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Sample records for additionally cellular uptake

  1. Cellular uptake of metallated cobalamins.

    PubMed

    Tran, Mai Thanh Quynh; Stürup, Stefan; Lambert, Ian Henry; Gammelgaard, Bente; Furger, Evelyne; Alberto, Roger

    2016-03-01

    Cellular uptake of vitamin B12-cisplatin conjugates was estimated via detection of their metal constituents (Co, Pt, and Re) by inductively coupled plasma mass spectrometry (ICP-MS). Vitamin B12 (cyano-cob(iii)alamin) and aquo-cob(iii)alamin [Cbl-OH2](+), which differ in the β-axial ligands (CN(-) and H2O, respectively), were included as control samples. The results indicated that B12 derivatives delivered cisplatin to both cellular cytosol and nuclei with an efficiency of one third compared to the uptake of free cisplatin cis-[Pt(II)Cl2(NH3)2]. In addition, uptake of charged B12 derivatives including [Cbl-OH2](+), [{Co}-CN-{cis-PtCl(NH3)2}](+), [{Re}-{Co}-CN-{cis-PtCl(NH3)2}](+), and [{Co}-CN-{trans-Pt(Cyt)(NH3)2}](2+) (Cyt = cytarabin) was high compared to neutral B12, which implied the existence of an additional internalization pathway for charged B12 vitamin analogs. The affinities of the charged B12 derivatives to the B12 transporters HC, IF and TC were similar to that of native vitamin B12. PMID:26739575

  2. Substrate stiffness regulates cellular uptake of nanoparticles.

    PubMed

    Huang, Changjin; Butler, Peter J; Tong, Sheng; Muddana, Hari S; Bao, Gang; Zhang, Sulin

    2013-04-10

    Nanoparticle (NP)-bioconjugates hold great promise for more sensitive disease diagnosis and more effective anticancer drug delivery compared with existing approaches. A critical aspect in both applications is cellular internalization of NPs, which is influenced by NP properties and cell surface mechanics. Despite considerable progress in optimization of the NP-bioconjugates for improved targeting, the role of substrate stiffness on cellular uptake has not been investigated. Using polyacrylamide (PA) hydrogels as model substrates with tunable stiffness, we quantified the relationship between substrate stiffness and cellular uptake of fluorescent NPs by bovine aortic endothelial cells (BAECs). We found that a stiffer substrate results in a higher total cellular uptake on a per cell basis, but a lower uptake per unit membrane area. To obtain a mechanistic understanding of the cellular uptake behavior, we developed a thermodynamic model that predicts that membrane spreading area and cell membrane tension are two key factors controlling cellular uptake of NPs, both of which are modulated by substrate stiffness. Our experimental and modeling results not only open up new avenues for engineering NP-based cancer cell targets for more effective in vivo delivery but also contribute an example of how the physical environment dictates cellular behavior and function.

  3. 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.

  4. Cellular uptake of neutral phosphorodiamidate morpholino oligomers.

    PubMed

    Iversen, Patrick L; Aird, Katherine M; Wu, Rebecca; Morse, Michael M; Devi, Gayathri R

    2009-09-01

    Phosphorodiamidate morpholino oligomers (PMO), which have a neutral chemistry, are extensively being used as tools for selective inhibition of gene expression in cell culture models and are currently in human clinical trials. Unlike phosphorothioates (PS ODN) and other charged oligonucleotides, little is known about the uptake characteristics of neutral oligomers. The purpose of this study was to understand the kinetics of PMO transport in cells and correlate with antisense activity. In contrast to primary cells and some transformed cell lines which were uptake permissive, established cancer cell lines showed very poor uptake with an occasional diffuse intracellular pattern. Differential PMO uptake was also observed in immune cells, with dendritic cells and monocytes showing highest uptake compared to T and B cells. In addition, PMO localization was observed to be heterogeneous within a population of uptake permissive cells. Unassisted PMO delivery targeting specific genes was correlated with functional antisense efficacy in experiments showing correction of pre-mRNA missplicing and inhibition of target enzyme activity in cells in culture. PMO internalization in uptake-permissive cells was identified to be specific, saturable, and energy-dependent, suggesting a receptor mediated uptake mechanism. Understanding PMO transport should facilitate the design of more effective synthetic antisense oligomers as therapeutic agents.

  5. Cellular fatty acid uptake: a pathway under construction.

    PubMed

    Su, Xiong; Abumrad, Nada A

    2009-03-01

    Membrane uptake of long-chain fatty acids (FAs) is the first step in cellular FA utilization and a point of metabolic regulation. CD36 facilitates a major fraction of FA uptake by key tissues. This review highlights the contribution of CD36 to pathophysiology in rodents and humans. Novel concepts regarding regulation of CD36-facilitated uptake are discussed (i.e. the role of membrane rafts and caveolae, CD36 recycling between intracellular depots and the membrane, and chemical modifications of the protein that impact its turnover and recruitment). Importantly, CD36 membrane levels and turnover are abnormal in diabetes, resulting in dysfunctional FA utilization. In addition, variants in the CD36 gene were shown recently to influence susceptibility for the metabolic syndrome, which greatly increases the risk of diabetes and heart disease. PMID:19185504

  6. Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation

    SciTech Connect

    Hazawa, Masaharu; Tomiyama, Kenichi; Saotome-Nakamura, Ai; Obara, Chizuka; Yasuda, Takeshi; Gotoh, Takaya; Tanaka, Izumi; Yakumaru, Haruko; Ishihara, Hiroshi; Tajima, Katsushi

    2014-04-18

    Highlights: • Radiation increases cellular uptake of exosomes. • Radiation induces colocalization of CD29 and CD81. • Exosomes selectively bind the CD29/CD81 complex. • Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation. - Abstract: Exosomes mediate intercellular communication, and mesenchymal stem cells (MSC) or their secreted exosomes affect a number of pathophysiologic states. Clinical applications of MSC and exosomes are increasingly anticipated. Radiation therapy is the main therapeutic tool for a number of various conditions. The cellular uptake mechanisms of exosomes and the effects of radiation on exosome–cell interactions are crucial, but they are not well understood. Here we examined the basic mechanisms and effects of radiation on exosome uptake processes in MSC. Radiation increased the cellular uptake of exosomes. Radiation markedly enhanced the initial cellular attachment to exosomes and induced the colocalization of integrin CD29 and tetraspanin CD81 on the cell surface without affecting their expression levels. Exosomes dominantly bound to the CD29/CD81 complex. Knockdown of CD29 completely inhibited the radiation-induced uptake, and additional or single knockdown of CD81 inhibited basal uptake as well as the increase in radiation-induced uptake. We also examined possible exosome uptake processes affected by radiation. Radiation-induced changes did not involve dynamin2, reactive oxygen species, or their evoked p38 mitogen-activated protein kinase-dependent endocytic or pinocytic pathways. Radiation increased the cellular uptake of exosomes through CD29/CD81 complex formation. These findings provide essential basic insights for potential therapeutic applications of exosomes or MSC in combination with radiation.

  7. Cellular uptake: lessons from supramolecular organic chemistry.

    PubMed

    Gasparini, Giulio; Bang, Eun-Kyoung; Montenegro, Javier; Matile, Stefan

    2015-07-01

    The objective of this Feature Article is to reflect on the importance of established and emerging principles of supramolecular organic chemistry to address one of the most persistent problems in life sciences. The main topic is dynamic covalent chemistry on cell surfaces, particularly disulfide exchange for thiol-mediated uptake. Examples of boronate and hydrazone exchange are added for contrast, comparison and completion. Of equal importance are the discussions of proximity effects in polyions and counterion hopping, and more recent highlights on ring tension and ion pair-π interactions. These lessons from supramolecular organic chemistry apply to cell-penetrating peptides, particularly the origin of "arginine magic" and the "pyrenebutyrate trick," and the currently emerging complementary "disulfide magic" with cell-penetrating poly(disulfide)s. They further extend to the voltage gating of neuronal potassium channels, gene transfection, and the delivery of siRNA. The collected examples illustrate that the input from conceptually innovative chemistry is essential to address the true challenges in biology beyond incremental progress and random screening.

  8. Variable Nanoparticle-Cell Adhesion Strength Regulates Cellular Uptake

    NASA Astrophysics Data System (ADS)

    Yuan, Hongyan; Li, Ju; Bao, Gang; Zhang, Sulin

    2010-09-01

    In receptor-mediated endocytosis, cells exercise biochemical control over the mechanics of adhesion to engulf foreign particles, featuring a variable adhesion strength. Here we present a thermodynamic model with which we elucidate that the variable adhesion strength critically governs the cellular uptake, yielding an uptake phase diagram in the space of ligand density and particle size. We identify from the diagram an endocytosed phase with markedly high uptake, encompassed by a lower and an upper phase boundary that are set, respectively, by the enthalpic and entropic limits of the adhesion strength. The phase diagram may provide useful guidance to the rational design of nanoparticle-based therapeutic and diagnostic agents.

  9. Insight into nanoparticle cellular uptake and intracellular targeting

    PubMed Central

    Yameen, Basit; Choi, Won Il; Vilos, Cristian; Swami, Archana; Shi, Jinjun; Farokhzad, Omid C.

    2014-01-01

    Collaborative efforts from the fields of biology, materials science, and engineering are leading to exciting progress in the development of nanomedicines. Since the targets of many therapeutic agents are localized in subcellular compartments, modulation of nanoparticle-cell interactions for an efficient cellular uptake through the plasma membrane, and the development of nanomedicines for precise delivery to subcellular compartments remain formidable challenges. The cellular internalization routes have a determining effect on the post-internalization fate and intracellular localization of nanoparticles. This review highlights the cellular uptake routes most relevant to the field of non-targeted nanomedicine, and presents an account of ligand targeted nanoparticles for receptor mediated cellular internalization as a strategy for modulating the cellular uptake of nanoparticles. Ligand targeted nanoparticles have been the main impetus behind the progress of nanomedicines towards the clinic. This strategy has even resulted in a remarkable development towards effective oral delivery of nanomedicines that can overcome the intestinal epithelial cellular barrier. A detailed overview of the recent developments towards subcellular targeting that is emerging as a platform for the next generation organelle specific nanomedicines is also provided. Each section of the review includes prospect, potential, and concrete expectations from the field of targeted nanomedicines and strategies to meet those expectations. PMID:24984011

  10. 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

  11. 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.

