiated tumours. After 17-18 fractions, however, the tumour content of 3H is lower than that of non-irradiated tumours. The wave-like pattern of uptake could be due either to capillary occlusion brought about by radiation induced cellular swelling and oedema followed by re-opening of the capillaries during periods of decreased cellularity, or to some mechanism of recovery from radiation damage during the week-end rest period.

The uptake of 3H-vincristine by a mouse carcinoma during a course of fractionated radiotherapy.

PubMed Central

Zanelli, G. D.; Rota, L.; Trovo, M.; Grigoletto, E.; Roncadin, M.

1989-01-01

The variations in uptake of 3H-vincristine sulphate, given as a bolus i.v. injection, by a transplantable murine tumour during a realistic course of fractionated daily gamma-radiation of 25 x 2.0 Gy have been investigated. Maximum levels of 3H in the tumours are found when the tracer is injected 4h after irradiation and the tumours are dissected out 1 h after injection. During the course of daily irradiation the pattern of uptake varies considerably but reproducibly. There are peaks of uptake after 7, 13 and 22 fractions of 2.0 Gy when the amount of 3H in the tumours is as much as three times that found in non-irradiated tumours. After 17-18 fractions, however, the tumour content of 3H is lower than that of non-irradiated tumours. The wave-like pattern of uptake could be due either to capillary occlusion brought about by radiation induced cellular swelling and oedema followed by re-opening of the capillaries during periods of decreased cellularity, or to some mechanism of recovery from radiation damage during the week-end rest period. PMID:2789937

The cellular uptake and transport of zein nanoparticles: Effect of sodium caseinate

USDA-ARS?s Scientific Manuscript database

Cellular evaluation of zein nanoparticles has not been studied systematically due to their poor redispersibility. Caseinate (CAS) stabilized zein nanoparticles have been recently developed with better redispersibility in salt solutions. In this study, zein-CAS nanoparticles were prepared with differ...

Proteomic Analysis of Serum Opsonins Impacting Biodistribution and Cellular Association of Porous Silicon Microparticles

PubMed Central

Serda, Rita E.; Blanco, Elvin; Mack, Aaron; Stafford, Susan J.; Amra, Sarah; Li, Qingpo; van de Ven, Anne L.; Tanaka, Takemi; Torchilin, Vladimir P.; Wiktorowicz, John E.; Ferrari, Mauro

2014-01-01

Mass transport of drug delivery vehicles is guided by particle properties, such as shape, composition and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light chain variable region, fibrinogen, and complement component 1 compared to their anionic counterparts. The anionic-surface favored equal accumulation of microparticles in the liver and spleen, while cationic-surfaces favored preferential accumulation in the spleen. Immunohistochemistry supported macrophage internalization of both anionic and cationic silicon microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution. PMID:21303614

Optimization of a Biomimetic Apatite Nanoparticle Delivery System for Non-viral Gene Transfection---a Simulated Body Fluid Approach

NASA Astrophysics Data System (ADS)

Das, Debobrato

Current methods for gene delivery utilize nanocarriers such as liposomes and viral vectors that may produce in vivo toxicity, immunogenicity, or mutagenesis. Moreover, these common high-cost systems have a low efficacy of gene-vehicle transport across the cell plasma membrane followed by inadequate release and weak intracellular stability of the genetic sequence. Thus, this study aims to maximize gene transfection while minimizing cytotoxicity by utilizing supersaturated blood-plasma ions derived from simulated body fluids (SBF). With favorable electrostatic interactions to create biocompatible calcium-phosphate nanoparticles (NPs) derived from biomimetic apatite (BA), results suggest that the SBF system, though naturally sensitive to reaction conditions, after optimization can serve as a tunable and versatile platform for the delivery of various types of nucleic acids. From a systematic exploration of the effects of nucleation pH, incubation temperature, and time on transfection efficiency, the study proposes distinct characteristic trends in SBF BA-NP morphology, cellular uptake, cell viability, and gene modulation. Specifically, with aggressive nucleation and growth of BA-NPs in solution (observed via scanning electron microscopy), the ensuing microenvironment imposes a more toxic cellular interaction (indicated by alamarBlue and BCA assays), limiting particle uptake (fluorescence experiments) and subsequent gene knockdown (quantitative loss of function assays). Controlled precipitation of BA-NPs function to increase particle accessibility by surrounding cells, and subsequently enhance uptake and transfection efficiency. By closely examining such trends, an optimal fabrication condition of pH 6.5-37C can be observed where particle growth is more tamed and less chaotic, providing improved, favorable cellular interactions that increase cell uptake and consequently maximize gene transfection, without compromising cellular viability.

Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells.

PubMed

Yogalingam, Gouri; Lee, Amanda R; Mackenzie, Donald S; Maures, Travis J; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y; Hague, Chuck; Christianson, Terri; Bell, Sean M; LeBowitz, Jonathan H

2017-03-10

Neutrophil myeloperoxidase (MPO) catalyzes the H 2 O 2 -dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N -retinylidene- N -retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N -retinylidene- N -retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells*

PubMed Central

Yogalingam, Gouri; Lee, Amanda R.; Mackenzie, Donald S.; Maures, Travis J.; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y.; Hague, Chuck; Christianson, Terri; Bell, Sean M.; LeBowitz, Jonathan H.

2017-01-01

Neutrophil myeloperoxidase (MPO) catalyzes the H2O2-dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N-retinylidene-N-retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N-retinylidene-N-retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. PMID:28115520

Quantitative assessment of surface functionality effects on microglial uptake and retention of PAMAM dendrimers

NASA Astrophysics Data System (ADS)

Liaw, Kevin; Gök, Ozgul; DeRidder, Louis B.; Kannan, Sujatha; Kannan, Rangaramanujam M.

2018-04-01

Dendrimers are a promising class of polymeric nanoparticles for delivery of therapeutics and diagnostics. Polyamidoamine (PAMAM) dendrimers have shown significant efficacy in many animal models, with performance dependent on surface functionalities. Understanding the effects of end groups on biological interactions is critical for rational design of dendrimer-mediated therapies. In this study, we quantify the cellular trafficking kinetics (endocytosis and exocytosis) of generation 4 neutral (D4-OH), cationic (D4-NH2), anionic (D3.5-COOH), and generation 6 neutral (D6-OH) PAMAM dendrimers to investigate the nanoscale effects of surface functionality and size on cellular interactions. Resting and LPS-activated microglia were studied due to their central roles in dendrimer therapies for central nervous system disorders. D4-OH exhibits greater cellular uptake and lower retention than the larger D6-OH. D4-OH and D3.5-COOH exhibit similar trafficking kinetics, while D4-NH2 exhibits significant membrane interactions, resulting in faster cell association but lower internalization. Cationic charge may also enhance vesicular escape for greater cellular retention and preferential partitioning to nuclei. LPS activation further improves uptake of dendrimers, with smaller and cationic dendrimers experiencing the greatest increases in uptake compared to resting microglia. These studies have implications for the dependence of trafficking pathway on dendrimer properties and inform the design of dendrimer constructs tailored to specific therapeutic needs. Cationic dendrimers are ideal for delivering genetic materials to nuclei, but toxicity may be a limiting factor. Smaller, neutral dendrimers are best suited for delivering high levels of therapeutics in acute neuroinflammation, while larger or cationic dendrimers provide robust retention for sustained release of therapeutics in longer-term diseases.

Clusterin in the protein corona plays a key role in the stealth effect of nanoparticles against phagocytes.

PubMed

Aoyama, Michihiko; Hata, Katsutomo; Higashisaka, Kazuma; Nagano, Kazuya; Yoshioka, Yasuo; Tsutsumi, Yasuo

2016-11-25

In biological fluids, nanoparticles interact with biological components such as proteins, and a layer called the "protein corona" forms around the nanoparticles. It is believed that the composition of the protein corona affects the cellular uptake and in vivo biodistribution of nanoparticles; however, the key proteins of the protein corona that control the biological fate of nanoparticles remain unclear. Recently, it was reported that clusterin binding to pegylated nanoparticles is important for the stealth effect of pegylated nanoparticles in phagocytes. However, the effect of clusterin on non-pegylated nanoparticles is unknown, although it is known that clusterin is present in the protein corona of non-pegylated nanoparticles. Here, we assessed the stealth effect of clusterin in the corona of non-pegylated silver nanoparticles and silica nanoparticles. We found that serum- and plasma-protein corona inhibited the cellular uptake of silver nanoparticles and silica nanoparticles in phagocytes and that the plasma-protein corona showed a greater stealth effect compared with the serum-protein corona. Clusterin was present in both the serum- and plasma-protein corona, but was present at a higher level in the plasma-protein corona than in the serum-protein corona. Clusterin binding to silver nanoparticles and silica nanoparticles suppressed the cellular uptake of nanoparticles in human macrophage-like cells (THP-1 cells). Although further studies are required to determine how clusterin suppresses non-specific cellular uptake in phagocytes, our data suggest that clusterin plays a key role in the stealth effect of not only pegylated nanoparticles but also non-pegylated nanoparticles. Copyright © 2016 Elsevier Inc. All rights reserved.

Zinc starvation induces autophagy in yeast

PubMed Central

Kawamata, Tomoko; Horie, Tetsuro; Matsunami, Miou; Sasaki, Michiko; Ohsumi, Yoshinori

2017-01-01

Zinc is an essential nutrient for all forms of life. Within cells, most zinc is bound to protein. Because zinc serves as a catalytic or structural cofactor for many proteins, cells must maintain zinc homeostasis under severely zinc-deficient conditions. In yeast, the transcription factor Zap1 controls the expression of genes required for uptake and mobilization of zinc, but to date the fate of existing zinc-binding proteins under zinc starvation remains poorly understood. Autophagy is an evolutionarily conserved cellular degradation/recycling process in which cytoplasmic proteins and organelles are sequestered for degradation in the vacuole/lysosome. In this study, we investigated how autophagy functions under zinc starvation. Zinc depletion induced non-selective autophagy, which is important for zinc-limited growth. Induction of autophagy by zinc starvation was not directly related to transcriptional activation of Zap1. Instead, TORC1 inactivation directed zinc starvation-induced autophagy. Abundant zinc proteins, such as Adh1, Fba1, and ribosomal protein Rpl37, were degraded in an autophagy-dependent manner. But the targets of autophagy were not restricted to zinc-binding proteins. When cellular zinc is severely depleted, this non-selective autophagy plays a role in releasing zinc from the degraded proteins and recycling zinc for other essential purposes. PMID:28264932

Facile Synthesis of Uniform Virus-like Mesoporous Silica Nanoparticles for Enhanced Cellular Internalization

PubMed Central

2017-01-01

The low-efficiency cellular uptake property of current nanoparticles greatly restricts their application in the biomedical field. Herein, we demonstrate that novel virus-like mesoporous silica nanoparticles can easily be synthesized, showing greatly superior cellular uptake property. The unique virus-like mesoporous silica nanoparticles with a spiky tubular rough surface have been successfully synthesized via a novel single-micelle epitaxial growth approach in a low-concentration-surfactant oil/water biphase system. The virus-like nanoparticles’ rough surface morphology results mainly from the mesoporous silica nanotubes spontaneously grown via an epitaxial growth process. The obtained nanoparticles show uniform particle size and excellent monodispersity. The structural parameters of the nanoparticles can be well tuned with controllable core diameter (∼60–160 nm), tubular length (∼6–70 nm), and outer diameter (∼6–10 nm). Thanks to the biomimetic morphology, the virus-like nanoparticles show greatly superior cellular uptake property (invading living cells in large quantities within few minutes, <5 min), unique internalization pathways, and extended blood circulation duration (t1/2 = 2.16 h), which is much longer than that of conventional mesoporous silica nanoparticles (0.45 h). Furthermore, our epitaxial growth strategy can be applied to fabricate various virus-like mesoporous core–shell structures, paving the way toward designed synthesis of virus-like nanocomposites for biomedicine applications. PMID:28852697

Temporal and mechanistic tracking of cellular uptake dynamics with novel surface fluorophore-bound nanodiamonds.

PubMed

Schrand, Amanda M; Lin, Jonathan B; Hens, Suzanne Ciftan; Hussain, Saber M

2011-02-01

Nanoparticles (NPs) offer promise for a multitude of biological applications including cellular probes at the bio-interface for targeted delivery of anticancer substances, Raman and fluorescent-based imaging and directed cell growth. Nanodiamonds (NDs), in particular, have several advantages compared to other carbon-based nanomaterials - including a rich surface chemistry useful for chemical conjugation, high biocompatibility with little reactive oxygen species (ROS) generation, physical and chemical stability that affords sterilization, high surface area to volume ratio, transparency and a high index of refraction. The visualization of ND internalization into cells is possible via photoluminescence, which is produced by direct dye conjugation or high energy irradiation that creates nitrogen vacancy centers. Here, we explore the kinetics and mechanisms involved in the intracellular uptake and localization of novel, highly-stable, fluorophore-conjugated NDs. Examination in a neuronal cell line (N2A) shows ND localization to early endosomes and lysosomes with eventual release into the cytoplasm. The addition of endocytosis and exocytosis inhibitors allows for diminished uptake and increased accumulation, respectively, which further corroborates cellular behavior in response to NDs. Ultimately, the ability of the NDs to travel throughout cellular compartments of varying pH without degradation of the surface-conjugated fluorophore or alteration of cell viability over extended periods of time is promising for their use in biomedical applications as stable, biocompatible, fluorescent probes.

A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): A pilot study in a canine model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukumoto, Shinya; Hanazono, Kiwamu; Fu, Dah-Renn

2013-09-13

Highlights: •LAT1 is highly expressed in tumors but at low levels in normal tissues. •We examine LAT1 expression and function in malignant melanoma (MM). •LAT1 expression in MM tissues and cell lines is higher than those in normal tissues. •LAT1 selective inhibitors inhibit amino acid uptake and cell growth in MM cells. •New chemotherapeutic protocols including LAT1 inhibitors are effective for treatment. -- Abstract: L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transportermore » recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25 MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P < 0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [{sup 3}H]L-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P < 0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM.« less

Selective Inhibition of K+, Na+, Cl−, and PO43− Uptake in Zea mays L. by Bipolaris (Helminthosporium) maydis Race T Pathotoxin

PubMed Central

Mertz, Stuart M.; Arntzen, Charles J.

1977-01-01

Pathotoxin preparations were obtained from either axenic culture filtrate of race T of Bipolaris maydis (Nisikado) Shoemaker (new culture media and toxin purification procedures are described) or extracts of maize leaves infected with the fungus. The toxins (10−6 to 10−8m) caused inhibition of [86Rb]K+ uptake in leaf discs and apical root segments of Zea mays L. cv W64A Texas (Tcms) and normal (N) cytoplasms. Significant inhibition was measurable as early as 5 min after adding toxin. In Tcms per cent inhibition was increased by increasing toxin concentration and time in toxin, by using solution at pH 5 rather than pH 7, by decreasing external KCl concentration over the range 50 to 0.1 mm (in the presence of 0.5 mm CaSO4), or by exposing leaf discs to light rather than dark during the uptake period in toxin. Root uptake of 22Na+ and 36Cl− was inhibited to a lesser extent than K+. Inhibition of 32PO43− uptake occurred after 40 min when cyclosis had ceased. When combined with data in the literature, our data indicate that the plasmalemma is the probable primary site of toxin action in N and Tcms maize. Comparison of the effects of toxin on K+ uptake in N and Tcms maize suggests the existence of more than one mode of toxin action: a weak disruptive effect in N and Tcms, and in addition, specific membrane sites in Tcms involved in monovalent ion uptake. Six genotypes in N or Tcms cytoplasm which exhibited different degrees of disease susceptibility in the field showed a corresponding gradation of susceptibility to the toxin when a K+ uptake bioassay was used. This correlation is strong evidence that the sites of toxin action affecting K+ transport have characteristics closely related to cellular factors regulating susceptibility to fungal attack. PMID:16660094

Glucose-functionalized Au nanoprisms for optoacoustic imaging and near-infrared photothermal therapy

NASA Astrophysics Data System (ADS)

Han, Jishu; Zhang, Jingjing; Yang, Meng; Cui, Daxiang; de La Fuente, Jesus M.

2015-12-01

Targeted imaging and tumor therapy using nanomaterials has stimulated research interest recently, but the high cytotoxicity and low cellular uptake of nanomaterials limit their bioapplication. In this paper, glucose (Glc) was chosen to functionalize Au nanoprisms (NPrs) for improving the cytotoxicity and cellular uptake of Au@PEG-Glc NPrs into cancer cells. Glucose is a primary source of energy at the cellular level and at cellular membranes for cell recognition. A coating of glucose facilitates the accumulation of Au@PEG-Glc NPrs in a tumor region much more than Au@PEG NPrs. Due to the high accumulation and excellent photoabsorbing property of Au@PEG-Glc NPrs, enhanced optoacoustic imaging of a tumor in vivo was achieved, and visualization of the tumor further guided cancer treatment. Based on the optical-thermal conversion performance of Au@PEG-Glc NPrs, the tumor in vivo was effectively cured through photothermal therapy. The current work demonstrates the great potential of Au@PEG-Glc NPrs in optoacoustic imaging and photothermal cancer therapy in future.Targeted imaging and tumor therapy using nanomaterials has stimulated research interest recently, but the high cytotoxicity and low cellular uptake of nanomaterials limit their bioapplication. In this paper, glucose (Glc) was chosen to functionalize Au nanoprisms (NPrs) for improving the cytotoxicity and cellular uptake of Au@PEG-Glc NPrs into cancer cells. Glucose is a primary source of energy at the cellular level and at cellular membranes for cell recognition. A coating of glucose facilitates the accumulation of Au@PEG-Glc NPrs in a tumor region much more than Au@PEG NPrs. Due to the high accumulation and excellent photoabsorbing property of Au@PEG-Glc NPrs, enhanced optoacoustic imaging of a tumor in vivo was achieved, and visualization of the tumor further guided cancer treatment. Based on the optical-thermal conversion performance of Au@PEG-Glc NPrs, the tumor in vivo was effectively cured through photothermal therapy. The current work demonstrates the great potential of Au@PEG-Glc NPrs in optoacoustic imaging and photothermal cancer therapy in future. Electronic supplementary information (ESI) available: The evolution of the UV-vis absorption of Au NPrs by centrifugation, TEM image of PEG-capped Au NPrs, the UV-vis absorption of glucose, cytotoxicity of Au@PEG-Glc NPrs, gastric cell viabilities versus the concentration of Au@PEG-Glc NPrs and gastric cell viabilities filled with 80 μg Au@PEG-Glc NPrs versus the irradiation time, optoacoustic signals of Au NPr solution and Au@PEG NPrs. See DOI: 10.1039/c5nr06261f

Uptake of silver nanoparticles by monocytic THP-1 cells depends on particle size and presence of serum proteins

NASA Astrophysics Data System (ADS)

Kettler, Katja; Giannakou, Christina; de Jong, Wim H.; Hendriks, A. Jan; Krystek, Petra

2016-09-01

Human health risks by silver nanoparticle (AgNP) exposure are likely to increase due to the increasing number of NP-containing products and demonstrated adverse effects in various cell lines. Unfortunately, results from (toxicity) studies are often based on exposure dose and are often measured only at a fixed time point. NP uptake kinetics and the time-dependent internal cellular concentration are often not considered. Macrophages are the first line of defense against invading foreign agents including NPs. How macrophages deal with the particles is essential for potential toxicity of the NPs. However, there is a considerable lack of uptake studies of particles in the nanometer range and macrophage-like cells. Therefore, uptake rates were determined over 24 h for three different AgNPs sizes (20, 50 and 75 nm) in medium with and without fetal calf serum. Non-toxic concentrations of 10 ng Ag/mL for monocytic THP-1 cells, representing realistic exposure concentration for short-term exposures, were chosen. The uptake of Ag was higher in medium without fetal calf serum and showed increasing uptake for decreasing NP sizes, both on NP mass and on number basis. Internal cellular concentrations reached roughly 32/10 %, 25/18 % and 21/15 % of the nominal concentration in the absence of fetal calf serum/with fetal calf serum for 20-, 50- and 75-nm NPs, respectively. Our research shows that uptake kinetics in macrophages differ for various NP sizes. To increase the understanding of the mechanism of NP toxicity in cells, the process of uptake (timing) should be considered.

Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry

PubMed Central

2013-01-01

Background The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface. Results Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells. Conclusion The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs. PMID:23388071

Cellular membrane trafficking of mesoporous silica nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, I-Ju

This dissertation mainly focuses on the investigation of the cellular membrane trafficking of mesoporous silica nanoparticles. We are interested in the study of endocytosis and exocytosis behaviors of mesoporous silica nanoparticles with desired surface functionality. The relationship between mesoporous silica nanoparticles and membrane trafficking of cells, either cancerous cells or normal cells was examined. Since mesoporous silica nanoparticles were applied in many drug delivery cases, the endocytotic efficiency of mesoporous silica nanoparticles needs to be investigated in more details in order to design the cellular drug delivery system in the controlled way. It is well known that cells can engulfmore » some molecules outside of the cells through a receptor-ligand associated endocytosis. We are interested to determine if those biomolecules binding to cell surface receptors can be utilized on mesoporous silica nanoparticle materials to improve the uptake efficiency or govern the mechanism of endocytosis of mesoporous silica nanoparticles. Arginine-glycine-aspartate (RGD) is a small peptide recognized by cell integrin receptors and it was reported that avidin internalization was highly promoted by tumor lectin. Both RGD and avidin were linked to the surface of mesoporous silica nanoparticle materials to investigate the effect of receptor-associated biomolecule on cellular endocytosis efficiency. The effect of ligand types, ligand conformation and ligand density were discussed in Chapter 2 and 3. Furthermore, the exocytosis of mesoporous silica nanoparticles is very attractive for biological applications. The cellular protein sequestration study of mesoporous silica nanoparticles was examined for further information of the intracellular pathway of endocytosed mesoporous silica nanoparticle materials. The surface functionality of mesoporous silica nanoparticle materials demonstrated selectivity among the materials and cancer and normal cell lines. We aimed to determine the specific organelle that mesoporous silica nanoparticles could approach via the identification of harvested proteins from exocytosis process. Based on the study of endo- and exocytosis behavior of mesoporous silica nanoparticle materials, we can design smarter drug delivery vehicles for cancer therapy that can be effectively controlled. The destination, uptake efficiency and the cellular distribution of mesoporous silica nanoparticle materials can be programmable. As a result, release mechanism and release rate of drug delivery systems can be a well-controlled process. The deep investigation of an endo- and exocytosis study of mesoporous silica nanoparticle materials promotes the development of drug delivery applications.« less

Loss of intracellular lipid binding proteins differentially impacts saturated fatty acid uptake and nuclear targeting in mouse hepatocytes

PubMed Central

Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Martin, Gregory G.; Landrock, Kerstin K.; Landrock, Danilo; Payne, H. Ross; Kier, Ann B.

2012-01-01

The liver expresses high levels of two proteins with high affinity for long-chain fatty acids (LCFAs): liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2). Real-time confocal microscopy of cultured primary hepatocytes from gene-ablated (L-FABP, SCP-2/SCP-x, and L-FABP/SCP-2/SCP-x null) mice showed that the loss of L-FABP reduced cellular uptake of 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazo)-aminostearic acid (a fluorescent-saturated LCFA analog) by ∼50%. Importantly, nuclear targeting of the LCFA was enhanced when L-FABP was upregulated (SCP-2/SCP-x null) but was significantly reduced when L-FABP was ablated (L-FABP null), thus impacting LCFA nuclear targeting. These effects were not associated with a net decrease in expression of key membrane proteins involved in LCFA or glucose transport. Since hepatic LCFA uptake and metabolism are closely linked to glucose uptake, the effect of glucose on L-FABP-mediated LCFA uptake and nuclear targeting was examined. Increasing concentrations of glucose decreased cellular LCFA uptake and even more extensively decreased LCFA nuclear targeting. Loss of L-FABP exacerbated the decrease in LCFA nuclear targeting, while loss of SCP-2 reduced the glucose effect, resulting in enhanced LCFA nuclear targeting compared with control. Simply, ablation of L-FABP decreases LCFA uptake and even more extensively decreases its nuclear targeting. PMID:22859366

Muscarinic receptor stimulation of D-aspartate uptake into human SH-SY5Y neuroblastoma cells is attenuated by hypoosmolarity.

PubMed

Foster, Daniel J; Heacock, Anne M; Fisher, Stephen K

2010-04-01

In addition to its function as an excitatory neurotransmitter, glutamate plays a major role as an osmolyte within the central nervous system (CNS). Accordingly, mechanisms that regulate glutamate release and uptake are of physiological importance not only during conditions in which cell volume remains constant but also when cells are subjected to hypoosmotic stress. In the present study, the ability of muscarinic cholinergic receptors (mAChRs) to regulate the uptake of glutamate (monitored as D-aspartate) into human SH-SY5Y neuroblastoma cells under isotonic or hypotonic conditions has been examined. In isotonic media, agonist activation of mAChRs resulted in a significant increase (250-300% of control) in the uptake of D-aspartate and, concurrently, a cellular redistribution of the excitatory amino acid transporter 3 (EAAT3) to the plasma membrane. mAChR-mediated increases in d-aspartate uptake were potently blocked by the EAAT3 inhibitor l-beta-threo-benzyl-aspartate. In hypotonic media, the ability of mAChR activation to facilitate D-aspartate uptake was significantly attenuated (40-50%), and the cellular distribution of EAAT3 was disrupted. Reduction of mAChR-stimulated D-aspartate uptake under hypoosmotic conditions could be fully reversed upon re-exposure of the cells to isotonic media. Under both isotonic and hypotonic conditions, mAChR-mediated increases in D-aspartate uptake depended on cytoskeletal integrity, protein kinase C and phosphatidylinositol 3-kinase activities, and the availability of intracellular Ca2+. In contrast, dependence on extracellular Ca2+ was observed only under isotonic conditions. The results suggest that, although the uptake of D-aspartate into SH-SY5Y cells is enhanced after mAChR activation, this process is markedly attenuated by hypoosmolarity.

Effect of adenosine on the growth of human T-lymphocyte leukemia cell line MOLT-4.

PubMed

Streitová, Denisa; Weiterová, Lenka; Hofer, Michal; Holá, Jirina; Horváth, Viktor; Kozubík, Alois; Znojil, Vladimír

2007-09-01

Adenosine has been observed to suppress the growth of MOLT-4 human leukemia cells in vitro. Changes in the cell cycle, especially increased percentage of cells in S phase, prolonged generation time, and induction of apoptosis at higher adenosine concentrations have been found to be responsible for the growth suppression. Dipyridamole, a drug inhibiting the cellular uptake of adenosine, reversed partially but significantly the adenosine-induced growth suppression. It follows from these results that the action of adenosine on the MOLT-4 cells comprises its cellular uptake and intracellular operation. These findings present new data on anticancer efficacy of adenosine.

Improved Cellular Specificity of Plasmonic Nanobubbles versus Nanoparticles in Heterogeneous Cell Systems

PubMed Central

Lukianova-Hleb, Ekaterina Y.; Ren, Xiaoyang; Constantinou, Pamela E.; Danysh, Brian P.; Shenefelt, Derek L.; Carson, Daniel D.; Farach-Carson, Mary C.; Kulchitsky, Vladimir A.; Wu, Xiangwei; Wagner, Daniel S.; Lapotko, Dmitri O.

2012-01-01

The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level. PMID:22509318

Improved cellular specificity of plasmonic nanobubbles versus nanoparticles in heterogeneous cell systems.

PubMed

Lukianova-Hleb, Ekaterina Y; Ren, Xiaoyang; Constantinou, Pamela E; Danysh, Brian P; Shenefelt, Derek L; Carson, Daniel D; Farach-Carson, Mary C; Kulchitsky, Vladimir A; Wu, Xiangwei; Wagner, Daniel S; Lapotko, Dmitri O

2012-01-01

The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level.

Improving the biopharmaceutical attributes of mangiferin using vitamin E-TPGS co-loaded self-assembled phosholipidic nano-mixed micellar systems.

PubMed

Khurana, Rajneet Kaur; Gaspar, Balan Louis; Welsby, Gail; Katare, O P; Singh, Kamalinder K; Singh, Bhupinder

2018-06-01

The current research work encompasses the development, characterization, and evaluation of self-assembled phospholipidic nano-mixed miceller system (SPNMS) of a poorly soluble BCS Class IV xanthone bioactive, mangiferin (Mgf) functionalized with co-delivery of vitamin E TPGS. Systematic optimization using I-optimal design yielded self-assembled phospholipidic nano-micelles with a particle size of < 60 nm and > 80% of drug release in 15 min. The cytotoxicity and cellular uptake studies performed using MCF-7 and MDA-MB-231 cell lines demonstrated greater kill and faster cellular uptake. The ex vivo intestinal permeability revealed higher lymphatic uptake, while in situ perfusion and in vivo pharmacokinetic studies indicated nearly 6.6- and 3.0-folds augmentation in permeability and bioavailability of Mgf. In a nutshell, vitamin E functionalized SPNMS of Mgf improved the biopharmaceutical performance of Mgf in rats for enhanced anticancer potency.

Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1.

PubMed

Marr, A K; Jenssen, H; Moniri, M Roshan; Hancock, R E W; Panté, N

2009-01-01

Although both lactoferrin (Lf), a component of the innate immune system of living organisms, and its N-terminal pepsin cleavage product lactoferricin (Lfcin) have anti-herpes activity, the precise mechanisms by which Lf and Lfcin bring about inhibition of herpes infections are not fully understood. In the present study, experiments were carried out to characterize the activity of bovine Lf and Lfcin (BLf and BLfcin) against the Herpes simplex virus-1 (HSV-1). HSV-1 cellular uptake and intracellular trafficking were studied by immunofluorescence microscopy. In comparison to the untreated infected control cells, both the BLf- and BLfcin-treated cells showed a significant reduction in HSV-1 cellular uptake. The few virus particles that were internalized appeared to have a delayed intracellular trafficking. Thus, in addition to their interference with the uptake of the virus into host cells, Lf and Lfcin also exert their antiviral effect intracellularly.

g-force induced giant efficiency of nanoparticles internalization into living cells

PubMed Central

Ocampo, Sandra M.; Rodriguez, Vanessa; de la Cueva, Leonor; Salas, Gorka; Carrascosa, Jose. L.; Josefa Rodríguez, María; García-Romero, Noemí; Luis, Jose; Cuñado, F.; Camarero, Julio; Miranda, Rodolfo; Belda-Iniesta, Cristobal; Ayuso-Sacido, Angel

2015-01-01

Nanotechnology plays an increasingly important role in the biomedical arena. Iron oxide nanoparticles (IONPs)-labelled cells is one of the most promising approaches for a fast and reliable evaluation of grafted cells in both preclinical studies and clinical trials. Current procedures to label living cells with IONPs are based on direct incubation or physical approaches based on magnetic or electrical fields, which always display very low cellular uptake efficiencies. Here we show that centrifugation-mediated internalization (CMI) promotes a high uptake of IONPs in glioblastoma tumour cells, just in a few minutes, and via clathrin-independent endocytosis pathway. CMI results in controllable cellular uptake efficiencies at least three orders of magnitude larger than current procedures. Similar trends are found in human mesenchymal stem cells, thereby demonstrating the general feasibility of the methodology, which is easily transferable to any laboratory with great potential for the development of improved biomedical applications. PMID:26477718

Neomycin inhibits PDGF-induced IP3 formation and DNA synthesis but not PDGF-stimulated uptake of inorganic phosphate in C3H/10T1/2 fibroblasts.

PubMed

Vassbotn, F S; Langeland, N; Holmsen, H

1990-09-01

Porcine PDGF was found to increase [3H]inositol trisphosphate, [3H]thymidine incorporation and 32P-labelling of polyphosphoinositides in C3H/10T1/2 Cl 8 fibroblasts. These responses to PDGF stimulation were all inhibited by 5 mM neomycin, a polycationic aminoglycoside formerly known to inhibit polyphosphoinositide turnover. PDGF also markedly increased the cellular uptake of inorganic [32P]Pi. This response of PDGF was not inhibited by neomycin (5 mM). Thus, neomycin inhibited PDGF-induced IP3 formation, 32P-labelling of polyphosphoinositides and DNA synthesis, but not cellular uptake of inorganic phosphate. These effects of neomycin suggest a bifurcation of the initial part of the PDGF-induced signal transduction, separating at the receptor level or before phospholipase C activation.

Effect of PEG molecular weight on stability, T₂ contrast, cytotoxicity, and cellular uptake of superparamagnetic iron oxide nanoparticles (SPIONs).

PubMed

Park, Yoonjee C; Smith, Jared B; Pham, Tuan; Whitaker, Ragnhild D; Sucato, Christopher A; Hamilton, James A; Bartolak-Suki, Elizabeth; Wong, Joyce Y

2014-07-01

Superparamagnetic iron oxide nanoparticles (SPIONs) are currently unavailable as MRI contrast agents for detecting atherosclerosis in the clinical setting because of either low signal enhancement or safety concerns. Therefore, a new generation of SPIONs with increased circulation time, enhanced image contrast, and less cytotoxicity is essential. In this study, monodisperse SPIONs were synthesized and coated with polyethylene glycol (PEG) of varying molecular weights. The resulting PEGylated SPIONs were characterized, and their interactions with vascular smooth muscle cells (VSMCs) were examined. SPIONs were tested at different concentrations (100 and 500 ppm Fe) for stability, T2 contrast, cytotoxicity, and cellular uptake to determine an optimal formulation for in vivo use. We found that at 100 ppm Fe, the PEG 2K SPIONs showed adequate stability and magnetic contrast, and exhibited the least cytotoxicity and nonspecific cellular uptake. An increase in cell viability was observed when the SPION-treated cells were washed with PBS after 1h incubation compared to 5 and 24h incubation without washing. Our investigation provides insight into the potential safe application of SPIONs in the clinic. Copyright © 2014 Elsevier B.V. All rights reserved.

Multifunctional organic–inorganic hybrid nanoparticles and nanosheets based on chitosan derivative and layered double hydroxide: cellular uptake mechanism and application for topical ocular drug delivery

PubMed Central

Chi, Huibo; Gu, Yan; Xu, Tingting; Cao, Feng

2017-01-01

To study the cellular uptake mechanism of multifunctional organic–inorganic hybrid nanoparticles and nanosheets, new chitosan–glutathione–valine–valine-layered double hydroxide (CG-VV-LDH) nanosheets with active targeting to peptide transporter-1 (PepT-1) were prepared, characterized and further compared with CG-VV-LDH nanoparticles. Both organic–inorganic hybrid nanoparticles and nanosheets showed a sustained release in vitro and prolonged precorneal retention time in vivo, but CG-VV-LDH nanoparticles showed superior permeability in the isolated cornea of rabbits than CG-VV-LDH nanosheets. Furthermore, results of cellular uptake on human corneal epithelial primary cells (HCEpiC) and retinal pigment epithelial (ARPE-19) cells indicated that both clathrin-mediated endocytosis and active transport of PepT-1 are involved in the internalization of CG-VV-LDH nanoparticles and CG-VV-LDH nanosheets. In summary, the CG-VV-LDH nanoparticle may be a promising carrier as a topical ocular drug delivery system for the treatment of ocular diseases of mid-posterior segments, while the CG-VV-LDH nanosheet may be suitable for the treatment of ocular surface diseases. PMID:28280329

Surface modification of solid lipid nanoparticles for oral delivery of curcumin: Improvement of bioavailability through enhanced cellular uptake, and lymphatic uptake.

PubMed

Baek, Jong-Suep; Cho, Cheong-Weon

2017-08-01

Curcumin has been reported to exhibit potent anticancer effects. However, poor solubility, bioavailability and stability of curcumin limit its in vivo efficacy for the cancer treatment. Solid lipid nanoparticles (SLN) are a promising delivery system for the enhancement of bioavailability of hydrophobic drugs. However, burst release of drug from SLN in acidic environment limits its usage as oral delivery system. Hence, we prepared N-carboxymethyl chitosan (NCC) coated curcumin-loaded SLN (NCC-SLN) to inhibit the rapid release of curcumin in acidic environment and enhance the bioavailability. The NCC-SLN exhibited suppressed burst release in simulated gastric fluid while sustained release was observed in simulated intestinal fluid. Furthermore, NCC-SLN exhibited increased cytotoxicity and cellular uptake on MCF-7 cells. The lymphatic uptake and oral bioavailability of NCC-SLN were found to be 6.3-fold and 9.5-fold higher than that of curcumin solution, respectively. These results suggest that NCC-SLN could be an efficient oral delivery system for curcumin. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel approach for targeted delivery to motoneurons using cholera toxin-B modified protocells

PubMed Central

Gonzalez Porras, Maria A.; Durfee, Paul N.; Gregory, Ashley M.; Sieck, Gary C.; Brinker, C. Jeffrey; Mantilla, Carlos B.

2017-01-01

Background Trophic interactions between muscle fibers and motoneurons at the neuromuscular junction (NMJ) play a critical role in determining motor function throughout development, ageing, injury, or disease. Treatment of neuromuscular disorders is hindered by the inability to selectively target motoneurons with pharmacological and genetic interventions. New method We describe a novel delivery system to motoneurons using mesoporous silica nanoparticles encapsulated within a lipid bilayer (protocells) and modified with the atoxic subunit B of the cholera toxin (CTB) that binds to gangliosides present on neuronal membranes. Results CTB modified protocells showed significantly greater motoneuron uptake compared to unmodified protocells after 24 h of treatment (60% vs. 15%, respectively). CTB-protocells showed specific uptake by motoneurons compared to muscle cells and demonstrated cargo release of a surrogate drug. Protocells showed a lack of cytotoxicity and unimpaired cellular proliferation. In isolated diaphragm muscle-phrenic nerve preparations, preferential axon terminal uptake of CTB-modified protocells was observed compared to uptake in surrounding muscle tissue. A larger proportion of axon terminals displayed uptake following treatment with CTB-protocells compared to unmodified protocells (40% vs. 6%, respectively). Comparison with existing method(s) Current motoneuron targeting strategies lack the functionality to load and deliver multiple cargos. CTB-protocells capitalizes on the advantages of liposomes and mesoporous silica nanoparticles allowing a large loading capacity and cargo release. The ability of CTB-protocells to target motoneurons at the NMJ confers a great advantage over existing methods. Conclusions CTB-protocells constitute a viable targeted motoneuron delivery system for drugs and genes facilitating various therapies for neuromuscular diseases. PMID:27641118

A novel approach for targeted delivery to motoneurons using cholera toxin-B modified protocells.

PubMed

Gonzalez Porras, Maria A; Durfee, Paul N; Gregory, Ashley M; Sieck, Gary C; Brinker, C Jeffrey; Mantilla, Carlos B

2016-11-01

Trophic interactions between muscle fibers and motoneurons at the neuromuscular junction (NMJ) play a critical role in determining motor function throughout development, ageing, injury, or disease. Treatment of neuromuscular disorders is hindered by the inability to selectively target motoneurons with pharmacological and genetic interventions. We describe a novel delivery system to motoneurons using mesoporous silica nanoparticles encapsulated within a lipid bilayer (protocells) and modified with the atoxic subunit B of the cholera toxin (CTB) that binds to gangliosides present on neuronal membranes. CTB modified protocells showed significantly greater motoneuron uptake compared to unmodified protocells after 24h of treatment (60% vs. 15%, respectively). CTB-protocells showed specific uptake by motoneurons compared to muscle cells and demonstrated cargo release of a surrogate drug. Protocells showed a lack of cytotoxicity and unimpaired cellular proliferation. In isolated diaphragm muscle-phrenic nerve preparations, preferential axon terminal uptake of CTB-modified protocells was observed compared to uptake in surrounding muscle tissue. A larger proportion of axon terminals displayed uptake following treatment with CTB-protocells compared to unmodified protocells (40% vs. 6%, respectively). Current motoneuron targeting strategies lack the functionality to load and deliver multiple cargos. CTB-protocells capitalizes on the advantages of liposomes and mesoporous silica nanoparticles allowing a large loading capacity and cargo release. The ability of CTB-protocells to target motoneurons at the NMJ confers a great advantage over existing methods. CTB-protocells constitute a viable targeted motoneuron delivery system for drugs and genes facilitating various therapies for neuromuscular diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Highlights in Endocytosis of Nanostructured Systems.

PubMed

Voltan, Aline R; Alarcon, Kaila M; Fusco-Almeida, Ana M; Soares, Christiane P; Mendes-Giannini, Maria J S; Chorilli, Marlus

2017-01-01

The focus of this review is the cellular internalisation mechanism of nanostructured systems (NSs) and their endosomal escape for targeted drug delivery. Endocytosis is a cellular process of internalisation of different molecules and foreign microorganisms. It is currently being studied for drug delivery through nanostructured systems. The most commonly studied routes of cellular uptake are phagocytosis, macro-pinocytosis, clathrinmediated endocytosis, caveolin-mediated endocytosis, and clathrin and caveolinindependent endocytosis. The mechanism utilised by NSs for cellular entry depends on factors such as cell type and its physicochemical properties. Currently, with the development of drugs-loaded onto NSs, it has been possible to increase the therapeutic index against few diseases. The NSs can deliver the active drug at locations that conventional drugs cannot, thereby minimising unwanted side effects. On cellular entry of NSs, there is a possibility of an endosomal escape of the contents into the cytoplasm, a mechanism that can be exploited so that NSs can migrate intra-cellularly and deliver the drug to the target of interest. Designing endolysosomal escape strategy is not an easy task, but it is critical for the optimal pharmacological action on the target tissue. The cellular uptake of drugs is a very important factor in therapy. Although NSs have emerged as effective drug delivery vehicle for treatment of diseases, it is crucial to understand the mechanism of NSs endocytosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Surface bioengineering of diatomite based nanovectors for efficient intracellular uptake and drug delivery.

PubMed

Terracciano, Monica; Shahbazi, Mohammad-Ali; Correia, Alexandra; Rea, Ilaria; Lamberti, Annalisa; De Stefano, Luca; Santos, Hélder A

2015-12-21

Diatomite is a natural porous silica material of sedimentary origin. Due to its peculiar properties, it can be considered as a valid surrogate of synthetic porous silica for nano-based drug delivery. In this work, we exploit the potential of diatomite nanoparticles (DNPs) for drug delivery with the aim of developing a successful dual-biofunctionalization method by polyethylene glycol (PEG) coverage and cell-penetrating peptide (CPP) bioconjugation, to improve the physicochemical and biological properties of the particles, to enhance the intracellular uptake in cancer cells, and to increase the biocompatibility of 3-aminopropyltriethoxysilane (APT) modified-DNPs. DNPs-APT-PEG-CPP showed hemocompatibility for up to 200 μg mL(-1) after 48 h of incubation with erythrocytes, with a hemolysis value of only 1.3%. The cytotoxicity of the modified-DNPs with a concentration up to 200 μg mL(-1) and incubation with MCF-7 and MDA-MB-231 breast cancer cells for 24 h, demonstrated that PEGylation and CPP-bioconjugation can strongly reduce the cytotoxicity of DNPs-APT. The cellular uptake of the modified-DNPs was also evaluated using the above mentioned cancer cell lines, showing that the CPP-bioconjugation can considerably increase the DNP cellular uptake. Moreover, the dual surface modification of DNPs improved both the loading of a poorly water-soluble anticancer drug, sorafenib, with a loading degree up to 22 wt%, and also enhanced the drug release profiles in aqueous solutions. Overall, this work demonstrates that the biofunctionalization of DNPs is a promising platform for drug delivery applications in cancer therapy as a result of its enhanced stability, biocompatibility, cellular uptake, and drug release profiles.

Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hossain, Ekhtear; Ota, Akinobu, E-mail: aota@aichi-med-u.ac.jp; Karnan, Sivasundaram

Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, anmore » anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis. - Highlights: • Sodium arsenite (SA) increases LOX-1 expression in mouse aortic endothelial cells. • SA enhances cellular uptake of oxidized LDL in dose-dependent manner. • SA-induced ROS generation enhances phosphorylation of NF-κB. • SA upregulates LOX-1 expression through ROS-activated NF-κB signaling pathway.« less

Biological effects of simple changes in functionality on rhodium metalloinsertors

PubMed Central

Weidmann, Alyson G.; Komor, Alexis C.; Barton, Jacqueline K.

2013-01-01

DNA mismatch repair (MMR) is crucial to ensuring the fidelity of the genome. The inability to correct single base mismatches leads to elevated mutation rates and carcinogenesis. Using metalloinsertors–bulky metal complexes that bind with high specificity to mismatched sites in the DNA duplex–our laboratory has adopted a new chemotherapeutic strategy through the selective targeting of MMR-deficient cells, that is, those that have a propensity for cancerous transformation. Rhodium metalloinsertors display inhibitory effects selectively in cells that are deficient in the MMR machinery, consistent with this strategy. However, a highly sensitive structure–function relationship is emerging with the development of new complexes that highlights the importance of subcellular localization. We have found that small structural modifications, for example a hydroxyl versus a methyl functional group, can yield profound differences in biological function. Despite similar binding affinities and selectivities for DNA mismatches, only one metalloinsertor shows selective inhibition of cellular proliferation in MMR-deficient versus -proficient cells. Studies of whole-cell, nuclear and mitochondrial uptake reveal that this selectivity depends upon targeting DNA mismatches in the cell nucleus. PMID:23776288

Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

PubMed

Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

2016-01-01

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

Phloretin attenuates hyperuricemia-induced endothelial dysfunction through co-inhibiting inflammation and GLUT9-mediated uric acid uptake.

PubMed

Liu, Shuyun; Yuan, Yujia; Zhou, Yijie; Zhao, Meng; Chen, Younan; Cheng, Jingqiu; Lu, Yanrong; Liu, Jingping

2017-10-01

Hyperuricemia is an important risk factor for cardiovascular and renal diseases. Phloretin had shown antioxidant and anti-inflammatory properties, but its role in endothelial injury is rarely reported. In this study, we aimed to investigate the protective effect of phloretin on UA-induced injury in human umbilical vein endothelial cells. The effects of UA and phloretin on cell viability, inflammation, THP-1 monocyte adhesion, endothelial cell tube formation, GLUT9 expression and UA uptake in human umbilical vein endothelial cells were evaluated. The changes of nuclear factor-kappa B/extracellular regulated protein kinases signalling were also analysed. Our results showed that UA reduced cell viability and tube formation, and increased inflammation and monocytes adhesion in human umbilical vein endothelial cells in a dose-dependent manner. In contrast, phloretin significantly attenuated pro-inflammatory factors expression and endothelial injury induced by UA. Phloretin inhibited the activation of extracellular regulated protein kinases/nuclear factor-kappa B pathway, and reduced GLUT9 and it mediated UA uptake in human umbilical vein endothelial cells. These results indicated that phloretin attenuated UA-induced endothelial injury via a synergic mechanism including direct anti-inflammatory effect and lowering cellular UA uptake. Our study suggested that phloretin might be a promising therapy for hyperuricemia-related cardiovascular diseases. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

E4orf1 Enhances Glucose Uptake Independent of Proximal Insulin Signaling

PubMed Central

Na, Ha-Na; Hegde, Vijay; Dubuisson, Olga; Dhurandhar, Nikhil V.

2016-01-01

Impaired proximal insulin signaling is often present in diabetes. Hence, approaches to enhance glucose disposal independent of proximal insulin signaling are desirable. Evidence indicates that Adenovirus-derived E4orf1 protein may offer such an approach. This study determined if E4orf1 improves insulin sensitivity and downregulates proximal insulin signaling in vivo and enhances cellular glucose uptake independent of proximal insulin signaling in vitro. High fat fed mice were injected with a retrovirus plasmid expressing E4orf1, or a null vector. E4orf1 significantly improved insulin sensitivity in response to a glucose load. Yet, their proximal insulin signaling in fat depots was impaired, as indicated by reduced tyrosine phosphorylation of insulin receptor (IR), and significantly increased abundance of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). In 3T3-L1 pre-adipocytes E4orf1 expression impaired proximal insulin signaling. Whereas, treatment with rosiglitazone reduced ENPP1 abundance. Unaffected by IR-KD (insulin receptor knockdown) with siRNA, E4orf1 significantly up-regulated distal insulin signaling pathway and enhanced cellular glucose uptake. In vivo, E4orf1 impairs proximal insulin signaling in fat depots yet improves glycemic control. This is probably explained by the ability of E4orf1 to promote cellular glucose uptake independent of proximal insulin signaling. E4orf1 may provide a therapeutic template to enhance glucose disposal in the presence of impaired proximal insulin signaling. PMID:27537838

E4orf1 Enhances Glucose Uptake Independent of Proximal Insulin Signaling.

PubMed

Na, Ha-Na; Hegde, Vijay; Dubuisson, Olga; Dhurandhar, Nikhil V

2016-01-01

Impaired proximal insulin signaling is often present in diabetes. Hence, approaches to enhance glucose disposal independent of proximal insulin signaling are desirable. Evidence indicates that Adenovirus-derived E4orf1 protein may offer such an approach. This study determined if E4orf1 improves insulin sensitivity and downregulates proximal insulin signaling in vivo and enhances cellular glucose uptake independent of proximal insulin signaling in vitro. High fat fed mice were injected with a retrovirus plasmid expressing E4orf1, or a null vector. E4orf1 significantly improved insulin sensitivity in response to a glucose load. Yet, their proximal insulin signaling in fat depots was impaired, as indicated by reduced tyrosine phosphorylation of insulin receptor (IR), and significantly increased abundance of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). In 3T3-L1 pre-adipocytes E4orf1 expression impaired proximal insulin signaling. Whereas, treatment with rosiglitazone reduced ENPP1 abundance. Unaffected by IR-KD (insulin receptor knockdown) with siRNA, E4orf1 significantly up-regulated distal insulin signaling pathway and enhanced cellular glucose uptake. In vivo, E4orf1 impairs proximal insulin signaling in fat depots yet improves glycemic control. This is probably explained by the ability of E4orf1 to promote cellular glucose uptake independent of proximal insulin signaling. E4orf1 may provide a therapeutic template to enhance glucose disposal in the presence of impaired proximal insulin signaling.

Photocytotoxicity of mTHPC (Temoporfin) Loaded Polymeric Micelles Mediated by Lipase Catalyzed Degradation

PubMed Central

Hofman, Jan-Willem; Carstens, Myrra G.; van Zeeland, Femke; Helwig, Conny; Flesch, Frits M.; Hennink, Wim E.

2008-01-01

Purpose To study the in vitro photocytotoxicity and cellular uptake of biodegradable polymeric micelles loaded with the photosensitizer mTHPC, including the effect of lipase-catalyzed micelle degradation. Methods Micelles of mPEG750-b-oligo(ɛ-caprolactone)5 (mPEG750-b-OCL5) with a hydroxyl (OH), benzoyl (Bz) or naphthoyl (Np) end group were formed and loaded with mTHPC by the film hydration method. The cellular uptake of the loaded micelles, and their photocytotoxicity on human neck squamous carcinoma cells in the absence and presence of lipase were compared with free and liposomal mTHPC (Fospeg®). Results Micelles composed of mPEG750-b-OCL5 with benzoyl and naphtoyl end groups had the highest loading capacity up to 30% (w/w), likely due to π–π interactions between the aromatic end group and the photosensitizer. MTHPC-loaded benzoylated micelles (0.5 mg/mL polymer) did not display photocytotoxicity or any mTHPC-uptake by the cells, in contrast to free and liposomal mTHPC. After dilution of the micelles below the critical aggregation concentration (CAC), or after micelle degradation by lipase, photocytotoxicity and cellular uptake of mTHPC were restored. Conclusion The high loading capacity of the micelles, the high stability of mTHPC-loaded micelles above the CAC, and the lipase-induced release of the photosensitizer makes these micelles very promising carriers for photodynamic therapy in vivo. PMID:18597164

Targeted delivery of polyoxometalate nanocomposites.

PubMed

Geisberger, Georg; Paulus, Susann; Gyenge, Emina Besic; Maake, Caroline; Patzke, Greta R

2011-10-04

Polyoxometalate/carboxymethyl chitosan nanocomposites with an average diameter of 130 nm are synthesized and labeled with fluorescein isothiocyanate (FITC) for a combined drug-carrier and cellular-monitoring approach. [Eu(β(2) -SiW(11) O(39) )(2) ](13-) /CMC nanospheres as a representative example do not display cytotoxicity for POM concentrations up to 2 mg mL(-1) . Cellular uptake of fluoresecently labelled {EuSiW(11) O(39) }/FITC-CMC nanoparticles is monitored with confocal laser scanning microscopy. Nanoparticle uptake occurs after incubation times of around 1 h and no cyctotoxic effects are observed upon prolonged treatment. The preferential location of the POM/CMC nanocomposites in the perinuclear region is furthermore verified with transmission electron microscopy investigations on unlabeled nanoparticles. Therefore, this approach is a promising dual strategy for the safe cellular transfer and monitoring of bioactive POMs. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The effect of tunicamycin on the glucose uptake, growth, and cellular adhesion in the protozoan parasite Crithidia fasciculata.

PubMed

Rojas, Robert; Segovia, Christopher; Trombert, Annette Nicole; Santander, Javier; Manque, Patricio

2014-10-01

Crithidia fasciculata represents a very interesting model organism to study biochemical, cellular, and genetic processes unique to members of the family of the Trypanosomatidae. Thus, C. fasciculata parasitizes several species of insects and has been widely used to test new therapeutic strategies against parasitic infections. By using tunicamycin, a potent inhibitor of glycosylation in asparaginyl residues of glycoproteins (N-glycosylation), we demonstrate that N-glycosylation in C. fasciculata cells is involved in modulating glucose uptake, dramatically impacting growth, and cell adhesion. C. fasciculata treated with tunicamycin was severely affected in their ability to replicate and to adhere to polystyrene substrates and losing their ability to aggregate into small and large groups. Moreover, under tunicamycin treatment, the parasites were considerably shorter and rounder and displayed alterations in cytoplasmic vesicles formation. Furthermore, glucose uptake was significantly impaired in a tunicamycin dose-dependent manner; however, no cytotoxic effect was observed. Interestingly, this effect was reversible. Thus, when tunicamycin was removed from the culture media, the parasites recovered its growth rate, cell adhesion properties, and glucose uptake. Collectively, these results suggest that changes in the tunicamycin-dependent glycosylation levels can influence glucose uptake, cell growth, and adhesion in the protozoan parasite C. fasciculata.

Cellular uptake and intracellular trafficking of PEG-b-PLA polymeric micelles.

PubMed

Zhang, Zhen; Xiong, Xiaoqin; Wan, Jiangling; Xiao, Ling; Gan, Lu; Feng, Youmei; Xu, Huibi; Yang, Xiangliang

2012-10-01

Besides as an inert carrier for hydrophobic anticancer agents, polymeric micelles composed of di-block copolymer poly(ethylene glycol)-poly(lactic acid) (PEG-b-PLA) function as biological response modifiers including reversal of multidrug resistance in cancer. However, the uptake mechanisms and the subsequent intracellular trafficking remain to be elucidated. In this paper, we found that the uptake of PEG-b-PLA polymeric micelles incorporating nile red (M-NR) was significantly inhibited by both dynamin inhibitor dynasore and dynamin-2 dominant negative mutant (dynamin-2 K44A). Exogenously expressed caveolin-1 colocalized with M-NR and upregulated M-NR internalization in HepG2 cells expressing low level of endogenous caveolin-1, while caveolin-1 dominant negative mutant (caveolin-1 Y14F) significantly downregulated M-NR internalization in C6 cells expressing high level of endogenous caveolin-1. Exogenously expressed clathrin light chain A (clathrin LCa) did not mainly colocalize with the internalized M-NR and had no effect on M-NR uptake. These results suggested that dynamin- and caveolin-dependent but clathrin-independent endocytosis was involved in M-NR cellular uptake. We further found that M-NR colocalized with lysosome and microtubulin after internalization. Copyright © 2012 Elsevier Ltd. All rights reserved.

Activity of a sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and mechanism of ascorbate uptake

PubMed Central

Luo, Shuanghui; Wang, Zhiying; Kansara, Viral; Pal, Dhananjay; Mitra, Ashim. K.

2008-01-01

The objective of this research was to functionally characterize sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and to study the effect of substituted benzene derivatives on the intracellular accumulation of ascorbic acid (AA). Mechanism of AA uptake and transport was delineated. Uptake of [14C]ascorbic acid ([14C]AA) was studied in the absence and presence of excess unlabelled AA, anion transporter inhibitors, and a series of mono- and di- substituted benzenes. Transepithelial transport of [14C]AA across polarized cell membrane has been studied for the first time. Role of cellular protein kinase mediated pathways on the regulation of AA uptake has been investigated. The cellular localizations of SVCTs were observed using confocal microscopy. Uptake of AA was found to be saturable with a Km of 83.2 μM and Vmax of 94.2 pmol/min/mg protein for SVCT1. The process was pH, sodium, temperature, and energy dependent. It was under the regulation of cellular protein kinase C (PKC) and Ca2+/CaM mediated pathways. [14C]AA uptake was significantly inhibited in the presence of excess unlabelled AA and a series of electron-withdrawing group i.e. halogen- and nitro- substituted benzene derivatives. AA appears to translocate across polarized cell membrane from apical to basal side (A−B) as well as basal to apical side (B−A) at a similar permeability. It appears that SVCT1 was mainly expressed on the apical side and SVCT2 may be located on both apical and basal sides. In conclusion, SVCT has been functionally characterized in MDCK-MDR1 cells. The interference of a series of electrophile substituted benzenes on the AA uptake process may be explained by their structural similarity. SVCT may be targeted to facilitate the delivery of drugs with low bioavailability by conjugating with AA and its structural analogs. MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of AA conjugated prodrugs. PMID:18417304

Dual-Functional Nanographene Oxide as Cancer-Targeted Drug-Delivery System to Selectively Induce Cancer-Cell Apoptosis.

PubMed

Zhou, Binwei; Huang, Yanyu; Yang, Fang; Zheng, Wenjie; Chen, Tianfeng

2016-04-05

Construction of bioresponsive drug-delivery nanosystems could enhance the anticancer efficacy of anticancer agents and reduce their toxic side effects. Herein, by using transferrin (Tf) as a surface decorator, we constructed a cancer-targeted nanographene oxide (NGO) nanosystem for use in drug delivery. This nanosystem (Tf-NGO@HPIP) drastically enhanced the cellular uptake, retention, and anticancer efficacy of loaded drugs but showed much lower toxicity to normal cells. The nanosystem was internalized through receptor-mediated endocytosis and triggered pH-dependent drug release in acidic environments and in the presence of cellular enzymes. Moreover, Tf-NGO@HPIP effectively induced cancer-cell apoptosis through activation of superoxide-mediated p53 and MAPK pathways along with inactivation of ERK and AKT. Taken together, this study demonstrates a good strategy for the construction of bioresponsive NGO drug-delivery nanosystems and their use as efficient anticancer drug carriers. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular variation of the nonribosomal peptide-polyketide siderophore yersiniabactin through biosynthetic and metabolic engineering.

PubMed

Ahmadi, Mahmoud Kamal; Fawaz, Samar; Fang, Lei; Yu, Zhipeng; Pfeifer, Blaine A

2016-05-01

The production of the mixed nonribosomal peptide-polyketide natural product yersiniabactin (Ybt) has been established using E. coli as a heterologous host. In this study, precursor-directed biosynthesis was used to generate five new analogs of Ybt, demonstrating the flexibility of the heterologous system and the biosynthetic process in allowing compound diversity. A combination of biosynthetic and cellular engineering was then used to influence the production metrics of the resulting analogs. First, the cellular levels and activity of FadL, a hydrocarbon transport protein, were tested for subsequent influence upon exogenous precursor uptake and Ybt analog production with a positive correlation observed between FadL over-production and analog formation. Next, a Ybt biosynthetic editing enzyme was removed from the heterologous system which decreased native compound production but increased analog formation. A final series of experiments enhanced endogenous anthranilate towards complete pathway formation of the associated analog which showed a selective ability to bind gold. © 2015 Wiley Periodicals, Inc.

One-step Melt Synthesis of Water Soluble, Photoluminescent, Surface-Oxidized Silicon Nanoparticles for Cellular Imaging Applications

PubMed Central

Manhat, Beth A.; Brown, Anna L.; Black, Labe A.; Ross, J.B. Alexander; Fichter, Katye; Vu, Tania; Richman, Erik

2012-01-01

We have developed a versatile, one-step melt synthesis of water-soluble, highly emissive silicon nanoparticles using bi-functional, low-melting solids (such as glutaric acid) as reaction media. Characterization through transmission electron microscopy, selected area electron diffraction, X-ray photoelectron spectroscopy, and Raman spectroscopy shows that the one-step melt synthesis produces nanoscale Si cores surrounded by a silicon oxide shell. Analysis of the nanoparticle surface using FT-IR, zeta potential, and gel electrophoresis indicates that the bi-functional ligand used in the one-step synthesis is grafted onto the nanoparticle, which allows for tuning of the particle surface charge, solubility, and functionality. Photoluminescence spectra of the as-prepared glutaric acid-synthesized silicon nanoparticles show an intense blue-green emission with a short (ns) lifetime suitable for biological imaging. These nanoparticles are found to be stable in biological media and have been used to examine cellular uptake and distribution in live N2a cells. PMID:23139440

Transferrin-functionalized nanographene oxide for delivery of platinum complexes to enhance cancer-cell selectivity and apoptosis-inducing efficacy.

PubMed

Zhu, Hai; Zhou, Binwei; Chan, Leung; Du, Yanxin; Chen, Tianfeng

2017-01-01

Rational design and construction of delivery nanosystems for anticancer metal complexes is a crucial strategy to improve solubility under physiological conditions and permeability and retention behavior in tumor cells. Therefore, in this study, we designed and synthesize a transferrin (Tf)-conjugated nanographene oxide (NGO) nanosystem as a cancer-targeted nanocarrier of Pt complexes (Tf-NGO@Pt). This nanodelivery system exhibited good solubility under physiological conditions. Moreover, Tf-NGO@Pt showed higher anticancer efficacy against MCF human breast cancer cells than the free Pt complex, and effectively inhibited cancer-cell migration and invasion, with involvement of reactive oxygen species overproduction. In addition, nanolization also enhanced the penetration ability and inhibitory effect of the Pt complex toward MCF7 breast cancer-cell tumor spheroids. The enhancement of anticancer efficacy was positively correlated with increased cellular uptake and cellular drug retention. This study provides a new strategy to facilitate the future application of metal complexes in cancer therapy.

Size-Dependent Regulation of Intracellular Trafficking of Polystyrene Nanoparticle-Based Drug-Delivery Systems.

PubMed

Wang, Ting; Wang, Lu; Li, Xiaoming; Hu, Xingjie; Han, Yuping; Luo, Yao; Wang, Zejun; Li, Qian; Aldalbahi, Ali; Wang, Lihua; Song, Shiping; Fan, Chunhai; Zhao, Yun; Wang, Maolin; Chen, Nan

2017-06-07

Nanoparticles (NPs) have shown great promise as intracellular imaging probes or nanocarriers and are increasingly being used in biomedical applications. A detailed understanding of how NPs get "in and out" of cells is important for developing new nanomaterials with improved selectivity and less cytotoxicity. Both physical and chemical characteristics have been proven to regulate the cellular uptake of NPs. However, the exocytosis process and its regulation are less explored. Herein, we investigated the size-regulated endocytosis and exocytosis of carboxylated polystyrene (PS) NPs. PS NPs with a smaller size were endocytosed mainly through the clathrin-dependent pathway, whereas PS NPs with a larger size preferred caveolae-mediated endocytosis. Furthermore, our results revealed exocytosis of larger PS NPs and tracked the dynamic process at the single-particle level. These results indicate that particle size is a key factor for the regulation of intracellular trafficking of NPs and provide new insight into the development of more effective cellular nanocarriers.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiba, T.; Fisher, S.K.; Park, J.

The potential role of inositol phospholipid turnover in mediating acid secretion was examined in a preparation enriched for isolated canine gastric parietal cells. The stimulatory effects of carbamoylcholine (carbachol) and gastrin on parietal cell uptake of ({sup 14}C)aminopyrine were linked to dose- and time-dependent selective reduction in cellular phosphatidylinositol content, although the specific fatty acid composition of the phosphoinositides was not altered. Analysis of ({sup 3}H)inositol phosphates accumulated in cells prelabeled with ({sup 3}H)inositol revealed an increase in labeled inositol trisphosphate by 5 min of incubation with either carbachol or gastrin. Furthermore, after preincubation of parietal cells in medium containingmore » ({sup 32}P)orthophosphate, the two secretagogues elicited a time-dependent decrease in {sup 32}P labeling of phosphatidylinositol 4,5-bisphosphate and concomitant increase in labeling of phosphatidic acid. These data demonstrate that the acid secretagogue actions of carbachol and gastrin are correlated with turnover of cellular inositol phospholipids in a preparation consisting predominantly of parietal cells.« less

Mechanistic aspects of fluorescent gold nanocluster internalization by live HeLa cells

NASA Astrophysics Data System (ADS)

Yang, Linxiao; Shang, Li; Nienhaus, G. Ulrich

2013-01-01

We have studied cellular uptake of ultrasmall fluorescent gold nanoclusters (AuNCs) by HeLa cells by confocal fluorescence microscopy in combination with quantitative image analysis. Water solubilized, lipoic acid-protected AuNCs, which had an overall hydrodynamic diameter of 3.3 nm and emitted fluorescence in the near-infrared region at ~700 nm, were observed to accumulate on the cell membrane prior to internalization. The internalization mechanisms were analyzed using inhibitors known to interfere with specific pathways. Cellular uptake of AuNCs is energy-dependent and involves multiple mechanisms: clathrin-mediated endocytosis and macropinocytosis appear to play a significant role, whereas the caveolin-mediated pathway contributes only to a lesser extent. Co-labeling of different cell organelles showed that intracellular trafficking of AuNCs mainly follows through endosomal pathways. The AuNCs were ultimately transferred to lysosomes; they were completely excluded from the nucleus even after 24 h.We have studied cellular uptake of ultrasmall fluorescent gold nanoclusters (AuNCs) by HeLa cells by confocal fluorescence microscopy in combination with quantitative image analysis. Water solubilized, lipoic acid-protected AuNCs, which had an overall hydrodynamic diameter of 3.3 nm and emitted fluorescence in the near-infrared region at ~700 nm, were observed to accumulate on the cell membrane prior to internalization. The internalization mechanisms were analyzed using inhibitors known to interfere with specific pathways. Cellular uptake of AuNCs is energy-dependent and involves multiple mechanisms: clathrin-mediated endocytosis and macropinocytosis appear to play a significant role, whereas the caveolin-mediated pathway contributes only to a lesser extent. Co-labeling of different cell organelles showed that intracellular trafficking of AuNCs mainly follows through endosomal pathways. The AuNCs were ultimately transferred to lysosomes; they were completely excluded from the nucleus even after 24 h. Electronic supplementary information (ESI) available: Effect of serum on the AuNC uptake by HeLa cells and colocalization result of AuNCs with the cell nucleus for 2-24 h. See DOI: 10.1039/c2nr33147k

Characterization of iron uptake from transferrin by murine endothelial cells.

PubMed

Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

2000-01-01

Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB.

Understanding the tissue effects of tribo-corrosion: uptake, distribution, and speciation of cobalt and chromium in human bone cells.

PubMed

Shah, Karan M; Quinn, Paul D; Gartland, Alison; Wilkinson, J Mark

2015-01-01

Cobalt and chromium species are released in the local tissues as a result of tribo-corrosion, and affect bone cell survival and function. However we have little understanding of the mechanisms of cellular entry, intracellular distribution, and speciation of the metals that result in impaired bone health. Here we used synchrotron based X-ray fluorescence (XRF), X-ray absorption spectroscopy (XAS), and fluorescent-probing approaches of candidate receptors P2X7R and divalent metal transporter-1 (DMT-1), to better understand the entry, intra-cellular distribution and speciation of cobalt (Co) and chromium (Cr) in human osteoblasts and primary human osteoclasts. We found that both Co and Cr were most highly localized at nuclear and perinuclear sites in osteoblasts, suggesting uptake through cell membrane transporters, and supported by a finding that P2X7 receptor blockade reduced cellular entry of Co. In contrast, metal species were present at discrete sites corresponding to the basolateral membrane in osteoclasts, suggesting cell entry by endocytosis and trafficking through a functional secretory domain. An intracellular reduction of Cr6+ to Cr3+ was the only redox change observed in cells treated with Co2+, Cr3+, and Cr6+. Our data suggest that the cellular uptake and processing of Co and Cr differs between osteoblasts and osteoclasts. © 2014 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society.

Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake.

PubMed

Halamoda-Kenzaoui, Blanka; Ceridono, Mara; Colpo, Pascal; Valsesia, Andrea; Urbán, Patricia; Ojea-Jiménez, Isaac; Gioria, Sabrina; Gilliland, Douglas; Rossi, François; Kinsner-Ovaskainen, Agnieszka

2015-01-01

Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO2 NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO2 NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays.

The effect of agglomeration state of silver and titanium dioxide nanoparticles on cellular response of HepG2, A549 and THP-1 cells.

PubMed

Lankoff, Anna; Sandberg, Wiggo J; Wegierek-Ciuk, Aneta; Lisowska, Halina; Refsnes, Magne; Sartowska, Bożena; Schwarze, Per E; Meczynska-Wielgosz, Sylwia; Wojewodzka, Maria; Kruszewski, Marcin

2012-02-05

Nanoparticles (NPs) occurring in the environment rapidly agglomerate and form particles of larger diameters. The extent to which this abates the effects of NPs has not been clarified. The motivation of this study was to examine how the agglomeration/aggregation state of silver (20nm and 200nm) and titanium dioxide (21nm) nanoparticles may affect the kinetics of cellular binding/uptake and ability to induce cytotoxic responses in THP1, HepG2 and A549 cells. Cellular binding/uptake, metabolic activation and cell death were assessed by the SSC flow cytometry measurements, the MTT-test and the propidium iodide assay. The three types of particles were efficiently taken up by the cells, decreasing metabolic activation and increasing cell death in all the cell lines. The magnitude of the studied endpoints depended on the agglomeration/aggregation state of particles, their size, time-point and cell type. Among the three cell lines tested, A549 cells were the most sensitive to these particles in relation to cellular binding/uptake. HepG2 cells showed a tendency to be more sensitive in relation to metabolic activation. THP-1 cells were the most resistant to all three types of particles in relation to all endpoints tested. Our findings suggest that particle features such as size and agglomeration status as well as the type of cells may contribute to nanoparticles biological impact. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake

PubMed Central

Halamoda-Kenzaoui, Blanka; Ceridono, Mara; Colpo, Pascal; Valsesia, Andrea; Urbán, Patricia; Ojea-Jiménez, Isaac; Gioria, Sabrina; Gilliland, Douglas; Rossi, François; Kinsner-Ovaskainen, Agnieszka

2015-01-01

Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO2 NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO2 NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays. PMID:26517371

Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethylenimine: preparation, characterization, intracellular trafficking, and cellular distribution

PubMed Central

Fan, Wei; Wu, Xin; Ding, Baoyue; Gao, Jing; Cai, Zhen; Zhang, Wei; Yin, Dongfeng; Wang, Xiang; Zhu, Quangang; Liu, Jiyong; Ding, Xueying; Gao, Shen

2012-01-01

Background Cationic copolymers consisting of polycations linked to nonionic amphiphilic block polymers have been evaluated as nonviral gene delivery systems, and a large number of different polymers and copolymers of linear, branched, and dendrimeric architectures have been tested in terms of their suitability and efficacy for in vitro and in vivo transfection. However, the discovery of new potent materials still largely relies on empiric approaches rather than a rational design. The authors investigated the relationship between the polymers’ structures and their biological performance, including DNA compaction, toxicity, transfection efficiency, and the effect of cellular uptake. Methods This article reports the synthesis and characterization of a series of cationic copolymers obtained by grafting polyethyleneimine with nonionic amphiphilic surfactant polyether-Pluronic® consisting of hydrophilic ethylene oxide and hydrophobic propylene oxide blocks. Transgene expression, cytotoxicity, localization of plasmids, and cellular uptake of these copolymers were evaluated following in vitro transfection of HeLa cell lines with various individual components of the copolymers. Results Pluronics can exhibit biological activity including effects on enhancing DNA cellular uptake, nuclear translocation, and gene expression. The Pluronics with a higher hydrophilic-lipophilic balance value lead to homogeneous distribution in the cytoplasm; those with a lower hydrophilic-lipophilic balance value prefer to localize in the nucleus. Conclusion This Pluronic-polyethyleneimine system may be worth exploring as components in the cationic copolymers as the DNA or small interfering RNA/microRNA delivery system in the near future. PMID:22403492

The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts.

PubMed

Yhee, Ji Young; Yoon, Hong Yeol; Kim, Hyunjoon; Jeon, Sangmin; Hergert, Polla; Im, Jintaek; Panyam, Jayanth; Kim, Kwangmeyung; Nho, Richard Seonghun

2017-01-01

Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6-78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts.

Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization.

PubMed

Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

2015-01-01

Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in the lysosomes with time.

The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts

PubMed Central

Yhee, Ji Young; Yoon, Hong Yeol; Kim, Hyunjoon; Jeon, Sangmin; Hergert, Polla; Im, Jintaek; Panyam, Jayanth; Kim, Kwangmeyung; Nho, Richard Seonghun

2017-01-01

Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6–78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts. PMID:28860768

Physiological and proteomic responses of different willow clones (Salix fragilis x alba) exposed to dredged sediment contaminated by heavy metals.

PubMed

Evlard, Aricia; Sergeant, Kjell; Ferrandis, Salvador; Printz, Bruno; Renaut, Jenny; Guignard, Cedric; Paul, Roger; Hausman, Jean-Francois; Campanella, Bruno

2014-01-01

High biomass producing species are considered as tools for remediation of contaminated soils. Willows (Salix spp.) are prominent study subjects in this regard. In this study, different willow clones (Salix fragilis x alba) were planted on heavy-metal polluted dredging sludge. A first objective was assessment of the biomass production for these clones. Using a Gupta statistic, four clones were identified as high biomass producers (HBP). For comparison, a group of four clones with lowest biomass production were selected (LBP). A second objective was to compare metal uptake as well as the physiological and proteomic responses of these two groups. All these complementary data's allow us to have a better picture of the health of the clones that would be used in phytoremediation programs. Cd, Zn, and Ni total uptake was higher in the HBPs but Pb total uptake was higher in LBPs. Our proteomic and physiological results showed that the LBPs were able to maintain cellular activity as much as the HBPs although the oxidative stress response was more pronounced in the LBPs. This could be due to the high Pb content found in this group although a combined effect of the other metals cannot be excluded.

Multifunctional PLGA Nanobubbles as Theranostic Agents: Combining Doxorubicin and P-gp siRNA Co-Delivery Into Human Breast Cancer Cells and Ultrasound Cellular Imaging.

PubMed

Yang, Hong; Deng, Liwei; Li, Tingting; Shen, Xue; Yan, Jie; Zuo, Liangming; Wu, Chunhui; Liu, Yiyao

2015-12-01

Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. One of the effective approaches to overcome MDR is to use nanoparticle-mediated the gene silence of chemotherapeutic export proteins by RNA interference to increase drug accumulation in drug resistant cancer cells. In this work, a new co-delivery system, DOX-PLGA/PEI/P-gp shRNA nanobubbles (NBs) around 327 nm, to overcome doxorubicin (DOX) resistance in MCF-7 human breast cancer was designed and developed. Positively charged polyethylenimine (PEI) were modified onto the surface of DOX-PLGA NBs through DCC/NHS crosslinking, and could efficiently condense P-gp shRNA into DOX-PLGA/PEI NBs at vector/shRNA weight ratios of 70:1 and above. An in vitro release profile demonstrated an efficient DOX release (more than 80%) from DOX-PLGA/PEI NBs at pH 4.4, suggesting a pH-responsive drug release for the multifunctionalized NBs. Cellular experimental results further showed that DOX-PLGA/PEI/P-gp shRNA NBs could facilitate cellular uptake of DOX into cells and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The IC50 of DOX-PLGA NBs against MCF-7/ADR cells was 2-fold lower than that of free DOX. The increased cellular uptake and nuclear accumulation of DOX delivered by DOX-PLGA/PEI/P-gp shRNA NBs in MCF-7/ADR cells was confirmed by fluorescence microscopy and fluorescence spectrophotometry, and might be owning to the down-regulation of P-gp and reduced the efflux of DOX. The cellular uptake mechanism of DOX-PLGA/PEI/P-gp shRNA NBs indicated that the macropinocytosis was one of the pathways for the uptake of NBs by MCF-7/ADR cells, which was also an energy-dependent process. Furthermore, the in vitro cellular ultrasound imaging suggested that the employment of the DOX-PLGA/PEI/P-gp shRNA NBs could efficiently enhance ultrasound imaging of cancer cells. These results demonstrated that the developed DOX-PLGA/PEI/P-gp shRNA NBs is a potential, safe and efficient theranotic agent for cancer therapy and diagnostics.

Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway.

PubMed

Lakhan, Ram; Said, Hamid M

2017-04-01

Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr 78 Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway.

Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway

PubMed Central

Lakhan, Ram

2017-01-01

Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr78Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway. PMID:28052864

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells.

PubMed

Lee, Hyang Yeon; Lee, Jae Jeong; Park, Jongmin; Park, Seung Bum

2011-01-03

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probing the nanoscale interaction forces and elastic properties of organic and inorganic materials using force-distance (F-D) spectroscopy

NASA Astrophysics Data System (ADS)

Vincent, Abhilash

Due to their therapeutic applications such as radical scavenging, MRI contrast imaging, Photoluminescence imaging, drug delivery, etc., nanoparticles (NPs) have a significant importance in bio-nanotechnology. The reason that prevents the utilizing NPs for drug delivery in medical field is mostly due to their biocompatibility issues (incompatibility can lead to toxicity and cell death). Changes in the surface conditions of NPs often lead to NP cytotoxicity. Investigating the role of NP surface properties (surface charges and surface chemistry) on their interactions with biomolecules (Cells, protein and DNA) could enhance the current understanding of NP cytotoxicity. Hence, it is highly beneficial to the nanotechnology community to bring more attention towards the enhancement of surface properties of NPs to make them more biocompatible and less toxic to biological systems. Surface functionalization of NPs using specific ligand biomolecules have shown to enhance the protein adsorption and cellular uptake through more favorable interaction pathways. Cerium oxide NPs (CNPs also known as nanoceria) are potential antioxidants in cell culture models and understanding the nature of interaction between cerium oxide NPs and biological proteins and cells are important due to their therapeutic application (especially in site specific drug delivery systems). The surface charges and surface chemistry of CNPs play a major role in protein adsorption and cellular uptake. Hence, by tuning the surface charges and by selecting proper functional molecules on the surface, CNPs exhibiting strong adhesion to biological materials can be prepared. By probing the nanoscale interaction forces acting between CNPs and protein molecules using Atomic Force Microscopy (AFM) based force-distance (F-D) spectroscopy, the mechanism of CNP-protein adsorption and CNP cellular uptake can be understood more quantitatively. The work presented in this dissertation is based on the application of AFM in studying the interaction forces as well as the mechanical properties of nanobiomaterials. The research protocol employed in the earlier part of the dissertation is specifically aimed to understand the operation of F-D spectroscopy technique. The elastic properties of thin films of silicon dioxide NPs were investigated using F-D spectroscopy in the high force regime of few 100 nN to 1 microN. Here, sol-gel derived porous nanosilica thin films of varying surface morphology, particle size and porosity were prepared through acid and base catalyzed process. AFM nanoindentation experiments were conducted on these films using the F-D spectroscopy mode and the nanoscale elastic properties of these films were evaluated. The major contribution of this dissertation is a study exploring the interaction forces acting between CNPs and transferrin proteins in picoNewton scale regime using the force-distance spectroscopy technique. This study projects the importance of obtaining appropriate surface charges and surface chemistry so that the NP can exhibit enhanced protein adsorption and NP cellular uptake.

The Role of Extracellular Binding Proteins in the Cellular Uptake of Drugs: Impact on Quantitative In Vitro-to-In Vivo Extrapolations of Toxicity and Efficacy in Physiologically Based Pharmacokinetic-Pharmacodynamic Research.

PubMed

Poulin, Patrick; Burczynski, Frank J; Haddad, Sami

2016-02-01

A critical component in the development of physiologically based pharmacokinetic-pharmacodynamic (PBPK/PD) models for estimating target organ dosimetry in pharmacology and toxicology studies is the understanding of the uptake kinetics and accumulation of drugs and chemicals at the cellular level. Therefore, predicting free drug concentrations in intracellular fluid will contribute to our understanding of concentrations at the site of action in cells in PBPK/PD research. Some investigators believe that uptake of drugs in cells is solely driven by the unbound fraction; conversely, others argue that the protein-bound fraction contributes a significant portion of the total amount delivered to cells. Accordingly, the current literature suggests the existence of a so-called albumin-mediated uptake mechanism(s) for the protein-bound fraction (i.e., extracellular protein-facilitated uptake mechanisms) at least in hepatocytes and cardiac myocytes; however, such mechanism(s) and cells from other organs deserve further exploration. Therefore, the main objective of this present study was to discuss further the implication of potential protein-facilitated uptake mechanism(s) on drug distribution in cells under in vivo conditions. The interplay between the protein-facilitated uptake mechanism(s) and the effects of a pH gradient, metabolism, transport, and permeation limitation potentially occurring in cells was also discussed, as this should violate the basic assumption on similar free drug concentration in cells and plasma. This was made because the published equations used to calculate drug concentrations in cells in a PBPK/PD model did not consider potential protein-facilitated uptake mechanism(s). Consequently, we corrected some published equations for calculating the free drug concentrations in cells compared with plasma in PBPK/PD modeling studies, and we proposed a refined strategy for potentially performing more accurate quantitative in vitro-to-in vivo extrapolations (IVIVEs) of toxicity (efficacy) at the cellular level from data generated in cell assays. Overall, this present study may help to optimize the human dose prediction in preclinical and clinical studies, while prescribing drugs with narrow therapeutic windows that are highly bound to extracellular proteins and/or highly ionized at the physiological pH. This may facilitate building a more accurate safety (efficacy) profile for such drugs. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

PubMed

Ramanathan, S; Qiu, B; Pooyan, S; Zhang, G; Stein, S; Leibowitz, M J; Sinko, P J

2001-12-13

We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential for enhancing the cellular uptake and transport of small peptide therapeutic agents.

The in vivo disposition and in vitro transmembrane transport of two model radiometabolites of DOTA-conjugated receptor-specific peptides labelled with (177) Lu.

PubMed

Volková, Marie; Mandíková, Jana; Bárta, Pavel; Navrátilová, Lucie; Lázníčková, Alice; Trejtnar, František

2015-01-01

In vivo metabolism of the radiolabelled receptor-specific peptides has been described; however, information regarding the pharmacokinetic behaviour of the degradation products within the body is very scarce. The present study was designed to obtain new knowledge on the disposition and elimination of low-molecular radiometabolites of receptor-specific peptides in the organism and to reveal the potential involvement of selected membrane transport mechanisms in the cellular uptake of radiometabolites, especially in the kidney. The study compared pharmacokinetics of two radiometabolites: a final metabolite of somatostatin analogues, (177)Lu-DOTA-DPhe, and a tripeptide metabolite of (177)Lu-DOTA-minigastrin 11, (177)Lu-DOTA-DGlu-Ala-Tyr. Their pharmacokinetics was compared with that of respective parent (177)Lu-radiopeptide. Both radiometabolites exhibited relative rapid clearing from most body tissues in rats in vivo along with predominant renal excretion. The long-term renal retention of the smaller radiometabolite (177)Lu-DOTA-DPhe was lower than that of (177)Lu-DOTA-DGlu-Ala-Tyr. An uptake of (177)Lu-DOTA-DPhe by human renal influx transporter organic cation transporter 2 was found in vitro in a cellular model. The study brings the first experimental data on the in vivo pharmacokinetics of radiometabolites of receptor-specific somatostatin and gastrin analogues. The found results may indicate a negative correlation between the degree of decomposition of the parent peptide chain and the renal retention of the metabolite. Copyright © 2015 John Wiley & Sons, Ltd.

Mannosylated protamine as a novel DNA vaccine carrier for effective induction of anti-tumor immune responses.

PubMed

Zeng, Zhaoyan; Dai, Shuang; Jiao, Yan; Jiang, Lei; Zhao, Yuekui; Wang, Bo; Zong, Li

2016-06-15

Gene immunotherapy has been developed as a promising strategy for inhibition of tumor growth. In the study, mannosylated protamine sulphate (MPS) was used as a novel DNA vaccine carrier to enhance transfection efficiency and anti-tumor immune responses. Anti-GRP DNA vaccine (pGRP) was selected as a model gene and condensed by MPS to form MPS/pGRP nanoparticles. The cellular uptake and transfection efficiency of MPS/pGRP nanoparticles in macrophages were evaluated. The effect of the nanoparticles in enhancing GRP-specific humoral immune response was then evaluated by nasal vaccination of nanoparticles in mice. The results demonstrated that both the cellular uptake and transfection efficiency of MPS nanoparticles in macrophages were higher than those of protamine nanoparticles. MPS/pGRP nanoparticles stimulated the production of higher titers (3.9×10(3)) of specific antibodies against GRP than those of protamine/pGRP nanoparticles (6.4×10(2), p<0.01) and intramuscular injection pGRP solution (2.5×10(3), p<0.05). Furthermore, the inhibitory rate in MPS/pGRP nanoparticles group (65.80%) was significantly higher than that in protamine/pGRP nanoparticles group (35.13%) and pGRP solution group (43.39%). Hence, it is evident that MPS is an efficient targeting gene delivery carrier which could improve in vitro transfection efficiency as well as anti-tumor immunotherapy in mice. Copyright © 2016 Elsevier B.V. All rights reserved.

Biological Evaluation of 99mTc-HYNIC-EDDA/tricine-(Ser)-D4 Peptide for Tumor Targeting.

PubMed

Kazemi, Ziba; Zahmatkesh, Mona Haddad; Abedi, Seyed Mohammad; Hosseinimehr, Seyed Jalal

2017-08-24

D4 small peptide (Leu-Ala-Arg-Leu-Leu-Thr) was selected as an appropriate agent for specific targeting of epidermal growth factor receptor (EGFR). The aim of study was to investigate the 99mTc-labeled D4 peptide for non-small cell lung tumor targeting. HYNIC-(Ser)3-D4 peptide was labeled with 99mTc using mixture of tricine and ethylenediamine diacetic acid (EDDA) as co-ligands. The in vitro cellular uptake of radiolabeled peptide was evaluated by blocking test on human non-small cell lung cancer (A-549) cell line and its biodistribution was evaluated in A-549 xenografted nude mice. This conjugated peptide was labeled with 99mTc in high radiochemical purity and it was highly stable in buffer and serum. The un-blocked to blocked cellular radioactivity ratio was 4- fold that showed a specific binding of this radiolabeled peptide on A-549 cell. Animal biodistribution in A-549 xenografted nude mice showed rapid clearance from blood and other non-target organs. Tumor uptake values as %ID/g (percentage of injection dose per gram of tissue) were 2.47% and 1.30% at 1 and 4 h after injection. This study showed the 99mTc-EDDA/tricine-HYNIC-(Ser)3-D4 peptide had tumor targeting on the non-small cell lung tumor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Comparative evaluation of transmembrane ion transport due to monopolar and bipolar nanosecond, high-intensity electroporation pulses based on full three-dimensional analyses

NASA Astrophysics Data System (ADS)

Hu, Q.; Joshi, R. P.

2017-07-01

Electric pulse driven membrane poration finds applications in the fields of biomedical engineering and drug/gene delivery. Here we focus on nanosecond, high-intensity electroporation and probe the role of pulse shape (e.g., monopolar-vs-bipolar), multiple electrode scenarios, and serial-versus-simultaneous pulsing, based on a three-dimensional time-dependent continuum model in a systematic fashion. Our results indicate that monopolar pulsing always leads to higher and stronger cellular uptake. This prediction is in agreement with experimental reports and observations. It is also demonstrated that multi-pronged electrode configurations influence and increase the degree of cellular uptake.

Interaction of nanoparticles with proteins: relation to bio-reactivity of the nanoparticle.

PubMed

Saptarshi, Shruti R; Duschl, Albert; Lopata, Andreas L

2013-07-19

Interaction of nanoparticles with proteins is the basis of nanoparticle bio-reactivity. This interaction gives rise to the formation of a dynamic nanoparticle-protein corona. The protein corona may influence cellular uptake, inflammation, accumulation, degradation and clearance of the nanoparticles. Furthermore, the nanoparticle surface can induce conformational changes in adsorbed protein molecules which may affect the overall bio-reactivity of the nanoparticle. In depth understanding of such interactions can be directed towards generating bio-compatible nanomaterials with controlled surface characteristics in a biological environment. The main aim of this review is to summarise current knowledge on factors that influence nanoparticle-protein interactions and their implications on cellular uptake.

Mechanisms of pH-Sensitivity and Cellular Internalization of PEOz-b-PLA Micelles with Varied Hydrophilic/Hydrophobic Ratios and Intracellular Trafficking Routes and Fate of the Copolymer.