  12. 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...

  13. Insight into nanoparticle cellular uptake and intracellular targeting.

    PubMed

    Yameen, Basit; Choi, Won Il; Vilos, Cristian; Swami, Archana; Shi, Jinjun; Farokhzad, Omid C

    2014-09-28

    Collaborative efforts from the fields of biology, materials science, and engineering are leading to exciting progress in the development of nanomedicines. Since the targets of many therapeutic agents are localized in subcellular compartments, modulation of nanoparticle-cell interactions for efficient cellular uptake through the plasma membrane and the development of nanomedicines for precise delivery to subcellular compartments remain formidable challenges. Cellular internalization routes determine the post-internalization fate and intracellular localization of nanoparticles. This review highlights the cellular uptake routes most relevant to the field of non-targeted nanomedicine and presents an account of ligand-targeted nanoparticles for receptor-mediated cellular internalization as a strategy for modulating the cellular uptake of nanoparticles. Ligand-targeted nanoparticles have been the main impetus behind the progress of nanomedicines towards the clinic. This strategy has already resulted in remarkable progress towards effective oral delivery of nanomedicines that can overcome the intestinal epithelial barrier. A detailed overview of the recent developments in subcellular targeting as a novel platform for next-generation organelle-specific nanomedicines is also provided. Each section of the review includes prospects, potential, and concrete expectations from the field of targeted nanomedicines and strategies to meet those expectations.

  14. Estimation of stream nutrient uptake from nutrient addition experiments

    SciTech Connect

    Payn, Robert

    2005-09-01

    Nutrient uptake in streams is often quantified by determining nutrient uptake length. However, current methods for measuring nutrient uptake length are often impractical, expensive, or demonstrably incorrect. We have developed a new method to estimate ambient nutrient uptake lengths using field experiments involving several levels of nutrient addition. Data analysis involves plotting nutrient addition uptake lengths versus added concentration and extrapolating to the negative ambient concentration. This method is relatively easy, inexpensive, and based on sound theoretical development. It is more accurate than the commonly used method involving a single nutrient addition. The utility of the method is supported by field studies directly comparing our new method with isotopic tracer methods for determining uptake lengths of phosphorus, ammonium, and nitrate. Our method also provides parameters for comparing potential nutrient limitation among streams.

  15. Cellular uptake and cytotoxicity of octahedral rhenium cluster complexes.

    PubMed

    Choi, Soo-Jin; Brylev, Konstantin A; Xu, Jing-Zhe; Mironov, Yuri V; Fedorov, Vladimir E; Sohn, Youn Soo; Kim, Sung-Jin; Choy, Jin-Ho

    2008-11-01

    Cellular uptake behavior of a novel class of octahedral rhenium cluster compounds, hexahydroxo complexes K(4)[{Re(6)S(8)}(OH)(6)].8H(2)O (1) and K(4)[{Re(6)Se(8)}(OH)(6)].8H(2)O (2), was evaluated in human cervical adenocarcinoma HeLa cells. Confocal microscopy and flow cytometry studies demonstrated that rhenium cluster 1 was not internalized into cell, while rhenium cluster 2 was. Conjugation of a polymer to rhenium cluster 1, namely the derivative K(4)[{Re(6)S(8)}(OH)(5)L] (3) (L is amphiphilic diblock copolymer MPEG550-CH(2)CONH-GlyPheLeuGlyPheLeu-COO(-)), considerably enhanced cellular uptake in a concentration-dependent manner and was predominantly localized in the cytoplasm and nucleus upon incubation time. The uptake of rhenium cluster 2 was mediated by energy-dependent endocytosis, whereas rhenium cluster 3 was directly ingested into cells by cell-fusion-like mechanism. According to the cytotoxicity evaluation test, both rhenium clusters 2 and 3 did not exhibit acute cytotoxic effects up to 50 microM, at the practical concentration level of biological applications. It is, therefore, expected that the rhenium cluster complexes can be promising potential candidates as diagnostic agents for medical treatment.

  16. Quantitatively understanding cellular uptake of gold nanoparticles via radioactivity analysis

    PubMed Central

    Shao, Xia; Schnau, Paul; Qian, Wei; Wang, Xueding

    2015-01-01

    The development of multifunctional gold nanoparticles (AuNPs) underwent an explosion in the last two decades. However, many questions regarding detailed surface chemistry and how they are affecting the behaviors of AuNPs in vivo and in vitro still need to be addressed before AuNPs can be widely adapted into clinical settings. In this work, radioactivity analysis was employed for quantitative evaluation of I-125 radiolabeled AuNPs uptakes by cancer cells. Facilitated with this new method, we have conducted initial bioevaluation of surfactant-free AuNPs produced by femtosecond laser ablation. Cellular uptake of AuNPs as a function of the RGD density on the AuNP surface, as well as a function of time, has been quantified. The radioactivity analysis may shed light on the dynamic interactions of AuNPs with cancer cells, and help achieve optimized designs of AuNPs for future clinical applications. PMID:26505012

  17. The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles

    PubMed Central

    Fröhlich, Eleonore

    2012-01-01

    Many types of nanoparticles (NPs) are tested for use in medical products, particularly in imaging and gene and drug delivery. For these applications, cellular uptake is usually a prerequisite and is governed in addition to size by surface characteristics such as hydrophobicity and charge. Although positive charge appears to improve the efficacy of imaging, gene transfer, and drug delivery, a higher cytotoxicity of such constructs has been reported. This review summarizes findings on the role of surface charge on cytotoxicity in general, action on specific cellular targets, modes of toxic action, cellular uptake, and intracellular localization of NPs. Effects of serum and intercell type differences are addressed. Cationic NPs cause more pronounced disruption of plasma-membrane integrity, stronger mitochondrial and lysosomal damage, and a higher number of autophagosomes than anionic NPs. In general, nonphagocytic cells ingest cationic NPs to a higher extent, but charge density and hydrophobicity are equally important; phagocytic cells preferentially take up anionic NPs. Cells do not use different uptake routes for cationic and anionic NPs, but high uptake rates are usually linked to greater biological effects. The different uptake preferences of phagocytic and nonphagocytic cells for cationic and anionic NPs may influence the efficacy and selectivity of NPs for drug delivery and imaging. PMID:23144561

  18. Investigation and characterization of the cellular uptake of nanoparticles

    NASA Astrophysics Data System (ADS)

    Siebein, Kerry Norine

    The focus of this study was to determine the effect of surface coatings on the cellular uptake of nanoparticles and their fate inside cells and tissue using correlative microscopy. The nanoparticle properties and cellular uptake, including unique identification of the composition, locations and distribution of nanoparticles in cells, were determined using multiple microscopy techniques. The effect of coatings on the properties of platinum nanoparticles and their uptake by BEAS cells was undertaken to determine their relationship to the expression of heme oxygenease (HO-1) enzyme. The 1.3M PVP platinum nanoparticles produced very fine and well dispersed nanoparticles that were observed in the lysosomes of the BEAS cells and the other nanoparticles studied were present in large agglomerates. The effect of polyethylene glycol (PEG) coating on the circulation time, agglomeration and accumulation of gold nanoparticles in the liver of mice was studied. A new approach to measuring the PEG coating thickness using high resolution TEM and negative staining techniques was introduced. The amount and distribution of gold in sections of the liver was determined using darkfield reflected light microscopy. Image analysis was used to determine the size, number and area fraction of agglomerates in the sections. The three dimensional distribution of gold nanoparticles in a single cell of the liver was obtained using ion abrasion scanning electron microscopy. The uncoated gold nanoparticles were taken up almost immediately by the Kupffer cells while the PEG coated nanoparticles were taken up after 2 hours. The native gold was observed in large, tightly packed agglomerates in lysosomes inside the cells, while the PEG coated nanoparticles were observed lining the inner surfaces of the lysosomes. Differences in the agglomeration of the gold nanoparticles had not been previously observed. The effect of surface charge on the fate of QDs ingested by daphnia magna (water fleas) was explored

  19. Cellular uptake and in vivo distribution of polyhistidine peptides.

    PubMed

    Iwasaki, Takashi; Tokuda, Yoshihisa; Kotake, Ayaka; Okada, Hiroyuki; Takeda, Shuji; Kawano, Tsuyoshi; Nakayama, Yuji

    2015-07-28

    Cell-penetrating peptides (CPPs) are arginine/lysine-rich sequences, and they are effectively internalized into cells. In this process, positive charge is crucial. In the present study, we found polyhistidine peptides (PHPs), as the novel CPP, which are efficiently internalized into cells in a positive charge-independent manner. Interestingly, cellular uptake of the PHPs increased as the chain length increased, reaching a maximum uptake at H16 (HHHHHHHHHHHHHHHH-NH2). This H16 peptide showed up to 14.6-fold higher cell-penetrating capacity against HT1080 human fibrosarcoma cells relative to a major CPP, the octa-arginine (RRRRRRRR-NH2) peptide. Cellular uptake of the H16 peptide is mainly due to macropinocytosis and most of the H16 peptide localizes in the lysosome and Golgi apparatus. However, a cytoplasmic pro-apoptotic domain (KLAKLAKKLAKLAK-NH2) conjugated to the H16 peptide showed cytotoxic effects. This indicates that a proportion of the H16 peptide escapes from the macropinosome to the cytoplasm. In a protein transduction study, green fluorescence protein fused to the H16 peptide (GFP-H16) was purified by Ni-NTA chromatography, detected using an anti-His-tag antibody and internalized into cells. This serial process reveals that H16 functions as a His-tag and protein transduction domain. Furthermore, in vivo distribution analysis showed that the H16 peptide accumulates immediately in tumor tissue and is retained up to 132h following injection into the tumor (HT1080 human fibrosarcoma)-bearing mice. This is the first observation of a His-polymer being internalize into cells efficiently. The findings suggest that PHPs are novel CPPs. In particular, the H16 peptide represents a promising drug delivery carrier candidate in medical and biotechnological fields.