PubMed

Wang, Dishi; Zhou, Yanxia; Li, Xinru; Qu, Xiaoyou; Deng, Yunqiang; Wang, Ziqi; He, Chuyu; Zou, Yang; Jin, Yiguang; Liu, Yan

2017-03-01

pH-responsive polymeric micelles have shown promise for the targeted and intracellular delivery of antitumor agents. The present study aimed to elucidate the possible mechanisms of pH-sensitivity and cellular internalization of PEOz-b-PLA micelles in detail, further unravel the effect of hydrophilic/hydrophobic ratio of the micelles on their cellular internalization, and examine the intracellular trafficking routes and fate of PEOz-b-PLA after internalization of the micelles. The results of variations in the size and Zeta potential of PEOz-b-PLA micelles and cross-sectional area of PEOz-b-PLA molecules with pH values suggested that electrostatic repulsion between PEOz chains resulting from ionization of the tertiary amide groups along PEOz chain at pH lower than its pK a was responsible for pH-sensitivity of PEOz-b-PLA micelles. Furthermore, the studies on internalization of PEOz-b-PLA micelles by MCF-7 cells revealed that the uptake of PEOz-b-PLA micelles was strongly influenced by their structural features, and showed that PEOz-b-PLA micelles with hydrophilic/hydrophobic ratio of 1.7-2.0 exhibited optimal cellular uptake. No evident alteration in cellular uptake of PEOz-b-PLA micelles was detected by flow cytometry upon the existence of EIPA and chlorpromazine. However, the intracellular uptake of the micelles in the presence of MβCD and genistein was effectively inhibited. Hence, the internalization of such micelles by MCF-7 cells appeared to proceed mainly through caveolae/lipid raft-mediated endocytosis without being influenced by their hydrophilic/hydrophobic ratio. Confocal micrographs revealed that late endosomes, mitochondria and endoplasmic reticulum were all involved in the intracellular trafficking of PEOz-b-PLA copolymers following their internalization via endocytosis, and then part of them was excreted from tumor cells to extracellular medium. These findings provided valuable information for developing desired PEOz-b-PLA micelles to improve their therapeutic efficacy and reducing the potential safety risks associated with their intracellular accumulation.

Development and Evaluation of Amphotericin B Loaded Iron Oxide Nanoparticles for Targeted Drug Delivery to Systemic Fungal Infections

NASA Astrophysics Data System (ADS)

Balabathula, Pavan

A targeted nanotheronostic drug delivery system to diagnose and treat life threatening invasive fungal infections (IFIs) such as cryptococcal meningitis was designed, developed, characterized, and evaluated. To address the development processes, first, iron oxide nanoparticles (IONP) (34-40 nm) coated with bovine serum albumin (BSA), loaded and targeted with amphotericin B (AMB) (AMB-IONP) was formulated by applying a layer by layer approach. Several designs (A, B, C, D, & E) of AMB-IONP were developed and their physicochemical properties such as drug loading with HPLC method, particle size, poly dispersity index (PDI), and zeta-potential using dynamic light scattering (DLS) technique, morphology with transmission electronic microscopy (TEM), and in vitro drug release profile with dialysis method were evaluated. Second, uptake (with fluorescence microscopy and flow cytometry) and killing efficacy (with susceptibility testing) of AMB-IONP in fungal clinical isolates of Candida species were evaluated and compared with standard drug AMB deoxycholate (AMB-D) data. Third, the cellular uptake mechanisms with endocytosis inhibitors and intracellular trafficking using TEM for design D were evaluated in selected isolates. Fourth, a stable lyophilized AMB-IONP formulation was developed and was suitable for clinical trials. A validated isocratic HPLC method was developed and validated for the quantitative determination of AMB. Design D was determined to be the lead formulation with drug loading of 13.6+/-6.9 of AMB/mg of IONP. The size, zeta-potential, and PDI for all formulation designs were found to be in an optimum range for a nanomedicine with ≤36 nm, ˜ -20 mV, and ≤0.2, respectively. The TEM images confirmed that the nanoparticles were monodispersed and spherical in shape. The drug release profile indicated a burst release up to 3 hours for designs A and B, followed by a sustained drug release profile up to 72 hours. Designs C and D (with and without glutaraldehyde) also had a sustained drug release profile up to 72 hours. The major mechanisms of drug release from these formulations were determined to be Fickian and non-Fickian diffusion with first order and Higuchi kinetic models as best fit. The cellular uptake profile for design D exhibited a time dependent uptake with maximum uptake at 0.5 and 4 hours for C. albicans and C. glabrata, respectively. All designs exhibited improved efficacy over AMB-D in the susceptibility testing conducted on clinical isolates of Candida. Design D was found to have an enhanced killing ability and was 16-25 fold more efficacious than AMB-D. An in vitro cellular association study found the uptake mechanism was energy dependent. An endocytosis inhibitor evaluation determined the major particle uptake pathway for C. albicans was lipid-raft mediated endocytosis, whereas for C. glabrata, it was clathrin-, caveolar-, and lipid-raft-mediated endocytosis. TEM and confocal images provided evidence the AMB-IONP were localized at or near the cell wall and membrane wall and inside the cytoplasm, nucleus and endolysosomal vesicles for tested isolates. The lyophilized formulation of AMB-IONP was successfully prepared using an appropriate amount (1:16 to the weight of IONP) of the lyoprotectant, sucrose. A short term stability study of both formulations (lyophilized and aqueous dispersion) at 5°C and 25°C for up to two months showed the lyophilized form was stable. In conclusion, a targeted nanotheronostic drug delivery system (AMB-IONP) was successfully designed, developed, characterized and evaluated as a potential drug product for IFIs treatment.

An updated model for nitrate uptake modelling in plants. I. Functional component: cross-combination of flow–force interpretation of nitrate uptake isotherms, and environmental and in planta regulation of nitrate influx

PubMed Central

Le Deunff, Erwan; Malagoli, Philippe

2014-01-01

Background and Aims In spite of major breakthroughs in the last three decades in the identification of root nitrate uptake transporters in plants and the associated regulation of nitrate transport activities, a simplified and operational modelling approach for nitrate uptake is still lacking. This is due mainly to the difficulty in linking the various regulations of nitrate transport that act at different levels of time and on different spatial scales. Methods A cross-combination of a Flow–Force approach applied to nitrate influx isotherms and experimentally determined environmental and in planta regulation is used to model nitrate in oilseed rape, Brassica napus. In contrast to ‘Enzyme–Substrate’ interpretations, a Flow–Force modelling approach considers the root as a single catalytic structure and does not infer hypothetical cellular processes among nitrate transporter activities across cellular layers in the mature roots. In addition, this approach accounts for the driving force on ion transport based on the gradient of electrochemical potential, which is more appropriate from a thermodynamic viewpoint. Key Results and Conclusions Use of a Flow–Force formalism on nitrate influx isotherms leads to the development of a new conceptual mechanistic basis to model more accurately N uptake by a winter oilseed rape crop under field conditions during the whole growth cycle. This forms the functional component of a proposed new structure–function mechanistic model of N uptake. PMID:24638820

The effect of static magnetic fields and tat peptides on cellular and nuclear uptake of magnetic nanoparticles.

PubMed

Smith, Carol-Anne M; de la Fuente, Jesus; Pelaz, Beatriz; Furlani, Edward P; Mullin, Margaret; Berry, Catherine C

2010-05-01

Magnetic nanoparticles are widely used in bioapplications such as imaging (MRI), targeted delivery (drugs/genes) and cell transfection (magnetofection). Historically, the impermeable nature of both the plasma and nuclear membranes hinder potential. Researchers combat this by developing techniques to enhance cellular and nuclear uptake. Two current popular methods are using external magnetic fields to remotely control particle direction or functionalising the nanoparticles with a cell penetrating peptide (e.g. tat); both of which facilitate cell entry. This paper compares the success of both methods in terms of nanoparticle uptake, analysing the type of magnetic forces the particles experience, and determines gross cell response in terms of morphology and structure and changes at the gene level via microarray analysis. Results indicated that both methods enhanced uptake via a caveolin dependent manner, with tat peptide being the more efficient and achieving nuclear uptake. On comparison to control cells, many groups of gene changes were observed in response to the particles. Importantly, the magnetic field also caused many change in gene expression, regardless of the nanoparticles, and appeared to cause F-actin alignment in the cells. Results suggest that static fields should be modelled and analysed prior to application in culture as cells clearly respond appropriately. Furthermore, the use of cell penetrating peptides may prove more beneficial in terms of enhancing uptake and maintaining cell homeostasis than a magnetic field. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid presenting cell uptake mediated by endocytosis

NASA Astrophysics Data System (ADS)

Feuser, Paulo Emilio; Jacques, Amanda Virtuoso; Arévalo, Juan Marcelo Carpio; Rocha, Maria Eliane Merlin; dos Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H. Hermes

2016-04-01

The encapsulation of superparamagnetic nanoparticles (MNPs) in polymeric nanoparticles (NPs) with modified surfaces can improve targeted delivery and induce cell death by hyperthermia. The goals of this study were to synthesize and characterize surface modified superparamagnetic poly(methyl methacrylate) with folic acid (FA) prepared by miniemulsion polymerization (MNPsPMMA-FA) and to evaluate their in vitro cytotoxicity and cellular uptake in non-tumor cells, murine fibroblast (L929) cells and tumor cells that overexpressed folate receptor (FR) β, and chronic myeloid leukemia cells in blast crisis (K562). Lastly, hemolysis assays were performed on human red blood cells. MNPsPMMA-FA presented an average mean diameter of 135 nm and a saturation magnetization (Ms) value of 37 emu/g of iron oxide, as well as superparamagnetic behavior. The MNPsPMMA-FA did not present cytotoxicity in L929 and K562 cells. Cellular uptake assays showed a higher uptake of MNPsPMMA-FA than MNPsPMMA in K562 cells when incubated at 37 °C. On the other hand, MNPsPMMA-FA showed a low uptake when endocytosis mechanisms were blocked at low temperature (4 °C), suggesting that the MNPsPMMA-FA uptake was mediated by endocytosis. High concentrations of MNPsPMMA-FA showed hemocompatibility when incubated for 24 h in human red blood cells. Therefore, our results suggest that these carrier systems can be an excellent alternative in targeted drug delivery via FR.

Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle-cell interaction

NASA Astrophysics Data System (ADS)

Lu, Yi-Ching; Luo, Pei-Chun; Huang, Chun-Wan; Leu, Yann-Lii; Wang, Tzu-Hao; Wei, Kuo-Chen; Wang, Hsin-Ell; Ma, Yunn-Hwa

2014-08-01

Nanoparticles may serve as carriers in targeted therapeutics; interaction of the nanoparticles with a biological system may determine their targeting effects and therapeutic efficacy. Epigallocatechin-3-gallate (EGCG), a major component of tea catechins, has been conjugated with nanoparticles and tested as an anticancer agent. We investigated whether EGCG may enhance nanoparticle uptake by tumor cells. Cellular uptake of a dextran-coated magnetic nanoparticle (MNP) was determined by confocal microscopy, flow cytometry or a potassium thiocyanate colorimetric method. We demonstrated that EGCG greatly enhanced interaction and/or internalization of MNPs (with or without polyethylene glycol) by glioma cells, but not vascular endothelial cells. The enhancing effects are both time- and concentration-dependent. Such effects may be induced by a simple mix of MNPs with EGCG at a concentration as low as 1-3 μM, which increased MNP uptake 2- to 7-fold. In addition, application of magnetic force further potentiated MNP uptake, suggesting a synergetic effect of EGCG and magnetic force. Because the effects of EGCG were preserved at 4 °C, but not when EGCG was removed from the culture medium prior to addition of MNPs, a direct interaction of EGCG and MNPs was implicated. Use of an MNP-EGCG composite produced by adsorption of EGCG and magnetic separation also led to an enhanced uptake. The results reveal a novel interaction of a food component and nanocarrier system, which may be potentially amenable to magnetofection, cell labeling/tracing, and targeted therapeutics.

Stepwise pH-responsive nanoparticles for enhanced cellular uptake and on-demand intracellular release of doxorubicin.

PubMed

Chen, Wei-Liang; Li, Fang; Tang, Yan; Yang, Shu-di; Li, Ji-Zhao; Yuan, Zhi-Qiang; Liu, Yang; Zhou, Xiao-Feng; Liu, Chun; Zhang, Xue-Nong

2017-01-01

Physicochemical properties, including particle size, zeta potential, and drug release behavior, affect targeting efficiency, cellular uptake, and antitumor effect of nanocarriers in a formulated drug-delivery system. In this study, a novel stepwise pH-responsive nanodrug delivery system was developed to efficiently deliver and significantly promote the therapeutic effect of doxorubicin (DOX). The system comprised dimethylmaleic acid-chitosan-urocanic acid and elicited stepwise responses to extracellular and intracellular pH. The nanoparticles (NPs), which possessed negative surface charge under physiological conditions and an appropriate nanosize, exhibited advantageous stability during blood circulation and enhanced accumulation in tumor sites via enhanced permeability and retention effect. The tumor cellular uptake of DOX-loaded NPs was significantly promoted by the first-step pH response, wherein surface charge reversion of NPs from negative to positive was triggered by the slightly acidic tumor extracellular environment. After internalization into tumor cells, the second-step pH response in endo/lysosome acidic environment elicited the on-demand intracellular release of DOX from NPs, thereby increasing cytotoxicity against tumor cells. Furthermore, stepwise pH-responsive NPs showed enhanced antiproliferation effect and reduced systemic side effect in vivo. Hence, the stepwise pH-responsive NPs provide a promising strategy for efficient delivery of antitumor agents.

Stepwise pH-responsive nanoparticles for enhanced cellular uptake and on-demand intracellular release of doxorubicin

PubMed Central

Chen, Wei-liang; Li, Fang; Tang, Yan; Yang, Shu-di; Li, Ji-zhao; Yuan, Zhi-qiang; Liu, Yang; Zhou, Xiao-feng; Liu, Chun; Zhang, Xue-nong

2017-01-01

Physicochemical properties, including particle size, zeta potential, and drug release behavior, affect targeting efficiency, cellular uptake, and antitumor effect of nanocarriers in a formulated drug-delivery system. In this study, a novel stepwise pH-responsive nanodrug delivery system was developed to efficiently deliver and significantly promote the therapeutic effect of doxorubicin (DOX). The system comprised dimethylmaleic acid-chitosan-urocanic acid and elicited stepwise responses to extracellular and intracellular pH. The nanoparticles (NPs), which possessed negative surface charge under physiological conditions and an appropriate nanosize, exhibited advantageous stability during blood circulation and enhanced accumulation in tumor sites via enhanced permeability and retention effect. The tumor cellular uptake of DOX-loaded NPs was significantly promoted by the first-step pH response, wherein surface charge reversion of NPs from negative to positive was triggered by the slightly acidic tumor extracellular environment. After internalization into tumor cells, the second-step pH response in endo/lysosome acidic environment elicited the on-demand intracellular release of DOX from NPs, thereby increasing cytotoxicity against tumor cells. Furthermore, stepwise pH-responsive NPs showed enhanced antiproliferation effect and reduced systemic side effect in vivo. Hence, the stepwise pH-responsive NPs provide a promising strategy for efficient delivery of antitumor agents. PMID:28652730

Heart cells in culture: a model of myocardial iron overload and chelation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Link, G.; Pinson, A.; Hershko, C.

1985-08-01

The effect of iron loading and chelation was studied in heart cell cultures obtained from newborn rats. Radioactive iron uptake per 2 X 10(6) cells/24 hr was 3.8% for /sup 59/Fe-transferrin, 15.8% for /sup 59/Fe-ferric ammonium citrate (FeAC) at 20 micrograms Fe/ml in 20% serum, and 37.1% for /sup 59/FeAC at 20 micrograms Fe/ml in serum-free medium. About one third of the cellular radioactive iron was in ferritin and the rest in an insoluble lysosomal fraction. Iron uptake was almost completely inhibited by reducing the incubation temperature from 37 degrees C to 10 degrees C. Intracellular concentrations of malonyldialdehyde (MDA)more » were doubled after 15 minutes of iron loading and reached maximal concentrations at 3 hours. Conversely, iron mobilization by deferoxamine at concentrations ranging from 0.025 mmol/L to 0.3 mmol/L resulted in normalization of cellular MDA concentrations, in direct proportion to the amounts of iron removed. These findings indicate that cultured myocardial cells are able to assimilate large amounts of nontransferrin iron and that iron uptake and mobilization are associated with striking changes in lipid peroxidation as manifested by the respective increase and decrease in cellular MDA concentrations.« less

Uptake and cellular distribution, in four plant species, of fluorescently labeled mesoporous silica nanoparticles.

PubMed

Sun, Dequan; Hussain, Hashmath I; Yi, Zhifeng; Siegele, Rainer; Cresswell, Tom; Kong, Lingxue; Cahill, David M

2014-08-01

We report the uptake of MSNs into the roots and their movement to the aerial parts of four plant species and their quantification using fluorescence, TEM and proton-induced x - ray emission (micro - PIXE) elemental analysis. Monodispersed mesoporous silica nanoparticles (MSNs) of optimal size and configuration were synthesized for uptake by plant organs, tissues and cells. These monodispersed nanoparticles have a size of 20 nm with interconnected pores with an approximate diameter of 2.58 nm. There were no negative effects of MSNs on seed germination or when transported to different organs of the four plant species tested in this study. Most importantly, for the first time, a combination of confocal laser scanning microscopy, transmission electron microscopy and proton-induced X-ray emission (micro-PIXE) elemental analysis allowed the location and quantification MSNs in tissues and in cellular and sub-cellular locations. Our results show that MSNs penetrated into the roots via symplastic and apoplastic pathways and then via the conducting tissues of the xylem to the aerial parts of the plants including the stems and leaves. The translocation and widescale distribution of MSNs in plants will enable them to be used as a new delivery means for the transport of different sized biomolecules into plants.

Effects of Nanoparticle Size on Cellular Uptake and Liver MRI with PVP-Coated Iron Oxide Nanoparticles

PubMed Central

Huang, Jing; Bu, Lihong; Xie, Jin; Chen, Kai; Cheng, Zhen; Li, Xingguo; Chen, Xiaoyuan

2010-01-01

The effect of nanoparticle size (30–120 nm) on magnetic resonance imaging (MRI) of hepatic lesions in vivo has been systematically examined using polyvinylpyrrolidone (PVP)-coated iron oxide nanoparticles (PVP-IOs). Such biocompatible PVP-IOs with different sizes were synthesized by a simple one-pot pyrolysis method. These PVP-IOs exhibited good crystallinity and high T2 relaxivities, and the relaxivity increased with the size of the magnetic nanoparticles. It was found that cellular uptake changed with both size and surface physiochemical properties, and that PVP-IO-37 with a core size of 37 nm and hydrodynamic particle size of 100 nm exhibited higher cellular uptake rate and greater distribution than other PVP-IOs and Feridex. We systematically investigated the effect of nanoparticle size on MRI of normal liver and hepatic lesions in vivo. The physical and chemical properties of the nanoparticles influenced their pharmacokinetic behavior, which ultimately determined their ability to accumulate in the liver. The contrast enhancement of PVP-IOs within the liver was highly dependent on the overall size of the nanoparticles, and the 100 nm PVP-IO-37 nanoparticles exhibited the greatest enhancement. These results will have implications in designing engineered nanoparticles that are optimized as MR contrast agents or for use in therapeutics. PMID:21043459

Scavenger receptor B1 (SR-B1) profoundly excludes high density lipoprotein (HDL) apolipoprotein AII as it nibbles HDL-cholesteryl ester.

PubMed

Gillard, Baiba K; Bassett, G Randall; Gotto, Antonio M; Rosales, Corina; Pownall, Henry J

2017-05-26

Reverse cholesterol transport (transfer of macrophage-cholesterol in the subendothelial space of the arterial wall to the liver) is terminated by selective high density lipoprotein (HDL)-cholesteryl ester (CE) uptake, mediated by scavenger receptor class B, type 1 (SR-B1). We tested the validity of two models for this process: "gobbling," i.e. one-step transfer of all HDL-CE to the cell and "nibbling," multiple successive cycles of SR-B1-HDL association during which a few CEs transfer to the cell. Concurrently, we compared cellular uptake of apoAI with that of apoAII, which is more lipophilic than apoAI, using HDL-[ 3 H]CE labeled with [ 125 I]apoAI or [ 125 I]apoAII. The studies were conducted in CHO-K1 and CHO-ldlA7 cells (LDLR -/- ) with (CHO-SR-B1) and without SR-B1 overexpression and in human Huh7 hepatocytes. Relative to CE, both apoAI and apoAII were excluded from uptake by all cells. However, apoAII was more highly excluded from uptake (2-4×) than apoAI. To distinguish gobbling versus nibbling mechanisms, media from incubations of HDL with CHO-SR-B1 cells were analyzed by non-denaturing PAGE, size-exclusion chromatography, and the distribution of apoAI, apoAII, cholesterol, and phospholipid among HDL species as a function of incubation time. HDL size gradually decreased, i.e. nibbling, with the concurrent release of lipid-free apoAI; apoAII was retained in an HDL remnant. Our data support an SR-B1 nibbling mechanism that is similar to that of streptococcal serum opacity factor, which also selectively removes CE and releases apoAI, leaving an apoAII-rich remnant. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Preparation strategy and illumination of three-dimensional cell cultures in light sheet-based fluorescence microscopy

NASA Astrophysics Data System (ADS)

Bruns, Thomas; Schickinger, Sarah; Wittig, Rainer; Schneckenburger, Herbert

2012-10-01

A device for selective plane illumination microscopy (SPIM) of three-dimensional multicellular spheroids, in culture medium under stationary or microfluidic conditions, is described. Cell spheroids are located in a micro-capillary and a light sheet, for illumination, is generated in an optical setup adapted to a conventional inverse microscope. Layers of the sample, of about 10 μm or less in diameter, are, thus, illuminated selectively and imaged by high resolution fluorescence microscopy. SPIM is operated at low light exposure even if a larger number of layers is imaged and is easily combined with laser scanning microscopy. Chinese hamster ovary cells expressing a membrane-associated green fluorescent protein are used for preliminary tests, and the uptake of the fluorescent marker, acridine orange via a microfluidic system, is visualized to demonstrate its potential in cancer research such as for the detection of cellular responses to anticancer drugs.

Semen amyloids participate in spermatozoa selection and clearance.

PubMed

Roan, Nadia R; Sandi-Monroy, Nathallie; Kohgadai, Nargis; Usmani, Shariq M; Hamil, Katherine G; Neidleman, Jason; Montano, Mauricio; Ständker, Ludger; Röcker, Annika; Cavrois, Marielle; Rosen, Jared; Marson, Kara; Smith, James F; Pilcher, Christopher D; Gagsteiger, Friedrich; Sakk, Olena; O'Rand, Michael; Lishko, Polina V; Kirchhoff, Frank; Münch, Jan; Greene, Warner C

2017-06-27

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens.

Effect of Thiols, Zinc, and Redox Conditions on Hg Uptake in Shewanella oneidensis

DOE PAGES

Szczuka, Aleksandra; Morel, Francois M. M.; Schaefer, Jeffra K.

2015-05-18

Mercury uptake in bacteria represents a key first step in the production and accumulation Of methylmercury in biota. Previous experiments with mercury methylating bacteria have shown that Hg uptake is enhanced by some thiols, in particular cysteine, and that it is an energy-dependent process through heavy Metal TA transporters. In this study, we examine Hg uptake in the nonmethylating facultative aerobe, Shewanella oneidensis, under both anaerobic and aerobic conditions. Our results demonstrate similar characteristics of the Hg uptake system to those of the Hg methylating strains: uptake is enhanced in the presence of some thiols but not others; uptake ismore » energy dependent as evidenced by inhibition by a protonophore; and uptake is inhibited by high Zn(II) concentrations. Initial cellular uptake rates in S. oneidensis were remarkably similar under aerobic and fumarate-reducing conditions. In conclusion, these data support a similar Hg(II) uptake mechanism within the proteobacteria of accidental Hg(II) transport through heavy metal transporters with similar rates of uptake but differences in the ability to take up Hg bound to different thiols.« less

Antimetabolic Effects of Polyphenols in Breast Cancer Cells: Focus on Glucose Uptake and Metabolism.

PubMed

Keating, Elisa; Martel, Fátima

2018-01-01

In the last years, metabolic reprogramming became a new key hallmark of tumor cells. One of its components is a deviant energetic metabolism, known as Warburg effect-an aerobic lactatogenesis- characterized by elevated rates of glucose uptake and consumption with high-lactate production even in the presence of oxygen. Because many cancer cells display a greater sensitivity to glucose deprivation-induced cytotoxicity than normal cells, inhibitors of glucose cellular uptake (facilitative glucose transporter 1 inhibitors) and oxidative metabolism (glycolysis inhibitors) are potential therapeutic targets in cancer treatment. Polyphenols, abundantly contained in fruits and vegetables, are dietary components with an established protective role against cancer. Several molecular mechanisms are involved in the anticancer effect of polyphenols, including effects on apoptosis, cell cycle regulation, plasma membrane receptors, signaling pathways, and epigenetic mechanisms. Additionally, inhibition of glucose cellular uptake and metabolism in cancer cell lines has been described for several polyphenols, and this effect was shown to be associated with their anticarcinogenic effect. This work will review data showing an antimetabolic effect of polyphenols and its involvement in the chemopreventive/chemotherapeutic potential of these dietary compounds, in relation to breast cancer.

Lentiviral Nef suppresses iron uptake in a strain specific manner through inhibition of Transferrin endocytosis

PubMed Central

2014-01-01

Background Increased cellular iron levels are associated with high mortality in HIV-1 infection. Moreover iron is an important cofactor for viral replication, raising the question whether highly divergent lentiviruses actively modulate iron homeostasis. Here, we evaluated the effect on cellular iron uptake upon expression of the accessory protein Nef from different lentiviral strains. Results Surface Transferrin receptor (TfR) levels are unaffected by Nef proteins of HIV-1 and its simian precursors but elevated in cells expressing Nefs from most other primate lentiviruses due to reduced TfR internalization. The SIV Nef-mediated reduction of TfR endocytosis is dependent on an N-terminal AP2 binding motif that is not required for downmodulation of CD4, CD28, CD3 or MHCI. Importantly, SIV Nef-induced inhibition of TfR endocytosis leads to the reduction of Transferrin uptake and intracellular iron concentration and is accompanied by attenuated lentiviral replication in macrophages. Conclusion Inhibition of Transferrin and thereby iron uptake by SIV Nef might limit viral replication in myeloid cells. Furthermore, this new SIV Nef function could represent a virus-host adaptation that evolved in natural SIV-infected monkeys. PMID:24383984

Synthesis and in vitro biochemical evaluation of oxime bond-linked daunorubicin–GnRH-III conjugates developed for targeted drug delivery

PubMed Central

Schuster, Sabine; Biri-Kovács, Beáta; Szeder, Bálint; Farkas, Viktor; Buday, László; Szabó, Zsuzsanna; Halmos, Gábor

2018-01-01

Gonadotropin releasing hormone-III (GnRH-III), a native isoform of the human GnRH isolated from sea lamprey, specifically binds to GnRH receptors on cancer cells enabling its application as targeting moieties for anticancer drugs. Recently, we reported on the identification of a novel daunorubicin–GnRH-III conjugate (GnRH-III–[4Lys(Bu), 8Lys(Dau=Aoa)] with efficient in vitro and in vivo antitumor activity. To get a deeper insight into the mechanism of action of our lead compound, the cellular uptake was followed by confocal laser scanning microscopy. Hereby, the drug daunorubicin could be visualized in different subcellular compartments by following the localization of the drug in a time-dependent manner. Colocalization studies were carried out to prove the presence of the drug in lysosomes (early stage) and on its site of action (nuclei after 10 min). Additional flow cytometry studies demonstrated that the cellular uptake of the bioconjugate was inhibited in the presence of the competitive ligand triptorelin indicating a receptor-mediated pathway. For comparative purpose, six novel daunorubicin–GnRH-III bioconjugates have been synthesized and biochemically characterized in which 6Asp was replaced by D-Asp, D-Glu and D-Trp. In addition to the analysis of the in vitro cytostatic effect and cellular uptake, receptor binding studies with 125I-triptorelin as radiotracer and degradation of the GnRH-III conjugates in the presence of rat liver lysosomal homogenate have been performed. All derivatives showed high binding affinities to GnRH receptors and displayed in vitro cytostatic effects on HT-29 and MCF-7 cancer cells with IC50 values in a low micromolar range. Moreover, we found that the release of the active drug metabolite and the cellular uptake of the bioconjugates were strongly affected by the amino acid exchange which in turn had an impact on the antitumor activity of the bioconjugates. PMID:29719573

Novel theranostic zinc phthalocyanine-phospholipid complex self-assembled nanoparticles for imaging-guided targeted photodynamic treatment with controllable ROS production and shape-assisted enhanced cellular uptake.

PubMed

Ma, Jinyuan; Li, Yang; Liu, Guihua; Li, Ai; Chen, Yilin; Zhou, Xinyi; Chen, Dengyue; Hou, Zhenqing; Zhu, Xuan

2018-02-01

The novel drug delivery system based on self-assembly of zinc phthalocyanine-soybean phosphatidylcholine (ZnPc-SPC) complex was developed by a co-solvent method followed by a nanoprecipitaion technique. DSPE-PEG-methotrexate (DSPE-PEG-MTX) was introduced on the surface of ZnPc-SPC self-assembled nanoparticles (ZS) to endow them with folate receptor-targeting property. NMR, XRD, FTIR, and UV-vis-NIR analysis demonstrated the weak molecular interaction between ZnPc and SPC. The ZS functionalized with DSPE-PEG-MTX (ZSPM) was successfully constructed with an average particle size of ∼170nm, a narrow size distribution, and could remain physiologically stable for at least 7days. In vitro cellular uptake and cytotoxicity studies demonstrated that ZSPM exhibited stronger cellular uptake efficacy and photodynamic cytotoxicity against HeLa and MCF-7 cells than ZS functionalized with DSPE-mPEG (ZSP) and free ZnPc. More importantly, ZSPM showed the enhanced accumulation effect at the tumor region compared with ZSP by the active-plus-passive targeting via enhanced permeability and retention (EPR) effect and folate receptor-mediated endocytosis. Furthermore, in vivo antitumor effect and histological analysis demonstrated the superior tumor growth inhibition effect of ZSPM. In addition, the needle-shape ZSP (ZSPN) exhibited better in vitro cellular uptake and in vivo tumor accumulation compared with ZSP due to the shape-assisted effect. Moreover, the interesting off-on switch effect of reactive oxygen species (ROS) production of ZnPc-SPC complex-based nanoparticles was discovered to achieve photodynamic treatment in a controllable way. These findings suggested that the ZnPc-SPC complex-based self-assembled nanoparticles could serve as a promising and effective formulation to achieve tumor-targeting fluorescence imaging and enhanced photodynamic treatment. Copyright © 2017. Published by Elsevier B.V.

Zinc starvation induces autophagy in yeast.

PubMed

Kawamata, Tomoko; Horie, Tetsuro; Matsunami, Miou; Sasaki, Michiko; Ohsumi, Yoshinori

2017-05-19

Zinc is an essential nutrient for all forms of life. Within cells, most zinc is bound to protein. Because zinc serves as a catalytic or structural cofactor for many proteins, cells must maintain zinc homeostasis under severely zinc-deficient conditions. In yeast, the transcription factor Zap1 controls the expression of genes required for uptake and mobilization of zinc, but to date the fate of existing zinc-binding proteins under zinc starvation remains poorly understood. Autophagy is an evolutionarily conserved cellular degradation/recycling process in which cytoplasmic proteins and organelles are sequestered for degradation in the vacuole/lysosome. In this study, we investigated how autophagy functions under zinc starvation. Zinc depletion induced non-selective autophagy, which is important for zinc-limited growth. Induction of autophagy by zinc starvation was not directly related to transcriptional activation of Zap1. Instead, TORC1 inactivation directed zinc starvation-induced autophagy. Abundant zinc proteins, such as Adh1, Fba1, and ribosomal protein Rpl37, were degraded in an autophagy-dependent manner. But the targets of autophagy were not restricted to zinc-binding proteins. When cellular zinc is severely depleted, this non-selective autophagy plays a role in releasing zinc from the degraded proteins and recycling zinc for other essential purposes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Biological Functionalization of Drug Delivery Carriers to Bypass Size Restrictions of Receptor-Mediated Endocytosis Independently from Receptor Targeting

PubMed Central

Ansar, Maria; Serrano, Daniel; Papademetriou, Iason; Bhowmick, Tridib Kumar; Muro, Silvia

2014-01-01

Targeting of drug carriers to cell-surface receptors involved in endocytosis is commonly used for intracellular drug delivery. However, most endocytic receptors mediate uptake via clathrin or caveolar pathways associated with ≤200-nm vesicles, restricting carrier design. We recently showed that endocytosis mediated by intercellular adhesion molecule 1 (ICAM-1), which differs from clathrin- and caveolar-mediated pathways, allows uptake of nano- and micro-carriers in cell culture and in vivo due to recruitment of cellular sphingomyelinases to the plasmalemma. This leads to ceramide generation at carrier binding sites and formation of actin stress-fibers, enabling engulfment and uptake of a wide size-range of carriers. Here we adapted this paradigm to enhance uptake of drug carriers targeted to receptors associated with size-restricted pathways. We coated sphingomyelinase onto model (polystyrene) submicro- and micro-carriers targeted to clathrin-associated mannose-6-phosphate receptor. In endothelial cells, this provided ceramide enrichment at the cell surface and actin stress-fiber formation, modifying the uptake pathway and enhancing carrier endocytosis without affecting targeting, endosomal transport, cell-associated degradation, or cell viability. This improvement depended on the carrier size and enzyme dose, and similar results were observed for other receptors (transferrin receptor) and cell types (epithelial cells). This phenomenon also enhanced tissue accumulation of carriers after intravenous injection in mice. Hence, it is possible to maintain targeting toward a selected receptor while bypassing natural size-restrictions of its associated endocytic route by functionalization of drug carriers with biological elements mimicking the ICAM-1 pathway. This strategy holds considerable promise to enhance flexibility of design of targeted drug delivery systems. PMID:24237309

Biological functionalization of drug delivery carriers to bypass size restrictions of receptor-mediated endocytosis independently from receptor targeting.

PubMed

Ansar, Maria; Serrano, Daniel; Papademetriou, Iason; Bhowmick, Tridib Kumar; Muro, Silvia

2013-12-23

Targeting of drug carriers to cell-surface receptors involved in endocytosis is commonly used for intracellular drug delivery. However, most endocytic receptors mediate uptake via clathrin or caveolar pathways associated with ≤200-nm vesicles, restricting carrier design. We recently showed that endocytosis mediated by intercellular adhesion molecule 1 (ICAM-1), which differs from clathrin- and caveolae-mediated pathways, allows uptake of nano- and microcarriers in cell culture and in vivo due to recruitment of cellular sphingomyelinases to the plasmalemma. This leads to ceramide generation at carrier binding sites and formation of actin stress-fibers, enabling engulfment and uptake of a wide size-range of carriers. Here we adapted this paradigm to enhance uptake of drug carriers targeted to receptors associated with size-restricted pathways. We coated sphingomyelinase onto model (polystyrene) submicro- and microcarriers targeted to clathrin-associated mannose-6-phosphate receptor. In endothelial cells, this provided ceramide enrichment at the cell surface and actin stress-fiber formation, modifying the uptake pathway and enhancing carrier endocytosis without affecting targeting, endosomal transport, cell-associated degradation, or cell viability. This improvement depended on the carrier size and enzyme dose, and similar results were observed for other receptors (transferrin receptor) and cell types (epithelial cells). This phenomenon also enhanced tissue accumulation of carriers after intravenous injection in mice. Hence, it is possible to maintain targeting toward a selected receptor while bypassing natural size restrictions of its associated endocytic route by functionalization of drug carriers with biological elements mimicking the ICAM-1 pathway. This strategy holds considerable promise to enhance flexibility of design of targeted drug delivery systems.

Gastrin stimulates renal dopamine production by increasing the renal tubular uptake of l-DOPA.

PubMed

Jiang, Xiaoliang; Zhang, Yanrong; Yang, Yu; Yang, Jian; Asico, Laureano D; Chen, Wei; Felder, Robin A; Armando, Ines; Jose, Pedro A; Yang, Zhiwei

2017-01-01

Gastrin is a peptide hormone that is involved in the regulation of sodium balance and blood pressure. Dopamine, which is also involved in the regulation of sodium balance and blood pressure, directly or indirectly interacts with other blood pressure-regulating hormones, including gastrin. This study aimed to determine the mechanisms of the interaction between gastrin and dopamine and tested the hypothesis that gastrin produced in the kidney increases renal dopamine production to keep blood pressure within the normal range. We show that in human and mouse renal proximal tubule cells (hRPTCs and mRPTCs, respectively), gastrin stimulates renal dopamine production by increasing the cellular uptake of l-DOPA via the l-type amino acid transporter (LAT) at the plasma membrane. The uptake of l-DOPA in RPTCs from C57Bl/6J mice is lower than in RPTCs from normotensive humans. l-DOPA uptake in renal cortical slices is also lower in salt-sensitive C57Bl/6J than in salt-resistant BALB/c mice. The deficient renal cortical uptake of l-DOPA in C57Bl/6J mice may be due to decreased LAT-1 activity that is related to its decreased expression at the plasma membrane, relative to BALB/c mice. We also show that renal-selective silencing of Gast by the renal subcapsular injection of Gast siRNA in BALB/c mice decreases renal dopamine production and increases blood pressure. These results highlight the importance of renal gastrin in stimulating renal dopamine production, which may give a new perspective in the prevention and treatment of hypertension. Copyright © 2017 the American Physiological Society.

56Co-labelled radioactive Fe3O4 nanoparticles for in vitro uptake studies on Balb/3T3 and Caco-2 cell lines

NASA Astrophysics Data System (ADS)

Marmorato, P.; Simonelli, F.; Abbas, K.; Kozempel, J.; Holzwarth, U.; Franchini, F.; Ponti, J.; Gibson, N.; Rossi, F.

2011-12-01

Magnetite nanoparticles (Fe3O4 NPs) are manufactured nanomaterials increasingly used in healthcare for different medical applications ranging from diagnosis to therapy. This study deals with the irradiation of Fe3O4 NPs with a proton beam in order to produce 56Co as radiolabel and also with the possible use of nuclear techniques for the quantification of Fe3O4 NPs in biological systems. Particular attention has been focused on the size distribution (in the range of 100 nm) and the surface charge of the NPs characterizing them before and after the irradiation process in order to verify if these essential properties would be preserved during irradiation. Moreover, X-ray diffraction studies have been performed on radioactive and non-radioactive NPs, to assess if major changes in NPs structure might occur due to thermal and/or radiation effects. The radiation emitted from the radiolabels has been used to quantify the cellular uptake of the NPs in in vitro studies. As for the biological applications two cell lines have been selected: immortalized mouse fibroblast cell line (Balb/3T3) and human epithelial colorectal adenocarcinoma cell line (Caco-2). The cell uptake has been quantified by radioactivity measurements of the 56Co radioisotope performed with high resolution γ-ray spectrometry equipment. This study has showed that, under well-established irradiation conditions, Fe3O4 NPs do not undergo significant structural modifications and thus the obtained results are in line with the uptake studies carried out with the same non-radioactive nanomaterials (NMs). Therefore, the radiolabelling method can be fruitfully applied to uptake studies because of the low-level exposure where higher sensitivity is required.

Hybrid ZnPc@TiO2 nanostructures for targeted photodynamic therapy, bioimaging and doxorubicin delivery.

PubMed

Flak, Dorota; Yate, Luis; Nowaczyk, Grzegorz; Jurga, Stefan

2017-09-01

In this study ZnPc@TiO 2 hybrid nanostructures, both nanoparticles and nanotubes, as potential photosensitizers for the photodynamic therapy, fluorescent bioimaging agents, as well as anti-cancer drug nanocarriers, were prepared via zinc phthalocyanine (ZnPc) deposition on TiO 2 . In order to provide the selectivity of prepared hybrid nanostructures towards cancer cells they were modified with folic acid molecules (FA). The efficient attachment of both ZnPc and FA molecules was confirmed with dynamic light scattering (DLS), zeta potential measurements and X-ray photoelectron spectroscopy (XPS). It was presented that ZnPc and FA attachment has a strong effect on fluorescence emission properties of TiO 2 nanostructures, which can be further used for their simultaneous visualization upon cellular uptake. ZnPc@TiO 2 and FA/ZnPc@TiO 2 hybrid nanotubes were then employed as doxorubicin nanocarriers. It was demonstrated that doxorubicin can be easily loaded on these hybrid nanostructures via an electrostatic interaction and then released. In vitro cytotoxicity and photo-cytotoxic activity studies showed that prepared hybrid nanostructures were selectively targeting to cancer cells. Doxorubicin loaded hybrid nanostructures were significantly more cytotoxic than un-loaded ones and their cytotoxic effect was even more severe upon irradiation. The cellular uptake of prepared hybrid nanostructures and their localization in cells was monitored in vitro in 2D cell culture and tumor-like 3D multicellular culture environment with fluorescent confocal microscopy. These hybrid nanostructures preferentially penetrated into human cervical cancer cells (HeLa) than into normal fibroblasts (MSU-1.1) and were mainly localized within the cell cytoplasm. HeLa cells spheroids were also efficiently labelled by prepared hybrid nanostructures. Fluorescent imaging of Hela cells treated with doxorubicin loaded hybrid nanostructures showed that doxorubicin was effectively delivered into cells, released and evenly distributed in the cytoplasm. In conclusion, prepared hybrid nanostructures exhibit high potential as selective bioimaging agents next to their photodynamic activity and drug delivery ability. Copyright © 2017 Elsevier B.V. All rights reserved.

Improving cell penetration of helical peptides stabilized by N-terminal crosslinked aspartic acids.

PubMed

Zhao, Hui; Jiang, Yanhong; Tian, Yuan; Yang, Dan; Qin, Xuan; Li, Zigang

2017-01-04

Cell penetration and nucleus translocation efficiency are important for the cellular activities of peptide therapeutics. For helical peptides stabilized by N-terminal crosslinked aspartic acid, correlations between their penetration efficiency/nucleus translocation and physicochemical properties were studied. An increase in hydrophobicity and isoelectric point will promote cellular uptake and nucleus translocation of stabilized helices.

Effect of binary organic solvents together with emulsifier on particle size and in vitro behavior of paclitaxel-encapsulated polymeric lipid nanoparticles.

PubMed

Qin, Shuzhi; Sun, Xiangshi; Li, Feng; Yu, Kongtong; Zhou, Yulin; Liu, Na; Zhao, Chengguo; Teng, Lesheng; Li, Youxin

2017-12-21

Biodegradable nanoparticles with diameters between 100 nm and 500 nm are of great interest in the contexts of targeted delivery. The present work provides a review concerning the effect of binary organic solvents together with emulsifier on particle size as well as the influence of particle size on the in vitro drug release and uptake behavior. The polymeric lipid nanoparticles (PLNs) with different particle sizes were prepared by using binary solvent dispersion method. Various formulation parameters such as binary organic solvent composition and emulsifier types were evaluated on the basis of their effects on particle size and size distribution. PLNs had a strong dependency on the surface tension, intrinsic viscosity and volatilization rate of binary organic solvents and the hydrophilicity/hydrophobicity of emulsifiers. Acetone-methanol system together with pluronic F68 as emulsifier was proved to obtain the smallest particle size. Then the PLNs with different particle sizes were used to investigate how particle size at nanoscale affects interacted with tumor cells. As particle size got smaller, cellular uptake increased in tumor cells and PLNs with particle size of ~120 nm had the highest cellular uptake and fastest release rate. The paclitaxel (PTX)-loaded PLNs showed a size-dependent inhibition of tumor cell growth, which was commonly influenced by cellular uptake and PTX release. The PLNs would provide a useful means to further elucidate roles of particle size on delivery system of hydrophobic drugs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Mesoporous silica nanoparticles loading doxorubicin reverse multidrug resistance: performance and mechanism

NASA Astrophysics Data System (ADS)

Shen, Jianan; He, Qianjun; Gao, Yu; Shi, Jianlin; Li, Yaping

2011-10-01

Multidrug resistance (MDR) is one of the major obstacles for successful chemotherapy in cancer. One of the effective approaches to overcome MDR is to use nanoparticle-mediated drug delivery to increase drug accumulation in drug resistant cancer cells. In this work, we first report that the performance and mechanism of an inorganic engineered delivery system based on mesoporous silica nanoparticles (MSNs) loading doxorubicin (DMNs) to overcome the MDR of MCF-7/ADR (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The experimental results showed that DMNs could enhance the cellular uptake of doxorubicin (DOX) and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells. The IC50 of DMNs against MCF-7/ADR cells was 8-fold lower than that of free DOX. However, an improved effect of DOX in DMNs against MCF-7 cells (a DOX-sensitive cancer cell line) was not found. The increased cellular uptake and nuclear accumulation of DOX delivered by DMNs in MCF-7/ADR cells was confirmed by confocal laser scanning microscopy, and could result from the down-regulation of P-gp and bypassing the efflux action by MSNs themselves. The cellular uptake mechanism of DMNs indicated that the macropinocytosis was one of the pathways for the uptake of DMNs by MCF-7/ADR cells. The in vivo biodistribution showed that DMNs induced a higher accumulation of DOX in drug resistant tumors than free DOX. These results suggested that MSNs could be an effective delivery system to overcome multidrug resistance.

The Receptor-Binding Domain in the VP1u Region of Parvovirus B19.

PubMed

Leisi, Remo; Di Tommaso, Chiarina; Kempf, Christoph; Ros, Carlos

2016-02-24

Parvovirus B19 (B19V) is known as the human pathogen causing the mild childhood disease erythema infectiosum. B19V shows an extraordinary narrow tissue tropism for erythroid progenitor cells in the bone marrow, which is determined by a highly restricted uptake. We have previously shown that the specific internalization is mediated by the interaction of the viral protein 1 unique region (VP1u) with a yet unknown cellular receptor. To locate the receptor-binding domain (RBD) within the VP1u, we analyzed the effect of truncations and mutations on the internalization capacity of the recombinant protein into UT7/Epo cells. Here we report that the N-terminal amino acids 5-80 of the VP1u are necessary and sufficient for cellular binding and internalization; thus, this N-terminal region represents the RBD required for B19V uptake. Using site-directed mutagenesis, we further identified a cluster of important amino acids playing a critical role in VP1u internalization. In silico predictions and experimental results suggest that the RBD is structured as a rigid fold of three α-helices. Finally, we found that dimerization of the VP1u leads to a considerably enhanced cellular binding and internalization. Taken together, we identified the RBD that mediates B19V uptake and mapped functional and structural motifs within this sequence. The findings reveal insights into the uptake process of B19V, which contribute to understand the pathogenesis of the infection and the neutralization of the virus by the immune system.

The Receptor-Binding Domain in the VP1u Region of Parvovirus B19

PubMed Central

Leisi, Remo; Di Tommaso, Chiarina; Kempf, Christoph; Ros, Carlos

2016-01-01

Parvovirus B19 (B19V) is known as the human pathogen causing the mild childhood disease erythema infectiosum. B19V shows an extraordinary narrow tissue tropism for erythroid progenitor cells in the bone marrow, which is determined by a highly restricted uptake. We have previously shown that the specific internalization is mediated by the interaction of the viral protein 1 unique region (VP1u) with a yet unknown cellular receptor. To locate the receptor-binding domain (RBD) within the VP1u, we analyzed the effect of truncations and mutations on the internalization capacity of the recombinant protein into UT7/Epo cells. Here we report that the N-terminal amino acids 5–80 of the VP1u are necessary and sufficient for cellular binding and internalization; thus, this N-terminal region represents the RBD required for B19V uptake. Using site-directed mutagenesis, we further identified a cluster of important amino acids playing a critical role in VP1u internalization. In silico predictions and experimental results suggest that the RBD is structured as a rigid fold of three α-helices. Finally, we found that dimerization of the VP1u leads to a considerably enhanced cellular binding and internalization. Taken together, we identified the RBD that mediates B19V uptake and mapped functional and structural motifs within this sequence. The findings reveal insights into the uptake process of B19V, which contribute to understand the pathogenesis of the infection and the neutralization of the virus by the immune system. PMID:26927158

Dual-Ligand Modified Polymer-Lipid Hybrid Nanoparticles for Docetaxel Targeting Delivery to Her2/neu Overexpressed Human Breast Cancer Cells.

PubMed

Yang, Zhe; Tang, Wenxin; Luo, Xingen; Zhang, Xiaofang; Zhang, Chao; Li, Hao; Gao, Di; Luo, Huiyan; Jiang, Qing; Liu, Jie

2015-08-01

In this study, a dual-ligand polymer-lipid hybrid nanoparticle drug delivery vehicle comprised of an anti-HER2/neu peptide (AHNP) mimic with a modified HIV-1 Tat (mTAT) was established for the targeted treatment of Her2/neu-overexpressing cells. The resultant dual-ligand hybrid nanoparticles (NPs) consisted of a poly(lactide-co-glycolide) core, a near 90% surface coverage of the lipid monolayer, and a 5.7 nm hydrated polyethylene glycol shell. Ligand density optimization study revealed that cellular uptake efficiency of the hybrid NPs could be manipulated by controlling the surface-ligand densities. Furthermore, the cell uptake kinetics and mechanism studies showed that the dual-ligand modifications of hybrid NPs altered the cellular uptake pathway from caveolae-mediated endocytosis (CvME) to the multiple endocytic pathways, which would significantly enhance the NP internalization. Upon the systemic investigation of the cellular uptake behavior of dual-ligand hybrid NPs, docetaxel (DTX), a hydrophobic anticancer drug, was successfully encapsulated into dual-ligand hybrid NPs with high drug loading for Her2/neu-overexpressing SK-BR-3 breast cancer cell treatment. The DTX-loaded dual-ligand hybrid NPs showed a decreased burst release and a more gradual sustained drug release property. Because of the synergistic effect of dual-ligand modification, DTX-loaded dual-ligand hybrid NPs exerted substantially better therapeutic potency against SK-BR-3 cancer cells than other NP formulations and free DTX drugs. These results demonstrate that the dual-ligand hybrid NPs could be a promising vehicle for targeted drug delivery to treat breast cancer.

Melanin targeting for intracellular drug delivery: Quantification of bound and free drug in retinal pigment epithelial cells.

PubMed

Rimpelä, Anna-Kaisa; Hagström, Marja; Kidron, Heidi; Urtti, Arto

2018-05-31

Melanin binding affects drug distribution and retention in pigmented ocular tissues, thereby affecting drug response, duration of activity and toxicity. Therefore, it is a promising possibility for drug targeting and controlled release in the pigmented cells and tissues. Intracellular unbound drug concentrations determine pharmacological and toxicological actions, but analyses of unbound vs. total drug concentrations in pigmented cells are lacking. We studied intracellular binding and cellular drug uptake in pigmented retinal pigment epithelial cells and in non-pigmented ARPE-19 cells with five model drugs (chloroquine, propranolol, timolol, diclofenac, methotrexate). The unbound drug fractions in pigmented cells were 0.00016-0.73 and in non-pigmented cells 0.017-1.0. Cellular uptake (i.e. distribution ratio Kp), ranged from 1.3 to 6300 in pigmented cells and from 1.0 to 25 in non-pigmented cells. Values for intracellular bioavailability, F ic , were similar in both cells types (although larger variation in pigmented cells). In vitro melanin binding parameters were used to predict intracellular unbound drug fraction and cell uptake. Comparison of predictions with experimental data indicates that other factors (e.g. ion-trapping, lipophilicity-related binding to other cell components) also play a role. Melanin binding is a major factor that leads to cellular uptake and unbound drug fractions of a range of 3-4 orders of magnitude indicating that large reservoirs of melanin bound drug can be generated in the cells. Understanding melanin binding has important implications on retinal drug targeting, efficacy and toxicity. Copyright © 2017. Published by Elsevier B.V.

Dominant oceanic bacteria secure phosphate using a large extracellular buffer

PubMed Central

Zubkov, Mikhail V.; Martin, Adrian P.; Hartmann, Manuela; Grob, Carolina; Scanlan, David J.

2015-01-01

The ubiquitous SAR11 and Prochlorococcus bacteria manage to maintain a sufficient supply of phosphate in phosphate-poor surface waters of the North Atlantic subtropical gyre. Furthermore, it seems that their phosphate uptake may counter-intuitively be lower in more productive tropical waters, as if their cellular demand for phosphate decreases there. By flow sorting 33P-phosphate-pulsed 32P-phosphate-chased cells, we demonstrate that both Prochlorococcus and SAR11 cells exploit an extracellular buffer of labile phosphate up to 5–40 times larger than the amount of phosphate required to replicate their chromosomes. Mathematical modelling is shown to support this conclusion. The fuller the buffer the slower the cellular uptake of phosphate, to the point that in phosphate-replete tropical waters, cells can saturate their buffer and their phosphate uptake becomes marginal. Hence, buffer stocking is a generic, growth-securing adaptation for SAR11 and Prochlorococcus bacteria, which lack internal reserves to reduce their dependency on bioavailable ambient phosphate. PMID:26198420

Protein Corona Influences Cellular Uptake of Gold Nanoparticles by Phagocytic and Nonphagocytic Cells in a Size-Dependent Manner.

PubMed

Cheng, Xiaju; Tian, Xin; Wu, Anqing; Li, Jianxiang; Tian, Jian; Chong, Yu; Chai, Zhifang; Zhao, Yuliang; Chen, Chunying; Ge, Cuicui

2015-09-23

The interaction at nanobio is a critical issue in designing safe nanomaterials for biomedical applications. Recent studies have reported that it is nanoparticle-protein corona rather than bare nanoparticle that determines the nanoparticle-cell interactions, including endocytic pathway and biological responses. Here, we demonstrate the effects of protein corona on cellular uptake of different sized gold nanoparticles in different cell lines. The experimental results show that protein corona significantly decreases the internalization of Au NPs in a particle size- and cell type-dependent manner. Protein corona exhibits much more significant inhibition on the uptake of large-sized Au NPs by phagocytic cell than that of small-sized Au NPs by nonphagocytic cell. The endocytosis experiment indicates that different endocytic pathways might be responsible for the differential roles of protein corona in the interaction of different sized Au NPs with different cell lines. Our findings can provide useful information for rational design of nanomaterials in biomedical application.

Relationship between peroxisome proliferator-activated receptor alpha activity and cellular concentration of 14 perfluoroalkyl substances in HepG2 cells.

PubMed

Rosenmai, Anna Kjerstine; Ahrens, Lutz; le Godec, Théo; Lundqvist, Johan; Oskarsson, Agneta

2018-02-01

Peroxisome proliferator-activated receptor alpha (PPARα) is a molecular target for perfluoroalkyl substances (PFASs). Little is known about the cellular uptake of PFASs and how it affects the PPARα activity. We investigated the relationship between PPARα activity and cellular concentration in HepG2 cells of 14 PFASs, including perfluoroalkyl carboxylates (PFCAs), perfluoroalkyl sulfonates and perfluorooctane sulfonamide (FOSA). Cellular concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry and PPARα activity was determined in transiently transfected cells by reporter gene assay. Cellular uptake of the PFASs was low (0.04-4.1%) with absolute cellular concentrations in the range 4-2500 ng mg -1 protein. Cellular concentration of PFCAs increased with perfluorocarbon chain length up to perfluorododecanoate. PPARα activity of PFCAs increased with chain length up to perfluorooctanoate. The maximum induction of PPARα activity was similar for short-chain (perfluorobutanoate and perfluoropentanoate) and long-chain PFCAs (perfluorododecanoate and perfluorotetradecanoate) (approximately twofold). However, PPARα activities were induced at lower cellular concentrations for the short-chain homologs compared to the long-chain homologs. Perfluorohexanoate, perfluoroheptanoate, perfluorooctanoate, perfluorononanoate (PFNA) and perfluorodecanoate induced PPARα activities >2.5-fold compared to controls. The concentration-response relationships were positive for all the tested compounds, except perfluorooctane sulfonate PFOS and FOSA, and were compound-specific, as demonstrated by differences in the estimated slopes. The relationships were steeper for PFCAs with chain lengths up to and including PFNA than for the other studied PFASs. To our knowledge, this is the first report establishing relationships between PPARα activity and cellular concentration of a broad range of PFASs. Copyright © 2017 John Wiley & Sons, Ltd.

Not all protein-mediated single-wall carbon nanotube dispersions are equally bioactive

NASA Astrophysics Data System (ADS)

Holt, Brian D.; McCorry, Mary C.; Boyer, Patrick D.; Dahl, Kris Noel; Islam, Mohammad F.

2012-11-01

Single-wall carbon nanotubes (SWCNTs) have been dispersed with proteins to increase biocompatibility and specificity, but examinations of dispersion parameters on functional cellular uptake are required for utilization of SWCNTs in biological applications. Here we correlate conditions of SWCNT dispersion with various proteins to uptake these SWCNTs in NIH-3T3 fibroblasts and J774A.1 macrophage-like cells. We varied protein types (bovine serum albumin - BSA, lysozyme - LSZ, and γ-globulins - γG), protein : SWCNT ratio and sonication time. Each protein created stable, high yield (~25%) dispersions in water while preserving intrinsic SWCNT fluorescence, but SWCNT-LSZ flocculated in media and SWCNT-γG formed clusters in both water and media, drastically altering cellular internalization. Dispersion quality and yield improved with increased protein : SWCNT - without substantial effects from depletion attraction, even at 100 : 1 protein : SWCNT - and slightly increased internalized SWCNTs for both NIH-3T3 and J774A.1 cells. Longer sonication time (12 versus 2 h) improved the dispersion yield and quality but caused minor damage to SWCNTs and altered protein structure. Cell association of SWCNT-BSA was homogenous and unaltered by sonication time. Bulk assay showed that cell association of SWCNT-LSZ and SWCNT-γG was altered with 12 versus 2 h sonication, but imaging of individual cells showed that these differences are likely from precipitation of clusters of SWCNT-LSZ and SWCNT-γG in media onto cells. Hence, the quality of SWCNT-protein dispersions in water does not necessarily correlate with bulk cellular uptake, and quantification at the level of individual cells is required to determine delivery efficacy.Single-wall carbon nanotubes (SWCNTs) have been dispersed with proteins to increase biocompatibility and specificity, but examinations of dispersion parameters on functional cellular uptake are required for utilization of SWCNTs in biological applications. Here we correlate conditions of SWCNT dispersion with various proteins to uptake these SWCNTs in NIH-3T3 fibroblasts and J774A.1 macrophage-like cells. We varied protein types (bovine serum albumin - BSA, lysozyme - LSZ, and γ-globulins - γG), protein : SWCNT ratio and sonication time. Each protein created stable, high yield (~25%) dispersions in water while preserving intrinsic SWCNT fluorescence, but SWCNT-LSZ flocculated in media and SWCNT-γG formed clusters in both water and media, drastically altering cellular internalization. Dispersion quality and yield improved with increased protein : SWCNT - without substantial effects from depletion attraction, even at 100 : 1 protein : SWCNT - and slightly increased internalized SWCNTs for both NIH-3T3 and J774A.1 cells. Longer sonication time (12 versus 2 h) improved the dispersion yield and quality but caused minor damage to SWCNTs and altered protein structure. Cell association of SWCNT-BSA was homogenous and unaltered by sonication time. Bulk assay showed that cell association of SWCNT-LSZ and SWCNT-γG was altered with 12 versus 2 h sonication, but imaging of individual cells showed that these differences are likely from precipitation of clusters of SWCNT-LSZ and SWCNT-γG in media onto cells. Hence, the quality of SWCNT-protein dispersions in water does not necessarily correlate with bulk cellular uptake, and quantification at the level of individual cells is required to determine delivery efficacy. Electronic supplementary information (ESI) available: Images of protein dispersions, comparison of absorbance and NIR fluorescence peak shifts, gross quantification of cellular uptake of SWCNTs, and summary of protein secondary structure as a function of sonication time in the presence of SWCNTs. See DOI: 10.1039/c2nr31928d

Delivery of kinesin spindle protein targeting siRNA in solid lipid nanoparticles to cellular models of tumor vasculature

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ying, Bo; Campbell, Robert B., E-mail: robert.campbell@mcphs.edu

2014-04-04

Highlights: • siRNA-lipid nanoparticles are solid particles not lipid bilayers with aqueous core. • High, but not low, PEG content can prevent nanoparticle encapsulation of siRNA. • PEG reduces cellular toxicity of cationic nanoparticles in vitro. • PEG reduces zeta potential while improving gene silencing of siRNA nanoparticles. • Kinesin spindle protein can be an effective target for tumor vascular targeting. - Abstract: The ideal siRNA delivery system should selectively deliver the construct to the target cell, avoid enzymatic degradation, and evade uptake by phagocytes. In the present study, we evaluated the importance of polyethylene glycol (PEG) on lipid-based carriermore » systems for encapsulating, and delivering, siRNA to tumor vessels using cellular models. Lipid nanoparticles containing different percentage of PEG were evaluated based on their physical chemical properties, density compared to water, siRNA encapsulation, toxicity, targeting efficiency and gene silencing in vitro. siRNA can be efficiently loaded into lipid nanoparticles (LNPs) when DOTAP is included in the formulation mixture. However, the total amount encapsulated decreased with increase in PEG content. In the presence of siRNA, the final formulations contained a mixed population of particles based on density. The major population which contains the majority of siRNA exhibited a density of 4% glucose, and the minor fraction associated with a decreased amount of siRNA had a density less than PBS. The inclusion of 10 mol% PEG resulted in a greater amount of siRNA associated with the minor fraction. Finally, when kinesin spindle protein (KSP) siRNA was encapsulated in lipid nanoparticles containing a modest amount of PEG, the proliferation of endothelial cells was inhibited due to the efficient knock down of KSP mRNA. The presence of siRNA resulted in the formation of solid lipid nanoparticles when prepared using the thin film and hydration method. LNPs with a relatively modest amount of PEG can sufficiently encapsulate siRNA, improve cellular uptake and the efficiency of gene silencing.« less

Cellular Origin of [18F]FDG-PET Imaging Signals During Ceftriaxone-Stimulated Glutamate Uptake: Astrocytes and Neurons.

PubMed

Dienel, Gerald A; Behar, Kevin L; Rothman, Douglas L

2017-12-01

Ceftriaxone stimulates astrocytic uptake of the excitatory neurotransmitter glutamate, and it is used to treat glutamatergic excitotoxicity that becomes manifest during many brain diseases. Ceftriaxone-stimulated glutamate transport was reported to drive signals underlying [ 18 F]fluorodeoxyglucose-positron emission tomographic ([ 18 F]FDG-PET) metabolic images of brain glucose utilization and interpreted as supportive of the notion of lactate shuttling from astrocytes to neurons. This study draws attention to critical roles of astrocytes in the energetics and imaging of brain activity, but the results are provocative because (1) the method does not have cellular resolution or provide information about downstream pathways of glucose metabolism, (2) neuronal and astrocytic [ 18 F]FDG uptake were not separately measured, and (3) strong evidence against lactate shuttling was not discussed. Evaluation of potential metabolic responses to ceftriaxone suggests lack of astrocytic specificity and significant contributions by pre- and postsynaptic neuronal compartments. Indeed, astrocytic glycolysis may not make a strong contribution to the [ 18 F]FDG-PET signal because partial or complete oxidation of one glutamate molecule on its uptake generates enough ATP to fuel uptake of 3 to 10 more glutamate molecules, diminishing reliance on glycolysis. The influence of ceftriaxone on energetics of glutamate-glutamine cycling must be determined in astrocytes and neurons to elucidate its roles in excitotoxicity treatment.

Enzyme-triggered delivery of chlorambucil from conjugates based on the cell-penetrating peptide BP16.

PubMed

Soler, Marta; González-Bártulos, Marta; Figueras, Eduard; Ribas, Xavi; Costas, Miquel; Massaguer, Anna; Planas, Marta; Feliu, Lidia

2015-02-07

The undecapeptide KKLFKKILKKL-NH2 (BP16) is a non-toxic cell-penetrating peptide (CPP) that is mainly internalized into cancer cells through a clathrin dependent endocytic mechanism and localizes in late endosomes. Moreover, this CPP is able to enhance the cellular uptake of chlorambucil (CLB) improving its cytotoxicity. In this work, we further explored the cell-penetrating properties of BP16 and those of its arginine analogue BP308. We investigated the influence on the cytotoxicity and on the cellular uptake of conjugating CLB at the N- or the C-terminal end of these undecapeptides. The effect of incorporating the cathepsin B-cleavable sequence Gly-Phe-Leu-Gly in CLB-BP16 and CLB-BP308 conjugates was also evaluated. The activity of CLB was significantly improved when conjugated at the N- or the C-terminus of BP16, or at the N-terminus of BP308. While CLB alone was not active (IC50 of 73.7 to >100 μM), the resulting conjugates displayed cytotoxic activity against CAPAN-1, MCF-7, PC-3, 1BR3G and SKMEL-28 cell lines with IC50 values ranging from 8.7 to 25.5 μM. These results were consistent with the internalization properties observed for the corresponding 5(6)-carboxyfluorescein-labeled conjugates. The presence of the tetrapeptide Gly-Phe-Leu-Gly at either the N- or the C-terminus of CLB-BP16 conjugates further increased the efficacy of CLB (IC50 of 3.6 to 16.2 μM), which could be attributed to its selective release in the lysosomal compartment. Enzymatic assays with cathepsin B showed the release of CLB-Gly-OH from these sequences within a short time. Therefore, the combination of BP16 with an enzymatic cleavable sequence can be used as a drug delivery system for the effective uptake and release of drugs in cancer cells.

On the importance of electrostatic interactions between cell penetrating peptides and membranes: a pathway toward tumor cell selectivity?

PubMed

Jobin, Marie-Lise; Alves, Isabel D

2014-12-01

Cell-penetrating peptides (CPPs) are small molecules of major interest due to their ability to efficiently transport cargos across cell membranes in a receptor- and energy-independent way and without being cytotoxic to cells. Since their discovery 20 years ago their potential interest in drug delivery and diagnosis became undeniable. CPPs are being used to deliver inside cells a large variety of cargos such as proteins, DNA, antibodies, imaging agents and nanoparticle drug carriers. Their cellular uptake mechanisms are still debated and may vary depending on their structure, nature and size of cargo they transport and type of cell line targeted. CPPs are generally rich in positively charged residues, thus they are prone to establish electrostatic interactions with anionic membrane components (sugars and lipids). Understanding the molecular basis of CPP membrane interaction and cellular uptake is crucial to improve their in vivo efficiency target-specificity. A great number of studies demonstrated the high potential of CPPs to translocate efficiently therapeutic cargos into cells and some peptides are even in clinical phase studies. Although these molecules seem perfect for a therapeutic or diagnosis purpose, they still possess a small but non negligible drawback: a complete lack of cell type specificity. Tumor cells have recently been shown to over-express certain glycosaminoglycans at the cell membrane surface and to possess a higher amount of anionic lipids in their outer leaflet than healthy cells. Such molecules confer the cell membrane an enhanced anionic character, property that could be used by CPPs to selectively target these cells. Moreover previous studies demonstrate the importance of electrostatic interactions between basic residues in the peptide, especially Arg, and the lipid headgroups and glycosaminoglycans in the cell membrane. Electrostatic interactions put at stake in this process might be one of the keys to resolve the puzzle of CPP cell type specificity. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Detention of copper by sulfur nanoparticles inhibits the proliferation of A375 malignant melanoma and MCF-7 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Hao; Zhang, Yikai; Zheng, Shanyuan

Selective induction of cell death or growth inhibition of cancer cells is the future of chemotherapy. Clinical trials have found that cancer tissues are enriched with copper. Based on this finding, many copper-containing compounds and complexes have been designed to “copper” cancer cells using copper as bait. However, recent studies have demonstrated that copper boosts tumor development, and copper deprivation from serum was shown to effectively inhibit the promotion of cancer. Mechanistically, copper is an essential cofactor for mitogen-activated protein kinase (MAPK)/extracellular activating kinase (ERK) kinase (MEK), a central molecule in the BRAF/MEK/ERK pathway. Therefore, depleting copper from cancer cellsmore » by directly sequestering copper has a wider field for research and potential for combination therapy. Based on the affinity between sulfur and copper, we therefore designed sulfur nanoparticles (Nano-S) that detain copper, achieving tumor growth restriction. We found that spherical Nano-S could effectively bind copper and form a tighter surficial structure. Moreover, this Nano-S detention of copper effectively inhibited the proliferation of A375 melanoma and MCF-7 breast cancer cells with minimum toxicity to normal cells. Mechanistic studies revealed that Nano-S triggered inactivation of the MEK-ERK pathway followed by inhibition of the proliferation of the A375 and MCF-7 cells. In addition, lower Nano-S concentrations and shorter exposure stimulated the expression of a copper transporter as compensation, which further increased the cellular uptake and anticancer activities of cisplatin. Collectively, our results highlight the potential of Nano-S as an anticancer agent or adjuvant through its detention of copper. - Highlights: • Nano-S selectively inhibited the mitosis of A375 and MCF-7 cells by depleting copper. • Nano-S inactivated MEK/ERK pathway through the detention of copper. • Nano-S improved the cellular uptake and anticancer activities of cisplatin.« less

Polymeric nanocarriers for transport modulation across the pulmonary epithelium: dendrimers, polymeric nanoparticles, and their nanoblends.

PubMed

Bharatwaj, Balaji; Dimovski, Radovan; Conti, Denise S; da Rocha, Sandro R P

2014-05-01

The purpose of this study was to (a) Determine the cellular transport and uptake of amine-terminated generation 3 (G3) poly(amido amine) (PAMAM) dendrimers across an in vitro model of the pulmonary epithelium, and the ability to modulate their transport by forming nanoblends of the dendrimers with biodegradable solid polymeric nanoparticles (NPs) and (b) to formulate dendrimer nanocarriers in portable oral inhalation devices and evaluate their aerosol characteristics. To that end, fluorescein isothiocyanate (FITC)-labeled G3 PAMAM dendrimer nanocarriers (DNCs) were synthesized, and also encapsulated within poly lactide-co-glycolide nanoparticles (NPs). Transport and uptake of both DNCs encapsulated within NPs (nanoblends) and unencapsulated DNCs were tracked across polarized monolayers of airway epithelial cells, Calu-3. DNCs were also formulated as core-shell microparticles in pressurized metered-dose inhalers (pMDIs) and their aerodynamic properties evaluated by Andersen cascade impaction. The apparent permeability of DNCs across the airway epithelial model was similar to that of a paracellular marker of comparable molar mass--order of 10(-7) cm s(-1). The transport and cellular internalization of the DNCs can be modulated by formulating them as nanoblends. The transport of the DNCs across the lung epithelium was completely suppressed within the time of the experiment (5 h) when formulated as blends. The encapsulation also prevents saturation of the cellular internalization profile. Nanoblending may be a potential strategy to modulate the rate of transport and cellular uptake of DNCs, and thus be used as a design strategy to achieve enhanced local or systemic drug delivery.

d-Fructose-Decorated Poly(ethylene imine) for Human Breast Cancer Cell Targeting.

PubMed

Englert, Christoph; Pröhl, Michael; Czaplewska, Justyna A; Fritzsche, Carolin; Preußger, Elisabeth; Schubert, Ulrich S; Traeger, Anja; Gottschaldt, Michael

2017-08-01

The high affinity of GLUT5 transporter for d-fructose in breast cancer cells has been discussed intensely. In this contribution, high molar mass linear poly(ethylene imine) (LPEI) is functionalized with d-fructose moieties to combine the selectivity for the GLUT5 transporter with the delivery potential of PEI for genetic material. The four-step synthesis of a thiol-group bearing d-fructose enables the decoration of a cationic polymer backbone with d-fructose via thiol-ene photoaddition. The functionalization of LPEI is confirmed by 2D NMR techniques, elemental analysis, and size exclusion chromatography. Importantly, a d-fructose decoration of 16% renders the polymers water-soluble and eliminates the cytotoxicity of PEI in noncancer L929 cells, accompanied by a reduced unspecific cellular uptake of the genetic material. In contrast, the cytotoxicity as well as the cell specific uptake is increased for triple negative MDA-MB-231 breast cancer cells. Therefore, the introduction of d-fructose shows superior potential for cell targeting, which can be assumed to be GLUT5 dependent. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Y-shaped biotin-conjugated poly (ethylene glycol)-poly (epsilon-caprolactone) copolymer for the targeted delivery of curcumin.

PubMed

Zhu, Wenxia; Song, Zhimei; Wei, Peng; Meng, Ning; Teng, Fangfang; Yang, Fengying; Liu, Na; Feng, Runliang

2015-04-01

In order to improve curcumin's low water-solubility and selective delivery to cancer, we reported ligand-mediated micelles based on a Y-shaped biotin-poly (ethylene glycol)-poly (epsilon-caprolactone)2 (biotin-PEG-PCL2) copolymer. Its structure was characterized by (1)H NMR. The blank and drug-loaded micelles obtained by way of thin-film hydration were characterized by dynamic light scattering, X-ray diffraction, infrared spectroscopy and hemolytic test. Curcumin was loaded into micelles with a high encapsulating efficiency (93.83%). Curcumin's water-solubility was enhanced 170,400 times higher than free curcumin. Biotin-PEG-PCL2 micelles showed slower drug release in vitro than H2N-PEG-PCL2 micelles. In vitro cellular uptake and cytotoxicity tests showed that higher dosage of curcumin might overcome the effect of slow release on cytotoxicities because of its higher uptake induced by biotin, resulting in higher anticancer activities against MDA-MB-436 cells. In brief, Y-shaped biotin-PEG-PCL2 is a promising delivery carrier for anticancer drug. Copyright © 2014 Elsevier Inc. All rights reserved.

Differential cellular and subcellular distribution of glutamate transporters in the cat retina.

PubMed

Fyk-Kolodziej, Bozena; Qin, Pu; Dzhagaryan, Arturik; Pourcho, Roberta G

2004-01-01

Retrieval of glutamate from extracellular sites in the retina involves at least five excitatory amino acid transporters. Immunocytochemical analysis of the cat retina indicates that each of these transporters exhibits a selective distribution which may reflect its specific function. The uptake of glutamate into Muller cells or astrocytes appears to depend upon GLAST and EAAT4, respectively. Staining for EAAT4 was also seen in the pigment epithelium. The remaining transporters are neuronal with GLT-1alpha localized to a number of cone bipolar, amacrine, and ganglion cells and GLT-1v in cone photoreceptors and several populations of bipolar cells. The EAAC1 transporter was found in horizontal, amacrine, and ganglion cells. Staining for EAAT5 was seen in the axon terminals of both rod and cone photoreceptors as well as in numerous amacrine and ganglion cells. Although some of the glutamate transporter molecules are positioned for presynaptic or postsynaptic uptake at glutamatergic synapses, others with localizations more distant from such contacts may serve in modulatory roles or provide protection against excitoxic or oxidative damage.

Reconciling the Krogh and Ussing interpretations of epithelial chloride transport - presenting a novel hypothesis for the physiological significance of the passive cellular chloride uptake.

PubMed

Larsen, Erik Hviid

2011-07-01

In 1937, August Krogh discovered a powerful active Cl(-) uptake mechanism in frog skin. After WWII, Hans Ussing continued the studies on the isolated skin and discovered the passive nature of the chloride uptake. The review concludes that the two modes of transport are associated with a minority cell type denoted as the γ-type mitochondria-rich (MR) cell, which is highly specialized for epithelial Cl(-) uptake whether the frog is in the pond of low [NaCl] or the skin is isolated and studied by Ussing chamber technique. One type of apical Cl(-) channels of the γ-MR cell is activated by binding of Cl(-) to an external binding site and by membrane depolarization. This results in a tight coupling of the uptake of Na(+) by principal cells and Cl(-) by MR cells. Another type of Cl(-) channels (probably CFTR) is involved in isotonic fluid uptake. It is suggested that the Cl(-) channels serve passive uptake of Cl(-) from the thin epidermal film of fluid produced by mucosal glands. The hypothesis is evaluated by discussing the turnover of water and ions of the epidermal surface fluid under terrestrial conditions. The apical Cl(-) channels close when the electrodiffusion force is outwardly directed as it is when the animal is in the pond. With the passive fluxes eliminated, the Cl(-) flux is governed by active transport and evidence is discussed that this is brought about by an exchange of cellular HCO(3) (-) with Cl(-) of the outside bath driven by an apical H(+) V-ATPase. © 2011 The Author. Acta Physiologica © 2011 Scandinavian Physiological Society.

Blending of diblock and triblock copolypeptide amphiphiles yields cell penetrating vesicles with low toxicity.

PubMed

Rodriguez, April R; Choe, Uh-Joo; Kamei, Daniel T; Deming, Timothy J

2015-01-01

We prepared dual hydrophilic triblock copolypeptide vesicles that form both micron and nanometer scale vesicles in aqueous media. The incorporation of terminal homoarginine segments into methionine sulfoxide-based vesicles was found to significantly enhance their cellular uptake compared to a non-ionic control. We also demonstrated that diblock and triblock copolypeptides with similar hydrophobic domains were found to mix well and form vesicle populations with uniform compositions. Blending of amphiphiles in vesicle nanocarriers was found to impart these materials with many advantageous properties, including good cellular uptake while maintaining minimal toxicity, as well as biological responsiveness to promote vesicle disruption and release of encapsulated cargos. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The influence of cellular uptake on gold nanorods photostability and photoacoustic conversion efficiency

NASA Astrophysics Data System (ADS)

Cavigli, Lucia; Ratto, Fulvio; Tatini, Francesca; Matteini, Paolo; Cini, Alberto; Giovannelli, Ilaria; de Angelis, Marella; Rossi, Francesca; Centi, Sonia; Pini, Roberto

2015-03-01

Their intense optical absorbance in the near-infrared window and chemical versatility make gold nanorods attractive for biomedical applications, such as photothermal therapies and photoacoustic imaging. However, their limited photostability remains a drawback of practical concern. In fact, when gold nanorods are irradiated with nanosecond laser pulses in resonance with their plasmon oscillations, there may occur reshaping into spherical particles or even fragmentation at higher optical fluences, which cause substantial modifications of their optical features with a loss of photoacoustic conversion efficiency. In this contribution, we focus on how the gold nanorods photostability is affected when these particles are modified for cellular uptake, by investigating their stability and photoacoustic conversion efficiency under near infrared pulsed irradiation at different laser fluences.

Graphitic and oxidised high pressure high temperature (HPHT) nanodiamonds induce differential biological responses in breast cancer cell lines.

PubMed

Woodhams, Benjamin; Ansel-Bollepalli, Laura; Surmacki, Jakub; Knowles, Helena; Maggini, Laura; de Volder, Michael; Atatüre, Mete; Bohndiek, Sarah

2018-06-19

Nanodiamonds have demonstrated potential as powerful sensors in biomedicine, however, their translation into routine use requires a comprehensive understanding of their effect on the biological system being interrogated. Under normal fabrication processes, nanodiamonds are produced with a graphitic carbon shell, but are often oxidized in order to modify their surface chemistry for targeting to specific cellular compartments. Here, we assessed the biological impact of this purification process, considering cellular proliferation, uptake, and oxidative stress for graphitic and oxidized nanodiamond surfaces. We show for the first time that oxidized nanodiamonds possess improved biocompatibility compared to graphitic nanodiamonds in breast cancer cell lines, with graphitic nanodiamonds inducing higher levels of oxidative stress despite lower uptake.

A glutamine-rich carrier efficiently delivers anti-CD47 siRNA driven by "glutamine trap" to inhibit lung cancer cell growth.

PubMed

Wu, JiaMin; Li, Zhi; Yang, Zeping; Guo, Ling; Zhang, Ye; Deng, Huihui; Wang, Cuifeng; Feng, Min

2018-06-25

It is not efficient enough using the current approaches for tumor-selective drug delivery based on the EPR effect and ligand-receptor interactions, and they have largely failed to translate into the clinic. So it is urgent to explore an enhanced strategy for effective delivery of anticancer agents. Clinically, many cancers require large amounts of glutamine for their continued growth and survival, resulting in circulating glutamine extraction by the tumor being much greater than that for any organs, behaving as a "glutamine trap". In the present study, we sought to elucidate whether the glutamine trap effect could be exploited to deliver therapeutic agents to selectively kill cancer cells. Here, a macromolecular glutamine analog, glutamine-functionalized branched polyethylenimine (GPI), was constructed as the carrier to deliver anti-CD47 siRNA for the blockage of CD47 "don't eat me" signals on cancer cells. The GPI/siRNA glutamine-rich polyplexes exhibited remarkably high levels of cellular uptake by glutamine-dependent lung cancer cells, wild-type A549 cells (A549WT) and its cisplatin-resistant cells (A549DDP), specifically under glutamine-depleted conditions. It was noted that the glutamine transporter ASCT2 was highly expressed both on A549WT and A549DDP, but almost no expression in normal human lung fibroblasts cells. Inhibition of ASCT2 significantly prevented the internalization of GPI polyplexes. These findings raised the intriguing possibility that the glutamine-rich GPI polyplexes utilize the ASCT2 pathway to selectively facilitate their cellular uptake by cancer cells. GPI further delivered anti-CD47 siRNA efficiently both in vitro and in vivo to down-regulate the intratumoral mRNA and protein expression levels of CD47. CD47 functions as a "don't eat me" signal and binds to the immunoreceptor SIRPα inducing evasion of phagocytic clearance. GPI/anti-CD47 siRNA polyplexes achieved significant antitumor activities both on A549WT and A549DDP tumor-bearing nude mice. Notably, it had no adverse effect on CD47-expressing red blood cells and platelets, likely due to selective delivery. Therefore, the glutamine-rich carrier GPI driven by the glutamine trap effect provides a promising new strategy for designing anticancer drug delivery systems.

Resveratrol self-emulsifying system increases the uptake by endothelial cells and improves protection against oxidative stress-mediated death.

PubMed

Amri, Ahmed; Le Clanche, Solenn; Thérond, Patrice; Bonnefont-Rousselot, Dominique; Borderie, Didier; Lai-Kuen, René; Chaumeil, Jean-Claude; Sfar, Souad; Charrueau, Christine

2014-04-01

The aim of the present study was to develop and characterize a resveratrol self-emulsifying drug delivery system (Res-SEDDS), and to compare the uptake of resveratrol by bovine aortic endothelial cells (BAECs), and the protection of these cells against hydrogen peroxide-mediated cell death, versus a control resveratrol ethanolic solution. Three Res-SEDDSs were prepared and evaluated. The in vitro self-emulsification properties of these formulations, the droplet size and the zeta potential of the nanoemulsions formed on adding them to water under mild agitation conditions were studied, together with their toxicity on BAECs. An optimal atoxic formulation (20% Miglyol® 812, 70% Montanox® 80, 10% ethanol 96% v/v) was selected and further studied. Pre-incubation of BAECs for 180 min with 25 μM resveratrol in the nanoemulsion obtained from the selected SEDDS significantly increased the membrane and intracellular concentrations of resveratrol (for example, 0.076±0.015 vs. ethanolic solution 0.041±0.016 nmol/mg of protein after 60 min incubation, p<0.05). Resveratrol intracellular localization was confirmed by fluorescence confocal microscopy. Resveratrol nanoemulsion significantly improved the endothelial cell protection from H2O2-induced injury (750, 1000 and 1500 μM H2O2) in comparison with incubation with the control resveratrol ethanolic solution (for example, 55±6% vs. 38±5% viability after 1500 μM H2O2 challenge, p<0.05). In conclusion, formulation of resveratrol as a SEDDS significantly improved its cellular uptake and potentiated its antioxidant properties on BAECs. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA Tetrahedron Delivery Enhances Doxorubicin-Induced Apoptosis of HT-29 Colon Cancer Cells

NASA Astrophysics Data System (ADS)

Zhang, Guiyu; Zhang, Zhiyong; Yang, Junen

2017-08-01

As a nano-sized drug carrier with the advantage of modifiability and proper biocompatibility, DNA tetrahedron (DNA tetra) delivery is hopeful to enhance the inhibitory efficiency of nontargeted anticancer drugs. In this investigation, doxorubicin (Dox) was assembled to a folic acid-modified DNA tetra via click chemistry to prepare a targeted antitumor agent. Cellular uptake efficiency was measured via fluorescent imaging. Cytotoxicity, inhibition efficiency, and corresponding mechanism on colon cancer cell line HT-29 were evaluated by MTT assay, cell proliferation curve, western blot, and flow cytometry. No cytotoxicity was induced by DNA tetra, but the cellular uptake ratio increased obviously resulting from the DNA tetra-facilitated penetration through cellular membrane. Accordingly, folic acid-DNA tetra-Dox markedly increased the antitumor efficiency with increased apoptosis levels. In details, 100 μM was the effective concentration and a 6-h incubation period was needed for apoptosis induction. In conclusion, nano-sized DNA tetrahedron was a safe and effective delivery system for Dox and correspondingly enhanced the anticancer efficiency.

Screening of medicinal plants for PPPAR-alpha and PPAR-gamma activation and evaluation of their effects on glucose uptake and 3T3-L1 adipogenesis

USDA-ARS?s Scientific Manuscript database

Medicinal plants are a rich source of ligands for nuclear receptors. The present study was aimed to screen a collection of plant extracts for PPAR-alpha/gamma activating properties and identify the active extract that can stimulate cellular glucose uptake without enhancing the adipogenesis. A report...

Dendritic polymer imaging systems for the evaluation of conjugate uptake and cleavage

NASA Astrophysics Data System (ADS)

Krüger, Harald R.; Nagel, Gregor; Wedepohl, Stefanie; Calderón, Marcelo

2015-02-01

Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening.Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening. Electronic supplementary information (ESI) available: Including detailed synthetic procedures of the dye and conjugate synthesis, as well as cellular uptake and inhibitor studies. See DOI: 10.1039/c4nr04467c

Effects of soil pH and aluminum on plant respiration

Treesearch

Rakesh Minocha; Subhash C. Minocha

2005-01-01

Interactions among external (soil) pH, cellular pH, and their effects on respiratory metabolism are complex. While the effects of changes in the apoplastic pH on the cytosolic pH are not clearly understood, pH directly affects enzymatic reactions in the cell, and pH-regulated ion uptake has profound indirect effects on cellular respiratory metabolism. A major...

Tetanus toxoid-loaded cationic non-aggregated nanostructured lipid particles triggered strong humoral and cellular immune responses.

PubMed

Kaur, Amandeep; Jyoti, Kiran; Rai, Shweta; Sidhu, Rupinder; Pandey, Ravi Shankar; Jain, Upendra Kumar; Katyal, Anju; Madan, Jitender

2016-05-01

In the present investigation, non-aggregated cationic and unmodified nanoparticles (TT-C-NLPs4 and TT-NLPs1) were prepared of about 49.2 ± 6.8-nm and 40.8 ± 8.3-nm, respectively. In addition, spherical shape, crystalline architecture and cationic charge were also noticed. Furthermore, integrity and conformational stability of TT were maintained in both TT-C-NLPs4 and TT-NLPs1, as evidenced by symmetrical position of bands and superimposed spectra, respectively in SDS-PAGE and circular dichroism. Cellular uptake in RAW264.7 cells indicating the concentration-dependent internalisation of nanoparticles. Qualitatively, CLSM exhibited enhanced cellular uptake of non-aggregated TT-C-NLPs4 owing to interaction with negatively charged plasma membrane and clevaloe mediated/independent endocytosis. In last, in vivo immunisation with non-aggregated TT-C-NLPs4 elicited strong humoral (anti-TT IgG) and cellular (IFN-γ) immune responses at day 42, as compared to non-aggregated TT-NLPs1 and TT-Alum following booster immunisation at day 14 and 28. Thus, non-aggregated cationic lipid nanoparticles may be a potent immune-adjuvant for parenteral delivery of weak antigens.

Infection of Polarized Cultures of Human Intestinal Epithelial Cells with Hepatitis A Virus: Vectorial Release of Progeny Virions through Apical Cellular Membranes

PubMed Central

Blank, Christian A.; Anderson, David A.; Beard, Michael; Lemon, Stanley M.

2000-01-01

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract. We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30- to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% ± 12.5% of the total virus yield compared with 0.23% ± 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms. PMID:10864660

[Primary study on fluro [ 19F] berberine derivative for human hepatocellular carcinoma targetting in vitro].

PubMed

Zhang, Tong; Wu, Xiaoai; Cai, Huawei; Liang, Meng; Fan, Chengzhong

2017-04-01

[ 18 F]HX-01, a Fluorine-18 labeled berberine derivative, is a potential positron emission tomography (PET) tumor imaging agent, while [ 19 F]HX-01 is a nonradioactive reference substance with different energy state and has the same physical and chemical properties. In order to collect data for further study of [ 18 F]HX-01 PET imaging of hepatocellular carcinoma in vivo , this study compared the uptake of [ 19 F]HX-01 by human hepatocellular carcinoma and normal hepatocytes in vitro . The target compound, [ 19 F]HX-01, was synthesized in one step using berberrubine and 3-fluoropropyl 4-methylbenzenesulfonate. Cellular uptake and localization of [ 19 F]HX-01 were performed by a fluorescence microscope in human hepatocellular carcinoma HepG2, SMMC-7721 and human normal hepatocyte HL-7702. Cellular proliferation inhibition and cell cytotoxicity assay of the [ 19 F]HX-01 were conducted using cell counting kit-8 (CCK-8) on HepG2, SMMC-7721 and HL-7702 cells. Fluorescent microscopy showed that the combining ability of [ 19 F]HX-01 to the carcinoma SMMC-7721 and HepG2 was higher than that to the normal HL-7702. Cellular proliferation inhibition assay demonstrated that [ 19 F]HX-01 leaded to a dose-dependent inhibition on SMMC-7721, HepG2, and HL-7702 proliferation. Cell cytotoxicity assay presented that the cytotoxicity of [ 19 F]HX-01 to SMMC-7721 and HepG2 was obviously higher than that to HL-7702. This in vitro study showed that [ 19 F]HX-01 had a higher selectivity on human hepatocellular carcinoma cells (SMMC-7721, HepG2) but has less toxicity to normal hepatocytes (HL-7702). This could set up the idea that the radioactive reference substance [ 18 F]HX-01 may be worthy of further development as a potential molecular probe targeting human hepatocellular carcinoma using PET.