  20. Design of a bistable switch to control cellular uptake.

    PubMed

    Oyarzún, Diego A; Chaves, Madalena

    2015-12-01

    Bistable switches are widely used in synthetic biology to trigger cellular functions in response to environmental signals. All bistable switches developed so far, however, control the expression of target genes without access to other layers of the cellular machinery. Here, we propose a bistable switch to control the rate at which cells take up a metabolite from the environment. An uptake switch provides a new interface to command metabolic activity from the extracellular space and has great potential as a building block in more complex circuits that coordinate pathway activity across cell cultures, allocate metabolic tasks among different strains or require cell-to-cell communication with metabolic signals. Inspired by uptake systems found in nature, we propose to couple metabolite import and utilization with a genetic circuit under feedback regulation. Using mathematical models and analysis, we determined the circuit architectures that produce bistability and obtained their design space for bistability in terms of experimentally tuneable parameters. We found an activation-repression architecture to be the most robust switch because it displays bistability for the largest range of design parameters and requires little fine-tuning of the promoters' response curves. Our analytic results are based on on-off approximations of promoter activity and are in excellent qualitative agreement with simulations of more realistic models. With further analysis and simulation, we established conditions to maximize the parameter design space and to produce bimodal phenotypes via hysteresis and cell-to-cell variability. Our results highlight how mathematical analysis can drive the discovery of new circuits for synthetic biology, as the proposed circuit has all the hallmarks of a toggle switch and stands as a promising design to control metabolic phenotypes across cell cultures. PMID:26674196

  1. Silver nanoparticles: correlating nanoparticle size and cellular uptake with genotoxicity

    PubMed Central

    Butler, Kimberly S.; Peeler, David J.; Casey, Brendan J.; Dair, Benita J.; Elespuru, Rosalie K.

    2015-01-01

    The focus of this research was to develop a better understanding of the pertinent physico-chemical properties of silver nanoparticles (AgNPs) that affect genotoxicity, specifically how cellular uptake influences a genotoxic cell response. The genotoxicity of AgNPs was assessed for three potential mechanisms: mutagenicity, clastogenicity and DNA strand-break-based DNA damage. Mutagenicity (reverse mutation assay) was assessed in five bacterial strains of Salmonella typhimurium and Echerichia coli, including TA102 that is sensitive to oxidative DNA damage. AgNPs of all sizes tested (10, 20, 50 and 100nm), along with silver nitrate (AgNO3), were negative for mutagenicity in bacteria. No AgNPs could be identified within the bacteria cells using transmission electron microscopy (TEM), indicating these bacteria lack the ability to actively uptake AgNPs 10nm or larger. Clastogenicity (flow cytometry-based micronucleus assay) and intermediate DNA damage (DNA strand breaks as measured in the Comet assay) were assessed in two mammalian white blood cell lines: Jurkat Clone E6-1 and THP-1. It was observed that micronucleus and Comet assay end points were inversely correlated with AgNP size, with smaller NPs inducing a more genotoxic response. TEM results indicated that AgNPs were confined within intracellular vesicles of mammalian cells and did not penetrate the nucleus. The genotoxicity test results and the effect of AgNO3 controls suggest that silver ions may be the primary, and perhaps only, cause of genotoxicity. Furthermore, since AgNO3 was not mutagenic in the gram-negative bacterial Ames strains tested, the lack of bacterial uptake of the AgNPs may not be the major reason for the lack of genotoxicity observed. PMID:25964273

  2. Evaluation of Asymmetric Immunoliposomal Nanoparticles for Cellular Uptake

    PubMed Central

    Whittenton, Jeremiah; Pitchumani, Ramanan; Thevananther, Sundararajah; Mohanty, Kishore

    2013-01-01

    Effective and targeted in vivo delivery of polynucleotide therapeutics is the key for the treatment of many diseases. Asymmetric immunoliposomes can be used as vehicles to deliver polynucleotides effectively because the two leaflets of the bilayer can have different compositions, which enhance the delivery capacity. The formation and in vitro cellular uptake of asymmetric immunoliposomes containing polynucleotide cargoes were studied here. Maleimide functionalized DSPE-PEG (2000) were incorporated into the outer leaflet to produce asymmetric liposomes capable of covalently attaching antibodies. Thiolated antibodies from both human and rabbit origin were conjugated to produce asymmetric pendant-type immunoliposomes that retain their specificity towards detection antibodies through the formation process. Human IgG conjugated asymmetric immunoliposomes were readily internalized (> 20 per cell) by macrophage, HEPG2, and CV-1 monkey kidney cells. The cells internalized the liposomal nanoparticles by the endocytic pathway. The immunoliposome-encapsulated endosomes were intact for at least 5 days and sequestered the plasmid from expression by the cell. PMID:22742513

  3. Improved cellular uptake of functionalized single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Antonelli, A.; Serafini, S.; Menotta, M.; Sfara, C.; Pierigé, F.; Giorgi, L.; Ambrosi, G.; Rossi, L.; Magnani, M.

    2010-10-01

    Single-walled carbon nanotubes (SWNTs) due to their unique structural and physicochemical properties, have been proposed as delivery systems for a variety of diagnostic and therapeutic agents. However, SWNTs have proven difficult to solubilize in aqueous solution, limiting their use in biological applications. In an attempt to improve SWNTs' solubility, biocompatibility, and to increase cell penetration we have thoroughly investigated the construction of carbon scaffolds coated with aliphatic carbon chains and phospholipids to obtain micelle-like structures. At first, oxidized SWNTs (2370 ± 30 nmol mg - 1 of SWNTs) were covalently coupled with an alcoholic chain (stearyl alcohol, C18H37OH; 816 nmol mg - 1 of SWNTs). Subsequently, SWNTs-COOC18H37 derivatives were coated with phosphatidylethanolamine (PE) or -serine (PS) phospholipids obtaining micelle-like structures. We found that cellular uptake of these constructs by phagocytic cells occurs via an endocytotic mechanism for constructs larger than 400 nm while occurs via diffusion through the cell membrane for constructs up to 400 nm. The material that enters the cell by phagocytosis is actively internalized by macrophages and localizes inside endocytotic vesicles. In contrast the material that enters the cells by diffusion is found in the cell cytosol. In conclusion, we have realized new biomimetic constructs based on alkylated SWNTs coated with phospholipids that are efficiently internalized by different cell types only if their size is lower than 400 nm. These constructs are not toxic to the cells and could now be explored as delivery systems for non-permeant cargoes.

  4. Modeling of cellular arginine uptake by more than one transporter.

    PubMed

    Nel, Marietha J; Woodiwiss, Angela J; Candy, Geoffrey P

    2012-01-01

    Determining the kinetic constants of arginine uptake by endothelial cells mediated by more than one transporter from linearization of data as Eadie-Hofstee plots or modeling which does not include the concentration of trace radiolabeled amino acid used to measure uptake may not be correct. The initial rate of uptake of trace [³H]L-arginine by HUVECs and ECV₃₀₄ cells in the presence of a range of unlabeled arginine and modifiers was used in nonlinear models to calculate the constants of arginine uptake using GraphPad Prism. Theoretical plots of uptake derived from constants determined from Eadie-Hofstee graphs overestimated uptake, whereas those from the nonlinear modeling approach agreed with experimental data. The contribution of uptake by individual transporters could be modeled and showed that leucine inhibited the individual transporters differently and not necessarily competitively. N-Ethylmaleimide inhibited only y⁺ transport, and BCH may be a selective inhibitor of y⁺L transport. The absence of sodium reduced arginine uptake by y⁺L transport and reduced the K(m)', whereas reducing sodium decreased arginine uptake by y⁺ transport without affecting the K (m)'. The nonlinear modeling approach using raw data avoided the errors inherent in methods deriving constants from the linearization of the uptake processes following Michaelian kinetics. This study provides explanations for discrepancies in the literature and suggests that a nonlinear modeling approach better characterizes the kinetics of amino acid uptake into cells by more than one transporter.

  5. Cytotoxicity, cellular uptake, and cellular biotransformations of oxaliplatin in human colon carcinoma cells.

    PubMed

    Luo, F R; Wyrick, S D; Chaney, S G

    1998-01-01

    Biotransformation products of platinum anticancer drugs have been suggested to be responsible for drug efficacy and toxicity. This study was designed to determine whether the efficacy of the closely related 1,2-diaminocyclohexane-Pt (dach-Pt) compounds oxaliplatin and ormaplatin were determined primarily by the parent drugs or by one of their biotransformation products. Based on consideration of both in vitro cytotoxicity in human colon carcinoma cells (HT-29) and concentrations following oxaliplatin administration in vivo, our data suggest that the efficacy of oxaliplatin is primarily determined by the plasma levels of the parent drug, with the biotransformation products Pt(dach)Cl2, Pt(dach)(H2O)Cl, and Pt(dach)(H2O)2 making only minor contributions. The stable biotransformation products containing amino acids did not have any significant cytotoxicity. In contrast, our data suggest that the efficacy of ormaplatin is primarily determined by plasma levels of Pt(dach)Cl2. The cytotoxicity of oxaliplatin, Pt(dach)Cl2, and Pt(dach)(H2O)Cl was approximately proportional to their cellular uptake, whereas the cytotoxicity of ormaplatin, Pt(dach)(H2O)2, and Pt(dach)(Met) was less than predicted from their uptake. Treatment of HT-29 cells with equimolar external concentrations of Pt(dach)Cl2 and oxaliplatin resulted in the formation of twofold more Pt-DNA adducts following Pt(dach)Cl2 treatment than following oxaliplatin treatment. However, intracellular Pt(dach)Cl2 levels were 30-fold higher for Pt(dach)Cl2-treated cells than for oxaliplatin-treated cells. These data suggest that intracellular conversion of oxaliplatin to Pt(dach)Cl2 makes only a minor contribution to Pt-DNA adduct formation and the resultant cytotoxicity of oxaliplatin. PMID:10367941

  6. Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways.

    PubMed

    Contreras, Janette; Elnagar, Ahmed Y O; Hamm-Alvarez, Sarah F; Camarero, Julio A

    2011-10-30

    Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands. PMID:21906641

  7. Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways.