Preparation, in vitro and in vivo evaluation of polymeric nanoparticles based on hyaluronic acid-poly(butyl cyanoacrylate) and D-alpha-tocopheryl polyethylene glycol 1000 succinate for tumor-targeted delivery of morin hydrate

PubMed Central

Abbad, Sarra; Wang, Cheng; Waddad, Ayman Yahia; Lv, Huixia; Zhou, Jianping

2015-01-01

Herein, we describe the preparation of a targeted cellular delivery system for morin hydrate (MH), based on a low-molecular-weight hyaluronic acid-poly(butyl cyanoacrylate) (HA-PBCA) block copolymer. In order to enhance the therapeutic effect of MH, D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) was mixed with HA-PBCA during the preparation process. The MH-loaded HA-PBCA “plain” nanoparticle (MH-PNs) and HA-PBCA/TPGS “mixed” nanoparticles (MH-MNs) were concomitantly characterized in terms of loading efficiency, particle size, zeta potential, critical aggregation concentration, and morphology. The obtained MH-PNs and MH-MNs exhibited a spherical morphology with a negative zeta potential and a particle size less than 200 nm, favorable for drug targeting. Remarkably, the addition of TPGS resulted in about 1.6-fold increase in drug-loading. The in vitro cell viability experiment revealed that MH-MNs enhanced the cytotoxicity of MH in A549 cells compared with MH solution and MH-PNs. Furthermore, blank MNs containing TPGS exhibited selective cytotoxic effects against cancer cells without diminishing the viability of normal cells. In addition, the cellular uptake study indicated that MNs resulted in 2.28-fold higher cellular uptake than that of PNs, in A549 cells. The CD44 receptor competitive inhibition and the internalization pathway studies suggested that the internalization mechanism of the nanoparticles was mediated mainly by the CD44 receptors through a clathrin-dependent endocytic pathway. More importantly, MH-MNs exhibited a higher in vivo antitumor potency and induced more tumor cell apoptosis than did MH-PNs, following intravenous administration to S180 tumor-bearing mice. Overall, the results imply that the developed nanoparticles are promising vehicles for the targeted delivery of lipophilic anticancer drugs. PMID:25609946

Naringin attenuates the cytotoxicity of hepatotoxin microcystin-LR by the curious mechanisms to OATP1B1- and OATP1B3-expressing cells.

PubMed

Takumi, Shota; Ikema, Satoshi; Hanyu, Tamami; Shima, Yusuke; Kurimoto, Takashi; Shiozaki, Kazuhiro; Sugiyama, Yasumasa; Park, Ho-Dong; Ando, Seiichi; Furukawa, Tatsuhiko; Komatsu, Masaharu

2015-03-01

Microcystin-LR, which is an inhibitor of serine/threonine protein phosphatase (PP)1 and PP2A, induces liver injury by its selective uptake system into the hepatocyte. It is also thought that microcystin-LR induces reactive oxygen species (ROS). We tried to establish the chemical prevention of microcystin-LR poisoning. We investigated the effect of grapefruit flavanone glycoside naringin on cytotoxicity of microcystin-LR using human hepatocyte uptake transporter OATP1B3-expressing HEK293-OATP1B3 cells. We found cytotoxicity of microcystin-LR was attenuated by naringin in a dose dependent manner. The inhibition magnitude of total cellular serine/threonine protein phosphatase activity induced by microcystin-LR was suppressed by naringin. In addition, uptake of microcystin-LR into HEK293-OATP1B3 cells was inhibited by naringin. Furthermore, microcystin-LR induced phosphorylation of p53 was inhibited by naringin. Regardless of the difference in the exposure pattern of pre-processing and post-processing of naringin, the toxicity of microcystin-LR was comparable. These results suggested that naringin is promising remedy as well as preventive medicine for liver damage with microcystin-LR. In addition, involvement of ROS production after exposure to the sublethal concentrations of microcystin-LR in the onset of cytotoxicity was negligible. Therefore, inhibition of microcystin-LR uptake and the pathway other than ROS production would be involved in the effect of naringin on the attenuation of microcystin-LR toxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

Enhanced intracellular delivery and controlled drug release of magnetic PLGA nanoparticles modified with transferrin.

PubMed

Cui, Yan-Na; Xu, Qing-Xing; Davoodi, Pooya; Wang, De-Ping; Wang, Chi-Hwa

2017-06-01

Owing to the presence of multidrug resistance in tumor cells, conventional chemotherapy remains clinically intractable. To enhance the therapeutic efficacy of chemotherapeutic agents, targeting strategies based on magnetic polymeric nanoparticles modified with targeting ligands have gained significant attention in cancer therapy. In this study, we synthesized transferrin (Tf)-modified poly(D,L-lactic-co-glycolic acid) nanoparticles (PLGA NPs) loaded with paclitaxel (PTX) and superparamagnetic nanoparticle (MNP) using a solid-in-oil-in-water solvent evaporation method, followed by Tf adsorption on the surface of NPs. The Tf-modified magnetic PLGA NPs were characterized in terms of particle morphology and size, magnetic properties, encapsulation efficiency and drug release. Furthermore, the cytotoxicity and cellular uptake of the drug-loaded magnetic PLGA NPs were evaluated in both MCF-7 breast cancer and U-87 glioma cells in vitro. We found that Tf-modified PTX-MNP-PLGA NPs showed the highest cytotoxicity effect and cellular uptake efficiency under Tf receptor mediation in both MCF-7 and U-87 cells compared to unmodified PLGA NPs and free PTX. The cellular uptake efficiency of Tf-modified magnetic PLGA NPs appeared to be facilitated by the applied magnetic field, but the difference did not reach statistical significance. This study illustrates that this proposed formulation can be used as one new alternative treatment for patients bearing inaccessible tumors.

Enhanced intracellular delivery and controlled drug release of magnetic PLGA nanoparticles modified with transferrin

PubMed Central

Cui, Yan-na; Xu, Qing-xing; Davoodi, Pooya; Wang, De-ping; Wang, Chi-Hwa

2017-01-01

Owing to the presence of multidrug resistance in tumor cells, conventional chemotherapy remains clinically intractable. To enhance the therapeutic efficacy of chemotherapeutic agents, targeting strategies based on magnetic polymeric nanoparticles modified with targeting ligands have gained significant attention in cancer therapy. In this study, we synthesized transferrin (Tf)-modified poly(D,L-lactic-co-glycolic acid) nanoparticles (PLGA NPs) loaded with paclitaxel (PTX) and superparamagnetic nanoparticle (MNP) using a solid-in-oil-in-water solvent evaporation method, followed by Tf adsorption on the surface of NPs. The Tf-modified magnetic PLGA NPs were characterized in terms of particle morphology and size, magnetic properties, encapsulation efficiency and drug release. Furthermore, the cytotoxicity and cellular uptake of the drug-loaded magnetic PLGA NPs were evaluated in both MCF-7 breast cancer and U-87 glioma cells in vitro. We found that Tf-modified PTX-MNP-PLGA NPs showed the highest cytotoxicity effect and cellular uptake efficiency under Tf receptor mediation in both MCF-7 and U-87 cells compared to unmodified PLGA NPs and free PTX. The cellular uptake efficiency of Tf-modified magnetic PLGA NPs appeared to be facilitated by the applied magnetic field, but the difference did not reach statistical significance. This study illustrates that this proposed formulation can be used as one new alternative treatment for patients bearing inaccessible tumors. PMID:28552909

Evaluation of the protective effects of curcuminoid (curcumin and bisdemethoxycurcumin)-loaded liposomes against bone turnover in a cell-based model of osteoarthritis.

PubMed

Yeh, Chih-Chang; Su, Yu-Han; Lin, Yu-Jhe; Chen, Pin-Jyun; Shi, Chung-Sheng; Chen, Cheng-Nan; Chang, Hsin-I

2015-01-01

Curcumin (Cur) and bisdemethoxycurcumin (BDMC), extracted from Curcuma longa, are poorly water-soluble polyphenol compounds that have shown anti-inflammatory potential for the treatment of osteoarthritis. To increase cellular uptake of Cur and BDMC in bone tissue, soybean phosphatidylcholines were used for liposome formulation. In this study, curcuminoid (Cur and BDMC)-loaded liposomes were characterized in terms of particle size, encapsulation efficiency, liposome stability, and cellular uptake. The results show that there is about 70% entrapment efficiency of Cur and BDMC in liposomes and that particle sizes are stable after liposome formation. Both types of liposome can inhibit macrophage inflammation and osteoclast differential activities. In comparison with free drugs (Cur and BDMC), curcuminoid-loaded liposomes were less cytotoxic and expressed high cellular uptake of the drugs. Of note is that Cur-loaded liposomes can prevent liposome-dependent inhibition of osteoblast differentiation and mineralization, but BDMC-loaded liposomes could not. With interleukin (IL)-1β stimulation, curcuminoid-loaded liposomes can successfully downregulate the expression of inflammatory markers on osteoblasts, and show a high osteoprotegerin (OPG)/receptor activator of nuclear factor κB ligand (RANKL) ratio to prevent osteoclastogenesis. In the present study, we demonstrated that Cur and BDMC can be successfully encapsulated in liposomes and can reduce osteoclast activity and maintain osteoblast functions. Therefore, curcuminoid-loaded liposomes may slow osteoarthritis progression.

The effect of oil-water partition coefficient on the distribution and cellular uptake of liposome-encapsulated gold nanoparticles.

PubMed

Bao, Quan-Ying; Liu, Ai-Yun; Ma, Yu; Chen, Huan; Hong, Jin; Shen, Wen-Bin; Zhang, Can; Ding, Ya

2016-10-01

The shape, size, and surface features of nanoparticles greatly influence the structure and properties of resulting hybrid nanosystems. In this work, gold nanoparticles (GNPs) were modified via S-Au covalent bonding by glycol monomethyl ether thioctate with poly(ethylene glycol) methyl ether of different molecular weights (i.e., 350, 550, and 750Da). These modified GNPs (i.e., GNP350, GNP550, and GNP750) showed different oil-water partition coefficients (Kp), as detected using inductively coupled plasma-atomic emission spectroscopy. The different Kp values of the gold conjugates (i.e., 13.98, 2.11, and 0.036 for GNP350, GNP550, and GNP750, respectively) resulted in different conjugate localization within liposomes, as observed by transmission electron microscopy. In addition, the cellular uptake of hybrid liposomes co-encapsulating gold conjugates and Nile red was evaluated using intracellular fluorescence intensity. The results indicated that precise GNP localization in the hydrophilic or hydrophobic liposome cavity could be achieved by regulating the GNP oil-water partition coefficient via surface modification; such localization could further affect the properties and functions of hybrid liposomes, including their cellular uptake profiles. This study furthers the understanding not only of the interaction between liposomes and inorganic nanoparticles but also of adjusting liposome-gold hybrid nanostructure properties via the surface chemistry of gold materials. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of tripolyphosphate on cellular uptake and RNA interference efficiency of chitosan-based nanoparticles in Raw 264.7 macrophages.

PubMed

Xiao, Bo; Ma, Panpan; Ma, Lijun; Chen, Qiubing; Si, Xiaoying; Walter, Lewins; Merlin, Didier

2017-03-15

Tumor necrosis factor-α (TNF-α) is a major pro-inflammatory cytokine that is mainly secreted by macrophages during inflammation. Here, we synthesized a series of N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chlorides (HTCCs), and then used a complex coacervation technique or tripolyphosphate (TPP)-assisted ionotropic gelation strategy to complex the HTCCs with TNF-α siRNA (siTNF) to form nanoparticles (NPs). The resultant NPs had a desirable particle size (210-279nm), a slightly positive zeta potential (14-22mV), and negligible cytotoxicity against Raw 264.7 macrophages and colon-26 cells. Subsequent cellular uptake tests demonstrated that the introduction of TPP to the NPs markedly increased their cellular uptake efficiency (to nearly 100%) compared with TPP-free NPs, and yielded a correspondingly higher intracellular concentration of siRNA. Critically, in vitro gene silencing experiments revealed that all of the TPP-containing NPs showed excellent efficiency in inhibiting the mRNA expression level of TNF-α (by approximately 85-92%, which was much higher than that obtained using Oligofectamine/siTNF complexes). Collectively, these results obviously suggest that our non-toxic TPP-containing chitosan-based NPs can be exploited as efficient siTNF carriers for the treatment of inflammatory diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

γ-Oryzanol Enhances Adipocyte Differentiation and Glucose Uptake

PubMed Central

Jung, Chang Hwa; Lee, Da-Hye; Ahn, Jiyun; Lee, Hyunjung; Choi, Won Hee; Jang, Young Jin; Ha, Tae-Youl

2015-01-01

Recent studies show that brown rice improves glucose intolerance and potentially the risk of diabetes, although the underlying molecular mechanisms remain unclear. One of the phytochemicals found in high concentration in brown rice is γ-oryzanol (Orz), a group of ferulic acid esters of phytosterols and triterpene alcohols. Here, we found that Orz stimulated differentiation of 3T3-L1 preadipocytes and increased the protein expression of adipogenic marker genes such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT/enhanced binding protein alpha (C/EBPα). Moreover, Orz significantly increased the glucose uptake in insulin-resistant cells and translocation of glucose transporter type 4 (GLUT4) from the cytosol to the cell surface. To investigate the mechanism by which Orz stimulated cell differentiation, we examined its effects on cellular signaling of the mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth and proliferation. The Orz treatment increased mTORC1 kinase activity based on phosphorylation of 70-kDa ribosomal S6 kinase 1 (S6K1). The effect of Orz on adipocyte differentiation was dependent on mTORC1 activity because rapamycin blocks cell differentiation in Orz-treated cells. Collectively, our results indicate that Orz stimulates adipocyte differentiation, enhances glucose uptake, and may be associated with cellular signaling mediated by PPAR-γ and mTORC1. PMID:26083118

γ-Oryzanol Enhances Adipocyte Differentiation and Glucose Uptake.

PubMed

Jung, Chang Hwa; Lee, Da-Hye; Ahn, Jiyun; Lee, Hyunjung; Choi, Won Hee; Jang, Young Jin; Ha, Tae-Youl

2015-06-15

Recent studies show that brown rice improves glucose intolerance and potentially the risk of diabetes, although the underlying molecular mechanisms remain unclear. One of the phytochemicals found in high concentration in brown rice is γ-oryzanol (Orz), a group of ferulic acid esters of phytosterols and triterpene alcohols. Here, we found that Orz stimulated differentiation of 3T3-L1 preadipocytes and increased the protein expression of adipogenic marker genes such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT/enhanced binding protein alpha (C/EBPα). Moreover, Orz significantly increased the glucose uptake in insulin-resistant cells and translocation of glucose transporter type 4 (GLUT4) from the cytosol to the cell surface. To investigate the mechanism by which Orz stimulated cell differentiation, we examined its effects on cellular signaling of the mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth and proliferation. The Orz treatment increased mTORC1 kinase activity based on phosphorylation of 70-kDa ribosomal S6 kinase 1 (S6K1). The effect of Orz on adipocyte differentiation was dependent on mTORC1 activity because rapamycin blocks cell differentiation in Orz-treated cells. Collectively, our results indicate that Orz stimulates adipocyte differentiation, enhances glucose uptake, and may be associated with cellular signaling mediated by PPAR-γ and mTORC1.

Microwave gallium-68 radiochemistry for kinetically stable bis(thiosemicarbazone) complexes: structural investigations and cellular uptake under hypoxia.

PubMed

Alam, Israt S; Arrowsmith, Rory L; Cortezon-Tamarit, Fernando; Twyman, Frazer; Kociok-Köhn, Gabriele; Botchway, Stanley W; Dilworth, Jonathan R; Carroll, Laurence; Aboagye, Eric O; Pascu, Sofia I

2016-01-07

We report the microwave synthesis of several bis(thiosemicarbazones) and the rapid gallium-68 incorporation to give the corresponding metal complexes. These proved kinetically stable under 'cold' and 'hot' biological assays and were investigated using laser scanning confocal microscopy, flow cytometry and radioactive cell retention studies under normoxia and hypoxia. (68)Ga complex retention was found to be 34% higher in hypoxic cells than in normoxic cells over 30 min, further increasing to 53% at 120 min. Our data suggests that this class of gallium complexes show hypoxia selectivity suitable for imaging in living cells and in vivo tests by microPET in nude athymic mice showed that they are excreted within 1 h of their administration.

Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

PubMed Central

Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

2015-01-01

Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment. PMID:25976444

Curcumin-loaded solid lipid nanoparticles have prolonged in vitro antitumour activity, cellular uptake and improved in vivo bioavailability.

PubMed

Sun, Jiabei; Bi, Chao; Chan, Hok Man; Sun, Shaoping; Zhang, Qingwen; Zheng, Ying

2013-11-01

The aim of the present study was to blend liquid lipids with solid lipids to encapsulate curcumin in solid lipid nanoparticles (SLNs), thereby improving the dispersibility and chemical stability of curcumin, prolonging its antitumour activity and cellular uptake and enhancing its bioavailability. Curcumin-loaded SLNs (C-SLNs) were prepared by high-pressure homogenisation with liquid lipid Sefsol-218(®). The morphology, stability and release of curcumin in the optimised formulation were investigated. The anti-cancer activity of the formulation was evaluated in MCF-7 cells. Fluorescence spectrophotometry was used to quantify cellular uptake of the drug. The pharmacokinetic profiles of curcumin in SLNs after intravenous administration were studied in rats. Blending Sefsol-218(®) into a lipid matrix reduced the particle size without improving drug loading. An optimised formulation consisting of Dynasan 114(®), Sefsol-218(®), and Pluronic F68(®) (630:70:300, w/w) loaded with 0.8% drug was prepared. This formulation could be dispersed in water with a mean particle size of 152.8 ± 4.7 nm and a 90% entrapment efficiency. Curcumin displayed a two-phase sustained release profile from C-SLNs with improved chemical stability. Compared to the solubilised solution, C-SLNs exhibited prolonged inhibitory activity in cancer cells, as well as time-dependent increases in intracellular uptake. After intravenous administration to rats, the bioavailability of curcumin was increased by 1.25-fold. C-SLNs with improved dispersibility and chemical stability in an aqueous system have been successfully developed. C-SLNs may represent a potentially useful cancer therapeutic curcumin delivery system. Copyright © 2013 Elsevier B.V. All rights reserved.

Mesoporous silica nanoparticles functionalized with hyaluronic acid. Effect of the biopolymer chain length on cell internalization.

PubMed

Nairi, Valentina; Magnolia, Silvia; Piludu, Marco; Nieddu, Mariella; Caria, Cristian Antonio; Sogos, Valeria; Vallet-Regì, Maria; Monduzzi, Maura; Salis, Andrea

2018-02-12

Mesoporous silica nanoparticles (MSNs) were functionalized with amino groups (MSN-NH 2 ) and then with hyaluronic acid, a biocompatible biopolymer which can be recognized by CD44 receptors in tumor cells, to obtain a targeting drug delivery system. To this purpose, three hyaluronic acid samples differing for the molecular weight, namely HA S (8-15 kDa), HA M (30-50 kDa) and HA L (90-130 kDa), were used. The MSN-HA S , MSN-HA M , and MSN-HA L materials were characterized through zeta potential and dynamic light scattering measurements at pH = 7.4 and T = 37 °C to simulate physiological conditions. While zeta potential showed an increasing negative value with the increase of the HA chain length, an anomalous value of the hydrodynamic diameter was observed for MSN-HA L , which was smaller than that of MSN-HA S and MSN-HA M samples. The cellular uptake of MSN-HA samples on HeLa cells at 37 °C was studied by optical and electron microscopy. HA chain length affected significantly the cellular uptake that occurred at a higher extent for MSN-NH 2 and MSN-HA S than for MSN-HA M and MSN-HA L samples. Cellular uptake experiments carried out at 4 °C showed that the internalization process was inhibited for MSN-HA samples but not for MSN-NH 2 . This suggests the occurrence of two different mechanisms of internalization. For MSN-NH 2 the uptake is mainly driven by the attractive electrostatic interaction with membrane phospholipids, while MSN-HA internalization involves CD44 receptors overexpressed in HeLa cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Increased cortical expression of the zinc transporter SLC39A12 suggests a breakdown in zinc cellular homeostasis as part of the pathophysiology of schizophrenia

PubMed Central

Scarr, Elizabeth; Udawela, Madhara; Greenough, Mark A; Neo, Jaclyn; Suk Seo, Myoung; Money, Tammie T; Upadhyay, Aradhana; Bush, Ashley I; Everall, Ian P; Thomas, Elizabeth A; Dean, Brian

2016-01-01

Our expression microarray studies showed messenger RNA (mRNA) for solute carrier family 39 (zinc transporter), member 12 (SLC39A12) was higher in dorsolateral prefrontal cortex from subjects with schizophrenia (Sz) in comparison with controls. To better understand the significance of these data we ascertained whether SLC39A12 mRNA was altered in a number of cortical regions (Brodmann’s area (BA) 8, 9, 44) from subjects with Sz, in BA 9 from subjects with mood disorders and in rats treated with antipsychotic drugs. In addition, we determined whether inducing the expression of SLC39A12 resulted in an increased cellular zinc uptake. SLC39A12 variant 1 and 2 mRNA was measured using quantitative PCR. Zinc uptake was measured in CHO cells transfected with human SLC39A12 variant 1 and 2. In Sz, compared with controls, SLC39A12 variant 1 and 2 mRNA was higher in all cortical regions studied. The were no differences in levels of mRNA for either variant of SLC39A12 in BA 9 from subjects with mood disorders and levels of mRNA for Slc39a12 was not different in the cortex of rats treated with antipsychotic drugs. Finally, expressing both variants in CHO-K1 cells was associated with an increase in radioactive zinc uptake. As increased levels of murine Slc39a12 mRNA has been shown to correlate with increasing cellular zinc uptake, our data would be consistent with the possibility of a dysregulated zinc homeostasis in the cortex of subjects with schizophrenia due to altered expression of SLC39A12. PMID:27336053

Alteration of the intravenous and oral pharmacokinetics of valsartan via the concurrent use of gemfibrozil in rats.

PubMed

Yang, Seung Jun; Kim, Bong Jin; Mo, Lingxuan; Han, Hyo-Kyung

2016-07-01

The present study aimed to examine the potential pharmacokinetic drug interaction between valsartan and gemfibrozil. Compared with the control given valsartan (10 mg/kg) alone, the concurrent use of gemfibrozil (10 mg/kg) significantly (p < 0.05) increased the oral exposure of valsartan in rats. In the presence of gemfibrozil, the Cmax and AUC of oral valsartan increased by 1.7- and 2.5-fold, respectively. Consequently, the oral bioavailability of valsartan was significantly higher (p < 0.05) in the presence of gemfibrozil compared with that of the control group. Furthermore, the intravenous pharmacokinetics of valsartan (1 mg/kg) was also altered by pretreatment with oral gemfibrozil (10 mg/kg). The plasma clearance of valsartan was decreased by two-fold in the presence of gemfibrozil, while the plasma half-life was not altered. In contrast, both the oral and intravenous pharmacokinetics of gemfibrozil were not affected by the concurrent use of valsartan. The cellular uptake of valsartan and gemfibrozil was also investigated by using cells overexpressing OATP1B1 or OATP1B3. Gemfibrozil and gemfibrozil 1-O-β glucuronide inhibited the cellular uptake of valsartan with IC50 values (µm) of 39.3 and 20.4, respectively, in MDCK/OATP1B1, while they were less interactive with OATP1B3. The cellular uptake of gemfibrozil was not affected by co-incubation with valsartan in both cells. Taken together, the present study suggests the potential drug interaction between valsartan and gemfibrozil, at least in part, via the OATP1B1-mediated transport pathways during hepatic uptake. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Formulation and in vitro characterization of protein-loaded liposomes

NASA Astrophysics Data System (ADS)

Kuzimski, Lauren

Background/Objective: Protein-based drugs are increasingly used to treat a variety of conditions including cancer and cardio-vascular disease. Due to the immune system's innate ability to degrade the foreign particles quickly, protein-based treatments are generally short-lived. To address this limitation, the objective of the study was to: 1) develop protein-loaded liposomes; 2) characterize size, stability, encapsulation efficiency and rate of protein release; and 3) determine intracellular uptake and distribution; and 4) protein structural changes. Method: Liposomes were loaded with a fluorescent-albumin using freeze-thaw (F/T) methodology. Albumin encapsulation and release were quantified by fluorescence spectroscopic techniques. Flow cytometry was used to determine liposome uptake by macrophages. Epifluorescence microscopy was used to determine cellular distribution of liposomes. Stability was determined using dynamic light scattering by measuring liposome size over one month period. Protein structure was determined using circular dichroism (CD). Result: Encapsulation of albumin in liposome was ˜90% and was dependent on F/T rates, with fifteen cycles yielding the highest encapsulation efficacy (p < 0.05). Albumin-loaded liposomes demonstrated consistent size (<300nm). Release of encapsulated albumin in physiological buffer at 25°C was ˜60% in 72 h. Fluorescence imaging suggested an endosomal route of cellular entry for the FITC-albumin liposome with maximum uptake rates in immune cells (30% at 2hour incubation). CD suggested protein structure is minimally impacted by freeze-thaw methodology. Conclusion: Using F/T as a loading method, we were able to successfully achieve a protein-loaded liposome that was under 300nm, had encapsulation of ˜90%. Synthesized liposomes demonstrated a burst release of encapsulate protein (60%) at 72 hours. Cellular trafficking confirmed endosomal uptake, and minimal protein damage was noticed in CD.

The Effect of Inflammatory Status on Butyrate and Folate Uptake by Tumoral (Caco-2) and Non-Tumoral (IEC-6) Intestinal Epithelial Cells

PubMed Central

Couto, Mafalda R.; Gonçalves, Pedro; Catarino, Telmo A.; Martel, Fátima

2017-01-01

Objective Colorectal cancer (CRC) is the second leading cause of cancer death in occidental countries. Chronic inflammatory bowel disease (crohn’s disease and ulcerative colitis) is associated with an increased risk for CRC development. The aim of this work was to investigate the relationship between inflammatory status and absorption of nutrients with a role in CRC pathogenesis. Materials and Methods In this experimental study, we evaluated the in vitro effect of tumour necrosis factor-alpha (TNF-α), interferon-γ (IF-γ), and acetylsalicylic acid on 14C-butyrate (14C- BT), 3H-folic acid (3H-FA) uptake, and on proliferation, viability and differentiation of Caco-2 and IEC-6 cells in culture. Results The proinflammatory cytokines TNF-α and INF-γ were found to decrease uptake of a low concentration of 14C-BT (10 µM) by Caco-2 (tumoral) and IEC-6 (normal) intestinal epithelial cell lines. However, the effect of TNF-α and INF-γ in IEC-6 cells is most probably related to a cytotoxic and antiproliferative impact. In contrast, INF-γ increases uptake of a high concentration (10 mM) of 14C-BT in Caco-2 cells. The anticarcinogenic effect of BT (10 mM) in these cells is not affected by the presence of this cytokine. On the other hand, acetylsalicylic acid stimulates 14C-BT uptake by Caco-2 cells and potentiates its antiproliferative effect. Finally, both TNF-α and INF-γ cause a significant decrease in 3H-FA uptake by Caco-2 cells. Conclusion The inflammatory status has an impact upon cellular uptake of BT and FA, two nutrients with a role in CRC pathogenesis. Moreover, the anti-inflammatory acetylsalicylic acid potentiates the anticarcinogenic effect of BT in Caco-2 cells by increasing its cellular uptake. PMID:28580313

Cellular response to high pulse repetition rate nanosecond pulses varies with fluorescent marker identity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steelman, Zachary A., E-mail: zachary.steelman@duke.edu; Tolstykh, Gleb P.; Beier, Hope T.

Nanosecond electric pulses (nsEP's) are a well-studied phenomena in biophysics that cause substantial alterations to cellular membrane dynamics, internal biochemistry, and cytoskeletal structure, and induce apoptotic and necrotic cell death. While several studies have attempted to measure the effects of multiple nanosecond pulses, the effect of pulse repetition rate (PRR) has received little attention, especially at frequencies greater than 100 Hz. In this study, uptake of Propidium Iodide, FM 1–43, and YO-PRO-1 fluorescent dyes in CHO-K1 cells was monitored across a wide range of PRRs (5 Hz–500 KHz) using a laser-scanning confocal microscope in order to better understand how high frequency repetition ratesmore » impact induced biophysical changes. We show that frequency trends depend on the identity of the dye under study, which could implicate transmembrane protein channels in the uptake response due to their chemical selectivity. Finally, YO-PRO-1 fluorescence was monitored in the presence of Gadolinium (Gd{sup 3+}), Ruthenium Red, and in calcium-free solution to elucidate a mechanism for its unique frequency trend. - Highlights: • Pulse repetition rate (PRR) is understudied in nanosecond electric pulsing. • 200 V pulses were applied to CHO-K1 cells from 5 Hz to 500 KHz. • Pulsing was repeated using a variety of fluorophores and imaging conditions. • The response is highly dependent on the fluorophore and the imaging conditions. • This may implicate protein channels in the nanoporation response.« less

Magnetic separation of algae genetically modified for increased intracellular iron uptake

NASA Astrophysics Data System (ADS)

Buck, Amy; Moore, Lee R.; Lane, Christopher D.; Kumar, Anil; Stroff, Clayton; White, Nicolas; Xue, Wei; Chalmers, Jeffrey J.; Zborowski, Maciej

2015-04-01

Algae were investigated in the past as a potential source of biofuel and other useful chemical derivatives. Magnetic separation of algae by iron oxide nanoparticle binding to cells has been proposed by others for dewatering of cellular mass prior to lipid extraction. We have investigated feasibility of magnetic separation based on the presence of natural iron stores in the cell, such as the ferritin in Auxenochlorella protothecoides (A. protothecoides) strains. The A. protothecoides cell constructs were tested for inserted genes and for increased intracellular iron concentration by inductively coupled plasma atomic absorption (ICP-AA). They were grown in Sueoka's modified high salt media with added vitamin B1 and increasing concentration of soluble iron compound (FeCl3 EDTA, from 1× to 8× compared to baseline). The cell magnetic separation conditions were tested using a thin rectangular flow channel pressed against interpolar gaps of a permanent magnet forming a separation system of a well-defined fluid flow and magnetic fringing field geometry (up to 2.2 T and 1000 T/m) dubbed "magnetic deposition microscopy", or MDM. The presence of magnetic cells in suspension was detected by formation of characteristic deposition bands at the edges of the magnet interpolar gaps, amenable to optical scanning and microscopic examination. The results demonstrated increasing cellular Fe uptake with increasing Fe concentration in the culture media in wild type strain and in selected genetically-modified constructs, leading to magnetic separation without magnetic particle binding. The throughput in this study is not sufficient for an economical scale harvest.

Cyclic-RGDyC functionalized liposomes for dual-targeting of tumor vasculature and cancer cells in glioblastoma: An in vitro boron neutron capture therapy study.

PubMed

Kang, Weirong; Svirskis, Darren; Sarojini, Vijayalekshmi; McGregor, Ailsa L; Bevitt, Joseph; Wu, Zimei

2017-05-30

The efficacy of boron neutron capture therapy depends on the selective delivery of 10B to the target. Integrins αvβ3 are transmembrane receptors over-expressed in both glioblastoma cells and its neovasculature. In this study, a novel approach to dual-target glioblastoma vasculature and tumor cells was investigated. Liposomes (124 nm) were conjugated with a αvβ3 ligand, cyclic arginine-glycine-aspartic acid-tyrosine-cysteine peptide (c(RGDyC)-LP) (1% molar ratio) through thiol-maleimide coupling. Expression of αvβ3 in glioblastoma cells (U87) and human umbilical vein endothelial cells (HUVEC), representing tumor angiogenesis, was determined using Western Blotting with other cells as references. The results showed that both U87 and HUVEC had stronger expression of αvβ3 than other cell types, and the degree of cellular uptake of c(RGDyC)-LP correlated with the αvβ3-expression levels of the cells. In contrast, control liposomes without c(RGDyC) showed little cellular uptake, regardless of cell type. In an in vitro boron neutron capture therapy study, the c(RGDyC)-LP containing sodium borocaptate generated more rapid and significant lethal effects to both U87 and HUVEC than the control liposomes and drug solution. Interestingly, neutron irradiated U87 and HUVEC showed different types of subsequent cell death. In conclusion, this study has demonstrated the potential of a new dual-targeting strategy using c(RGDyC)-LP to improve boron neutron capture therapy for glioblastoma.

Cyclic-RGDyC functionalized liposomes for dual-targeting of tumor vasculature and cancer cells in glioblastoma: An in vitro boron neutron capture therapy study

PubMed Central

Kang, Weirong; Svirskis, Darren; Sarojini, Vijayalekshmi; McGregor, Ailsa L.; Bevitt, Joseph; Wu, Zimei

2017-01-01

The efficacy of boron neutron capture therapy depends on the selective delivery of 10B to the target. Integrins αvβ3 are transmembrane receptors over-expressed in both glioblastoma cells and its neovasculature. In this study, a novel approach to dual-target glioblastoma vasculature and tumor cells was investigated. Liposomes (124 nm) were conjugated with a αvβ3 ligand, cyclic arginine-glycine-aspartic acid-tyrosine-cysteine peptide (c(RGDyC)-LP) (1% molar ratio) through thiol-maleimide coupling. Expression of αvβ3 in glioblastoma cells (U87) and human umbilical vein endothelial cells (HUVEC), representing tumor angiogenesis, was determined using Western Blotting with other cells as references. The results showed that both U87 and HUVEC had stronger expression of αvβ3 than other cell types, and the degree of cellular uptake of c(RGDyC)-LP correlated with the αvβ3-expression levels of the cells. In contrast, control liposomes without c(RGDyC) showed little cellular uptake, regardless of cell type. In an in vitro boron neutron capture therapy study, the c(RGDyC)-LP containing sodium borocaptate generated more rapid and significant lethal effects to both U87 and HUVEC than the control liposomes and drug solution. Interestingly, neutron irradiated U87 and HUVEC showed different types of subsequent cell death. In conclusion, this study has demonstrated the potential of a new dual-targeting strategy using c(RGDyC)-LP to improve boron neutron capture therapy for glioblastoma. PMID:28402271

Magnetic separation of algae genetically modified for increased intracellular iron uptake.

PubMed

Buck, Amy; Moore, Lee R; Lane, Christopher D; Kumar, Anil; Stroff, Clayton; White, Nicolas; Xue, Wei; Chalmers, Jeffrey J; Zborowski, Maciej

2015-04-15

Algae were investigated in the past as a potential source of biofuel and other useful chemical derivatives. Magnetic separation of algae by iron oxide nanoparticle binding to cells has been proposed by others for dewatering of cellular mass prior to lipid extraction. We have investigated feasibility of magnetic separation based on the presence of natural iron stores in the cell, such as the ferritin in Auxenochlorella protothecoides ( A. p. ) strains. The A. p. cell constructs were tested for inserted genes and for increased intracellular iron concentration by inductively coupled plasma atomic absorption (ICP-AA). They were grown in Sueoka's modified high salt media with added vitamin B1 and increasing concentration of soluble iron compound (FeCl 3 EDTA, from 1× to 8× compared to baseline). The cell magnetic separation conditions were tested using a thin rectangular flow channel pressed against interpolar gaps of a permanent magnet forming a separation system of a well-defined fluid flow and magnetic fringing field geometry (up to 2.2 T and 1,000 T/m) dubbed "magnetic deposition microscopy", or MDM. The presence of magnetic cells in suspension was detected by formation of characteristic deposition bands at the edges of the magnet interpolar gaps, amenable to optical scanning and microscopic examination. The results demonstrated increasing cellular Fe uptake with increasing Fe concentration in the culture media in wild type strain and in selected genetically-modified constructs, leading to magnetic separation without magnetic particle binding. The throughput in this study is not sufficient for an economical scale harvest.

Magnetic separation of algae genetically modified for increased intracellular iron uptake

PubMed Central

Buck, Amy; Moore, Lee R.; Lane, Christopher D.; Kumar, Anil; Stroff, Clayton; White, Nicolas; Xue, Wei; Chalmers, Jeffrey J.; Zborowski, Maciej

2017-01-01

Algae were investigated in the past as a potential source of biofuel and other useful chemical derivatives. Magnetic separation of algae by iron oxide nanoparticle binding to cells has been proposed by others for dewatering of cellular mass prior to lipid extraction. We have investigated feasibility of magnetic separation based on the presence of natural iron stores in the cell, such as the ferritin in Auxenochlorella protothecoides (A. p.) strains. The A. p. cell constructs were tested for inserted genes and for increased intracellular iron concentration by inductively coupled plasma atomic absorption (ICP-AA). They were grown in Sueoka's modified high salt media with added vitamin B1 and increasing concentration of soluble iron compound (FeCl3 EDTA, from 1× to 8× compared to baseline). The cell magnetic separation conditions were tested using a thin rectangular flow channel pressed against interpolar gaps of a permanent magnet forming a separation system of a well-defined fluid flow and magnetic fringing field geometry (up to 2.2 T and 1,000 T/m) dubbed “magnetic deposition microscopy”, or MDM. The presence of magnetic cells in suspension was detected by formation of characteristic deposition bands at the edges of the magnet interpolar gaps, amenable to optical scanning and microscopic examination. The results demonstrated increasing cellular Fe uptake with increasing Fe concentration in the culture media in wild type strain and in selected genetically-modified constructs, leading to magnetic separation without magnetic particle binding. The throughput in this study is not sufficient for an economical scale harvest. PMID:29353957

Size and surface functionalization of iron oxide nanoparticles influence the composition and dynamic nature of their protein corona.

PubMed

Ashby, Jonathan; Pan, Songqin; Zhong, Wenwan

2014-09-10

Nanoparticles (NPs) adsorb proteins when in the biological matrix, and the resulted protein corona could affect NP-cell interactions. The corona has a dynamic nature with the adsorbed proteins constantly exchanging with the free proteins in the matrix at various rates. The rapidly exchanging proteins compose the soft corona, which responds more dynamically to environment changes than the hard corona established by the ones with slow exchange rates. In the present study, the corona formed on the superparamagnetic iron oxide NPs (SPIONs) in human serum was studied by flow field-flow fractionation and ultracentrifugation, which rapidly differentiated the corona proteins based on their exchange rates. By varying the surface hydrophobicity of the SPIONs with a core size around 10 nm, we found out that, the more hydrophobic surface ligand attracted proteins with higher surface hydrophobicity and formed a more dynamic corona with a larger portion of the involved proteins with fast exchange rates. Increasing the core diameter of the SPIONs but keeping the surface ligand the same could also result in a more dynamic corona. A brief investigation of the effect on the cellular uptake of SPIONs using one selected corona protein, transferrin, was conducted. The result showed that, only the stably bound transferrin could significantly enhance cellular uptake, while transferrin bound in a dynamic nature had negligible impact. Our study has led to a better understanding of the relationship between the particle properties and the dynamic nature of the corona, which can help with design of nanomaterials with higher biocompatibility and higher efficacy in biosystems for biomedical applications.

Size and Surface Functionalization of Iron Oxide Nanoparticles Influence the Composition and Dynamic Nature of Their Protein Corona

PubMed Central

2015-01-01

Nanoparticles (NPs) adsorb proteins when in the biological matrix, and the resulted protein corona could affect NP-cell interactions. The corona has a dynamic nature with the adsorbed proteins constantly exchanging with the free proteins in the matrix at various rates. The rapidly exchanging proteins compose the soft corona, which responds more dynamically to environment changes than the hard corona established by the ones with slow exchange rates. In the present study, the corona formed on the superparamagnetic iron oxide NPs (SPIONs) in human serum was studied by flow field-flow fractionation and ultracentrifugation, which rapidly differentiated the corona proteins based on their exchange rates. By varying the surface hydrophobicity of the SPIONs with a core size around 10 nm, we found out that, the more hydrophobic surface ligand attracted proteins with higher surface hydrophobicity and formed a more dynamic corona with a larger portion of the involved proteins with fast exchange rates. Increasing the core diameter of the SPIONs but keeping the surface ligand the same could also result in a more dynamic corona. A brief investigation of the effect on the cellular uptake of SPIONs using one selected corona protein, transferrin, was conducted. The result showed that, only the stably bound transferrin could significantly enhance cellular uptake, while transferrin bound in a dynamic nature had negligible impact. Our study has led to a better understanding of the relationship between the particle properties and the dynamic nature of the corona, which can help with design of nanomaterials with higher biocompatibility and higher efficacy in biosystems for biomedical applications. PMID:25144382

Pseudolaric acid B attenuates atherosclerosis progression and inflammation by suppressing PPARγ-mediated NF-κB activation.

PubMed

Li, Tan; Wang, Wei; Li, Yu-Xiu; Li, Xiao; Ji, Wen-Jie; Ma, Yong-Qiang; Chen, Hong; Zhao, Ji-Hong; Zhou, Xin

2018-06-01

Atherosclerosis is a progressive disease of large arteries characterized with chronic inflammation and aberrant immune response. Pseudolaric acid B (PB) has been found to exert multiple effects by inhibiting inflammatory response. However, there is no comprehensive assessment of the effects of PB on atherosclerosis using relevant in vivo and in vitro models. Male ApoE -/- mice were treated with PB orally with a high fat diet (HFD) to clarify its anti-atherosclerotic activities. RAW264.7 macrophage line, a well-accepted cell model of atherosclerosis, was used to investigate anti-inflammatory effects and molecular mechanisms of PB. PB significantly attenuated atherosclerotic lesions by modulating plasma lipid profiles as well as inhibiting inflammatory responses in macrophages of atherosclerotic mice. Meanwhile, PB markedly suppressed the expression of pro-inflammatory cytokines, and regulated cholesterol efflux related genes in oxidative low density lipoprotein (ox-LDL)-loaded macrophages. The cellular uptake of Dil-labeled ox-LDL was significantly inhibited by PB either. Moreover, the ability of PB to suppress nuclear factor kappa B (NF-κB) and activate peroxisome proliferator-activated receptor gamma (PPARγ) was confirmed using luciferase reporter assays. Conversely, the selective PPARγ antagonist GW9662 reversed the influence of PB in macrophages. Together, these findings indicate that PB exerts its protective effects on atherosclerosis by inhibiting macrophage-mediated inflammatory response and cellular ox-LDL uptake, and promoting cholesterol efflux by suppressing NF-κB activation PPARγ-dependently. Therefore, PB may be a promising agent for inflammatory and atherosclerotic diseases. Copyright © 2018. Published by Elsevier B.V.

The prion-ZIP connection: From cousins to partners in iron uptake

PubMed Central

Singh, Neena; Asthana, Abhishek; Baksi, Shounak; Desai, Vilok; Haldar, Swati; Hari, Sahi; Tripathi, Ajai K

2015-01-01

ABSTRACT Converging observations from disparate lines of inquiry are beginning to clarify the cause of brain iron dyshomeostasis in sporadic Creutzfeldt-Jakob disease (sCJD), a neurodegenerative condition associated with the conversion of prion protein (PrPC), a plasma membrane glycoprotein, from α-helical to a β-sheet rich PrP-scrapie (PrPSc) isoform. Biochemical evidence indicates that PrPC facilitates cellular iron uptake by functioning as a membrane-bound ferrireductase (FR), an activity necessary for the transport of iron across biological membranes through metal transporters. An entirely different experimental approach reveals an evolutionary link between PrPC and the Zrt, Irt-like protein (ZIP) family, a group of proteins involved in the transport of zinc, iron, and manganese across the plasma membrane. Close physical proximity of PrPC with certain members of the ZIP family on the plasma membrane and increased uptake of extracellular iron by cells that co-express PrPC and ZIP14 suggest that PrPC functions as a FR partner for certain members of this family. The connection between PrPC and ZIP proteins therefore extends beyond common ancestry to that of functional cooperation. Here, we summarize evidence supporting the facilitative role of PrPC in cellular iron uptake, and implications of this activity on iron metabolism in sCJD brains. PMID:26689487

Cytotoxicity and cellular uptake of different sized gold nanoparticles in ovarian cancer cells

NASA Astrophysics Data System (ADS)

Kumar, Dhiraj; Mutreja, Isha; Chitcholtan, Kenny; Sykes, Peter

2017-11-01

Nanomedicine has advanced the biomedical field with the availability of multifunctional nanoparticles (NPs) systems that can target a disease site enabling drug delivery and helping to monitor the disease. In this paper, we synthesised the gold nanoparticles (AuNPs) with an average size 18, 40, 60 and 80 nm, and studied the effect of nanoparticles size, concentration and incubation time on ovarian cancer cells namely, OVCAR5, OVCAR8, and SKOV3. The size measured by transmission electron microscopy images was slightly smaller than the hydrodynamic diameter; measured size by ImageJ as 14.55, 38.13, 56.88 and 78.56 nm. The cellular uptake was significantly controlled by the AuNPs size, concentration, and the cell type. The nanoparticles uptake increased with increasing concentration, and 18 and 80 nm AuNPs showed higher uptake ranging from 1.3 to 5.4 μg depending upon the concentration and cell type. The AuNPs were associated with a temporary reduction in metabolic activity, but metabolic activity remained more than 60% for all sample types; NPs significantly affected the cell proliferation activity in first 12 h. The increase in nanoparticle size and concentration induced the production of reactive oxygen species in 24 h.