    PubMed

    Contreras, Janette; Elnagar, Ahmed Y O; Hamm-Alvarez, Sarah F; Camarero, Julio A

    2011-10-30

    Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands.

  8. Purification, immunotoxic effects, and cellular uptake of trichothecene mycotoxins

    SciTech Connect

    Witt, M.F.

    1989-01-01

    Studies were carried out to better understand how the trichothecenes alter immune function in animals and humans. Deoxynivalenol (DON) was purified for use in animal feeding studies. Dietary exposure to DON for 8 weeks altered the serum immunoglobulin profile in mice and decreased the splenic plaque-forming cell response to the antigen sheep red blood cells. The uptake of ({sup 3}H)T-2 toxin by a murine B-cell hybridoma was studied in order to learn more about the way in which trichothecenes interact with immune cells. A simple procedure was developed for the laboratory production and purification of gram quantities of crystalline DON. When Fusarium graminearum R6576 was grown on rice, concentrations of 600 to 700 ppm DON accumulated after 13 to 18 days of incubation. A DON derivative, 15-acetylDON, was also found at concentrations of 100 to 300 ppm after 7 to 10 days. DON was purified from crude culture extracts by water-saturated silica gel chromatography. Alpha-({sup 3}H)T-2 toxin of 99% chemical and radiochemical purity was prepared for use in uptake studies. Both the rate of uptake of ({sup 3}H)T-2 toxin by hybridomas and the time required for accumulation of ({sup 3}H)T-2 to reach equilibrium were proportional to the concentration of ({sup 3}H)T-2. ({sup 3}H)T-2 toxin accumulated by hybridomas was proportional to the concentration of ({sup 3}H)T-2 between 10{sup {minus}8} and 10{sup {minus}3} M. The rate of uptake of ({sup 3}H)jT-2 toxin by hybridomas was inhibited by the trichothecenes T-2 toxin, DON, verrucarin A, and roridin A, as well as the antibiotic anisomycin. The kinetics and concentration dependence of accumulation, along with the inhibition patterns, suggest that uptake of ({sup 3}H)T-2 toxin by hybridomas is mediated by binding of toxin to ribosomes.

  9. Effects of physicochemical properties of zinc oxide nanoparticles on cellular uptake

    NASA Astrophysics Data System (ADS)

    Yu, J.; Baek, M.; Chung, H. E.; Choi, S. J.

    2011-07-01

    Zinc oxide (ZnO) nanoparticles have been used as a source of zinc, an essential trace element in food industry and also widely applied to various cosmetic products. However, there are few researches demonstrating that the cellular uptake behaviours of ZnO with respect to the physicochemical characteristics such as particle size and surface charge in human cells. In this study, we evaluated the cellular uptake of ZnO with two different sizes (20 and 70 nm) and different charges (positive and negative). Human lung epithelial cells were exposed to ZnO for a given time, and then the uptake amount of ZnO was measured with inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The results showed that the smaller sized ZnO could more easily enter the cells than the larger sized ZnO. In terms of surface charge, positively charged ZnO showed high cellular uptake compared to ZnO with negative charge. The internalization pathway of positively charged ZnO nanoparticles was determined to be primarily related to the energy-dependent endocytosis. It is, therefore, concluded that the particle size and surface charge of ZnO nanoparticles are critical factors influencing on their cellular uptake. Understanding the cellular uptake behaviours of nanoparticles with respect to physicochemical properties may be important to predict their toxicity potential on human.

  10. Bioinspired Cellular Structures: Additive Manufacturing and Mechanical Properties

    NASA Astrophysics Data System (ADS)

    Stampfl, J.; Pettermann, H. E.; Liska, R.

    Biological materials (e.g., wood, trabecular bone, marine skeletons) rely heavily on the use of cellular architecture, which provides several advantages. (1) The resulting structures can bear the variety of "real life" load spectra using a minimum of a given bulk material, featuring engineering lightweight design principles. (2) The inside of the structures is accessible to body fluids which deliver the required nutrients. (3) Furthermore, cellular architectures can grow organically by adding or removing individual struts or by changing the shape of the constituting elements. All these facts make the use of cellular architectures a reasonable choice for nature. Using additive manufacturing technologies (AMT), it is now possible to fabricate such structures for applications in engineering and biomedicine. In this chapter, we present methods that allow the 3D computational analysis of the mechanical properties of cellular structures with open porosity. Various different cellular architectures including disorder are studied. In order to quantify the influence of architecture, the apparent density is always kept constant. Furthermore, it is shown that how new advanced photopolymers can be used to tailor the mechanical and functional properties of the fabricated structures.

  11. Uptake Rate of Cationic Mitochondrial Inhibitor MKT-077 Determines Cellular Oxygen Consumption Change in Carcinoma Cells

    PubMed Central

    Chunta, John L.; Vistisen, Kerry S.; Yazdi, Zeinab; Braun, Rod D.

    2012-01-01

    Objective Since tumor radiation response is oxygen-dependent, radiosensitivity can be enhanced by increasing tumor oxygenation. Theoretically, inhibiting cellular oxygen consumption is the most efficient way to increase oxygen levels. The cationic, rhodacyanine dye-analog MKT-077 inhibits mitochondrial respiration and could be an effective metabolic inhibitor. However, the relationship between cellular MKT-077 uptake and metabolic inhibition is unknown. We hypothesized that rat and human mammary carcinoma cells would take up MKT-077, causing a decrease in oxygen metabolism related to drug uptake. Methods R3230Ac rat breast adenocarcinoma cells were exposed to MKT-077. Cellular MKT-077 concentration was quantified using spectroscopy, and oxygen consumption was measured using polarographic electrodes. MKT-077 uptake kinetics were modeled by accounting for uptake due to both the concentration and potential gradients across the plasma and mitochondrial membranes. These kinetic parameters were used to model the relationship between MKT-077 uptake and metabolic inhibition. MKT-077-induced changes in oxygen consumption were also characterized in MDA-MB231 human breast carcinoma cells. Results Cells took up MKT-077 with a time constant of ∼1 hr, and modeling showed that over 90% of intracellular MKT-077 was bound or sequestered, likely by the mitochondria. The uptake resulted in a rapid decrease in oxygen consumption, with a time constant of ∼30 minutes. Surprisingly the change in oxygen consumption was proportional to uptake rate, not cellular concentration. MKT-077 proved a potent metabolic inhibitor, with dose-dependent decreases of 45–73% (p = 0.003). Conclusions MKT-077 caused an uptake rate-dependent decrease in cellular metabolism, suggesting potential efficacy for increasing tumor oxygen levels and radiosensitivity in vivo. PMID:22616013

  12. Tuning Cellular Uptake of Molecular Probes by Rational Design of Their Assembly into Supramolecular Nanoprobes.

    PubMed

    Lock, Lye Lin; Reyes, Claudia D; Zhang, Pengcheng; Cui, Honggang

    2016-03-16

    Intracellular sensing of pathologically relevant biomolecules could provide essential information for accurate evaluation of disease staging and progression, yet the poor cellular uptake of water-soluble molecular probes limits their use as protease sensors. In other cases such as extracellular sensing, cellular uptake should be effectively inhibited. Self-assembly of molecular probes into supramolecular nanoprobes presents a potential strategy to alter their interaction mechanisms with cells to promote or reduce their cellular uptake. Here, we report on the design, synthesis, and assembly of peptide-based molecular beacons into supramolecular protease sensors of either spherical or filamentous shapes. We found that positively charged spherical nanobeacons demonstrate much higher cellular uptake efficiency than its monomeric form, thus making them most suitable for intracellular sensing of the lysosomal protease cathepsin B. Our results also suggest that assembly into filamentous nanobeacons significantly reduces their internalization by cancer cells, an important property that can be utilized for probing extracellular protease activities. These studies provide important guiding principles for rational design of supramolecular nanoprobes with tunable cellular uptake characteristics.

  13. Cellular uptake of a dexamethasone palmitate-low density lipoprotein complex by macrophages and foam cells.

    PubMed

    Tauchi, Yoshihiko; Chono, Sumio; Morimoto, Kazuhiro

    2003-04-01

    To evaluate the utility of a dexamethasone palmitate (DP)-low density lipoprotein (LDL) complex to transport drug into foam cells, the cellular uptake of DP-LDL complex by macrophages and foam cells was examined. The DP-LDL complex was prepared by incubation with DP and LDL, and the DP-LDL complex and murine macrophages were incubated. No cellular uptake of the DP-LDL complex by macrophages was found until 6 h after the start of incubation, but this gradually increased from 12 to 48 h. On the other hand, the cellular uptake of the oxidized DP-LDL complex was already apparent at 3 h after the start incubation, and then markedly increased until 48 h incubation along with that of the lipid emulsion (LE) containing DP (DP-LE). The cellular uptake of DP-LE by foam cells was significantly lower than that by macrophages. However, the cellular uptake of DP-LDL complex by foam cells was similar to that by macrophages. These findings suggest that the DP-LDL complex is oxidatively modified, and then incorporated into macrophages and foam cells through the scavenger receptor pathway. Since selective delivery of drugs into foam cells in the early stage of atherosclerosis is a useful protocol for antiatherosclerosis treatment, the DP-LDL complex appears to be a potentially useful drug-carrier complex for future antiatherosclerotic therapy.

  14. Cellular uptake and anticancer activity of carboxylated gallium corroles.

    PubMed

    Pribisko, Melanie; Palmer, Joshua; Grubbs, Robert H; Gray, Harry B; Termini, John; Lim, Punnajit

    2016-04-19

    We report derivatives of gallium(III) tris(pentafluorophenyl)corrole, 1 [Ga(tpfc)], with either sulfonic (2) or carboxylic acids (3, 4) as macrocyclic ring substituents: the aminocaproate derivative, 3 [Ga(ACtpfc)], demonstrated high cytotoxic activity against all NCI60 cell lines derived from nine tumor types and confirmed very high toxicity against melanoma cells, specifically the LOX IMVI and SK-MEL-28 cell lines. The toxicities of 1, 2, 3, and 4 [Ga(3-ctpfc)] toward prostate (DU-145), melanoma (SK-MEL-28), breast (MDA-MB-231), and ovarian (OVCAR-3) cancer cells revealed a dependence on the ring substituent: IC50values ranged from 4.8 to >200 µM; and they correlated with the rates of uptake, extent of intracellular accumulation, and lipophilicity. Carboxylated corroles 3 and 4, which exhibited about 10-fold lower IC50values (<20 µM) relative to previous analogs against all four cancer cell lines, displayed high efficacy (Emax= 0). Confocal fluorescence imaging revealed facile uptake of functionalized gallium corroles by all human cancer cells that followed the order: 4 > 3 > 2 > 1 (intracellular accumulation of gallium corroles was fastest in melanoma cells). We conclude that carboxylated gallium corroles are promising chemotherapeutics with the advantage that they also can be used for tumor imaging.