Glycyrrhetinic Acid Liposomes Containing Mannose-Diester Lauric Diacid-Cholesterol Conjugate Synthesized by Lipase-Catalytic Acylation for Liver-Specific Delivery.

PubMed

Chen, Jing; Chen, Yuchao; Cheng, Yi; Gao, Youheng

2017-09-24

Mannose-diester lauric diacid-cholesterol (Man-DLD-Chol), as a liposomal target ligand, was synthesized by lipase catalyzed in a non-aqueous medium. Its chemical structure was confirmed by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Glycyrrhetinic acid (GA) liposomes containing Man-DLD-Chol (Man-DLD-Chol-GA-Lp) were prepared by the film-dispersion method. We evaluated the characterizations of liposomes, drug-release in vitro, the hemolytic test, cellular uptake, pharmacokinetics, and the tissue distributions. The cellular uptake in vitro suggested that the uptake of Man-DLD-Chol-modified liposomes was significantly higher than that of unmodified liposomes in HepG2 cells. Pharmacokinetic parameters indicated that Man-DLD-Chol-GA-Lp was eliminated more rapidly than GA-Lp. In tissue distributions, the targeting efficiency (Te) of Man-DLD-Chol-GA-Lp on liver was 54.67%, relative targeting efficiency (R Te ) was 3.39, relative uptake rate (Re) was 4.78, and peak concentration ratio (Ce) was 3.46. All these results supported the hypothesis that Man-DLD-Chol would be an efficient liposomal carrier, and demonstrated that Man-DLD-Chol-GA-Lp has potential as a drug delivery for liver-targeting therapy.

Influence of inhibitors of serotonin uptake on intestinal epithelium and colorectal carcinomas.

PubMed

Tutton, P J; Barkla, D H

1982-08-01

Previous studies have shown that in certain tissues, including colonic carcinomas, cell proliferation may be promoted by serotonin, and indirect evidence suggests that the effects of this amine on colonic tumours involves a cellular-uptake mechanism. In the present study, two specific inhibitors of serotonin uptake, Citalopram and Fluoxetine, are examined for their effects on cell proliferation and tumour growth. Each of the agents was found to suppress cell division in dimethylhydrazine-induced colonic tumours in rats, and to retard the growth of 2 out of 3 lines of human colonic tumours propagated as xenografts in immune-deprived mice.

Steap4 Plays a Critical Role in Osteoclastogenesis in Vitro by Regulating Cellular Iron/Reactive Oxygen Species (ROS) Levels and cAMP Response Element-binding Protein (CREB) Activation*

PubMed Central

Zhou, Jian; Ye, Shiqiao; Fujiwara, Toshifumi; Manolagas, Stavros C.; Zhao, Haibo

2013-01-01

Iron is essential for osteoclast differentiation, and iron overload in a variety of hematologic diseases is associated with excessive bone resorption. Iron uptake by osteoclast precursors via the transferrin cycle increases mitochondrial biogenesis, reactive oxygen species production, and activation of cAMP response element-binding protein, a critical transcription factor downstream of receptor activator of NF-κB-ligand-induced calcium signaling. These changes are required for the differentiation of osteoclast precursors to mature bone-resorbing osteoclasts. However, the molecular mechanisms regulating cellular iron metabolism in osteoclasts remain largely unknown. In this report, we provide evidence that Steap4, a member of the six-transmembrane epithelial antigen of prostate (Steap) family proteins, is an endosomal ferrireductase with a critical role in cellular iron utilization in osteoclasts. Specifically, we show that Steap4 is the only Steap family protein that is up-regulated during osteoclast differentiation. Knocking down Steap4 expression in vitro by lentivirus-mediated short hairpin RNAs inhibits osteoclast formation and decreases cellular ferrous iron, reactive oxygen species, and the activation of cAMP response element-binding protein. These results demonstrate that Steap4 is a critical enzyme for cellular iron uptake and utilization in osteoclasts and, thus, indispensable for osteoclast development and function. PMID:23990467

Effects of vitamin E supplementation on cellular α-tocopherol concentrations of neutrophils in Holstein calves

PubMed Central

Higuchi, Hidetoshi; Ito, Erina; Iwano, Hidetoma; Oikawa, Shin; Nagahata, Hajime

2013-01-01

The effects of vitamin E supplementation on cellular α-tocopherol concentrations of neutrophils from Holstein calves and the mechanism of scavenger receptor class B type I (SR-BI)-mediated uptake of α-tocopherol were examined. Cellular α-tocopherol concentrations in vitamin E-treated calves increased from 3.5 ± 0.38 to 7.2 ± 0.84 μg/107 cells, respectively, within 14 d after vitamin E supplementation; these concentrations were significantly higher than those of control calves (P < 0.01). The expression indices of SR-BI [a major receptor that recognizes high-density lipoprotein (HDL)] mRNA in neutrophils were two to five times higher (P < 0.01) in neutrophils obtained from vitamin E-supplemented calves compared with those from control calves, and anti-SR-B1 antibody, ranging from 0.1 to 1.0 μg/mL, significantly (P < 0.01) decreased cellular α-tocopherol concentrations of neutrophils. Cytochalasin D and latrunculin B, major inhibitors of actin polymerization of neutrophils, significantly decreased cellular α-tocopherol concentrations of neutrophils (P < 0.01). Our results demonstrated that in vitamin E-supplemented calves: 1) α-tocopherol is mainly distributed with HDL, 2) α-tocopherol within HDL is recognized by SR-BI on the surface of neutrophils, and 3) rearrangement of the actin cytoskeleton is a crucial step for the uptake of α-tocopherol by neutrophils. PMID:24082403

Enhancing the cellular uptake of siRNA duplexes following noncovalent packaging with protein transduction domain peptides.

PubMed

Meade, Bryan R; Dowdy, Steven F

2008-03-01

The major limitation in utilizing information rich macromolecules for basic science and therapeutic applications is the inability of these large molecules to readily diffuse across the cellular membrane. While this restriction represents an efficient defense system against cellular penetration of unwanted foreign molecules and thus a crucial component of cell survival, overcoming this cellular characteristic for the intracellular delivery of macromolecules has been the focus of a large number of research groups worldwide. Recently, with the discovery of RNA interference, many of these groups have redirected their attention and have applied previously characterized cell delivery methodologies to synthetic short interfering RNA duplexes (siRNA). Protein transduction domain and cell penetrating peptides have been shown to enhance the delivery of multiple types of macromolecular cargo including peptides, proteins and antisense oligonucleotides and are now being utilized to enhance the cellular uptake of siRNA molecules. The dense cationic charge of these peptides that is critical for interaction with cell membrane components prior to internalization has also been shown to readily package siRNA molecules into stable nanoparticles that are capable of traversing the cell membrane. This review discusses the recent advances in noncovalent packaging of siRNA molecules with cationic peptides and the potential for the resulting complexes to successfully induce RNA interference within both in vitro and in vivo settings.

Fluorous Peptide Nucleic Acids: PNA Analogues with Fluorine in Backbone (γ-CF2-apg-PNA) Enhance Cellular Uptake.

PubMed

Ellipilli, Satheesh; Ganesh, Krishna N

2015-09-18

Fluorous PNA analogues possessing fluorine as inherent part of aminopropylglycine (apg) backbone (γ-CF2-apg PNA) have been synthesized and evaluated for biophysical and cell penetrating properties. These form duplexes of higher thermal stability with cRNA than cDNA, although destabilized compared to duplexes of standard aeg-PNA. Cellular uptake of the fluorinated γ-CF2-apg PNAs in NIH 3T3 and HeLa cells was 2-3-fold higher compared to that of nonfluorinated apg PNA, with NIH 3T3 cells showing better permeability compared to HeLa cells. The backbone fluorinated PNAs, which are first in this class, when combined with other chemical modifications may have potential for future PNA-based antisense agents.

Targeting dendritic cells through gold nanoparticles: A review on the cellular uptake and subsequent immunological properties.

PubMed

Ahmad, Suhana; Zamry, Anes Ateqah; Tan, Hern-Tze Tina; Wong, Kah Keng; Lim, JitKang; Mohamud, Rohimah

2017-11-01

Gold nanoparticles (NPs) have been proposed as a highly potential tool in immunotherapies due to its advantageous properties including customizable size and shapes, surface functionality and biocompatibility. Dendritic cells (DCs), the sentinels of immune response, have been of interest to be manipulated by using gold NPs for targeted delivery of immunotherapeutic agent. Researches done especially in human DCs showed a variation of gold NPs effects on cellular uptake and internalization, DC maturation and subsequent T cells priming as well as cytotoxicity. In this review, we describe the synthesis and physiochemical properties of gold NPs as well as the importance of gold NPs in immunotherapies through their actions on human DCs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Surface sulfonamide modification of poly(N-isopropylacrylamide)-based block copolymer micelles to alter pH and temperature responsive properties for controlled intracellular uptake.

PubMed

Cyphert, Erika L; von Recum, Horst A; Yamato, Masayuki; Nakayama, Masamichi

2018-06-01

Two different surface sulfonamide-functionalized poly(N-isopropylacrylamide)-based polymeric micelles were designed as pH-/temperature-responsive vehicles. Both sulfadimethoxine- and sulfamethazine-surface functionalized micelles were characterized to determine physicochemical properties, hydrodynamic diameters, zeta potentials, temperature-dependent size changes, and lower critical solution temperatures (LCST) in both pH 7.4 and 6.8 solutions (simulating both physiological and mild low pH conditions), and tested in the incorporation of a proof-of-concept hydrophobic antiproliferative drug, paclitaxel. Cellular uptake studies were conducted using bovine carotid endothelial cells and fluorescently labeled micelles to evaluate if there was enhanced cellular uptake of the micelles in a low pH environment. Both variations of micelles showed enhanced intracellular uptake under mildly acidic (pH 6.8) conditions at temperatures slightly above their LCST and minimal uptake at physiological (pH 7.4) conditions. Due to the less negative zeta potential of the sulfamethazine-surface micelles compared to sulfadimethoxine-surface micelles, and the proximity of their LCST to physiological temperature (37°C), the sulfamethazine variation was deemed more amenable for clinically relevant temperature and pH-stimulated applications. Nevertheless, we believe both polymeric micelle variations have the capacity to be implemented as an intracellular drug or gene delivery system in response to mildly acidic conditions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1552-1560, 2018. © 2018 Wiley Periodicals, Inc.

Zirconium phosphate nanoplatelets: a biocompatible nanomaterial for drug delivery to cancer

NASA Astrophysics Data System (ADS)

Saxena, Vipin; Diaz, Agustin; Clearfield, Abraham; Batteas, James D.; Hussain, Muhammad Delwar

2013-02-01

The objective of this study was to evaluate the biocompatibility of zirconium phosphate (ZrP) nanoplatelets (NPs), and their use in drug delivery. ZrP and doxorubicin-intercalated ZrP (DOX:ZrP) NPs were characterized by using X-Ray Powder Diffraction (XRPD), Thermogravimetric Analysis (TGA), Transmission Electron Micrography (TEM), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Biocompatibility of ZrP NPs was evaluated in human embryonic kidney (HEK-293), breast cancer (MCF-7), metastatic breast cancer (MDA-MB-231), ovarian cancer (OVCAR-3), resistant cancer (NCI-RES/ADR) cells and mouse macrophage (RAW 264.7) cell lines. Hemocompatibility of ZrP NPs was evaluated with human red blood cells. Simulated body fluid (SBF) of pH 7.4 was used to determine the in vitro release of doxorubicin from DOX:ZrP NPs. Cellular uptake and in vitro cytotoxicity studies of DOX:ZrP NPs were determined in MDA-MB-231. The ZrP nanomaterial can be prepared in the 100-200 nm size range with a platelet-like shape. The ZrP NPs themselves are biocompatible, hemocompatible and showed no toxicity to the macrophage cells. ZrP NPs can intercalate high loads (35% w/w) of doxorubicin between their layers. The release of DOX was sustained for about 2 weeks. DOX:ZrP NPs showed higher cellular uptake and increased cytotoxicity than free DOX in MDA-MB-231 cells. ZrP NPs are highly biocompatible, can intercalate large amounts of drugs and sustain the release of drugs. ZrP NPs improved the cellular uptake and cytotoxicity of DOX to MDA-MB-231 cells. ZrP NPs are promising nanocarriers for drug delivery in cancer therapy.The objective of this study was to evaluate the biocompatibility of zirconium phosphate (ZrP) nanoplatelets (NPs), and their use in drug delivery. ZrP and doxorubicin-intercalated ZrP (DOX:ZrP) NPs were characterized by using X-Ray Powder Diffraction (XRPD), Thermogravimetric Analysis (TGA), Transmission Electron Micrography (TEM), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Biocompatibility of ZrP NPs was evaluated in human embryonic kidney (HEK-293), breast cancer (MCF-7), metastatic breast cancer (MDA-MB-231), ovarian cancer (OVCAR-3), resistant cancer (NCI-RES/ADR) cells and mouse macrophage (RAW 264.7) cell lines. Hemocompatibility of ZrP NPs was evaluated with human red blood cells. Simulated body fluid (SBF) of pH 7.4 was used to determine the in vitro release of doxorubicin from DOX:ZrP NPs. Cellular uptake and in vitro cytotoxicity studies of DOX:ZrP NPs were determined in MDA-MB-231. The ZrP nanomaterial can be prepared in the 100-200 nm size range with a platelet-like shape. The ZrP NPs themselves are biocompatible, hemocompatible and showed no toxicity to the macrophage cells. ZrP NPs can intercalate high loads (35% w/w) of doxorubicin between their layers. The release of DOX was sustained for about 2 weeks. DOX:ZrP NPs showed higher cellular uptake and increased cytotoxicity than free DOX in MDA-MB-231 cells. ZrP NPs are highly biocompatible, can intercalate large amounts of drugs and sustain the release of drugs. ZrP NPs improved the cellular uptake and cytotoxicity of DOX to MDA-MB-231 cells. ZrP NPs are promising nanocarriers for drug delivery in cancer therapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr34242e

Impact of ocean phytoplankton diversity on phosphate uptake

PubMed Central

Lomas, Michael W.; Bonachela, Juan A.; Levin, Simon A.; Martiny, Adam C.

2014-01-01

We have a limited understanding of the consequences of variations in microbial biodiversity on ocean ecosystem functioning and global biogeochemical cycles. A core process is macronutrient uptake by microorganisms, as the uptake of nutrients controls ocean CO2 fixation rates in many regions. Here, we ask whether variations in ocean phytoplankton biodiversity lead to novel functional relationships between environmental variability and phosphate (Pi) uptake. We analyzed Pi uptake capabilities and cellular allocations among phytoplankton groups and the whole community throughout the extremely Pi-depleted western North Atlantic Ocean. Pi uptake capabilities of individual populations were well described by a classic uptake function but displayed adaptive differences in uptake capabilities that depend on cell size and nutrient availability. Using an eco-evolutionary model as well as observations of in situ uptake across the region, we confirmed that differences among populations lead to previously uncharacterized relationships between ambient Pi concentrations and uptake. Supported by novel theory, this work provides a robust empirical basis for describing and understanding assimilation of limiting nutrients in the oceans. Thus, it demonstrates that microbial biodiversity, beyond cell size, is important for understanding the global cycling of nutrients. PMID:25422472

The effect of nanoparticle size on in vivo pharmacokinetics and cellular interaction

PubMed Central

Hoshyar, Nazanin; Gray, Samantha; Han, Hongbin; Bao, Gang

2016-01-01

Nanoparticle-based technologies offer exciting new approaches to disease diagnostics and therapeutics. To take advantage of unique properties of nanoscale materials and structures, the size, shape and/or surface chemistry of nanoparticles need to be optimized, allowing their functionalities to be tailored for different biomedical applications. Here we review the effects of nanoparticle size on cellular interaction and in vivo pharmacokinetics, including cellular uptake, biodistribution and circulation half-life of nanoparticles. Important features of nanoparticle probes for molecular imaging and modeling of nanoparticle size effects are also discussed. PMID:27003448

Ecological Uptake and Depuration of Carbon Nanotubes by Lumbriculus variegatus

PubMed Central

Petersen, Elijah J.; Huang, Qingguo; Weber, Walter J.

2008-01-01

Background Carbon nanotubes represent a class of nanomaterials having broad application potentials and documented cellular uptake and ecotoxicological effects that raise the possibility that they may bioaccumulate in living organisms. Objectives Radioactively labeled nanotubes were synthesized using a novel methane chemical vapor deposition procedure. Single-walled carbon nanotubes (SWNTs), multiwalled carbon nanotubes (MWNTs), and pyrene were spiked to sediment samples, and the respective uptake and depuration of these nanotubes and pyrene were assessed by the oligochaete, Lumbriculus variegatus. Results 14C-labeled carbon nanotubes were developed for these experiments to overcome significant previous limitations for quantifying nanotube materials in environmental and biological media. Biota-sediment accumulation factors for SWNTs and MWNTs were observed to be almost an order of magnitude lower than those for pyrene, a four-ringed polycyclic aromatic hydrocarbon (PAH). The depuration behaviors of the oligochaete suggested that the nanotubes detected in these organisms were associated with sediments remaining in the organism guts and not absorbed into cellular tissues as was the pyrene. The results suggest that, unlike PAHs, purified carbon nanotubes do not readily absorb into organism tissues. PMID:18414633

Cationic nanoemulsions as potential carriers for intracellular delivery

PubMed Central

Khachane, P.V.; Jain, A.S.; Dhawan, V.V.; Joshi, G.V.; Date, A.A.; Mulherkar, R.; Nagarsenker, M.S.

2014-01-01

Successful cytosolic delivery enables opportunities for improved treatment of various genetic disorders, infectious diseases and cancer. Cationic nanoemulsions were designed using alternative excipients and evaluated for particle size, charge, effect of sterilization on its stability, DNA condensation potential and cellular uptake efficiency. Various concentrations of non-ionic and ionic stabilizers were evaluated to design formula for colloidally stable cationic nanoemulsion. The nanoemulsion comprised of 5% Capmul MCM, 0.5% didodecyldimethylammonium bromide (DDAB), 1% phospholipid, 1% Poloxamer 188 and 2.25% glycerol and possessed particle size of 81.6 ± 3.56 nm and 137.1 ± 1.57 nm before and after steam sterilization, respectively. DNA condensation studies were carried out at various nanoemulsion: DNA ratios ranging from 1:1 to 10:1. Cell uptake studies were conducted on human embryonic kidney (HEK) cell lines which are widely reported for transfection studies. The nanoemulsions showed excellent cellular uptake as evaluated by fluorescence microscopy and flow cytometry. Overall, a colloidally stable cationic nanoemulsion with good DNA condensation ability was successfully fabricated for efficient cytosolic delivery and potential for in vivo effectiveness. PMID:25972740

Distinct Akt phosphorylation states are required for insulin regulated Glut4 and Glut1-mediated glucose uptake.

PubMed

Beg, Muheeb; Abdullah, Nazish; Thowfeik, Fathima Shazna; Altorki, Nasser K; McGraw, Timothy E

2017-06-07

Insulin, downstream of Akt activation, promotes glucose uptake into fat and muscle cells to lower postprandial blood glucose, an enforced change in cellular metabolism to maintain glucose homeostasis. This effect is mediated by the Glut4 glucose transporter. Growth factors also enhance glucose uptake to fuel an anabolic metabolism required for tissue growth and repair. This activity is predominantly mediated by the Glut1. Akt is activated by phosphorylation of its kinase and hydrophobic motif (HM) domains. We show that insulin-stimulated Glut4-mediated glucose uptake requires PDPK1 phosphorylation of the kinase domain but not mTORC2 phosphorylation of the HM domain. Nonetheless, an intact HM domain is required for Glut4-mediated glucose uptake. Whereas, Glut1-mediated glucose uptake also requires mTORC2 phosphorylation of the HM domain, demonstrating both phosphorylation-dependent and independent roles of the HM domain in regulating glucose uptake. Thus, mTORC2 links Akt to the distinct physiologic programs related to Glut4 and Glut1-mediated glucose uptake.

A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans.

PubMed

Knowlton, Wendy M; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D; Jin, Yishi

2017-01-01

The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo , we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1 . Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7 , and isp-1 , and the putative oxidoreductase rad-8 . In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1 . Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the cellular adaptations used by neurons under conditions of injury.

A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans

PubMed Central

Knowlton, Wendy M.; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D.; Jin, Yishi

2017-01-01

The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo, we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1. Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7, and isp-1, and the putative oxidoreductase rad-8. In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1. Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the cellular adaptations used by neurons under conditions of injury. PMID:28539870

Effect of Tumor Microenvironment on Selective Uptake of Boric Acid in HepG2 Human Hepatoma Cells.

PubMed

Bai, Yu-Chi; Hsia, Yu-Chun; Lin, Yu-Ting; Chen, Kuan-Hao; Chou, Fong-In; Yang, Chia-Min; Chuang, Yung-Jen

2017-11-01

Feasibility and efficacy of boric acid (BA)-mediated boron neutron capture therapy (BNCT) was first demonstrated by eliminating hepatocellular carcinoma (HCC) in a rat model. Furthermore, selective uptake of BA by liver tumor cells was shown in a rabbit model. To gain further insight, this study aimed to investigate the mechanisms of transportation and selective uptake of BA in HepG2 liver tumor cells. Transportation of BA in HepG2 cells was analyzed by time-course assays and by analyzing the rate of diffusion versus the concentration of BA. The effect of different tumor conditions on BA uptake was studied by treating HepG2 cells with 25 μg 10 B/ml BA under different concentrations of glucose, at different pH and in the presence of water-soluble cholesterol. HepG2 cells mainly uptake BA by simple diffusion. Cell membrane permeability may also contribute to tumor-specific uptake of BA. The selective uptake of BA was achieved primarily by diffusion, while other factors, such as low pH and increased membrane fluidity, which are hallmarks of HCC, might further enhance BA uptake. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Functional analysis RaZIP1 transporter of the ZIP family from the ectomycorrhizal Zn-accumulating Russula atropurpurea.

PubMed

Leonhardt, Tereza; Sácký, Jan; Kotrba, Pavel

2018-04-01

A search of R. atropurpurea transcriptome for sequences encoding the transporters of the Zrt-, Irt-like Protein (ZIP) family, which are in eukaryotes integral to Zn supply into cytoplasm, allowed the identification of RaZIP1 cDNA with a predicted product belonging to ZIP I subfamily; it was subjected to functional studies in mutant Saccharomyces cerevisiae strains. The expression of RaZIP1, but not RaZIP1 H208A or RaZIP1 H232A mutants lacking conserved-among-ZIPs transmembrane histidyls, complemented Zn uptake deficiency in zrt1Δzrt2Δ yeasts. RaZIP1 substantially increased cellular Zn uptake in this strain and added to Zn sensitivity in zrc1Δcot1Δ mutant. The Fe uptake deficiency in ftr1Δ strain was not rescued and Mn uptake was insufficient for toxicity in Mn-sensitive pmr1Δ yeasts. By contrast, RaZIP1 increased Cd sensitivity in yap1Δ strain and conferred Cd transport activity in yeasts, albeit with substantially lower efficiency compared to Zn transport. In metal uptake assays, the accumulation of Zn in zrt1Δzrt2Δ strain remained unaffected by Cd, Fe, and Mn present in 20-fold molar excess over Zn. Immunofluorescence microscopy detected functional hemagglutinin-tagged HA::RaZIP1 on the yeast cell protoplast periphery. Altogether, these data indicate that RaZIP1 is a high-affinity plasma membrane transporter specialized in Zn uptake, and improve the understanding of the cellular and molecular biology of Zn in R. atropurpurea that is known for its ability to accumulate remarkably high concentrations of Zn.

Molecular and functional characterization of riboflavin specific transport system in rat brain capillary endothelial cells.

PubMed

Patel, Mitesh; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Mitra, Ashim K

2012-08-15

Riboflavin is an important water soluble vitamin (B2) required for metabolic reactions, normal cellular growth, differentiation and function. Mammalian brain cells cannot synthesize riboflavin and must import from systemic circulation. However, the uptake mechanism, cellular translocation and intracellular trafficking of riboflavin in brain capillary endothelial cells are poorly understood. The primary objective of this study is to investigate the existence of a riboflavin-specific transport system and delineate the uptake and intracellular regulation of riboflavin in immortalized rat brain capillary endothelial cells (RBE4). The uptake of [3H]-riboflavin is sodium, temperature and energy dependent but pH independent. [3H]-Riboflavin uptake is saturable with K(m) and V(max) values of 19 ± 3 μM and 0.235 ± 0.012 pmol/min/mg protein, respectively. The uptake process is inhibited by unlabelled structural analogs (lumiflavin, lumichrome) but not by structurally unrelated vitamins. Ca(++)/calmodulin and protein kinase A (PKA) pathways are found to play an important role in the intracellular regulation of [3H]-riboflavin. Apical and baso-lateral uptake of [3H]-riboflavin clearly indicates that a riboflavin specific transport system is predominantly localized on the apical side of RBE4 cells. A 628 bp band corresponding to a riboflavin transporter is revealed in RT-PCR analysis. These findings, for the first time report the existence of a specialized and high affinity transport system for riboflavin in RBE4 cells. The blood-brain barrier (BBB) is a major obstacle limiting drug transport inside the brain as it regulates drug permeation from systemic circulation. This transporter can be utilized for targeted delivery in enhancing brain permeation of highly potent drugs on systemic administration. Copyright © 2012 Elsevier B.V. All rights reserved.

Effects of the Monoamine Uptake Inhibitors RTI-112 and RTI-113 on Cocaine- and Food-Maintained Responding in Rhesus Monkeys

PubMed Central

SS, Negus; NK, Mello; HL, Kimmel; LL, Howell; FI, Carroll

2009-01-01

Cocaine blocks uptake of the monoamines dopamine, serotonin and norepinephrine, and monoamine uptake inhibitors constitute one class of drugs under consideration as candidate “agonist” medications for the treatment of cocaine abuse and dependence. The pharmacological selectivity of monoamine uptake inhibitors to block uptake of dopamine, serotonin and norepinephrine is one factor that may influence the efficacy and/or safety of these compounds as drug abuse treatment medications. To address this issue, the present study compared the effects of 7-day treatment with a non-selective monoamine uptake inhibitor (RTI-112) and a dopamine-selective uptake inhibitor (RTI-113) on cocaine- and food-maintained responding in rhesus monkeys. Monkeys (N=3) were trained to respond for cocaine injections (0.01 mg/kg/inj) and food pellets under a second-order schedule [FR2(VR16:S)] during alternating daily components of cocaine and food availability. Both RTI-112 (0.0032–0.01 mg/kg/hr) and RTI-113 (0.01–0.056 mg/kg/hr) produced dose-dependent, sustained and nearly complete elimination of cocaine self-administration. However, for both drugs, the potency to reduce cocaine self-administration was similar to the potency to reduce food-maintained responding. These findings do not support the hypothesis that pharmacological selectivity to block dopamine uptake is associated with behavioral selectivity to decrease cocaine- vs. food-maintained responding in rhesus monkeys. PMID:18755212

Manipulating the antigen-specific immune response by the hydrophobicity of amphiphilic poly(γ-glutamic acid) nanoparticles.

PubMed

Shima, Fumiaki; Akagi, Takami; Uto, Tomofumi; Akashi, Mitsuru

2013-12-01

The new generation vaccines are safe but poorly immunogenic, and thus they require the use of adjuvants. However, conventional vaccine adjuvants fail to induce potent cellular immunity, and their toxicity and side-effects hinder the clinical use. Therefore, a vaccine adjuvant which is safe and can induce an antigen-specific cellular immunity-biased immune response is urgently required. In the development of nanoparticle-based vaccine adjuvants, the hydrophobicity is one of the most important factors. It could control the interaction between the encapsulated antigens and/or nanoparticles with immune cells. In this study, nanoparticles (NPs) composed of amphiphilic poly(γ-glutamic acid)-graft-L-phenylalanine ethyl ester (γ-PGA-Phe) with various grafting degrees of hydrophobic side chains were prepared to evaluate the effect of hydrophobicity of vaccine carriers on the antigen encapsulation behavior, cellular uptake, activation of dendritic cells (DCs), and induction of antigen-specific cellular immunity-biased immune responses. These NPs could efficiently encapsulate antigens, and the uptake amount of the encapsulated antigen by DCs was dependent on the hydrophobicity of γ-PGA-Phe NPs. Moreover, the activation potential of the DCs and the induction of antigen-specific cellular immunity were correlated with the hydrophobicity of γ-PGA-Phe NPs. By controlling the hydrophobicity of antigen-encapsulated γ-PGA-Phe NPs, the activation potential of DCs was able to manipulate about 5 to 30-hold than the conventional vaccine, and the cellular immunity was about 10 to 40-hold. These results suggest that the hydrophobicity of NPs is a key factor for changing the interaction between NPs and immune cells, and thus the induction of cellular immunity-biased immune response could be achieved by controlling the hydrophobicity of them. Copyright © 2013 Elsevier Ltd. All rights reserved.

Human HOXA5 homeodomain enhances protein transduction and its application to vascular inflammation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ji Young; Park, Kyoung sook; Cho, Eun Jung

2011-07-01

Highlights: {yields} We have developed an E. coli protein expression vector including human specific gene sequences for protein cellular delivery. {yields} The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence. {yields} HOXA5-APE1/Ref-1 inhibited TNF-alpha-induced monocyte adhesion to endothelial cells. {yields} Human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins. -- Abstract: Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including humanmore » specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1 min and its transduction reached a maximum at 1 h within cell lysates. The cellular uptake of HOXA5-EGFP at 37 {sup o}C was greater than in 4 {sup o}C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100 nM) inhibited TNF-{alpha}-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.« less

Increasing the Accuracy of Volume and ADC Delineation for Heterogeneous Tumor on Diffusion-Weighted MRI: Correlation with PET/CT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gong, Nan-Jie; Wong, Chun-Sing, E-mail: drcswong@gmail.com; Chu, Yiu-Ching

2013-10-01

Purpose: To improve the accuracy of volume and apparent diffusion coefficient (ADC) measurements in diffusion-weighted magnetic resonance imaging (MRI), we proposed a method based on thresholding both the b0 images and the ADC maps. Methods and Materials: In 21 heterogeneous lesions from patients with metastatic gastrointestinal stromal tumors (GIST), gross lesion were manually contoured, and corresponding volumes and ADCs were denoted as gross tumor volume (GTV) and gross ADC (ADC{sub g}), respectively. Using a k-means clustering algorithm, the probable high-cellularity tumor tissues were selected based on b0 images and ADC maps. ADC and volume of the tissues selected using themore » proposed method were denoted as thresholded ADC (ADC{sub thr}) and high-cellularity tumor volume (HCTV), respectively. The metabolic tumor volume (MTV) in positron emission tomography (PET)/computed tomography (CT) was measured using 40% maximum standard uptake value (SUV{sub max}) as the lower threshold, and corresponding mean SUV (SUV{sub mean}) was also measured. Results: HCTV had excellent concordance with MTV according to Pearson's correlation (r=0.984, P<.001) and linear regression (slope = 1.085, intercept = −4.731). In contrast, GTV overestimated the volume and differed significantly from MTV (P=.005). ADC{sub thr} correlated significantly and strongly with SUV{sub mean} (r=−0.807, P<.001) and SUV{sub max} (r=−0.843, P<.001); both were stronger than those of ADC{sub g}. Conclusions: The proposed lesion-adaptive semiautomatic method can help segment high-cellularity tissues that match hypermetabolic tissues in PET/CT and enables more accurate volume and ADC delineation on diffusion-weighted MR images of GIST.« less

Improved antimicrobial therapy with cationic tetra- and octa-substituted phthalocyanines

NASA Astrophysics Data System (ADS)

Angelov, I.; Mantareva, V.; Kussovski, V.; Woehrle, D.; Borisova, E.; Avramov, L.

2008-12-01

Photodynamic therapy (PDT) today is an innovative and not yet widespread light-drug initiated treatment that is based on the photoactive compound irradiated with proper light to produce oxygen species that are toxic to the pathogenic biological objects- bacteria, viruses, tumor cells. The obstacles that limited the efficacy of PDT concern to the selectivity and multi-drug resistance prolong time for cellular release and side effects of skin photosensitivity for commercial porphyrin originated photosensitizers (PS). Now there are very intensive investigations for introducing in practice a new, with a least side effects PSs for PDT. The usefulness of the more extended macromolecules structured with proper substituents refers not only to the improved optical properties like far-red and with intensive absorption and emission capacity, but mainly to the ability for selective delivery and adhesion to the target cells, such as bacteria or other pathogens. The present study focuses on the charge effect of photodynamic agent on the uptake capacity toward gram-negative bacteria cells and their further photoinactivation. The multi-drug resistant microorganism Aeromanas hydrophilla, which is causing diseases to fishes and humans, is treated. The new octa-cationic phthalocyanines are designed to compare PDT efficacy to the efficacy of tetra-substituted derivatives with the same functional peripheral substituents. The higher cellular accumulation to the bacteria cells as a result of the high number of positive charges of photosensitizer, leading to the better adhesion to the cellular membranes and improved photoinactivation of bacteria causing superficial and intraorgan infections. These results set a base of a rationale design of covalently octa-substituted phthalocyanines with positive charge for a successful treatment of microorganisms.

SWEET sugar transporters for phloem transport and pathogen nutrition.

PubMed

Chen, Li-Qing

2014-03-01

Many intercellular solute transport processes require an apoplasmic step, that is, efflux from one cell and subsequent uptake by an adjacent cell. Cellular uptake transporters have been identified for many solutes, including sucrose; however, efflux transporters have remained elusive for a long time. Cellular efflux of sugars plays essential roles in many processes, such as sugar efflux as the first step in phloem loading, sugar efflux for nectar secretion, and sugar efflux for supplying symbionts such as mycorrhiza, and maternal efflux for filial tissue development. Furthermore, sugar efflux systems can be hijacked by pathogens for access to nutrition from hosts. Mutations that block recruitment of the efflux mechanism by the pathogen thus cause pathogen resistance. Until recently, little was known regarding the underlying mechanism of sugar efflux. The identification of sugar efflux carriers, SWEETs (Sugars Will Eventually be Exported Transporters), has shed light on cellular sugar efflux. SWEETs appear to function as uniporters, facilitating diffusion of sugars across cell membranes. Indeed, SWEETs probably mediate sucrose efflux from putative phloem parenchyma into the phloem apoplasm, a key step proceeding phloem loading. Engineering of SWEET mutants using transcriptional activator-like effector nuclease (TALEN)-based genomic editing allowed the engineering of pathogen resistance. The widespread expression of the SWEET family promises to provide insights into many other cellular efflux mechanisms.

Cell source determines the immunological impact of biomimetic nanoparticles.

PubMed

Evangelopoulos, Michael; Parodi, Alessandro; Martinez, Jonathan O; Yazdi, Iman K; Cevenini, Armando; van de Ven, Anne L; Quattrocchi, Nicoletta; Boada, Christian; Taghipour, Nima; Corbo, Claudia; Brown, Brandon S; Scaria, Shilpa; Liu, Xuewu; Ferrari, Mauro; Tasciotti, Ennio

2016-03-01

Recently, engineering the surface of nanotherapeutics with biologics to provide them with superior biocompatibility and targeting towards pathological tissues has gained significant popularity. Although the functionalization of drug delivery vectors with cellular materials has been shown to provide synthetic particles with unique biological properties, these approaches may have undesirable immunological repercussions upon systemic administration. Herein, we comparatively analyzed unmodified multistage nanovectors and particles functionalized with murine and human leukocyte cellular membrane, dubbed Leukolike Vectors (LLV), and the immunological effects that may arise in vitro and in vivo. Previously, LLV demonstrated an avoidance of opsonization and phagocytosis, in addition to superior targeting of inflammation and prolonged circulation. In this work, we performed a comprehensive evaluation of the importance of the source of cellular membrane in increasing their systemic tolerance and minimizing an inflammatory response. Time-lapse microscopy revealed LLV developed using a cellular coating derived from a murine (i.e., syngeneic) source resulted in an active avoidance of uptake by macrophage cells. Additionally, LLV composed of a murine membrane were found to have decreased uptake in the liver with no significant effect on hepatic function. As biomimicry continues to develop, this work demonstrates the necessity to consider the source of biological material in the development of future drug delivery carriers. Copyright © 2015. Published by Elsevier Ltd.

Targeting tumor highly-expressed LAT1 transporter with amino acid-modified nanoparticles: Toward a novel active targeting strategy in breast cancer therapy.

PubMed

Li, Lin; Di, Xingsheng; Wu, Mingrui; Sun, Zhisu; Zhong, Lu; Wang, Yongjun; Fu, Qiang; Kan, Qiming; Sun, Jin; He, Zhonggui

2017-04-01

Designing active targeting nanocarriers with increased cellular accumulation of chemotherapeutic agents is a promising strategy in cancer therapy. Herein, we report a novel active targeting strategy based on the large amino acid transporter 1 (LAT1) overexpressed in a variety of cancers. Glutamate was conjugated to polyoxyethylene stearate as a targeting ligand to achieve LAT1-targeting PLGA nanoparticles. The targeting efficiency of nanoparticles was investigated in HeLa and MCF-7 cells. Significant increase in cellular uptake and cytotoxicity was observed in LAT1-targeting nanoparticles compared to the unmodified ones. More interestingly, the internalized LAT1 together with targeting nanoparticles could recycle back to the cell membrane within 3 h, guaranteeing sufficient transporters on cell membrane for continuous cellular uptake. The LAT1 targeting nanoparticles exhibited better tumor accumulation and antitumor effects. These results suggested that the overexpressed LAT1 on cancer cells holds a great potential to be a high-efficiency target for the rational design of active-targeting nanosystems. Copyright © 2016 Elsevier Inc. All rights reserved.

Fundamentals of pulmonary drug delivery.

PubMed

Groneberg, D A; Witt, C; Wagner, U; Chung, K F; Fischer, A

2003-04-01

Aerosol administration of peptide-based drugs plays an important role in the treatment of pulmonary and systemic diseases and the unique cellular properties of airway epithelium offers a great potential to deliver new compounds. As the relative contributions from the large airways to the alveolar space are important to the local and systemic availability, the sites and mechanism of uptake and transport of different target compounds have to be characterized. Among the different respiratory cells, the ciliated epithelial cells of the larger and smaller airways and the type I and type II pneumocytes are the key players in pulmonary drug transport. With their diverse cellular characteristics, each of these cell types displays a unique uptake possibility. Next to the knowledge of these cellular aspects, the nature of aerosolized drugs, characteristics of delivery systems and the depositional and pulmonary clearance mechanisms display major targets to optimize pulmonary drug delivery. Based on the growing knowledge on pulmonary cell biology and pathophysiology due to modern methods of molecular biology, the future characterization of pulmonary drug transport pathways can lead to new strategies in aerosol drug therapy.

Enhanced splicing correction effect by an oligo-aspartic acid-PNA conjugate and cationic carrier complexes.

PubMed

Bae, Yun Mi; Kim, Myung Hee; Yu, Gwang Sig; Um, Bong Ho; Park, Hee Kyung; Lee, Hyun-il; Lee, Kang Taek; Suh, Yung Doug; Choi, Joon Sig

2014-02-10

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA. They recognize specific cellular nucleic acid sequences and form stable complexes with complementary DNA or RNA. Here, we designed an oligo-aspartic acid-PNA conjugate and showed its enhanced delivery into cells with high gene correction efficiency using conventional cationic carriers, such as polyethylenimine (PEI) and Lipofectamine 2000. The negatively charged oligo-aspartic acid-PNA (Asp(n)-PNA) formed complexes with PEI and Lipofectamine, and the resulting Asp(n)-PNA/PEI and Asp(n)-PNA/Lipofectamine complexes were introduced into cells. We observed significantly enhanced cellular uptake of Asp(n)-PNA by cationic carriers and detected an active splicing correction effect even at nanomolar concentrations. We found that the splicing correction efficiency of the complex depended on the kind of the cationic carriers and on the number of repeating aspartic acid units. By enhancing the cellular uptake efficiency of PNAs, these results may provide a novel platform technology of PNAs as bioactive substances for their biological and therapeutic applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Multi-functionality Redefined with Colloidal Carotene Carbon Nanoparticles for Synchronized Chemical Imaging, Enriched Cellular Uptake and Therapy.

PubMed

Misra, Santosh K; Mukherjee, Prabuddha; Chang, Huei-Huei; Tiwari, Saumya; Gryka, Mark; Bhargava, Rohit; Pan, Dipanjan

2016-07-11

Typically, multiplexing high nanoparticle uptake, imaging, and therapy requires careful integration of three different functions of a multiscale molecular-particle assembly. Here, we present a simpler approach to multiplexing by utilizing one component of the system for multiple functions. Specifically, we successfully synthesized and characterized colloidal carotene carbon nanoparticle (C(3)-NP), in which a single functional molecule served a threefold purpose. First, the presence of carotene moieties promoted the passage of the particle through the cell membrane and into the cells. Second, the ligand acted as a potent detrimental moiety for cancer cells and, finally, the ligands produced optical contrast for robust microscopic detection in complex cellular environments. In comparative tests, C(3)-NP were found to provide effective intracellular delivery that enables both robust detection at cellular and tissue level and presents significant therapeutic potential without altering the mechanism of intracellular action of β-carotene. Surface coating of C(3) with phospholipid was used to generate C(3)-Lipocoat nanoparticles with further improved function and biocompatibility, paving the path to eventual in vivo studies.

Multi-functionality Redefined with Colloidal Carotene Carbon Nanoparticles for Synchronized Chemical Imaging, Enriched Cellular Uptake and Therapy

PubMed Central

Misra, Santosh K.; Mukherjee, Prabuddha; Chang, Huei-Huei; Tiwari, Saumya; Gryka, Mark; Bhargava, Rohit; Pan, Dipanjan

2016-01-01

Typically, multiplexing high nanoparticle uptake, imaging, and therapy requires careful integration of three different functions of a multiscale molecular-particle assembly. Here, we present a simpler approach to multiplexing by utilizing one component of the system for multiple functions. Specifically, we successfully synthesized and characterized colloidal carotene carbon nanoparticle (C3-NP), in which a single functional molecule served a threefold purpose. First, the presence of carotene moieties promoted the passage of the particle through the cell membrane and into the cells. Second, the ligand acted as a potent detrimental moiety for cancer cells and, finally, the ligands produced optical contrast for robust microscopic detection in complex cellular environments. In comparative tests, C3-NP were found to provide effective intracellular delivery that enables both robust detection at cellular and tissue level and presents significant therapeutic potential without altering the mechanism of intracellular action of β-carotene. Surface coating of C3 with phospholipid was used to generate C3-Lipocoat nanoparticles with further improved function and biocompatibility, paving the path to eventual in vivo studies. PMID:27405011

Multi-functionality Redefined with Colloidal Carotene Carbon Nanoparticles for Synchronized Chemical Imaging, Enriched Cellular Uptake and Therapy

NASA Astrophysics Data System (ADS)

Misra, Santosh K.; Mukherjee, Prabuddha; Chang, Huei-Huei; Tiwari, Saumya; Gryka, Mark; Bhargava, Rohit; Pan, Dipanjan

2016-07-01

Typically, multiplexing high nanoparticle uptake, imaging, and therapy requires careful integration of three different functions of a multiscale molecular-particle assembly. Here, we present a simpler approach to multiplexing by utilizing one component of the system for multiple functions. Specifically, we successfully synthesized and characterized colloidal carotene carbon nanoparticle (C3-NP), in which a single functional molecule served a threefold purpose. First, the presence of carotene moieties promoted the passage of the particle through the cell membrane and into the cells. Second, the ligand acted as a potent detrimental moiety for cancer cells and, finally, the ligands produced optical contrast for robust microscopic detection in complex cellular environments. In comparative tests, C3-NP were found to provide effective intracellular delivery that enables both robust detection at cellular and tissue level and presents significant therapeutic potential without altering the mechanism of intracellular action of β-carotene. Surface coating of C3 with phospholipid was used to generate C3-Lipocoat nanoparticles with further improved function and biocompatibility, paving the path to eventual in vivo studies.

New nanomicelle curcumin formulation for ocular delivery: improved stability, solubility, and ocular anti-inflammatory treatment.

PubMed

Li, Mengshuang; Xin, Meng; Guo, Chuanlong; Lin, Guiming; Wu, Xianggen

2017-11-01

A stable topical ophthalmic curcumin formulation with high solubility, stability, and efficacy is needed for pharmaceutical use in clinics. The objective of this article was to describe a novel curcumin containing a nanomicelle formulation using a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol (PVCL-PVA-PEG) graft copolymer. Nanomicelle curcumin was formulated and optimized and then further evaluated for in vitro cytotoxicity/in vivo ocular irritation, in vitro cellular uptake/in vivo corneal permeation, and in vitro antioxidant activity/in vivo anti-inflammatory efficacy. The solubility, chemical stability, and antioxidant activity were greatly improved after the encapsulation of the PVCL-PVA-PEG nanomicelles. The nanomicelle curcumin ophthalmic solution was simple to prepare and the nanomicelles are stable to the storage conditions, and it had good cellular tolerance. Nanomicelle curcumin also had excellent ocular tolerance in rabbits. The use of nanomicelles significantly improved in vitro cellular uptake and in vivo corneal permeation as well as improved anti-inflammatory efficacy when compared with a free curcumin solution. These findings indicate that nanomicelles could be promising topical delivery systems for the ocular administration of curcumin.

Understanding the effect of alkyl chains of gemini cations on the physicochemical and cellular properties of polyurethane micelles.

PubMed

Pan, Zhicheng; Fang, Danxuan; Song, Yuanqing; Song, Nijia; Ding, Mingming; Li, Jiehua; Luo, Feng; Li, Jianshu; Tan, Hong; Fu, Qiang

2018-06-06

Cationic gemini quaternary ammonium (GQA) has been used as a cell internalization promoter to improve the permeability of the cell membrane and enhance the cellular uptake. However, the effect of the alkyl chain length on the cellular properties of nanocarriers has not been elucidated yet. In this study, we developed a series of polyurethane micelles containing GQAs with various alkyl chain lengths. The alteration of the gemini alkyl chain length was found to change the distribution of GQA surfactants in the micellar structure and affect the surface charge exposure, stability, and the protein absorption properties of nanocarriers. Moreover, we also clarified the role of the alkyl chain length in tumor cell internalization and macrophage uptake of polyurethane micelles. This work provides a new understanding on the effect of the GQA alkyl chain length on the physicochemical and biological properties of nanomedicines, and offers guidance on the rational design of effective drug delivery systems where the issue of functional group exposure at the micellar surface should be considered.

Semen amyloids participate in spermatozoa selection and clearance

PubMed Central

Roan, Nadia R; Sandi-Monroy, Nathallie; Kohgadai, Nargis; Usmani, Shariq M; Hamil, Katherine G; Neidleman, Jason; Montano, Mauricio; Ständker, Ludger; Röcker, Annika; Cavrois, Marielle; Rosen, Jared; Marson, Kara; Smith, James F; Pilcher, Christopher D; Gagsteiger, Friedrich; Sakk, Olena; O’Rand, Michael; Lishko, Polina V; Kirchhoff, Frank

2017-01-01

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens. DOI: http://dx.doi.org/10.7554/eLife.24888.001 PMID:28653619

Platinum nanozymes recover cellular ROS homeostasis in an oxidative stress-mediated disease model

NASA Astrophysics Data System (ADS)

Moglianetti, Mauro; de Luca, Elisa; Pedone, Deborah; Marotta, Roberto; Catelani, Tiziano; Sartori, Barbara; Amenitsch, Heinz; Retta, Saverio Francesco; Pompa, Pier Paolo

2016-02-01

In recent years, the use of nanomaterials as biomimetic enzymes has attracted great interest. In this work, we show the potential of biocompatible platinum nanoparticles (Pt NPs) as antioxidant nanozymes, which combine abundant cellular internalization and efficient scavenging activity of cellular reactive oxygen species (ROS), thus simultaneously integrating the functions of nanocarriers and antioxidant drugs. Careful toxicity assessment and intracellular tracking of Pt NPs proved their cytocompatibility and high cellular uptake, with compartmentalization within the endo/lysosomal vesicles. We have demonstrated that Pt NPs possess strong and broad antioxidant properties, acting as superoxide dismutase, catalase, and peroxidase enzymes, with similar or even superior performance than natural enzymes, along with higher adaptability to the changes in environmental conditions. We then exploited their potent activity as radical scavenging materials in a cellular model of an oxidative stress-related disorder, namely human Cerebral Cavernous Malformation (CCM) disease, which is associated with a significant increase in intracellular ROS levels. Noteworthily, we found that Pt nanozymes can efficiently reduce ROS levels, completely restoring the cellular physiological homeostasis.In recent years, the use of nanomaterials as biomimetic enzymes has attracted great interest. In this work, we show the potential of biocompatible platinum nanoparticles (Pt NPs) as antioxidant nanozymes, which combine abundant cellular internalization and efficient scavenging activity of cellular reactive oxygen species (ROS), thus simultaneously integrating the functions of nanocarriers and antioxidant drugs. Careful toxicity assessment and intracellular tracking of Pt NPs proved their cytocompatibility and high cellular uptake, with compartmentalization within the endo/lysosomal vesicles. We have demonstrated that Pt NPs possess strong and broad antioxidant properties, acting as superoxide dismutase, catalase, and peroxidase enzymes, with similar or even superior performance than natural enzymes, along with higher adaptability to the changes in environmental conditions. We then exploited their potent activity as radical scavenging materials in a cellular model of an oxidative stress-related disorder, namely human Cerebral Cavernous Malformation (CCM) disease, which is associated with a significant increase in intracellular ROS levels. Noteworthily, we found that Pt nanozymes can efficiently reduce ROS levels, completely restoring the cellular physiological homeostasis. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08358c

Influence of inhibitors of serotonin uptake on intestinal epithelium and colorectal carcinomas.

PubMed Central

Tutton, P. J.; Barkla, D. H.

1982-01-01

Previous studies have shown that in certain tissues, including colonic carcinomas, cell proliferation may be promoted by serotonin, and indirect evidence suggests that the effects of this amine on colonic tumours involves a cellular-uptake mechanism. In the present study, two specific inhibitors of serotonin uptake, Citalopram and Fluoxetine, are examined for their effects on cell proliferation and tumour growth. Each of the agents was found to suppress cell division in dimethylhydrazine-induced colonic tumours in rats, and to retard the growth of 2 out of 3 lines of human colonic tumours propagated as xenografts in immune-deprived mice. PMID:6983886

Radionuclide therapy using ¹³¹I-labeled anti-epidermal growth factor receptor-targeted nanoparticles suppresses cancer cell growth caused by EGFR overexpression.

PubMed

Li, Wei; Liu, Zhongyun; Li, Chengxia; Li, Ning; Fang, Lei; Chang, Jin; Tan, Jian

2016-03-01

Anti-epidermal growth factor receptor (EGFR)-targeted nanoparticles can be used to deliver a therapeutic and imaging agent to EGFR-overexpressing tumor cells. (131)I-labeled anti-EGFR nanoparticles derived from cetuximab were used as a tumor-targeting vehicle in radionuclide therapy. This paper describes the construction of the anti-EGFR nanoparticle EGFR-BSA-PCL. This nanoparticle was characterized for EGFR-targeted binding and cellular uptake in EGFR-overexpressing cancer cells by using flow cytometry and confocal microscopy. Anti-EGFR and non-targeted nanoparticles were labeled with (131)I using the chloramine-T method. Analyses of cytotoxicity and targeted cell killing with (131)I were performed using the MTT assay. The time-dependent cellular uptake of (131)I-labeled anti-EGFR nanoparticles proved the slow-release effects of nanoparticles. A radioiodine therapy study was also performed in mice. The EGFR-targeted nanoparticle EGFR-BSA-PCL and the non-targeted nanoparticle BSA-PCL were constructed; the effective diameters were approximately 100 nm. The results from flow cytometry and confocal microscopy revealed significant uptake of EGFR-BSA-PCL in EGFR-overexpressing tumor cells. Compared with EGFR-BSA-PCL, BSA-PCL could also bind to cells, but tumor cell retention was minimal and weak. In MTT assays, the EGFR-targeted radioactive nanoparticle (131)I-EGFR-BSA-PCL showed greater cytotoxicity and targeted cell killing than the non-targeted nanoparticle (131)I-BSA-PCL. The radioiodine uptake of both (131)I-labeled nanoparticles, (131)I-EGFR-BSA-PCL and (131)I-BSA-PCL, was rapid and reached maximal levels 4 h after incubation, but the (131)I uptake of (131)I-EGFR-BSA-PCL was higher than that of (131)I-BSA-PCL. On day 15, the average tumor volumes of the (131)I-EGFR-BSA-PCL and (131)I-BSA-PCL groups showed a slow growth relationship compared with that of the control group. The EGFR-targeted nanoparticle EGFR-BSA-PCL demonstrated superior cellular binding and uptake compared with those of the control BSA-PCL. The EGFR-targeted radioactive nanoparticle (131)I-EGFR-BSA-PCL exhibited favorable intracellular retention of (131)I. Radionuclide therapy using (131)I-EGFR-BSA-PCL, which showed excellent targeted cell killing, suppressed cancer cell growth caused by EGFR overexpression.

Multiphoton luminescent graphene quantum dots for in vivo tracking of human adipose-derived stem cells

NASA Astrophysics Data System (ADS)

Kim, Jin; Song, Sung Ho; Jin, Yoonhee; Park, Hyun-Ji; Yoon, Hyewon; Jeon, Seokwoo; Cho, Seung-Woo

2016-04-01

The applicability of graphene quantum dots (GQDs) for the in vitro and in vivo live imaging and tracking of different types of human stem cells is investigated. GQDs synthesized by the modified graphite intercalated compound method show efficient cellular uptake with improved biocompatibility and highly sensitive optical properties, indicating their feasibility as a bio-imaging probe for stem cell therapy.The applicability of graphene quantum dots (GQDs) for the in vitro and in vivo live imaging and tracking of different types of human stem cells is investigated. GQDs synthesized by the modified graphite intercalated compound method show efficient cellular uptake with improved biocompatibility and highly sensitive optical properties, indicating their feasibility as a bio-imaging probe for stem cell therapy. Electronic supplementary information (ESI) available: Additional results. See DOI: 10.1039/c6nr02143c

Spontaneous confocal Raman microscopy--a tool to study the uptake of nanoparticles and carbon nanotubes into cells

NASA Astrophysics Data System (ADS)

Romero, Gabriela; Rojas, Elena; Estrela-Lopis, Irina; Donath, Edwin; Moya, Sergio Enrique

2011-06-01

Confocal Raman microscopy as a label-free technique was applied to study the uptake and internalization of poly(lactide- co-glycolide) (PLGA) nanoparticles (NPs) and carbon nanotubes (CNTs) into hepatocarcinoma human HepG2 cells. Spontaneous confocal Raman spectra was recorded from the cells exposed to oxidized CNTs and to PLGA NPs. The Raman spectra showed bands arising from the cellular environment: lipids, proteins, nucleic acids, as well as bands characteristic for either PLGA NPs or CNTs. The simultaneous generation of Raman bands from the cell and nanomaterials from the same spot proves internalization, and also indicates the cellular region, where the nanomaterial is located. For PLGA NPs, it was found that they preferentially co-localized with lipid bodies, while the oxidized CNTs are located in the cytoplasm.

Chirality-dependent cellular uptake of chiral nanocarriers and intracellular delivery of different amounts of guest molecules

NASA Astrophysics Data System (ADS)

Kehr, Nermin Seda; Jose, Joachim

2017-12-01

We demonstrate the organic molecules loaded and chiral polymers coated periodic mesoporous organosilica (PMO) to generate chiral nanocarriers that we used to study chirality-dependent cellular uptake in serum and serum-free media and the subsequent delivery of different amounts of organic molecules into cells. Our results show that the amount of internalized PMO and thus the transported amount of organic molecules by nanocarrier PMO into cells was chirality dependent and controlled by hard/soft protein corona formation on the PMO surfaces. Therefore, this study demonstrate that chiral porous nanocarriers could potentially be used as advanced drug delivery systems which are able to use the specific chiral surface-protein interactions to influence/control the amount of (bio)active molecules delivered to cells in drug delivery and/or imaging applications.

Tetanus toxoid-loaded layer-by-layer nanoassemblies for efficient systemic, mucosal, and cellular immunostimulatory response following oral administration.

PubMed

Harde, Harshad; Agrawal, Ashish Kumar; Jain, Sanyog

2015-10-01

The present study reports the tetanus toxoid (TT)-loaded layer-by-layer nanoassemblies (layersomes) with enhanced protection, permeation, and presentation for comprehensive oral immunization. The stable and lyophilized TT-loaded layersomes were prepared by a thin-film hydration method followed by alternate layer-by-layer coating of an electrolyte. The developed system was assessed for in vitro stability of antigen and formulation, cellular uptake, ex vivo intestinal uptake, and immunostimulatory response using a suitable experimental protocol. Layersomes improved the stability in simulated biological media as well as protected the integrity/conformation and native 3D structure of TT as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorescence spectroscopy, respectively. The cell culture studies demonstrated a 3.8-fold higher permeation of layersomes in Caco-2 cells and an 8.5-fold higher uptake by antigen-presenting cells (RAW 264.7). The TT-loaded layersomes elicited a complete immunostimulatory profile consisting of higher systemic (serum IgG titer), mucosal (sIgA titer), and cellular (interleukin-2 (IL-2) and interferon-γ (IFN-γ) levels) immune response after peroral administration in mice. The modified TT inhibition assay further confirmed the elicitation of complete protective levels of anti-TT antibody (>0.1 IU/mL) by layersomes. In conclusion, the proposed strategy is expected to contribute significantly in the field of stable liposome technology for mass immunization through the oral route.

On the mechanism of Cr (VI)-induced carcinogenesis: dose dependence of uptake and cellular responses.

PubMed

Liu, K; Husler, J; Ye, J; Leonard, S S; Cutler, D; Chen, F; Wang, S; Zhang, Z; Ding, M; Wang, L; Shi, X

2001-06-01

Cr (VI) compounds are widely used industrial chemicals and are recognized human carcinogens. The mechanisms of carcinogenesis associated with these compounds remain to be investigated. The present study focused on dose-dependence of Cr (VI)-induced uptake and cellular responses. The results show that Cr (VI) is able to enter the cells (human lung epithelial cell line A549) at low concentration (< 10 microM) and that the Cr (VI) uptake appears to be a combination of saturable transport and passive diffusion. Electron spin resonance (ESR) trapping measurements showed that upon stimulation with Cr (VI), A549 cells were able to generate reactive oxygen species (ROS). The amount of ROS generated depended on the Cr (VI) concentration. ROS generation involved NADPH-dependent flavoenzymes. Cr (VI) affected the following cellular parameters in a dose-dependent manner, (a) activation of nuclear transcription factors NF-kappaB, and p53, (b) DNA damage, (c) induction of cell apoptosis, and (d) inhibition of cell proliferation. The activation of transcription factors was assessed by electrophoretic mobility shift assay and western blot analysis, DNA damage by single cell gel electrophoresis assay, cell apoptosis by DNA fragmentation assay, and cell proliferation by a non-radioactive ELISA kit. At the concentration range used in the present study, no thresholds were found in all of these cell responses to Cr (VI). The results may guide further research to better understand and evaluate the risk of Cr (VI)-induced carcinogenesis at low levels of exposure.

Design of curcumin-loaded PLGA nanoparticles formulation with enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo.

PubMed

Anand, Preetha; Nair, Hareesh B; Sung, Bokyung; Kunnumakkara, Ajaikumar B; Yadav, Vivek R; Tekmal, Rajeshwar R; Aggarwal, Bharat B

2010-02-01

Curcumin, a yellow pigment present in the spice turmeric (Curcuma longa), has been linked with antioxidant, anti-inflammatory, antiproliferative, anticancer, antidiabetic, antirheumatic, and antiviral effects, but its optimum potential is limited by its lack of solubility in aqueous solvents and poor oral bioavailability. We employed a polymer-based nanoparticle approach to improve bioavailability. Curcumin was encapsulated with 97.5% efficiency in biodegradable nanoparticulate formulation based on poly (lactide-co-glycolide) (PLGA) and a stabilizer polyethylene glycol (PEG)-5000. Dynamic laser light scattering and transmission electron microscopy indicated a particle diameter of 80.9 nm. This curcumin, renamed from hereon "as curcumin (NP)", was characterized for its biological activity. In vitro curcumin (NP) exhibited very rapid and more efficient cellular uptake than curcumin. Estrase staining revealed that curcumin (NP) was at least as potent as or more potent than curcumin in inducing apoptosis of leukemic cells and in suppressing proliferation of various tumor cell lines. When examined by electrophoretic gel shift mobility assay, curcumin (NP) was more active than curcumin in inhibiting TNF-induced NF-kappaB activation and in suppression of NF-kappaB-regulated proteins involved in cell proliferation (cyclin D1), invasion (MMP-9), and angiogenesis (VEGF). In mice, curcumin (NP) was more bioavailable and had a longer half-life than curcumin. Overall we demonstrate that curcumin-loaded PLGA nanoparticles formulation has enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo over curcumin.

Indocyanine Green-Encapsulated Hybrid Polymeric Nanomicelles for Photothermal Cancer Therapy.

PubMed

Jian, Wei-Hong; Yu, Ting-Wei; Chen, Chien-Ju; Huang, Wen-Chia; Chiu, Hsin-Cheng; Chiang, Wen-Hsuan

2015-06-09

Indocyanine green (ICG), an FDA approved medical near-infrared (NIR) imaging agent, has been extensively used in cancer theranosis. However, the limited aqueous photostability, rapid body clearance, and poor cellular uptake severely restrict its practical applications. For these problems to be overcome, ICG-encapsulated hybrid polymeric nanomicelles (PNMs) were developed in this work through coassociation of the amphiphilic diblock copolymer poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG) and hydrophobic electrostatic complexes composed of ICG molecules and branched poly(ethylenimine) (PEI). The ICG-encapsulated hybrid PNMs featured a hydrophobic PLGA/ICG/PEI core stabilized by hydrophilic PEG shells. The encapsulation of electrostatic ICG/PEI complexes into the compact PLGA-rich core not only facilitated the ICG loading but also promoted its aqueous optical stability. The effects of the chain length of PEI in combination with ICG on the physiochemical properties of PNMs and their drug leakage were also investigated. PEI(10k) (10 kDa) could form highly robust and dense complexes with ICG, and thus prominently reduced ICG outflow from the PNMs. The results of in vitro cellular uptake and cytotoxicity studies revealed that the ICG/PEI(10k)-loaded PNMs significantly promoted cellular uptake of ICG by HeLa cells due to their near-neutral surface, and thereby augmented the NIR-triggered hyperthermia effect in destroying cancer cells. These findings strongly indicate that the ICG/PEI10k-loaded PNMs have significant potential for attaining effective cancer imaging and photothermal therapy.

Camptothecin prodrug nanomicelle based on a boronate ester-linked diblock copolymer as the carrier of doxorubicin with enhanced cellular uptake.

PubMed

Gao, Ya; Xiao, Yi; Liu, Shiyuan; Yu, Jiahui

2018-02-01

A novel pH-sensitive polymeric prodrug of camptothecin (CPT) by polymerizing γ-camptothecin-glutamate N-carboxyanhydride (Glu (CPT)-NCA) on boronate ester-linked poly (ethyleneglycol) (PEG) directly via the amine-initiated ring open polymerization (ROP) has been developed. The resulting amphiphilic prodrug (mPEG-BC-PGluCPT) could self-assemble into nanoparticles and encapsulate doxorubicin (Dox) simultaneously in aqueous solution for dual-drug delivery. The formation of polymeric prodrug micelles (mPEG-BC@PGluCPT) was confirmed by the measurements of critical aggregation concentration (CAC), particle size, and morphology observations. The mPEG-BC@PGluCPT micelles were colloidally stable in solutions for two weeks. Polymeric prodrug micelles mPEG-BC@PGluCPT and Dox-loaded micelles mPEG-BC@PGluCPT⋅Dox showed sustained drug release profiles over 48 h. As expected, drug release was accelerated by the decreasement of pH value from 7.4 to 6.0, which demonstrated pH-dependent manner of drug release. Additionally, it was found that cellular uptake of mPEG-BC@PGluCPT⋅Dox micelles on HepG2 cells was higher than that on HL-7702 cells, especially in culture medium at pH 6.0. The enhanced cellular uptake of mPEG-BC@PGluCPT⋅Dox micelles under acidic condition on HepG2 cells resulted in the higher cytotoxicity of mPEG-BC@PGluCPT⋅Dox micelles at acidic pH than that at pH 7.4.

Dual-pH Sensitive Charge-reversal Nanocomplex for Tumor-targeted Drug Delivery with Enhanced Anticancer Activity.

PubMed

Zhou, Qing; Hou, Yilin; Zhang, Li; Wang, Jianlin; Qiao, Youbei; Guo, Songyan; Fan, Li; Yang, Tiehong; Zhu, Lin; Wu, Hong

2017-01-01

Poly(β-L-malic acid) (PMLA), a natural aliphatic polyester, has been proven to be a promising carrier for anti-cancer drugs. In spite of excellent bio-compatibility, the application of PMLA as the drug carrier for cancer therapy is limited by its low cellular uptake efficiency. The strong negative charge of PMLA impedes its uptake by cancer cells because of the electrostatic repulsion. In this study, a dual pH-sensitive charge-reversal PMLA-based nanocomplex (PMLA-PEI-DOX-TAT@PEG-DMMA) was developed for effective tumor-targeted drug delivery, enhanced cellular uptake, and intracellular drug release. The prepared nanocomplex showed a negative surface charge at the physiological pH, which could protect the nanocomplex from the attack of plasma proteins and recognition by the reticuloendothelial system, so as to prolong its circulation time. While at the tumor extracellular pH 6.8, the DMMA was hydrolyzed, leading to the charge reversal and exposure of the TAT on the polymeric micelles, thus enhancing the cellular internalization. Then, the polymeric micelles underwent dissociation and drug release in response to the acidic pH in the lyso/endosomal compartments of the tumor cell. Both in vitro and in vivo efficacy studies indicated that the nanocomplex significantly inhibited the tumor growth while the treatment showed negligible systemic toxicity, suggesting that the developed dual pH-sensitive PMLA-based nanocomplex would be a promising drug delivery system for tumor-targeted drug delivery with enhanced anticancer activity.

Dual-pH Sensitive Charge-reversal Nanocomplex for Tumor-targeted Drug Delivery with Enhanced Anticancer Activity

PubMed Central

Zhou, Qing; Hou, Yilin; Zhang, Li; Wang, Jianlin; Qiao, Youbei; Guo, Songyan; Fan, Li; Yang, Tiehong; Zhu, Lin; Wu, Hong

2017-01-01

Poly(β-L-malic acid) (PMLA), a natural aliphatic polyester, has been proven to be a promising carrier for anti-cancer drugs. In spite of excellent bio-compatibility, the application of PMLA as the drug carrier for cancer therapy is limited by its low cellular uptake efficiency. The strong negative charge of PMLA impedes its uptake by cancer cells because of the electrostatic repulsion. In this study, a dual pH-sensitive charge-reversal PMLA-based nanocomplex (PMLA-PEI-DOX-TAT@PEG-DMMA) was developed for effective tumor-targeted drug delivery, enhanced cellular uptake, and intracellular drug release. The prepared nanocomplex showed a negative surface charge at the physiological pH, which could protect the nanocomplex from the attack of plasma proteins and recognition by the reticuloendothelial system, so as to prolong its circulation time. While at the tumor extracellular pH 6.8, the DMMA was hydrolyzed, leading to the charge reversal and exposure of the TAT on the polymeric micelles, thus enhancing the cellular internalization. Then, the polymeric micelles underwent dissociation and drug release in response to the acidic pH in the lyso/endosomal compartments of the tumor cell. Both in vitro and in vivo efficacy studies indicated that the nanocomplex significantly inhibited the tumor growth while the treatment showed negligible systemic toxicity, suggesting that the developed dual pH-sensitive PMLA-based nanocomplex would be a promising drug delivery system for tumor-targeted drug delivery with enhanced anticancer activity. PMID:28638469

Platinum transfer from hCTR1 to Atox1 is dependent on the type of platinum complex.

PubMed

Wu, Xuelei; Yuan, Siming; Wang, Erqiong; Tong, Yang; Ma, Guolin; Wei, Kaiju; Liu, Yangzhong

2017-05-24

In spite of their wide application, the cellular uptake of platinum based anticancer drugs is still unclear. The copper transport protein, hCTR1, is proposed to facilitate the cellular uptake of cisplatin, whereas organic cation transport (OCT) is more important for oxaliplatin. It has been reported that both N-terminal and C-terminal metal binding motifs of hCTR1 are highly reactive to cisplatin, which is the initial step of protein assisted cellular uptake of cisplatin. It is still unknown how the platinum drugs in hCTR1 transfer to cytoplasmic media, and whether various platinum complexes possess different activities in this process. Herein, we investigated the reaction of the platinated C-terminal metal binding motif of hCTR1 (C8) with the down-stream protein Atox1. Results show that Atox1 is highly reactive to the platinated C8 adducts of cisplatin and transplatin, whereas the oxaliplatin/C8 adduct is much less reactive. The platinum transfer from C8 to Atox1 occurs in the reaction, which results in the protein unfolding of Atox1. These results demonstrated that the platinated intracellular-domain of hCTR1 is reactive to Atox1, and the reactivity is dependent on the ligand and the coordination structure of platinum complexes. The different reactivity is consistent with the hypothesis that hCTR1 is more significant in the transport of cisplatin than that of oxaliplatin.

Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in a rat liver cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barhoumi, Rola, E-mail: rmouneimne@cvm.tamu.edu; Mouneimne, Youssef; Ramos, Ernesto

2011-05-15

Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an 'advanced unmixing process', identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 h. The application of the advanced unmixing process resulted inmore » the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to the analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis.« less

Sodium-Glucose Transporter-2 (SGLT2; SLC5A2) Enhances Cellular Uptake of Aminoglycosides

PubMed Central

Jiang, Meiyan; Wang, Qi; Karasawa, Takatoshi; Koo, Ja-Won; Li, Hongzhe; Steyger, Peter S.

2014-01-01

Aminoglycoside antibiotics, like gentamicin, continue to be clinically essential worldwide to treat life-threatening bacterial infections. Yet, the ototoxic and nephrotoxic side-effects of these drugs remain serious complications. A major site of gentamicin uptake and toxicity resides within kidney proximal tubules that also heavily express electrogenic sodium-glucose transporter-2 (SGLT2; SLC5A2) in vivo. We hypothesized that SGLT2 traffics gentamicin, and promotes cellular toxicity. We confirmed in vitro expression of SGLT2 in proximal tubule-derived KPT2 cells, and absence in distal tubule-derived KDT3 cells. D-glucose competitively decreased the uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent analog of glucose, and fluorescently-tagged gentamicin (GTTR) by KPT2 cells. Phlorizin, an SGLT2 antagonist, strongly inhibited uptake of 2-NBDG and GTTR by KPT2 cells in a dose- and time-dependent manner. GTTR uptake was elevated in KDT3 cells transfected with SGLT2 (compared to controls); and this enhanced uptake was attenuated by phlorizin. Knock-down of SGLT2 expression by siRNA reduced gentamicin-induced cytotoxicity. In vivo, SGLT2 was robustly expressed in kidney proximal tubule cells of heterozygous, but not null, mice. Phlorizin decreased GTTR uptake by kidney proximal tubule cells in Sglt2+/− mice, but not in Sglt2−/− mice. However, serum GTTR levels were elevated in Sglt2−/− mice compared to Sglt2+/− mice, and in phlorizin-treated Sglt2+/− mice compared to vehicle-treated Sglt2+/− mice. Loss of SGLT2 function by antagonism or by gene deletion did not affect gentamicin cochlear loading or auditory function. Phlorizin did not protect wild-type mice from kanamycin-induced ototoxicity. We conclude that SGLT2 can traffic gentamicin and contribute to gentamicin-induced cytotoxicity. PMID:25268124

Tracking intracellular uptake and localisation of alkyne tagged fatty acids using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Jamieson, Lauren E.; Greaves, Jennifer; McLellan, Jayde A.; Munro, Kevin R.; Tomkinson, Nicholas C. O.; Chamberlain, Luke H.; Faulds, Karen; Graham, Duncan

2018-05-01

Intracellular uptake, distribution and metabolism of lipids are tightly regulated characteristics in healthy cells. An analytical technique capable of understanding these characteristics with a high level of species specificity in a minimally invasive manner is highly desirable in order to understand better how these become disrupted during disease. In this study, the uptake and distribution of three different alkyne tagged fatty acids in single cells were monitored and compared, highlighting the ability of Raman spectroscopy combined with alkyne tags for better understanding of the fine details with regard to uptake, distribution and metabolism of very chemically specific lipid species. This indicates the promise of using Raman spectroscopy directly with alkyne tagged lipids for cellular studies as opposed to subsequently clicking of a fluorophore onto the alkyne for fluorescence imaging.

Extraction protocol and liquid chromatography/tandem mass spectrometry method for determining micelle-entrapped paclitaxel at the cellular and subcellular levels: Application to a cellular uptake and distribution study.

PubMed

Zheng, Nan; Lian, Bin; Du, Wenwen; Xu, Guobing; Ji, Jiafu

2018-01-01

Paclitaxel-loaded polymeric micelles (PTX-PM) are commonly used as tumor-targeted nanocarriers and display outstanding antitumor features in clinic, but its accumulation and distribution in vitro are lack of investigation. It is probably due to the complex micellar system and its low concentration at the cellular or subcellular levels. In this study, we developed an improved extraction method, which was a combination of mechanical disruption and liquid-liquid extraction (LLE), to extract the total PTX from micelles in the cell lysate and subcellular compartments. An ultra-performance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) method was optimized to detect the low concentration of PTX at cellular and subcellular levels simultaneously, using docetaxel as internal standard (IS). The method was proved to release PTX totally from micelles (≥95.93%) with a consistent and reproducible extraction recovery (≥75.04%). Good linearity was obtained at concentrations ranging from 0.2 to 20ng/mL. The relative error (RE%) for accuracy varied from 0.68 to 7.56%, and the intra- and inter-precision (relative standard deviation, RSD%) was less than 8.64% and 13.14%, respectively. This method was fully validated and successfully applied to the cellular uptake and distribution study of PTX-loaded PLGA-PEG micelles in human breast cancer cells (MCF-7). Copyright © 2017 Elsevier B.V. All rights reserved.

Modeling the relationship between fluorodeoxyglucose uptake and tumor radioresistance as a function of the tumor microenvironment.

PubMed

Jeong, Jeho; Deasy, Joseph O

2014-01-01

High fluorodeoxyglucose positron emission tomography (FDG-PET) uptake in tumors has often been correlated with increasing local failure and shorter overall survival, but the radiobiological mechanisms of this uptake are unclear. We explore the relationship between FDG-PET uptake and tumor radioresistance using a mechanistic model that considers cellular status as a function of microenvironmental conditions, including proliferating cells with access to oxygen and glucose, metabolically active cells with access to glucose but not oxygen, and severely hypoxic cells that are starving. However, it is unclear what the precise uptake levels of glucose should be for cells that receive oxygen and glucose versus cells that only receive glucose. Different potential FDG uptake profiles, as a function of the microenvironment, were simulated. Predicted tumor doses for 50% control (TD50) in 2 Gy fractions were estimated for each assumed uptake profile and for various possible cell mixtures. The results support the hypothesis of an increased avidity of FDG for cells in the intermediate stress state (those receiving glucose but not oxygen) compared to well-oxygenated (and proliferating) cells.

The cellular uptake mechanism, intracellular transportation, and exocytosis of polyamidoamine dendrimers in multidrug-resistant breast cancer cells.

PubMed

Zhang, Jie; Liu, Dan; Zhang, Mengjun; Sun, Yuqi; Zhang, Xiaojun; Guan, Guannan; Zhao, Xiuli; Qiao, Mingxi; Chen, Dawei; Hu, Haiyang

2016-01-01

Polyamidoamine dendrimers, which can deliver drugs and genetic materials to resistant cells, are attracting increased research attention, but their transportation behavior in resistant cells remains unclear. In this paper, we performed a systematic analysis of the cellular uptake, intracellular transportation, and efflux of PAMAM-NH2 dendrimers in multidrug-resistant breast cancer cells (MCF-7/ADR cells) using sensitive breast cancer cells (MCF-7 cells) as the control. We found that the uptake rate of PAMAM-NH2 was much lower and exocytosis of PAMAM-NH2 was much greater in MCF-7/ADR cells than in MCF-7 cells due to the elimination of PAMAM-NH2 from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells. Macropinocytosis played a more important role in its uptake in MCF-7/ADR cells than in MCF-7 cells. PAMAM-NH2 aggregated and became more degraded in the lysosomal vesicles of the MCF-7/ADR cells than in those of the MCF-7 cells. The endoplasmic reticulum and Golgi complex were found to participate in the exocytosis rather than endocytosis process of PAMAM-NH2 in both types of cells. Our findings clearly showed the intracellular transportation process of PAMAM-NH2 in MCF-7/ADR cells and provided a guide of using PAMAM-NH2 as a drug and gene vector in resistant cells.

Coupled elasticity–diffusion model for the effects of cytoskeleton deformation on cellular uptake of cylindrical nanoparticles

PubMed Central

Wang, Jizeng; Li, Long

2015-01-01

Molecular dynamic simulations and experiments have recently demonstrated how cylindrical nanoparticles (CNPs) with large aspect ratios penetrate animal cells and inevitably deform cytoskeletons. Thus, a coupled elasticity–diffusion model was adopted to elucidate this interesting biological phenomenon by considering the effects of elastic deformations of cytoskeleton and membrane, ligand–receptor binding and receptor diffusion. The mechanism by which the binding energy drives the CNPs with different orientations to enter host cells was explored. This mechanism involved overcoming the resistance caused by cytoskeleton and membrane deformations and the change in configurational entropy of the ligand–receptor bonds and free receptors. Results showed that deformation of the cytoskeleton significantly influenced the engulfing process by effectively slowing down and even hindering the entry of the CNPs. Additionally, the engulfing depth was determined quantitatively. CNPs preferred or tended to vertically attack target cells until they were stuck in the cytoskeleton as implied by the speed of vertically oriented CNPs that showed much faster initial engulfing speeds than horizontally oriented CNPs. These results elucidated the most recent molecular dynamics simulations and experimental observations on the cellular uptake of carbon nanotubes and phagocytosis of filamentous Escherichia coli bacteria. The most efficient engulfment showed the stiffness-dependent optimal radius of the CNPs. Cytoskeleton stiffness exhibited more significant influence on the optimal sizes of the vertical uptake than the horizontal uptake. PMID:25411410

In vitro Cellular Uptake and Dimerization of Signal Transducer and Activator of Transcription-3 (STAT3) Identify the Photosensitizing and Imaging-Potential of Isomeric Photosensitizers Derived from Chlorophyll-a and Bacteriochlorophyll-a

PubMed Central

Srivatsan, Avinash; Wang, Yanfang; Joshi, Penny; Sajjad, Munawwar; Chen, Yihui; Liu, Chao; Thankppan, Krishnakumar; Missert, Joseph R.; Tracy, Erin; Morgan, Janet; Rigual, Nestor; Baumann, Heinz; Pandey, Ravindra K.

2011-01-01

Among the photosensitizers investigated, both ring-D and ring-B reduced chlorins containing the m-iodobenzyloxyethyl group at position-3 and a carboxylic acid functionality at position-172 showed highest uptake by tumor cells and light-dependent photo reaction that correlated with maximal tumor-imaging [positron emission tomography (PET) and fluorescence] and long-term photodynamic therapy (PDT) efficacy in BALB/c mice bearing Colon26 tumors. However, among the ring-D reduced compounds, the isomer containing 1′-m-iobenzyloxyethyl group at position-3 was more effective than the corresponding 8-(1′-m-iodobenzyloxyethyl) derivative. All photosensitizers showed maximum uptake by tumor tissue 24h after injection and the tumors exposed with light at low fluence and fluence rates (128 J/cm2, 14 mW/cm2) produced significantly enhanced tumor eradication than those exposed at higher fluence and fluence rate (135 J/cm,2 75mW/cm2). Interestingly, dose-dependent cellular uptake of the compounds and light-dependent STAT3 dimerization have emerged as sensitive rapid indicators for PDT efficacy in vitro and in vivo and could be used as in vitro/in vivo biomarkers for evaluating and optimizing the in vivo treatment parameters of the existing and new PDT candidates. PMID:21842893

In vitro dosimetry of agglomerates

NASA Astrophysics Data System (ADS)

Hirsch, V.; Kinnear, C.; Rodriguez-Lorenzo, L.; Monnier, C. A.; Rothen-Rutishauser, B.; Balog, S.; Petri-Fink, A.

2014-06-01

Agglomeration of nanoparticles in biological fluids is a pervasive phenomenon that leads to difficulty in the interpretation of results from in vitro exposure, primarily due to differing particokinetics of agglomerates to nanoparticles. Therefore, well-defined small agglomerates were designed that possessed different particokinetic profiles, and their cellular uptake was compared to a computational model of dosimetry. The approach used here paves the way for a better understanding of the impact of agglomeration on the nanoparticle-cell interaction.Agglomeration of nanoparticles in biological fluids is a pervasive phenomenon that leads to difficulty in the interpretation of results from in vitro exposure, primarily due to differing particokinetics of agglomerates to nanoparticles. Therefore, well-defined small agglomerates were designed that possessed different particokinetic profiles, and their cellular uptake was compared to a computational model of dosimetry. The approach used here paves the way for a better understanding of the impact of agglomeration on the nanoparticle-cell interaction. Electronic supplementary information (ESI) available: ITC data for tiopronin/Au-NP interactions, agglomeration kinetics at different pHs for tiopronin-coated Au-NPs, UV-Vis spectra in water, PBS and DMEM and temporal correlation functions for single Au-NPs and corresponding agglomerates, calculation of diffusion and sedimentation parameters, modelling of relative cell uptake based on the ISDD model and cytotoxicity of single Au-NPs and their agglomerates, and synthesis and cell uptake of large spherical Au-NPs. See DOI: 10.1039/c4nr00460d

Uptake in melanoma cells of N-(2-diethylaminoethyl)-2-iodobenzamide (BZA2), an imaging agent for melanoma staging: relation to pigmentation.

PubMed

Mansard, Sandrine; Papon, Janine; Moreau, Marie-France; Miot-Noirault, Elisabeth; Labarre, Pierre; Bayle, Martine; Veyre, Annie; Madelmont, Jean-Claude; Moins, Nicole

2005-07-01

N-(2-diethylaminoethyl)-2-iodobenzamide (BZA(2)) has been singled out as the most efficacious melanoma scintigraphy imaging agent. Our work was designed to assess the mechanisms of the specific affinity of the radioiodinated iodobenzamide for melanoma tissue. We studied the cellular uptake and retention of [(125)I]-BZA(2) on various cell lines. In vitro, cellular [(125)I]-BZA(2) uptake was related to the pigmentation status of the cells: higher in pigmented melanoma cell lines (M4 Beu, IPC 227, B 16) than in a nonpigmented one (M3 Dau) and nonmelanoma cell lines (MCF 7 and L 929). Two mechanisms were assessed: binding of the tracer to melanin or to sigma receptors of melanoma cells. First, the uptake of [(125)I]-BZA(2) after melanogenesis stimulation by alpha-melanocyte-stimulating hormone and l-tyrosine increased in the B 16 melanoma cell line both in vitro and in vivo according to melanin concentration. Moreover, the binding of [(125)I]-BZA(2) to synthetic melanin was dependent on melanin concentration and could be saturated. Second, no competition was evidenced on M4 Beu cells between [(125)I]-BZA(2) and haloperidol, a sigma ligand, at concentrations < or =10(-6) M. We show that the specificity and sensibility of BZA(2) as a melanoma scintigraphic imaging agent are mostly due to interactions with melanic pigments.

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

PubMed Central

Maejima, Ikuko; Takahashi, Atsushi; Omori, Hiroko; Kimura, Tomonori; Takabatake, Yoshitsugu; Saitoh, Tatsuya; Yamamoto, Akitsugu; Hamasaki, Maho; Noda, Takeshi; Isaka, Yoshitaka; Yoshimori, Tamotsu

2013-01-01

Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis. PMID:23921551

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury.

PubMed

Maejima, Ikuko; Takahashi, Atsushi; Omori, Hiroko; Kimura, Tomonori; Takabatake, Yoshitsugu; Saitoh, Tatsuya; Yamamoto, Akitsugu; Hamasaki, Maho; Noda, Takeshi; Isaka, Yoshitaka; Yoshimori, Tamotsu

2013-08-28

Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.

Molecular control of vertebrate iron homeostasis by iron regulatory proteins

PubMed Central

Wallander, Michelle L.; Leibold, Elizabeth A.; Eisenstein, Richard S.

2008-01-01

Both deficiencies and excesses of iron represent major public health problems throughout the world. Understanding the cellular and organismal processes controlling iron homeostasis is critical for identifying iron-related diseases and in advancing the clinical treatments for such disorders of iron metabolism. Iron regulatory proteins (IRPs) 1 and 2 are key regulators of vertebrate iron metabolism. These RNA binding proteins post-transcriptionally control the stability or translation of mRNAs encoding proteins involved in iron homeostasis thereby controlling the uptake, utilization, storage or export of iron. Recent evidence provides insight into how IRPs selectively control the translation or stability of target mRNAs, how IRP RNA binding activity is controlled by iron-dependent and iron-independent effectors, and the pathological consequences of dysregulation of the IRP system. PMID:16872694

Charge-switching amino acids-based cationic lipids for efficient gene delivery.

PubMed

Zheng, Li-Ting; Yi, Wen-Jing; Liu, Qiang; Su, Rong-Chuan; Zhao, Zhi-Gang

2015-12-15

A series of charge-switching amino acids-based cationic lipids 4a-4e bearing a benzyl ester at the terminus of the acyl chain, but differing in the polar-head group were prepared. The physicochemical properties of these lipids, including size, zeta potential and cellular uptake of the lipoplexes formed from with DNA, as well as the transfection efficiency (TE), were investigated. The results showed that the chemical structure of the cationic head-group clearly affects the physicochemical parameters of the amino acid-based lipids and especially the TE. The selected lipid, 4c gave 2.1 times higher TE than bPEI 25k in the presence of 10% serum in HeLa cells, with little toxicity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

DOE PAGES

Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; ...

2015-05-15

Fabrication of stimuli-triggered drug delivery vehicle is is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). In conclusion, the enzyme-activated intracellular deliverymore » of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment.« less

Green synthesis of multifunctional carbon dots from coriander leaves and their potential application as antioxidants, sensors and bioimaging agents.

PubMed

Sachdev, Abhay; Gopinath, P

2015-06-21

In the present study, a facile one-step hydrothermal treatment of coriander leaves for preparing carbon dots (CDs) has been reported. Optical and structural properties of the CDs have been extensively studied by UV-visible and fluorescence spectroscopic, microscopic (transmission electron microscopy, scanning electron microscopy) and X-ray diffraction techniques. Surface functionality and composition of the CDs have been illustrated by elemental analysis and Fourier transform infrared spectroscopy (FTIR). Quenching of the fluorescence of the CDs in the presence of metal ions is of prime significance, hence CDs have been used as a fluorescence probe for sensitive and selective detection of Fe(3+) ions. Eventually, biocompatibility and bioimaging aspects of CDs have been evaluated on lung normal (L-132) and cancer (A549) cell lines. Qualitative analysis of cellular uptake of CDs has been pursued through fluorescence microscopy, while quantitative analysis using a flow cytometer provided an insight into the concentration and cell-type dependent uptake of CDs. The article further investigates the antioxidant activity of CDs. Therefore, we have validated the practicality of CDs obtained from a herbal carbon source for versatile applications.

Fluorophore labeling of a cell-penetrating peptide induces differential effects on its cellular distribution and affects cell viability.