  15. Enhanced Cellular Uptake of Bowl-like Microcapsules.

    PubMed

    Li, Huiying; Zhang, Wenbo; Tong, Weijun; Gao, Changyou

    2016-05-11

    Among several properties of colloidal particles, shape is emerging as an important parameter for tailoring the interactions between particles and cells. In this study, bowl-like multilayer microcapsules were prepared by osmotic-induced invagination of their spherical counterparts in a concentrated polyelectrolyte solution. The internalization behaviors of bowl-like and spherical microcapsules were compared by coincubation with smooth muscle cells (SMCs) and macrophages. The bowl-like capsules tended to attach onto the cell membranes from the bend side and could be enwrapped by the membranes of SMCs, leading to a faster uptake rate and larger accumulation inside cells than those of their spherical counterparts. These results are important for understanding the shape-dependent internalization behavior, providing useful guidance for further materials design especially in biomedical applications. PMID:27119770

  16. The influence of cell penetrating peptide branching on cellular uptake of QDs

    NASA Astrophysics Data System (ADS)

    Breger, Joyce; Delehanty, James; Susumu, Kimihiro; Anderson, George; Muttenhaler, Markus; Dawson, Philip; Medintz, Igor

    2016-03-01

    Semiconductor quantum dots (QDs) serve as a valuable platform for understating the intricacies of nanoparticle cellular uptake and fate for the development of theranostics. Developing novel internalization peptides that maximize cellular uptake while minimizing the amount of peptide is important to allow space on the nanoparticle for other cargo (e.g. drugs). We have designed a range of branched, dendritic internalization peptides composed of polyarginine (Arg9) branches (1 to 16 repeats) attached a dendritic wedge based on the sequence WP9G2H6. By attaching these branched dendritic peptides to QD's, we can study the influence of branching on cellular uptake as a function of time, ratio, and degree of branching.

  17. Carotenoid and polyphenol bioaccessibility and cellular uptake from plum and cabbage varieties.

    PubMed

    Kaulmann, Anouk; André, Christelle M; Schneider, Yves-Jacques; Hoffmann, Lucien; Bohn, Torsten

    2016-04-15

    Plum and cabbage are rich in carotenoids and polyphenols. However, their bioactivity depends on their release and intestinal uptake. Four varieties of Brassicaceae (Duchy, Scots Kale, Kale, Kalorama) and Prunus (Cherry Plum, Plum 620, Ersinger, Italian Plum) were studied; bioaccessibility following in vitro digestion, cellular uptake (Caco-2 vs. co-culture cell model: Caco-2:HT-29-MTX (90:10%) and colonic fermentation were determined for carotenoids/polyphenols; the influence of certain kitchen preparations was likewise studied. Carotenoids were non-significantly influenced by the latter, while for polyphenols, boiling and steaming significantly reduced total phenolics (p<0.05). Carotenoid bioaccessibility did not differ significantly between Prunus vs. Brassicaceae varieties, but xanthophyll was higher than carotene bioaccessibility (p<0.01). Polyphenol bioaccessibility was low (<10%), possibly compromised by the cream containing test meal. Total carotenoid cellular uptake varied between varieties (0.3-4.1%), being higher for carotenes (4.1%) than for xanthophylls (1.6%, p<0.01), and were higher for the co-culture cell model compared to Caco-2 cells (p<0.01). Total carotenoid recovery in the colonic fraction varied from 4% to 25%. Lower bioaccessibility of carotenes thus appeared to be somewhat counterbalanced by higher cellular uptake. The potential positive role of the mucus layer for cellular uptake and the fate of the colonic digesta deserve further attention in the future.

  18. Carotenoid and polyphenol bioaccessibility and cellular uptake from plum and cabbage varieties.

    PubMed

    Kaulmann, Anouk; André, Christelle M; Schneider, Yves-Jacques; Hoffmann, Lucien; Bohn, Torsten

    2016-04-15

    Plum and cabbage are rich in carotenoids and polyphenols. However, their bioactivity depends on their release and intestinal uptake. Four varieties of Brassicaceae (Duchy, Scots Kale, Kale, Kalorama) and Prunus (Cherry Plum, Plum 620, Ersinger, Italian Plum) were studied; bioaccessibility following in vitro digestion, cellular uptake (Caco-2 vs. co-culture cell model: Caco-2:HT-29-MTX (90:10%) and colonic fermentation were determined for carotenoids/polyphenols; the influence of certain kitchen preparations was likewise studied. Carotenoids were non-significantly influenced by the latter, while for polyphenols, boiling and steaming significantly reduced total phenolics (p<0.05). Carotenoid bioaccessibility did not differ significantly between Prunus vs. Brassicaceae varieties, but xanthophyll was higher than carotene bioaccessibility (p<0.01). Polyphenol bioaccessibility was low (<10%), possibly compromised by the cream containing test meal. Total carotenoid cellular uptake varied between varieties (0.3-4.1%), being higher for carotenes (4.1%) than for xanthophylls (1.6%, p<0.01), and were higher for the co-culture cell model compared to Caco-2 cells (p<0.01). Total carotenoid recovery in the colonic fraction varied from 4% to 25%. Lower bioaccessibility of carotenes thus appeared to be somewhat counterbalanced by higher cellular uptake. The potential positive role of the mucus layer for cellular uptake and the fate of the colonic digesta deserve further attention in the future. PMID:26616956

  19. STRA6: role in cellular retinol uptake and efflux

    PubMed Central

    Kelly, Mary

    2015-01-01

    Distribution of vitamin A throughout the body is important to maintain retinoid function in peripheral tissues and to ensure optimal vision. A critical step of this process is the transport of vitamin A across cell membranes. Increasing evidence indicates that this process is mediated by a multidomian membrane protein that is encoded by the stimulated by retinoic acid 6 (STRA6) gene. Biochemical studies revealed that STRA6 is a transmembrane pore which transports vitamin A bidirectionally between extra- and intracellular retinoid binding proteins. Vitamin A accumulation in cells is driven by coupling of transport with vitamin A esterification. Loss-of-function studies in zebrafish and mouse models have unraveled the critical importance of STRA6 for vitamin A homeostasis of peripheral tissues. Impairment in vitamin A transport and uptake homeostasis are associated with diseases including type 2 diabetes and a microphthalmic syndrome known as Matthew Wood Syndrome. This review will discuss the advanced state of knowledge about STRA6’s biochemistry, biology and association with disease. PMID:26312242

  20. Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides.

    PubMed

    Allred, Benjamin E; Rupert, Peter B; Gauny, Stacey S; An, Dahlia D; Ralston, Corie Y; Sturzbecher-Hoehne, Manuel; Strong, Roland K; Abergel, Rebecca J

    2015-08-18

    Synthetic radionuclides, such as the transuranic actinides plutonium, americium, and curium, present severe health threats as contaminants, and understanding the scope of the biochemical interactions involved in actinide transport is instrumental in managing human contamination. Here we show that siderocalin, a mammalian siderophore-binding protein from the lipocalin family, specifically binds lanthanide and actinide complexes through molecular recognition of the ligands chelating the metal ions. Using crystallography, we structurally characterized the resulting siderocalin-transuranic actinide complexes, providing unprecedented insights into the biological coordination of heavy radioelements. In controlled in vitro assays, we found that intracellular plutonium uptake can occur through siderocalin-mediated endocytosis. We also demonstrated that siderocalin can act as a synergistic antenna to sensitize the luminescence of trivalent lanthanide and actinide ions in ternary protein-ligand complexes, dramatically increasing the brightness and efficiency of intramolecular energy transfer processes that give rise to metal luminescence. Our results identify siderocalin as a potential player in the biological trafficking of f elements, but through a secondary ligand-based metal sequestration mechanism. Beyond elucidating contamination pathways, this work is a starting point for the design of two-stage biomimetic platforms for photoluminescence, separation, and transport applications.

  1. Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides

    PubMed Central

    Allred, Benjamin E.; Rupert, Peter B.; Gauny, Stacey S.; An, Dahlia D.; Ralston, Corie Y.; Sturzbecher-Hoehne, Manuel; Strong, Roland K.; Abergel, Rebecca J.

    2015-01-01

    Synthetic radionuclides, such as the transuranic actinides plutonium, americium, and curium, present severe health threats as contaminants, and understanding the scope of the biochemical interactions involved in actinide transport is instrumental in managing human contamination. Here we show that siderocalin, a mammalian siderophore-binding protein from the lipocalin family, specifically binds lanthanide and actinide complexes through molecular recognition of the ligands chelating the metal ions. Using crystallography, we structurally characterized the resulting siderocalin–transuranic actinide complexes, providing unprecedented insights into the biological coordination of heavy radioelements. In controlled in vitro assays, we found that intracellular plutonium uptake can occur through siderocalin-mediated endocytosis. We also demonstrated that siderocalin can act as a synergistic antenna to sensitize the luminescence of trivalent lanthanide and actinide ions in ternary protein–ligand complexes, dramatically increasing the brightness and efficiency of intramolecular energy transfer processes that give rise to metal luminescence. Our results identify siderocalin as a potential player in the biological trafficking of f elements, but through a secondary ligand-based metal sequestration mechanism. Beyond elucidating contamination pathways, this work is a starting point for the design of two-stage biomimetic platforms for photoluminescence, separation, and transport applications. PMID:26240330

  2. Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides.

    PubMed

    Allred, Benjamin E; Rupert, Peter B; Gauny, Stacey S; An, Dahlia D; Ralston, Corie Y; Sturzbecher-Hoehne, Manuel; Strong, Roland K; Abergel, Rebecca J

    2015-08-18

    Synthetic radionuclides, such as the transuranic actinides plutonium, americium, and curium, present severe health threats as contaminants, and understanding the scope of the biochemical interactions involved in actinide transport is instrumental in managing human contamination. Here we show that siderocalin, a mammalian siderophore-binding protein from the lipocalin family, specifically binds lanthanide and actinide complexes through molecular recognition of the ligands chelating the metal ions. Using crystallography, we structurally characterized the resulting siderocalin-transuranic actinide complexes, providing unprecedented insights into the biological coordination of heavy radioelements. In controlled in vitro assays, we found that intracellular plutonium uptake can occur through siderocalin-mediated endocytosis. We also demonstrated that siderocalin can act as a synergistic antenna to sensitize the luminescence of trivalent lanthanide and actinide ions in ternary protein-ligand complexes, dramatically increasing the brightness and efficiency of intramolecular energy transfer processes that give rise to metal luminescence. Our results identify siderocalin as a potential player in the biological trafficking of f elements, but through a secondary ligand-based metal sequestration mechanism. Beyond elucidating contamination pathways, this work is a starting point for the design of two-stage biomimetic platforms for photoluminescence, separation, and transport applications. PMID:26240330

  3. 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.

  4. 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. PMID:27006261

  5. A physical model for the size-dependent cellular uptake of nanoparticles modified with cationic surfactants

    PubMed Central

    Xu, Airen; Yao, Mingfei; Xu, Guangkui; Ying, Jingyan; Ma, Weicheng; Li, Bo; Jin, Yi

    2012-01-01

    Background The aim of this work was to improve oral bioavailability. The uptake of a series of quaternary ammonium salt didodecyl dimethylammonium bromide (DMAB)-modified nanoparticles (with uniform sizes ranging from 50 nm to 300 nm) into heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2) and human colon adenocarcinoma cells (HT-29) was investigated. Methods Coumarin-6 (C6) loaded poly (lactide-co-glycolide) (PLGA) nanoparticles were prepared with DMAB using the emulsion solvent diffusion method. The physicochemical properties and cellular uptake of these nanoparticles were studied. Deserno’s model was applied to explain the experimental observations. Results The results showed that the surface modification of PLGA nanoparticles with DMAB notably improved the cellular uptake. The cellular uptake was size-dependent and had an optimal particle size of 100 nm. The experimental data was integrated numerically, and was in agreement with the theoretical model. Conclusion These results indicated that the interactions between the charged nanoparticles and the cells resulted from various forces (eg, electrostatic forces, hydrophobic forces, bending and stretching forces, and limited receptor-mediated endocytosis), and the uptake of the nanoparticles occurred as a result of competition. PMID:22848178

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Stapling monomeric GCN4 peptides allows for DNA binding and enhanced cellular uptake.

    PubMed

    Iyer, Abhishek; Van Lysebetten, Dorien; Ruiz García, Yara; Louage, Benoit; De Geest, Bruno G; Madder, Annemieke

    2015-04-01

    The basic DNA recognition region of the GCN4 protein comprising 23 amino acids has been modified to contain two optimally positioned cysteines which have been linked and stapled using cross-linkers of suitable lengths. This results in stapled peptides with a stabilized α-helical conformation which allows for DNA binding and concurrent enhancement of cellular uptake.

  8. 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.

  9. 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.

  10. Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle-cell interaction.

    PubMed

    Lu, Yi-Ching; Luo, Pei-Chun; Huang, Chun-Wan; Leu, Yann-Lii; Wang, Tzu-Hao; Wei, Kuo-Chen; Wang, Hsin-Ell; Ma, Yunn-Hwa

    2014-09-01

    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.

  11. Cellular Uptake and Internalization of Hyaluronan-based Doxorubicin and Cisplatin Conjugates

    PubMed Central

    Cai, Shuang; Alhowyan, Adel Ali B; Yang, Qiuhong; Forrest, W.C. Melanie; Shnayder, Yelizaveta; Forrest, M. Laird

    2015-01-01

    Background Hyaluronan (HA) is a ligand for the CD44 receptor which is crucial to cancer cell proliferation and metastasis. High levels of CD44 expression in many cancers have encouraged the development of HA-based carriers for anti-cancer therapeutics. Purpose The objective of this study was to determine whether HA conjugation of anticancer drugs impacts CD44-specific HA-drug uptake and disposition by human head and neck cancer cells. Methods The internalization and cellular disposition of hyaluronan-doxorubicin (HA-DOX), hyaluronan-cisplatin (HA-Pt), and hyaluronan-cyanine7 (HA-Cy7) conjugates were investigated by inhibiting endocytosis pathways, and by inhibiting the CD44–mediated internalization pathways that are known to mediate hyaluronan uptake in vitro. Results Cellular internalization of HA was regulated by CD44 receptors. In mouse xenografts, HA conjugation significantly enhanced tumor cell uptake compared to unconjugated drug. Discussion The results suggested that the main mechanism of HA-based conjugate uptake may be active transport via CD44 in conjunction with a clathrin–dependent endocytic pathway. Other HA receptors, hyaluronan–mediated motility receptor (RHAMM) and lymphatic vessel endothelial hyaluronan receptor (LYVE-1), did not play a significant role in conjugate uptake. Conclusions HA conjugation significantly increased CD44 mediated drug uptake and extended the residence time of drugs in tumor cells. PMID:24892741

  12. Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency

    NASA Astrophysics Data System (ADS)

    Yang, Zhe; Jiang, Zhaozhong; Cao, Zhong; Zhang, Chao; Gao, Di; Luo, Xingen; Zhang, Xiaofang; Luo, Huiyan; Jiang, Qing; Liu, Jie

    2014-08-01

    We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell

  13. Esterification of Ginsenoside Rh2 Enhanced Its Cellular Uptake and Antitumor Activity in Human HepG2 Cells.

    PubMed

    Chen, Fang; Deng, Ze-Yuan; Zhang, Bing; Xiong, Zeng-Xing; Zheng, Shi-Lian; Tan, Chao-Li; Hu, Jiang-Ning

    2016-01-13

    Our previous research had indicated that the octyl ester derivative of ginsenoside Rh2 (Rh2-O) might have a higher bioavailability than Rh2 in the Caco-2 cell line. The aim of this study was to investigate the cellular uptake and antitumor effects of Rh2-O in human HepG2 cells as well as its underlying mechanism compared with Rh2. Results showed that Rh2-O exhibited a higher cellular uptake (63.24%) than Rh2 (36.76%) when incubated with HepG2 cells for 24 h. Rh2-O possessed a dose- and time-dependent inhibitory effect against the proliferation of HepG2 cells. The IC50 value of Rh2-O for inhibition of HepG2 cell proliferation was 20.15 μM, which was roughly half the value of Rh2. Rh2-O induced apoptosis of HepG2 cells through a mitochondrial-mediated intrinsic pathway. In addition, the accumulation of ROS was detected in Rh2-O-treated HepG2 cells, which participated in the apoptosis of HepG2 cells. Conclusively, the findings above all suggested that Rh2-O as well as Rh2 inducing HepG2 cells apoptosis might involve similar mechanisms; however, Rh2-O had better antitumor activities than Rh2, probably due to its higher cellular uptake. PMID:26672619

  14. Multilayer Coating of Tetrandrine-loaded PLGA nanoparticles: Effect of surface charges on cellular uptake rate and drug release profile.

    PubMed

    Meng, Rui; Li, Ke; Chen, Zhe; Shi, Chen

    2016-02-01

    The effect of surface charges on the cellular uptake rate and drug release profile of tetrandrine-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (TPNs) was studied. Stabilizer-free nanoprecipitation method was used in this study for the synthesis of TPNs. A typical layer-by-layer approach was applied for multi-coating particles' surface with use of poly(styrene sulfonate) sodium salt (PSS) as anionic layer and poly(allylamine hydrochloride) (PAH) as cationic layer. The modified TPNs were characterized by different physicochemical techniques such as Zeta sizer, scanning electron microscopy and transmission electron microscopy. The drug loading efficiency, release profile and cellular uptake rate were evaluated by high performance liquid chromatography and confocal laser scanning microscopy, respectively. The resultant PSS/PAH/PSS/PAH/TPNs (4 layers) exhibited spherical-shaped morphology with the average size of 160.3±5.165 nm and zeta potential of-57.8 mV. The encapsulation efficiency and drug loading efficiency were 57.88% and 1.73%, respectively. Multi-layer coating of polymeric materials with different charges on particles' surface could dramatically influence the drug release profile of TPNs (4 layers vs. 3 layers). In addition, variable layers of surface coating could also greatly affect the cellular uptake rate of TPNs in A549 cells within 8 h. Overall, by coating particles' surface with those different charged polymers, precise control of drug release as well as cellular uptake rate can be achieved simultaneously. Thus, this approach provides a new strategy for controllable drug delivery. PMID:26838734

  15. 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

  16. Biocompatible transferrin-conjugated sodium hexametaphosphate-stabilized gold nanoparticles: synthesis, characterization, cytotoxicity and cellular uptake

    NASA Astrophysics Data System (ADS)

    Parab, Harshala J.; Huang, Jing-Hong; Lai, Tsung-Ching; Jan, Yi-Hua; Liu, Ru-Shi; Wang, Jui-Ling; Hsiao, Michael; Chen, Chung-Hsuan; Hwu, Yeu-Kuang; Tsai, Din Ping; Chuang, Shih-Yi; Pang, Jong-Hwei S.

    2011-09-01

    The feasibility of using gold nanoparticles (AuNPs) for biomedical applications has led to considerable interest in the development of novel synthetic protocols and surface modification strategies for AuNPs to produce biocompatible molecular probes. This investigation is, to our knowledge, the first to elucidate the synthesis and characterization of sodium hexametaphosphate (HMP)-stabilized gold nanoparticles (Au-HMP) in an aqueous medium. The role of HMP, a food additive, as a polymeric stabilizing and protecting agent for AuNPs is elucidated. The surface modification of Au-HMP nanoparticles was carried out using polyethylene glycol and transferrin to produce molecular probes for possible clinical applications. In vitro cell viability studies performed using as-synthesized Au-HMP nanoparticles and their surface-modified counterparts reveal the biocompatibility of the nanoparticles. The transferrin-conjugated nanoparticles have significantly higher cellular uptake in J5 cells (liver cancer cells) than control cells (oral mucosa fibroblast cells), as determined by inductively coupled plasma mass spectrometry. This study demonstrates the possibility of using an inexpensive and non-toxic food additive, HMP, as a stabilizer in the large-scale generation of biocompatible and monodispersed AuNPs, which may have future diagnostic and therapeutic applications.