PubMed

Birch, Ditlev; Christensen, Malene Vinther; Staerk, Dan; Franzyk, Henrik; Nielsen, Hanne Mørck

2017-12-01

Cell-penetrating peptides constitute efficient delivery vectors, and studies of their uptake and mechanism of translocation typically involve fluorophore-labeled conjugates. In the present study, the influence of a number of specific fluorophores on the physico-chemical properties and uptake-related characteristics of penetratin were studied. An array of seven fluorophores belonging to distinct structural classes was examined, and the impact of fluorophore labeling on intracellular distribution and cytotoxicity was correlated to the physico-chemical properties of the conjugates. Exposure of several mammalian cell types to fluorophore-penetratin conjugates revealed a strong structure-dependent reduction in viability (1.5- to 20-fold lower IC 50 values as compared to those of non-labeled penetratin). Also, the degree of less severe effects on membrane integrity, as well as intracellular distribution patterns differed among the conjugates. Overall, neutral hydrophobic fluorophores or negatively charged fluorophores conferred less cytotoxicity as compared to the effect exerted by positively charged, hydrophobic fluorophores. The latter conjugates, however, exhibited less membrane association and more clearly defined intracellular distribution patterns. Thus, selection of the appropriate flurophore is critical. Copyright © 2017 Elsevier B.V. All rights reserved.

Automated Synthesis of 18F-Fluoropropoxytryptophan for Amino Acid Transporter System Imaging

PubMed Central

Shih, I-Hong; Duan, Xu-Dong; Kong, Fan-Lin; Williams, Michael D.; Zhang, Yin-Han; Yang, David J.

2014-01-01

Objective. This study was to develop a cGMP grade of [18F]fluoropropoxytryptophan (18F-FTP) to assess tryptophan transporters using an automated synthesizer. Methods. Tosylpropoxytryptophan (Ts-TP) was reacted with K18F/kryptofix complex. After column purification, solvent evaporation, and hydrolysis, the identity and purity of the product were validated by radio-TLC (1M-ammonium acetate : methanol = 4 : 1) and HPLC (C-18 column, methanol : water = 7 : 3) analyses. In vitro cellular uptake of 18F-FTP and 18F-FDG was performed in human prostate cancer cells. PET imaging studies were performed with 18F-FTP and 18F-FDG in prostate and small cell lung tumor-bearing mice (3.7 MBq/mouse, iv). Results. Radio-TLC and HPLC analyses of 18F-FTP showed that the Rf and Rt values were 0.9 and 9 min, respectively. Radiochemical purity was >99%. The radiochemical yield was 37.7% (EOS 90 min, decay corrected). Cellular uptake of 18F-FTP and 18F-FDG showed enhanced uptake as a function of incubation time. PET imaging studies showed that 18F-FTP had less tumor uptake than 18F-FDG in prostate cancer model. However, 18F-FTP had more uptake than 18F-FDG in small cell lung cancer model. Conclusion. 18F-FTP could be synthesized with high radiochemical yield. Assessment of upregulated transporters activity by 18F-FTP may provide potential applications in differential diagnosis and prediction of early treatment response. PMID:25136592

Automated synthesis of 18F-fluoropropoxytryptophan for amino acid transporter system imaging.

PubMed

Shih, I-Hong; Duan, Xu-Dong; Kong, Fan-Lin; Williams, Michael D; Yang, Kevin; Zhang, Yin-Han; Yang, David J

2014-01-01

This study was to develop a cGMP grade of [(18)F]fluoropropoxytryptophan ((18)F-FTP) to assess tryptophan transporters using an automated synthesizer. Tosylpropoxytryptophan (Ts-TP) was reacted with K(18)F/kryptofix complex. After column purification, solvent evaporation, and hydrolysis, the identity and purity of the product were validated by radio-TLC (1M-ammonium acetate : methanol = 4 : 1) and HPLC (C-18 column, methanol : water = 7 : 3) analyses. In vitro cellular uptake of (18)F-FTP and (18)F-FDG was performed in human prostate cancer cells. PET imaging studies were performed with (18)F-FTP and (18)F-FDG in prostate and small cell lung tumor-bearing mice (3.7 MBq/mouse, iv). Radio-TLC and HPLC analyses of (18)F-FTP showed that the Rf and Rt values were 0.9 and 9 min, respectively. Radiochemical purity was >99%. The radiochemical yield was 37.7% (EOS 90 min, decay corrected). Cellular uptake of (18)F-FTP and (18)F-FDG showed enhanced uptake as a function of incubation time. PET imaging studies showed that (18)F-FTP had less tumor uptake than (18)F-FDG in prostate cancer model. However, (18)F-FTP had more uptake than (18)F-FDG in small cell lung cancer model. (18)F-FTP could be synthesized with high radiochemical yield. Assessment of upregulated transporters activity by (18)F-FTP may provide potential applications in differential diagnosis and prediction of early treatment response.

Characteristics of the uridine uptake system in normal and polyoma transformed hamster embryo cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lemkin, J.A.

1973-01-01

The lability of the uridine uptake system in the normal and polyoma transformed hamster embryo fibroblast was studied. The major areas investigated were: the kinetic parameters of uridine transport, a comparison of changes in cellular ATP content by factors which modulate uridine uptake, and a comparison of the qualitative and quantitative effects of the same modulating agent on uridine transport, cell growth, and cellular ATP content. Uridine uptake into cells in vitro was examined using tritiated uridine as a tracer to measure the amount of uridine incorporated into the acid soluble and acid-insoluble fractions of the cells studied. The ATPmore » content of the cells was determined by the firefly bioluminescence method. It was found that the K/sub t/ for uridine uptake into the normal hamster embryo cell and two polyoma transformed hamster embryo cell lines was identical. However, the V/sub max/ for uridine transport was higher in both polyoma transformed cell lines. Furthermore, the K/sub t/ in both the normal and transformed cell cultured in serum-less or serum-containing media was identical, although the V/sub max/ was higher in the serum-stimulated cell in both the normal and transformed cell. Stimulation of the normal cell with adenosine produced a different K/sub t/ for uridine transport. Preliminary investigations have demonstrated that treatment of the polyoma transformed with adenosine also induces a different K/sub t/ (not shown). The K/sub i/ for phloretin inhibition in serum-less and serum-stimulated normal and polyoma transformed cells was found to be identical in each case.« less

Improved mucoadhesion and cell uptake of chitosan and chitosan oligosaccharide surface-modified polymer nanoparticles for mucosal delivery of proteins.

PubMed

Dyawanapelly, Sathish; Koli, Uday; Dharamdasani, Vimisha; Jain, Ratnesh; Dandekar, Prajakta

2016-08-01

The main aim of the present study was to compare mucoadhesion and cellular uptake efficiency of chitosan (CS) and chitosan oligosaccharide (COS) surface-modified polymer nanoparticles (NPs) for mucosal delivery of proteins. We have developed poly (D, L-lactide-co-glycolide) (PLGA) NPs, surface-modified COS-PLGA NPs and CS-PLGA NPs, by using double emulsion solvent evaporation method, for encapsulating bovine serum albumin (BSA) as a model protein. Surface modification of NPs was confirmed using physicochemical characterization methods such as particle size and zeta potential, SEM, TEM and FTIR analysis. Both surface-modified PLGA NPs displayed a slow release of protein compared to PLGA NPs. Furthermore, we have explored the mucoadhesive property of COS as a material for modifying the surface of polymeric NPs. During in vitro mucoadhesion test, positively charged COS-PLGA NPs and CS-PLGA NPs exhibited enhanced mucoadhesion, compared to negatively charged PLGA NPs. This interaction was anticipated to improve the cell interaction and uptake of NPs, which is an important requirement for mucosal delivery of proteins. All nanoformulations were found to be safe for cellular delivery when evaluated in A549 cells. Moreover, intracellular uptake behaviour of FITC-BSA loaded NPs was extensively investigated by confocal laser scanning microscopy and flow cytometry. As we hypothesized, positively charged COS-PLGA NPs and CS-PLGA NPs displayed enhanced intracellular uptake compared to negatively charged PLGA NPs. Our results demonstrated that CS- and COS-modified polymer NPs could be promising carriers for proteins, drugs and nucleic acids via nasal, oral, buccal, ocular and vaginal mucosal routes.

Synthesis and evaluation of novel 18 F-labeled quinazoline derivatives with low lipophilicity for tumor PET imaging.

PubMed

Chong, Yan; Chang, Jin; Zhao, Wenwen; He, Yong; Li, Yuqiao; Zhang, Huabei; Qi, Chuanmin

2018-02-01

Four novel 18 F-labeled quinazoline derivatives with low lipophilicity, [ 18 F]4-(2-fluoroethoxy)-6,7-dimethoxyquinazoline ([ 18 F]I), [ 18 F]4-(3-((4-(2-fluoroethoxy)-7-methoxyquinazolin-6-yl)oxy)propyl)morpholine ([ 18 F]II), [ 18 F]4-(2-fluoroethoxy)-7-methoxy-6-(2-methoxyethoxy)quinazoline ([ 18 F]III), and [ 18 F]4-(2-fluoroethoxy)-6,7-bis(2-methoxyethoxy)quinazoline ([ 18 F]IV), were synthesized via a 2-step radiosynthesis procedure with an overall radiochemical yield of 10% to 38% (without decay correction) and radiochemical purities of >98%. The lipophilicity and stability of labeled compounds were tested in vitro. The log P values of the 4 radiotracers ranged from 0.52 to 1.07. We then performed ELISA to measure their affinities to EGFR-TK; ELISA assay results indicated that each inhibitor was specifically bounded to EGFR-TK in a dose-dependent manner. The EGFR-TK autophosphorylation IC 50 values of [ 18 F]I, [ 18 F]II, [ 18 F]III, and [ 18 F]IV were 7.732, 0.4698, 0.1174, and 0.1176 μM, respectively. All labeled compounds were evaluated via cellular uptake and blocking studies in HepG2 cell lines in vitro. Cellular uptake and blocking experiment results indicated that [ 18 F]I and [ 18 F]III had excellent cellular uptake at 120-minute postinjection in HepG2 carcinoma cells (51.80 ± 3.42%ID/mg protein and 27.31 ± 1.94%ID/mg protein, respectively). Additionally, biodistribution experiments in S180 tumor-bearing mice in vivo indicated that [ 18 F]I had a very fast clearance in blood and a relatively high uptake ratio of tumor to blood (4.76) and tumor to muscle (1.82) at 60-minute postinjection. [ 18 F]III had a quick clearance in plasma, and its highest uptake ratio of tumor to muscle was 2.55 at 15-minute postinjection. These experimental results and experiences were valuable for the further exploration of novel radiotracers of quinazoline derivatives. Copyright © 2017 John Wiley & Sons, Ltd.

Relative contribution of CTR1 and DMT1 in copper transport by the blood–CSF barrier: Implication in manganese-induced neurotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Gang; Department of Occupational and Environmental Health and the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi'an, Shanxi 710032; Chen, Jingyuan

2012-05-01

The homeostasis of copper (Cu) in the cerebrospinal fluid (CSF) is partially regulated by the Cu transporter-1 (CTR1) and divalent metal transporter-1 (DMT1) at the blood–CSF barrier (BCB) in the choroid plexus. Data from human and animal studies suggest an increased Cu concentration in blood, CSF, and brains following in vivo manganese (Mn) exposure. This study was designed to investigate the relative role of CTR1 and DMT1 in Cu transport under normal or Mn-exposed conditions using an immortalized choroidal Z310 cell line. Mn exposure in vitro resulted in an increased cellular {sup 64}Cu uptake and the up-regulation of both CTR1more » and DMT1. Knocking down CTR1 by siRNA counteracted the Mn-induced increase of {sup 64}Cu uptake, while knocking down DMT1 siRNA resulted in an increased cellular {sup 64}Cu uptake in Mn-exposed cells. To distinguish the roles of CTR1 and DMT1 in Cu transport, the Z310 cell-based tetracycline (Tet)-inducible CTR1 and DMT1 expression cell lines were developed, namely iZCTR1 and iZDMT1 cells, respectively. In iZCTR1 cells, Tet induction led to a robust increase (25 fold) of {sup 64}Cu uptake with the time course corresponding to the increased CTR1. Induction of DMT1 by Tet in iZDMT1 cells, however, resulted in only a slight increase of {sup 64}Cu uptake in contrast to a substantial increase in DMT1 mRNA and protein expression. These data indicate that CTR1, but not DMT1, plays an essential role in transporting Cu by the BCB in the choroid plexus. Mn-induced cellular overload of Cu at the BCB is due, primarily, to Mn-induced over-expression of CTR1. -- Highlights: ► This study compares the relative role of CTR1 and DMT1 in Cu transport by the BCB. ► Two novel tetracycline-inducible CTR1 and DMT1 expression cell lines are created. ► CTR1, but not DMT1, plays an essential role in transporting Cu by the BCB. ► Mn-induced cellular Cu overload is due to its induction of CTR1 rather than DMT1. ► Induction of CTR1 by Mn in the BCB contributes to an elevated Cu level in the CSF.« less

Cellular Microcultures: Programming Mechanical and Physicochemical Properties of 3D Hydrogel Cellular Microcultures via Direct Ink Writing (Adv. Healthcare Mater. 9/2016).

PubMed

McCracken, Joselle M; Badea, Adina; Kandel, Mikhail E; Gladman, A Sydney; Wetzel, David J; Popescu, Gabriel; Lewis, Jennifer A; Nuzzo, Ralph G

2016-05-01

R. Nuzzo and co-workers show on page 1025 how compositional differences in hydrogels are used to tune their cellular compliance by controlling their polymer mesh properties and subsequent uptake of the protein poly-l-lysine (green spheres in circled inset). The cover image shows pyramid micro-scaffolds prepared using direct ink writing (DIW) that differentially direct fibroblast and preosteoblast growth in 3D, depending on cell motility and surface treatment. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

PubMed Central

Chu, Kung-Hui; Alvarez-Cohen, Lisa

1998-01-01

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments. PMID:9726896

Evaluation of the In Vitro Effect of Gold Nanorod Aspect Ratio, Surface Charge and Chemistry on Cellular Association and Cytotoxicity

DTIC Science & Technology

2016-03-28

Synthesis of GNRs ..............................................................................................................3 3.2 PEG...chemistry we can enhance their biocompatibility while maintaining their cellular uptake. 3 3.0 METHODS 3.1 Synthesis of GNRs MTAB GNRs (MTAB-1...chlorauric acid (0.1 M) was combined at room temperature with a growth solution of CTAB (0.1 M), chlorauric acid (0.1 M) silver nitrate (0.1 M) ascorbic

Effects of X-shaped reduction-sensitive amphiphilic block copolymer on drug delivery.

PubMed

Xiao, Haijun; Wang, Lu

2015-01-01

To study the effects of X-shaped amphiphilic block copolymers on delivery of docetaxel (DTX) and the reduction-sensitive property on drug release, a novel reduction-sensitive amphiphilic copolymer, (PLGA)2-SS-4-arm-PEG2000 with a Gemini-like X-shape, was successfully synthesized. The formation of nanomicelles was proved with respect to the blue shift of the emission fluorescence as well as the fluorescent intensity increase of coumarin 6-loaded particles. The X-shaped polymers exhibited a smaller critical micelle concentration value and possessed higher micellar stability in comparison with those of linear ones. The size of X-shaped (PLGA)2-SS-4-arm-PEG2000 polymer nanomicelles (XNMs) was much smaller than that of nanomicelles prepared with linear polymers. The reduction sensitivity of polymers was confirmed by the increase of micellar sizes as well as the in vitro drug release profile of DTX-loaded XNMs (DTX/XNMs). Cytotoxicity assays in vitro revealed that the blank XNMs were nontoxic against A2780 cells up to a concentration of 50 µg/mL, displaying good biocompatibility. DTX/XNMs were more toxic against A2780 cells than other formulations in both dose- and time-dependent manners. Cellular uptake assay displayed a higher intracellular drug delivery efficiency of XNMs than that of nanomicelles prepared with linear polymers. Besides, the promotion of tubulin polymerization induced by DTX was visualized by immunofluorescence analysis, and the acceleration of apoptotic process against A2780 cells was also imaged using a fluorescent staining method. Therefore, this X-shaped reduction-sensitive (PLGA)2-SS-4-arm-PEG2000 copolymer could effectively improve the micellar stability and significantly enhance the therapeutic efficacy of DTX by increasing the cellular uptake and selectively accelerating the drug release inside cancer cells.

Enhanced anticancer activity and oral bioavailability of ellagic acid through encapsulation in biodegradable polymeric nanoparticles.

PubMed

Mady, Fatma M; Shaker, Mohamed A

2017-01-01

Despite the fact that various studies have investigated the clinical relevance of ellagic acid (EA) as a naturally existing bioactive substance in cancer therapy, little has been reported regarding the efficient strategy for improving its oral bioavailability. In this study, we report the formulation of EA-loaded nanoparticles (EA-NPs) to find a way to enhance its bioactivity as well as bioavailability after oral administration. Poly(ε-caprolactone) (PCL) was selected as the biodegradable polymer for the formulation of EA-NPs through the emulsion-diffusion-evaporation technique. The obtained NPs have been characterized by measuring particle size, zeta potential, Fourier transform infrared, differential scanning calorimetry, and X-ray diffraction. The entrapment efficiency and the release profile of EA was also determined. In vitro cellular uptake and cytotoxicity of the obtained NPs were evaluated using Caco-2 and HCT-116 cell lines, respectively. Moreover, in vivo study has been performed to measure the oral bioavailability of EA-NPs compared to free EA, using New Zealand white rabbits. NPs with distinct shape were obtained with high entrapment and loading efficiencies. Diffusion-driven release profile of EA from the prepared NPs was determined. EA-NP-treated HCT-116 cells showed relatively lower cell viability compared to free EA-treated cells. Fluorometric imaging revealed the cellular uptake and efficient localization of EA-NPs in the nuclear region of Caco-2 cells. In vivo testing revealed that the oral administration of EA-NPs produced a 3.6 times increase in the area under the curve compared to that of EA. From these results, it can be concluded that incorporation of EA into PCL as NPs enhances its oral bioavailability and activity.

Enhanced anticancer activity and oral bioavailability of ellagic acid through encapsulation in biodegradable polymeric nanoparticles

PubMed Central

Mady, Fatma M; Shaker, Mohamed A

2017-01-01

Despite the fact that various studies have investigated the clinical relevance of ellagic acid (EA) as a naturally existing bioactive substance in cancer therapy, little has been reported regarding the efficient strategy for improving its oral bioavailability. In this study, we report the formulation of EA-loaded nanoparticles (EA-NPs) to find a way to enhance its bioactivity as well as bioavailability after oral administration. Poly(ε-caprolactone) (PCL) was selected as the biodegradable polymer for the formulation of EA-NPs through the emulsion–diffusion–evaporation technique. The obtained NPs have been characterized by measuring particle size, zeta potential, Fourier transform infrared, differential scanning calorimetry, and X-ray diffraction. The entrapment efficiency and the release profile of EA was also determined. In vitro cellular uptake and cytotoxicity of the obtained NPs were evaluated using Caco-2 and HCT-116 cell lines, respectively. Moreover, in vivo study has been performed to measure the oral bioavailability of EA-NPs compared to free EA, using New Zealand white rabbits. NPs with distinct shape were obtained with high entrapment and loading efficiencies. Diffusion-driven release profile of EA from the prepared NPs was determined. EA-NP-treated HCT-116 cells showed relatively lower cell viability compared to free EA-treated cells. Fluorometric imaging revealed the cellular uptake and efficient localization of EA-NPs in the nuclear region of Caco-2 cells. In vivo testing revealed that the oral administration of EA-NPs produced a 3.6 times increase in the area under the curve compared to that of EA. From these results, it can be concluded that incorporation of EA into PCL as NPs enhances its oral bioavailability and activity. PMID:29066891

A safe and efficient hepatocyte-selective carrier system based on myristoylated preS1/21-47 domain of hepatitis B virus

NASA Astrophysics Data System (ADS)

Zhang, Quan; Zhang, Xuanmiao; Chen, Tijia; Wang, Xinyi; Fu, Yao; Jin, Yun; Sun, Xun; Gong, Tao; Zhang, Zhirong

2015-05-01

A safe and efficient liver targeted PEGylated liposome (PEG-Lip) based on N-terminal myristoylated preS1/21-47 (preS1/21-47myr) of hepatitis B virus was successfully developed. The study aimed to elucidate the cellular uptake mechanism of preS1/21-47myr modified PEG-Lip (preS1/21-47myr-PEG-Lip) in hepatogenic cells and the distribution behavior of preS1/21-47myr-PEG-Lip in Vr:CD1 (ICR) mice. The cellular uptake results showed that preS1/21-47myr-PEG-Lip was effectively taken up by hepatogenic cells (including primary hepatocytes and liver tumor cells) through a receptor-mediated endocytosis pathway compared with non-hepatogenic cells. After systemic administration to H22 hepatoma-bearing mice, preS1/21-47myr-PEG-Lip showed significant liver-specific delivery and an increase in the distribution of preS1/21-47myr-PEG-Lip in hepatic tumor. Furthermore, the antitumor effect of preS1/21-47myr-PEG-Lip loaded with paclitaxel (PTX) was remarkably stronger than that of PTX injection and PTX loaded liposomes (including common liposomes and PEG-Lip). In safety evaluation, no acute systemic toxicity and immunotoxicity were observed after intravenous injection of preS1/21-47myr-PEG-Lip. No liver toxicity was observed despite the dramatic increase of preS1/21-47myr-PEG-Lip in liver. Taken together, preS1/21-47myr-PEG-Lip represents a promising carrier system for targeted liver disease therapy and imaging.

Nitrogen monoxide decreases iron uptake from transferrin but does not mobilise iron from prelabelled neoplastic cells.

PubMed

Richardson, D R; Neumannova, V; Ponka, P

1995-05-12

The effect of congeners of nitrogen monoxide (NO) on iron (Fe) uptake from 59Fe-125I-transferrin (Tf) and release of 59Fe from prelabelled cells have been investigated in SK-MEL-28 human melanoma cells, human K562 cells and mouse MDW-4 cells. These studies have been initiated as it has been suggested that the tumoricidal effects of NO may be mediated by its acting to release Fe from cells (Hibbs et al., 1984 Biochem. Biophys. Res. Commun. 123, 716-723; Hibbs et al., 1988 Biochem. Biophys. Res. Commun. 157, 87-94). The nitrosonium ion (NO+) generator, sodium nitroprusside (SNP), decreased 59Fe uptake by melanoma cells to 57% of the control without decreasing 125I-Tf uptake after a 4-h incubation with 59Fe-125-Tf (1.25 microM). Longer incubations up to 24 h decreased 59Fe uptake and also 125I-Tf uptake. Two breakdown products of SNP, ferricyanide and cyanide, had no effect on 59Fe uptake. In addition, photolysis of the SNP solution prevented the inhibition of 59Fe uptake, suggesting that NO was the active agent. Two nitric oxide (NO.) producing agents, 3-morpholinosydnonimine (SIN), and S-nitroso-N-acetylpenicillamine (SNAP), also decreased 59Fe uptake from 59Fe-125I-Tf. Superoxide dismutase increased the efficacy of SIN, and the NO-scavenger, oxyhaemoglobin, prevented the inhibition of 59Fe uptake mediated by SNAP, again suggesting that NO was the active agent. Furthermore, dialysis studies demonstrated that none of the NO-generating agents could remove 59Fe from 59Fe-125I-Tf, suggesting that the decrease in cellular Fe uptake observed was not due to NO releasing Fe from the Fe-binding sites of Tf. Despite the ability of NO-producing agents at inhibiting 59Fe uptake by cells, they could not remove significant amounts of 59Fe from melanoma cells prelabelled with either 59Fe-citrate or 59Fe-125I-Tf. Similar data were obtained using K562 and MDW-4 cells. Interestingly, the NO+ generating agent, SNP, had no effect on [3H]thymidine uptake. However, when SNP was converted to an NO. generator by the addition of 1 mM ascorbate, its effect was similar to the NO. generator, SNAP, markedly reducing [3H]thymidine incorporation to 33% of the control value. The addition of unlabelled diferric Tf (0.625 microM) to SNAP ameliorated its inhibitory effect on cellular [3H]thymidine uptake, suggesting that the interaction of NO. with Fe was of importance in the inhibition observed. The results are discussed in the context of the cytostatic potential of NO via its binding to Fe.

Optimization of anti-cancer drugs and a targeting molecule on multifunctional gold nanoparticles.

PubMed

Rizk, Nahla; Christoforou, Nicolas; Lee, Sungmun

2016-05-06

Breast cancer is the most common and deadly cancer among women worldwide. Currently, nanotechnology-based drug delivery systems are useful for cancer treatment; however, strategic planning is critical in order to enhance the anti-cancer properties and reduce the side effects of cancer therapy. Here, we designed multifunctional gold nanoparticles (AuNPs) conjugated with two anti-cancer drugs, TGF-β1 antibody and methotrexate, and a cancer-targeting molecule, folic acid. First, optimum size and shape of AuNPs was selected by the highest uptake of AuNPs by MDA-MB-231, a metastatic human breast cancer cell line. It was 100 nm spherical AuNPs (S-AuNPs) that were used for further studies. A fixed amount (900 μl) of S-AuNP (3.8 × 10(8) particles/ml) was conjugated with folic acid-BSA or methotrexate-BSA. Methotrexate on S-AuNP induced cellular toxicity and the optimum amount of methotrexate-BSA (2.83 mM) was 500 μl. Uptake of S-AuNPs was enhanced by folate conjugation that binds to folate receptors overexpressed by MDA-MB-231 and the optimum uptake was at 500 μl of folic acid-BSA (2.83 mM). TGF-β1 antibody on S-AuNP reduced extracellular TGF-β1 of cancer cells by 30%. Due to their efficacy and tunable properties, we anticipate numerous clinical applications of multifunctional gold nanospheres in treating breast cancer.

Optimization of anti-cancer drugs and a targeting molecule on multifunctional gold nanoparticles

NASA Astrophysics Data System (ADS)

Rizk, Nahla; Christoforou, Nicolas; Lee, Sungmun

2016-05-01

Breast cancer is the most common and deadly cancer among women worldwide. Currently, nanotechnology-based drug delivery systems are useful for cancer treatment; however, strategic planning is critical in order to enhance the anti-cancer properties and reduce the side effects of cancer therapy. Here, we designed multifunctional gold nanoparticles (AuNPs) conjugated with two anti-cancer drugs, TGF-β1 antibody and methotrexate, and a cancer-targeting molecule, folic acid. First, optimum size and shape of AuNPs was selected by the highest uptake of AuNPs by MDA-MB-231, a metastatic human breast cancer cell line. It was 100 nm spherical AuNPs (S-AuNPs) that were used for further studies. A fixed amount (900 μl) of S-AuNP (3.8 × 108 particles/ml) was conjugated with folic acid-BSA or methotrexate-BSA. Methotrexate on S-AuNP induced cellular toxicity and the optimum amount of methotrexate-BSA (2.83 mM) was 500 μl. Uptake of S-AuNPs was enhanced by folate conjugation that binds to folate receptors overexpressed by MDA-MB-231 and the optimum uptake was at 500 μl of folic acid-BSA (2.83 mM). TGF-β1 antibody on S-AuNP reduced extracellular TGF-β1 of cancer cells by 30%. Due to their efficacy and tunable properties, we anticipate numerous clinical applications of multifunctional gold nanospheres in treating breast cancer.

A photo-responsive peptide- and asparagine-glycine-arginine (NGR) peptide-mediated liposomal delivery system.

PubMed

Xie, Xiangyang; Yang, Yanfang; Yang, Yang; Zhang, Hui; Li, Ying; Mei, Xingguo

2016-09-01

The conjugation of tunable peptides or materials with nanocarriers represents a promising approach for drug delivery to tumor cells. In this study, we report the development of a novel liposomal carrier system that exploits the cell surface binding synergism between photo-sensitive peptides (PSPs) and targeting ligands. The positive charges of the lysine residues on the cell-penetrating peptides (CPPs) were temporarily caged by the photolabile-protective groups (PG), thereby forming a PSP. Furthermore, this PSP enhances specific uptake into cancer cells after rapidly uncaging the PG via near-infrared (NIR) light illumination. In the circulatory system, the cell penetrability of PSP was hindered. In contrast, the asparagine-glycine-arginine (NGR) peptide moieties, selectively bind to CD13-positive tumors, were attached to the nanocarrier to facilitate the active accumulation of this liposomal carrier in tumor tissue. The dual-modified liposomes (PSP/NGR-L) were prepared by emulsification method, and the concentrations of DSPE-PEG 2000 -psCPP and DSPE-PEG 5000 -NGR in the liposomes were chosen to be 4% and 1% (molar ratio), respectively. The mean particle size of the PSP/NGR-L was about 95 nm, and the drug entrapment efficiency was more than 90%. Cellular uptake results demonstrated that the proposed PSP/NGR-L had an enhancement of cancer cell recognition and specific uptake. Furthermore, the PSP/NGR-L demonstrated a stronger antitumor efficacy in the HT-1080 tumor model in nude mice with the aid of NIR illumination.

Biological processing of dinuclear ruthenium complexes in eukaryotic cells.

PubMed

Li, Xin; Heimann, Kirsten; Dinh, Xuyen Thi; Keene, F Richard; Collins, J Grant

2016-10-20

The biological processing - mechanism of cellular uptake, effects on the cytoplasmic and mitochondrial membranes, intracellular sites of localisation and induction of reactive oxygen species - of two dinuclear polypyridylruthenium(ii) complexes has been examined in three eukaryotic cells lines. Flow cytometry was used to determine the uptake of [{Ru(phen)2}2{μ-bb12}](4+) (Rubb12) and [Ru(phen)2(μ-bb7)Ru(tpy)Cl](3+) {Rubb7-Cl, where phen = 1,10-phenanthroline, tpy = 2,2':6',2''-terpyridine and bbn = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane} in baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (HepG2) cell lines. The results demonstrated that the major uptake mechanism for Rubb12 and Rubb7-Cl was active transport, although with a significant contribution from carrier-assisted diffusion for Rubb12 and passive diffusion for Rubb7-Cl. Flow cytometry coupled with Annexin V/TO-PRO-3 double-staining was used to compare cell death by membrane damage or apoptosis. Rubb12 induced significant direct membrane damage, particularly with HepG2 cells, while Rubb7-Cl caused considerably less membrane damage but induced greater levels of apoptosis. Confocal microscopy, coupled with JC-1 assays, demonstrated that Rubb12 depolarises the mitochondrial membrane, whereas Rubb7-Cl had a much smaller affect. Cellular localisation experiments indicated that Rubb12 did not accumulate in the mitochondria, whereas significant mitochondrial accumulation was observed for Rubb7-Cl. The effect of Rubb12 and Rubb7-Cl on intracellular superoxide dismutase activity showed that the ruthenium complexes could induce cell death via a reactive oxygen species-mediated pathway. The results of this study demonstrate that Rubb12 predominantly kills eukaryotic cells by damaging the cytoplasmic membrane. As this dinuclear ruthenium complex has been previously shown to exhibit greater toxicity towards bacteria than eukaryotic cells, the results of the present study suggest that metal-based cationic oligomers can achieve selective toxicity against bacteria, despite exhibiting a non-specific membrane damage mechanism of action.

Quantification of intracellular payload release from polymersome nanoparticles

NASA Astrophysics Data System (ADS)

Scarpa, Edoardo; Bailey, Joanne L.; Janeczek, Agnieszka A.; Stumpf, Patrick S.; Johnston, Alexander H.; Oreffo, Richard O. C.; Woo, Yin L.; Cheong, Ying C.; Evans, Nicholas D.; Newman, Tracey A.

2016-07-01

Polymersome nanoparticles (PMs) are attractive candidates for spatio-temporal controlled delivery of therapeutic agents. Although many studies have addressed cellular uptake of solid nanoparticles, there is very little data available on intracellular release of molecules encapsulated in membranous carriers, such as polymersomes. Here, we addressed this by developing a quantitative assay based on the hydrophilic dye, fluorescein. Fluorescein was encapsulated stably in PMs of mean diameter 85 nm, with minimal leakage after sustained dialysis. No fluorescence was detectable from fluorescein PMs, indicating quenching. Following incubation of L929 cells with fluorescein PMs, there was a gradual increase in intracellular fluorescence, indicating PM disruption and cytosolic release of fluorescein. By combining absorbance measurements with flow cytometry, we quantified the real-time intracellular release of a fluorescein at a single-cell resolution. We found that 173 ± 38 polymersomes released their payload per cell, with significant heterogeneity in uptake, despite controlled synchronisation of cell cycle. This novel method for quantification of the release of compounds from nanoparticles provides fundamental information on cellular uptake of nanoparticle-encapsulated compounds. It also illustrates the stochastic nature of population distribution in homogeneous cell populations, a factor that must be taken into account in clinical use of this technology.

Interaction with culture medium components, cellular uptake and intracellular distribution of cobalt nanoparticles, microparticles and ions in Balb/3T3 mouse fibroblasts.

PubMed

Sabbioni, Enrico; Fortaner, Salvador; Farina, Massimo; Del Torchio, Riccardo; Petrarca, Claudia; Bernardini, Giovanni; Mariani-Costantini, Renato; Perconti, Silvia; Di Giampaolo, Luca; Gornati, Rosalba; Di Gioacchino, Mario

2014-02-01

The mechanistic understanding of nanotoxicity requires the physico-chemical characterisation of nanoparticles (NP), and their comparative investigation relative to the corresponding ions and microparticles (MP). Following this approach, the authors studied the dissolution, interaction with medium components, bioavailability in culture medium, uptake and intracellular distribution of radiolabelled Co forms (CoNP, CoMP and Co(2+)) in Balb/3T3 mouse fibroblasts. Co(2+) first saturates the binding sites of molecules in the extracellular milieu (e.g., albumin and histidine) and on the cell surface. Only after saturation, Co(2+) is actively uptaken. CoNP, instead, are predicted to be internalised by endocytosis. Dissolution of Co particles allows the formation of Co compounds (CoNP-rel), whose mechanism of cellular internalisation is unknown. Co uptake (ranking CoMP > CoNP > Co(2+)) reached maximum at 4 h. Once inside the cell, CoNP spread into the cytosol and organelles. Consequently, massive amounts of Co ions and CoNP-rel can reach subcellular compartments normally unexposed to Co(2+). This could explain the fact that the nuclear and mitochondrial Co concentrations resulted significantly higher than those obtained with Co(2+).

Monitoring Extracellular Vesicle Cargo Active Uptake by Imaging Flow Cytometry.

PubMed

Ofir-Birin, Yifat; Abou Karam, Paula; Rudik, Ariel; Giladi, Tal; Porat, Ziv; Regev-Rudzki, Neta

2018-01-01

Extracellular vesicles are essential for long distance cell-cell communication. They function as carriers of different compounds, including proteins, lipids and nucleic acids. Pathogens, like malaria parasites ( Plasmodium falciparum, Pf ), excel in employing vesicle release to mediate cell communication in diverse processes, particularly in manipulating the host response. Establishing research tools to study the interface between pathogen-derived vesicles and their host recipient cells will greatly benefit the scientific community. Here, we present an imaging flow cytometry (IFC) method for monitoring the uptake of malaria-derived vesicles by host immune cells. By staining different cargo components, we were able to directly track the cargo's internalization over time and measure the kinetics of its delivery. Impressively, we demonstrate that this method can be used to specifically monitor the translocation of a specific protein within the cellular milieu upon internalization of parasitic cargo; namely, we were able to visually observe how uptaken parasitic Pf -DNA cargo leads to translocation of transcription factor IRF3 from the cytosol to the nucleus within the recipient immune cell. Our findings demonstrate that our method can be used to study cellular dynamics upon vesicle uptake in different host-pathogen and pathogen-pathogen systems.

Modulation of Silica Nanoparticle Uptake into Human Osteoblast Cells by Variation of the Ratio of Amino and Sulfonate Surface Groups: Effects of Serum

PubMed Central

2015-01-01

To study the importance of the surface charge for cellular uptake of silica nanoparticles (NPs), we synthesized five different single- or multifunctionalized fluorescent silica NPs (FFSNPs) by introducing various ratios of amino and sulfonate groups into their surface. The zeta potential values of these FFSNPs were customized from highly positive to highly negative, while other physicochemical properties remained almost constant. Irrespective of the original surface charge, serum proteins adsorbed onto the surface, neutralized the zeta potential values, and prevented the aggregation of the tailor-made FFSNPs. Depending on the surface charge and on the absence or presence of serum, two opposite trends were found concerning the cellular uptake of FFSNPs. In the absence of serum, positively charged NPs were more strongly accumulated by human osteoblast (HOB) cells than negatively charged NPs. In contrast, in serum-containing medium, anionic FFSNPs were internalized by HOB cells more strongly, despite the similar size and surface charge of all types of protein-covered FFSNPs. Thus, at physiological condition, when the presence of proteins is inevitable, sulfonate-functionalized silica NPs are the favorite choice to achieve a desired high rate of NP internalization. PMID:26030456

A novel nanoparticle approach for imaging nutrient uptake by soil bacteria

NASA Astrophysics Data System (ADS)

O'Brien, S. L.; Whiteside, M. D.; Sholto-Douglas, D.; Antonopoulos, D. A.; Boyanov, M.; Durall, D. M.; Jones, M. D.; Lai, B.; O'Loughlin, E. J.; Kemner, K. M.

2014-12-01

The metabolic activities of soil microbes are the primary drivers of biogeochemical processes controlling the terrestrial carbon cycle, nutrient availability to plants, contaminant remediation, water quality, and other ecosystem services. However, we have a limited understanding of microbial metabolic processes such as nutrient uptake rates, substrate preferences, or how microbes and microbial metabolism are distributed throughout their habitat. Here we use a novel imaging technique with quantum dots (QDs, engineered semiconductor nanoparticles that produce size or composition-dependent fluorescence) to measure bacterial uptake of substrates of varying complexity. Cultures of two organisms differing in cell wall structure — Bacillus subtilis and Pseudomonas fluorescens — were grown in one of four ecologically relevant experimental conditions: nitrogen (N) limitation, phosphorus (P) limitation, N and P limitation, or no nutrient limitation. The cultures were then exposed to QDs with and without organic nutrients attached. X-ray fluorescence imaging was performed at 2ID-D at the Advanced Photon Source (APS) to determine the elemental distributions within both planktonic and surface-adhered (i.e, biofilms) cells. Uptake of unconjugated QDs was neglibible, and QDs conjugated to organic substrates varied depending on growth conditions and substrate, suggesting that they are a useful indicator of bacterial ecology. Cellular uptake was similar for the two bacterial species (2212 ± 273 nanoparticles per cm3 of cell volume for B. subtilis and 1682 ± 264 for P. fluorescens). On average, QD assimilation was six times greater when N or P was limiting, and cells took up about twice as much phosphoserine compared to other substrates, likely because it was the only compound providing both N and P. These results showed that regardless of their cell wall structure, bacteria can selectively take up quantifiable levels of QDs based on substrate and environmental conditions. APS images are consistent with those produced with confocal and optical microscopes, indicating that the XRF approach can detect bacterial uptake of CdSe-core QDs. These findings offer a new way to experimentally investigate basic bacterial ecology such as metabolic activity and biofilm development and function.

Histologic analysis of rabbit liver cancer treated by bulk ultrasound ablation

NASA Astrophysics Data System (ADS)

Karunakaran, Chandra Priya; Rudich, Steven M.; Alqadah, Amel; Burgess, Mark T.; Narmoneva, Daria A.; Mast, T. Douglas

2012-10-01

VX2 rabbit liver cancer, treated in vivo using bulk ultrasound ablation by miniaturized image-ablate arrays, was histologically analyzed using TTC vital stain and DAPI nucleic acid stain. VX2 cells were implanted into rabbit liver lobes and allowed to grow for 11-21 days. Liver lobes containing solid VX2 tumors were then treated with 4.8 MHz, 22.5-38.5 W/cm2 in situ intensity, unfocused ultrasound for exposure times of 20-120 s. After animal sacrifice, thermal lesions were bisected along the imaging/treatment plane, one face stained with TTC, and the other with DAPI. Levels of TTC uptake (no uptake, partial uptake, and complete uptake) in liver parenchyma corresponded to three discrete regions of tan, pink and red color. By processing images of DAPI-stained parenchymal tissue from these three regions, cellular damage was quantified. A viability index parameter incorporating the size and shape of DAPI-stained nuclei correlated significantly with levels of TTC uptake, and thus with local tissue viability. For ablation of normal liver, viability indices for parenchymal regions of no TTC uptake and partial TTC uptake were significantly different from those for viable tissue. For ablation of VX2 tumor, differences in viability index between regions of no TTC uptake and complete TTC uptake were smaller, but significant overall.

Industrial grade 2D molybdenum disulphide (MoS2): an in vitro exploration of the impact on cellular uptake, cytotoxicity, and inflammation

NASA Astrophysics Data System (ADS)

Moore, Caroline; Movia, Dania; Smith, Ronan J.; Hanlon, Damien; Lebre, Filipa; Lavelle, Ed C.; Byrne, Hugh J.; Coleman, Jonathan N.; Volkov, Yuri; McIntyre, Jennifer

2017-06-01

The recent surge in graphene research, since its liquid phase monolayer isolation and characterization in 2004, has led to advancements which are accelerating the exploration of alternative 2D materials such as molybdenum disulphide (MoS2), whose unique physico-chemical properties can be exploited in applications ranging from cutting edge electronic devices to nanomedicine. However, to assess any potential impact on human health and the environment, the need to understand the bio-interaction of MoS2 at a cellular and sub-cellular level is critical. Notably, it is important to assess such potential impacts of materials which are produced by large scale production techniques, rather than research grade materials. The aim of this study was to explore cytotoxicity, cellular uptake and inflammatory responses in established cell-lines that mimic different potential exposure routes (inhalation, A549; ingestion, AGS; monocyte, THP-1) following incubation with MoS2 flakes of varying sizes (50 nm, 117 nm and 177 nm), produced by liquid phase exfoliation. Using high content screening (HCS) and Live/Dead assays, it was established that 1 µg ml-1 (for the three different MoS2 sizes) did not induce toxic effects on any of the cell-lines. Confocal microscopy images revealed a normal cellular morphology in all cases. Transmission electron microscopy (TEM) confirmed the uptake of all MoS2 nanomaterials in all the cell-lines, the MoS2 ultimately locating in single membrane vesicles. At such sub-lethal doses, inflammatory responses are observed, however, associated, at least partially, with the presence of lipopolysaccharide endotoxin in nanomaterial suspensions and surfactant samples. Therefore, the inflammatory response of the cells to the MoS2 or endotoxin contamination was interrogated using a 10-plex ELISA which illustrates cytokine production. The experiments carried out using wild-type and endotoxin hyporesponsive bone marrow derived dendritic cells confirmed that the inflammatory responses result from a combination of endotoxin contamination, the MoS2 nanomaterials themselves, and the stabilizing surfactant.

Endocytosis via caveolae: alternative pathway with distinct cellular compartments to avoid lysosomal degradation?

PubMed Central

Kiss, Anna L; Botos, Erzsébet

2009-01-01

Endocytosis – the uptake of extracellular ligands, soluble molecules, protein and lipids from the extracellular surface – is a vital process, comprising multiple mechanisms, including phagocytosis, macropinocytosis, clathrin-dependent and clathrin-independent uptake such as caveolae-mediated and non-caveolar raft-dependent endocytosis. The best-studied endocytotic pathway for internalizing both bulk membrane and specific proteins is the clathrin-mediated endocytosis. Although many papers were published about the caveolar endocytosis, it is still not known whether it represents an alternative pathway with distinct cellular compartments to avoid lysosomal degradation or ligands taken up by caveolae can also be targeted to late endosomes/lysosomes. In this paper, we summarize data available about caveolar endocytosis. We are especially focussing on the intracellular route of caveolae and providing data supporting that caveolar endocytosis can join to the classical endocytotic pathway. PMID:19382909

Effect of Surface Properties on Liposomal siRNA Delivery

PubMed Central

Xia, Yuqiong; Tian, Jie; Chen, Xiaoyuan

2015-01-01

Liposomes are one of the most widely investigated carriers for siRNA delivery. The surface properties of liposomal carriers, including the surface charge, PEGylation, and ligand modification can significantly affect the gene silencing efficiency. Three barriers of systemic siRNA delivery (long blood circulation, efficient tumor penetration and efficient cellular uptake/endosomal escape) are analyzed on liposomal carriers with different surface charges, PEGylations and ligand modifications. Cationic formulations dominate siRNA delivery and neutral formulations also have good performance while anionic formulations are generally not proper for siRNA delivery. The PEG dilemma (prolonged blood circulation vs. reduced cellular uptake/endosomal escape) and the side effect of repeated PEGylated formulation (accelerated blood clearance) were discussed. Effects of ligand modification on cationic and neutral formulations were analyzed. Finally, we summarized the achievements in liposomal siRNA delivery, outlined existing problems and provided some future perspectives. PMID:26695117