  17. Uptake of dexamethasone incorporated into liposomes by macrophages and foam cells and its inhibitory effect on cellular cholesterol ester accumulation.

    PubMed

    Chono, Sumio; Morimoto, Kazuhiro

    2006-09-01

    To confirm the efficacy of dexamethasone incorporated into liposomes in the treatment of atherosclerosis, the uptake of dexamethasone-liposomes by macrophages and foam cells and its inhibitory effect on cellular cholesterol ester accumulation in these cells were investigated in-vitro. Dexamethasone-liposomes were prepared with egg yolk phosphatidylcholine, cholesterol and dicetylphosphate in a lipid molar ratio of 7/2/1 by the hydration method. This was adjusted to three different particle sizes to clarify the influence of particle size on the uptake by the macrophages and foam cells, and the inhibitory effect on cellular cholesterol ester accumulation. The distribution of particle sizes of dexamethasone-liposomes were 518.7+/-49.5 nm (L500), 202.2+/-23.1 nm (L200), and 68.6+/-6.5 nm (L70), respectively. For each size, dexamethasone concentration and dexamethasone/lipid molar ratio in dexamethasone-liposome suspension were 1 mg dexamethasone mL-1 and 0.134 mol dexamethasone mol-1 total lipids, respectively. The zeta potential was approximately -70 mV for all sizes. Dexamethasone-liposomes or free dexamethasone were added to the macrophages in the presence of oxidized low density lipoprotein (oxLDL) and foam cells, and then incubated at 37 degrees C. The uptake amount of dexamethasone by the macrophages and foam cells after a 24-h incubation was L500>L200>free dexamethasone>L70. The macrophages in the presence of oxLDL and foam cells were incubated with dexamethasone-liposomes or free dexamethasone for 24 h at 37 degrees C to evaluate the inhibitory effect on the cellular cholesterol ester accumulation. The cellular cholesterol ester level in the macrophages treated with oxLDL was significantly increased compared with that in macrophages without additives. L500, L200 and free dexamethasone significantly inhibited this cholesterol ester accumulation. L500, L200 and free dexamethasone also significantly reduced cellular cholesterol ester accumulation in foam cells. In

  18. Shape effect in cellular uptake of PEGylated nanoparticles: comparison between sphere, rod, cube and disk

    NASA Astrophysics Data System (ADS)

    Li, Ying; Kröger, Martin; Liu, Wing Kam

    2015-10-01

    PEGylated NPs for controllable cellular uptake and help establish quantitatively rules in designing NP-based vectors for targeted drug delivery. Electronic supplementary information (ESI) available: Additional simulation data, derivations, and relationships quoted in the main part of this work. See DOI: 10.1039/C5NR02970H

  19. Cellular uptake and activity of heparin functionalised cerium oxide nanoparticles in monocytes.

    PubMed

    Ting, S R Simon; Whitelock, John M; Tomic, Romana; Gunawan, Cindy; Teoh, Wey Yang; Amal, Rose; Lord, Megan S

    2013-06-01

    Cerium oxide nanoparticles (nanoceria) are effective in scavenging intracellular reactive oxygen species (ROS). In this study nanoceria synthesized by flame spray pyrolysis (dXRD = 12 nm) were functionalised with heparin via an organosilane linker, 3-aminopropyltriethoxysilane. Nanoceria were functionalised with approximately 130 heparin molecules per nanoparticle as determined by thermo gravimetric analysis. Heparin functionalised nanoceria were more effectively internalised by the human monocyte cell line, U937, and U937 cells that had been activated with phorbol 12 myristate 13-acetate (PMA) than bare nanoceria. The heparin functionalised nanoceria were also more effective in scavenging ROS than nanoceria in both activated and unactivated U937 cells. Heparin coupled nanoceria were found to be biologically active due to their ability to bind fibroblast growth factor 2 and signal through FGF receptor 1. Additionally, the heparin-coupled nanoceria, once internalised by the cells, were found to be degraded by 48 h. Together these data demonstrated that heparin enhanced the biological properties of nanoceria in terms of cellular uptake and ROS scavenging, while the nanoceria themselves were more effective at delivering heparin intracellularly than exposing cells to heparin in solution. PMID:23478040

  20. Cellular uptake and intracellular fate of protein releasing bacterial amyloids in mammalian cells.

    PubMed

    Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejandro; Garcia-Fruitós, Elena; Vázquez, Esther; Villaverde, Antonio

    2016-04-14

    Bacterial Inclusion Bodies (IBs) are amyloidal protein deposits that functionally mimic secretory granules from the endocrine system. When formed by therapeutically relevant proteins, they complement missing intracellular activities in jeopardized cell cultures, offering an intriguing platform for protein drug delivery in substitutive therapies. Despite the therapeutic potential of IBs, their capability to interact with eukaryotic cells, cross the cell membrane and release their functional building blocks into the cytosolic space remains essentially unexplored. We have systematically dissected the process by which bacterial amyloids interact with mammalian cells. An early and tight cell membrane anchorage of IBs is followed by cellular uptake of single or grouped IBs of variable sizes by macropinocytosis. Although an important fraction of the penetrating particles is led to lysosomal degradation, biologically significant amounts of protein are released into the cytosol. In addition, our data suggest the involvement of the bacterial cell folding modulator DnaK in the release of functional proteins from these amyloidal reservoirs. The mechanisms supporting the internalization of disintegrable protein nanoparticles revealed here offer clues to implement novel approaches for protein drug delivery based on controlled protein packaging as bacterial IBs.

  1. Biophysical determinants for cellular uptake of hydrocarbon-stapled peptide helices.

    PubMed

    Bird, Gregory H; Mazzola, Emanuele; Opoku-Nsiah, Kwadwo; Lammert, Margaret A; Godes, Marina; Neuberg, Donna S; Walensky, Loren D

    2016-10-01

    Hydrocarbon-stapled peptides are a class of bioactive alpha-helical ligands developed to dissect and target protein interactions. While there is consensus that stapled peptides can be effective chemical tools for investigating protein regulation, their broader utility for therapeutic modulation of intracellular interactions remains an active area of study. In particular, the design principles for generating cell-permeable stapled peptides are empiric, yet consistent intracellular access is essential to in vivo application. Here, we used an unbiased statistical approach to determine which biophysical parameters dictate the uptake of stapled-peptide libraries. We found that staple placement at the amphipathic boundary combined with optimal hydrophobic and helical content are the key drivers of cellular uptake, whereas excess hydrophobicity and positive charge at isolated amino acid positions can trigger membrane lysis at elevated peptide dosing. Our results provide a design roadmap for maximizing the potential to generate cell-permeable stapled peptides with on-mechanism cellular activity. PMID:27547919

  2. Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

    PubMed

    Oehlke, J; Birth, P; Klauschenz, E; Wiesner, B; Beyermann, M; Oksche, A; Bienert, M

    2002-08-01

    The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

  3. Size Dependent Cellular Uptake of Rod-like Bionanoparticles with Different Aspect Ratios

    PubMed Central

    Liu, Xiangxiang; Wu, Fengchi; Tian, Ye; Wu, Man; Zhou, Quan; Jiang, Shidong; Niu, Zhongwei

    2016-01-01

    Understanding the cellular internalization mechanism of nanoparticles is essential to study their biological fate. Especially, due to the anisotropic properties, rod-like nanoparticles have attracted growing interest for the enhanced internalization efficiency with respect to spherical nanoparticles. Here, to elucidate the effect of aspect ratio of rod-like nanoparticles on cellular uptake, tobacco mosaic virus (TMV), a typical rod-like bionanoparticle, is developed as a model. Nanorods with different aspect ratios can be obtained by ultrasound treatment and sucrose density gradient centrifugation. By incubating with epithelial and endothelial cells, we found that the rod-like bionanoparticles with various aspect ratios had different internalization pathways in different cell lines: microtubules transport in HeLa and clathrin-mediated uptake in HUVEC for TMV4 and TMV8; caveolae-mediated pathway and microtubules transport in HeLa and HUVEC for TMV17. Differently from most nanoparticles, for all the three TMV nano-rods with different aspect ratios, macropinocytosis takes no effect on the internalization in both cell types. This work provides a fundamental understanding of the influence of aspect ratio on cellular uptake decoupled from charge and material composition. PMID:27080246

  4. 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

  5. Cholesteryl to improve the cellular uptake of polymersomes within HeLa cells.

    PubMed

    Martin, Chloe; Marino, Nino; Curran, Ciara; McHale, Anthony P; Callan, John F; Callan, Bridgeen

    2016-09-10

    The need to develop a greater understanding of drug delivery systems has arisen through the development of alternative biological based therapeutics. Drug delivery systems need to adapt and respond to this increasing demand for cellular transportation of highly charged species. Polymersomal drug delivery systems have displayed great potential and versatility for such a task. In this manuscript we present the synthesis, characterisation and biological evaluation of six amphiphilic random co polymers with varying amounts of cholesteryl (0-39%wt) before the subsequent formation into polymersomes. The polymersomes were then analysed for size, zeta potential, encapsulation efficiency, release kinetics and cellular uptake. Results confirmed that the polymersome containing 12%wt cholesteryl polymer displayed a ten-fold increase in cellular uptake of Fitc-CM-dextran when compared to un-encapsulated drug, crossing the cellular membrane via endocytosis. The size of these vehicles ranged between 100 and 500nm, zeta potential was shown to be neutral at -0.82mV ±0.2 with encapsulation efficiencies in the region of 60%. The ease of adaptability and preparation of such systems renders them a viable alternative to liposomal drug delivery systems. PMID:27452422

  6. The effect of blood protein adsorption on cellular uptake of anatase TiO2 nanoparticles

    PubMed Central

    Allouni, Zouhir E; Gjerdet, Nils R; Cimpan, Mihaela R; Høl, Paul J

    2015-01-01

    Protein adsorption onto nanoparticles (NPs) in biological fluids has emerged as an important factor when testing biological responses to NPs, as this may influence both uptake and subsequent toxicity. The aim of the present study was to quantify the adsorption of proteins onto TiO2 NPs and to test the influence on cellular uptake. The surface composition of the particles was characterized by thermal analysis and by X-ray photoelectron spectroscopy. The adsorption of three blood proteins, ie, human serum albumin (HSA), γ-globulins (Glbs), and fibrinogen (Fib), onto three types of anatase NPs of different sizes was quantified for each protein. The concentration of the adsorbed protein was measured by ultraviolet-visible spectrophotometry using the Bradford method. The degree of cellular uptake was quantified by inductivity coupled plasma mass spectroscopy, and visualized by an ultra-high resolution imaging system. The proteins were adsorbed onto all of the anatase NPs. The quantity adsorbed increased with time and was higher for the smaller particles. Fib and Glbs showed the highest affinity to TiO2 NPs, while the lowest was seen for HSA. The adsorption of proteins affected the surface charge and the hydrodynamic diameter of the NPs in cell culture medium. The degree of particle uptake was highest in protein-free medium and in the presence HSA, followed by culture medium supplemented with Glbs, and lowest in the presence of Fib. The results indicate that the uptake of anatase NPs by fibroblasts is influenced by the identity of the adsorbed protein. PMID:25632230

  7. The effect of blood protein adsorption on cellular uptake of anatase TiO2 nanoparticles.

    PubMed

    Allouni, Zouhir E; Gjerdet, Nils R; Cimpan, Mihaela R; Høl, Paul J

    2015-01-01

    Protein adsorption onto nanoparticles (NPs) in biological fluids has emerged as an important factor when testing biological responses to NPs, as this may influence both uptake and subsequent toxicity. The aim of the present study was to quantify the adsorption of proteins onto TiO2 NPs and to test the influence on cellular uptake. The surface composition of the particles was characterized by thermal analysis and by X-ray photoelectron spectroscopy. The adsorption of three blood proteins, ie, human serum albumin (HSA), γ-globulins (Glbs), and fibrinogen (Fib), onto three types of anatase NPs of different sizes was quantified for each protein. The concentration of the adsorbed protein was measured by ultraviolet-visible spectrophotometry using the Bradford method. The degree of cellular uptake was quantified by inductivity coupled plasma mass spectroscopy, and visualized by an ultra-high resolution imaging system. The proteins were adsorbed onto all of the anatase NPs. The quantity adsorbed increased with time and was higher for the smaller particles. Fib and Glbs showed the highest affinity to TiO2 NPs, while the lowest was seen for HSA. The adsorption of proteins affected the surface charge and the hydrodynamic diameter of the NPs in cell culture medium. The degree of particle uptake was highest in protein-free medium and in the presence HSA, followed by culture medium supplemented with Glbs, and lowest in the presence of Fib. The results indicate that the uptake of anatase NPs by fibroblasts is influenced by the identity of the adsorbed protein. PMID:25632230

  8. Reduced plant uptake of pesticides with biochar additions to soil.

    PubMed

    Yu, Xiang-Yang; Ying, Guang-Guo; Kookana, Rai S

    2009-07-01

    We investigated the effectiveness of two types of biochars in reducing the bioavailability of two soil-applied insecticides (chlorpyrifos and carbofuran) to Spring onion (Allium cepa). The biochars prepared from the pyrolysis of Eucalyptus spp. wood chips at 450 and 850 degrees C (BC850) were thoroughly mixed into the soil to achieve 0%, 0.1%, 0.5% and 1% by soil weight. A spring onion crop was grown for 5 wk in the biochar-amended soils spiked with 50 mgkg(-1) of each pesticide. The loss of both pesticides due to degradation and or sequestration in soils decreased significantly with increasing amounts of biochars in soil. Over 35 d, 86-88% of the pesticides were lost from the control soil, whereas it was only 51% of carbofuran and 44% of chlorpyrifos from the soil amended with 1.0% BC850. Despite greater persistence of the pesticide residues in biochar-amended soils, the plant uptake of pesticides decreased markedly with increasing biochar content of the soil. With 1% of BC850 soil amendment, the total plant residues for chlorpyrifos and carbofuran decreased to 10% and 25% of that in the control treatment, respectively. The BC850 was particularly effective in reducing phytoavailability of both pesticides from soil, due to its high affinity for and ability to sequester pesticide residues.

  9. Reduced plant uptake of pesticides with biochar additions to soil.

    PubMed

    Yu, Xiang-Yang; Ying, Guang-Guo; Kookana, Rai S

    2009-07-01

    We investigated the effectiveness of two types of biochars in reducing the bioavailability of two soil-applied insecticides (chlorpyrifos and carbofuran) to Spring onion (Allium cepa). The biochars prepared from the pyrolysis of Eucalyptus spp. wood chips at 450 and 850 degrees C (BC850) were thoroughly mixed into the soil to achieve 0%, 0.1%, 0.5% and 1% by soil weight. A spring onion crop was grown for 5 wk in the biochar-amended soils spiked with 50 mgkg(-1) of each pesticide. The loss of both pesticides due to degradation and or sequestration in soils decreased significantly with increasing amounts of biochars in soil. Over 35 d, 86-88% of the pesticides were lost from the control soil, whereas it was only 51% of carbofuran and 44% of chlorpyrifos from the soil amended with 1.0% BC850. Despite greater persistence of the pesticide residues in biochar-amended soils, the plant uptake of pesticides decreased markedly with increasing biochar content of the soil. With 1% of BC850 soil amendment, the total plant residues for chlorpyrifos and carbofuran decreased to 10% and 25% of that in the control treatment, respectively. The BC850 was particularly effective in reducing phytoavailability of both pesticides from soil, due to its high affinity for and ability to sequester pesticide residues. PMID:19419749

  10. Unveiling the mechanism of uptake and sub-cellular distribution of cerium oxide nanoparticles†

    PubMed Central

    Singh, Sanjay; Kumar, Amit; Karakoti, Ajay; Seal, Sudipta; Self, William T.

    2011-01-01

    Cerium oxide nanoparticles (CNPs) have been recently studied for their potent superoxide scavenging properties in both cell and animal model systems. Data from these model systems have shown that exposure of cells to CNPs results in the protection against reactive oxygen species (ROS). Despite these exciting findings, very little is known regarding the uptake or subcellular distribution of these nanomaterials inside cells. In this study we utilized fluorophore (carboxyfluorescein) conjugated cerium oxide NPs (CCNPs) to study the mechanism of uptake and to elucidate the subcellular localization of CNPs using a keratinocyte model system. We observed rapid uptake (within 3 h) of CCNPs that was governed by energy-dependent, clathrin-mediated and caveolae-mediated endocytic pathways. We found CCNPs co-localized with mitochondria, lysosomes and endoplasmic reticulum as well as being abundant in the cytoplasm and the nucleus. Given the radical scavenging properties of cerium oxide and the widespread cellular disposition we observed, CNPs likely act as cellular antioxidants in multiple compartments of the cell imparting protection against a variety of oxidant injuries. PMID:20697616

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

    SciTech Connect

    Garcin, Pierre O.; Panté, Nelly

    2015-08-15

    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 and 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.

  12. Anchoring dipalmitoyl phosphoethanolamine to nanoparticles boosts cellular uptake and fluorine-19 magnetic resonance signal.

    PubMed

    Waiczies, Sonia; Lepore, Stefano; Sydow, Karl; Drechsler, Susanne; Ku, Min-Chi; Martin, Conrad; Lorenz, Dorothea; Schütz, Irene; Reimann, Henning M; Purfürst, Bettina; Dieringer, Matthias A; Waiczies, Helmar; Dathe, Margitta; Pohlmann, Andreas; Niendorf, Thoralf

    2015-02-12

    Magnetic resonance (MR) methods to detect and quantify fluorine ((19)F) nuclei provide the opportunity to study the fate of cellular transplants in vivo. Cells are typically labeled with (19)F nanoparticles, introduced into living organisms and tracked by (19)F MR methods. Background-free imaging and quantification of cell numbers are amongst the strengths of (19)F MR-based cell tracking but challenges pertaining to signal sensitivity and cell detection exist. In this study we aimed to overcome these limitations by manipulating the aminophospholipid composition of (19)F nanoparticles in order to promote their uptake by dendritic cells (DCs). As critical components of biological membranes, phosphatidylethanolamines (PE) were studied. Both microscopy and MR spectroscopy methods revealed a striking (at least one order of magnitude) increase in cytoplasmic uptake of (19)F nanoparticles in DCs following enrichment with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). The impact of enriching (19)F nanoparticles with PE on DC migration was also investigated. By manipulating the nanoparticle composition and as a result the cellular uptake we provide here one way of boosting (19)F signal per cell in order to overcome some of the limitations related to (19)F MR signal sensitivity. The boost in signal is ultimately necessary to detect and track cells in vivo.

  13. Anchoring Dipalmitoyl Phosphoethanolamine to Nanoparticles Boosts Cellular Uptake and Fluorine-19 Magnetic Resonance Signal

    NASA Astrophysics Data System (ADS)

    Waiczies, Sonia; Lepore, Stefano; Sydow, Karl; Drechsler, Susanne; Ku, Min-Chi; Martin, Conrad; Lorenz, Dorothea; Schütz, Irene; Reimann, Henning M.; Purfürst, Bettina; Dieringer, Matthias A.; Waiczies, Helmar; Dathe, Margitta; Pohlmann, Andreas; Niendorf, Thoralf

    2015-02-01

    Magnetic resonance (MR) methods to detect and quantify fluorine (19F) nuclei provide the opportunity to study the fate of cellular transplants in vivo. Cells are typically labeled with 19F nanoparticles, introduced into living organisms and tracked by 19F MR methods. Background-free imaging and quantification of cell numbers are amongst the strengths of 19F MR-based cell tracking but challenges pertaining to signal sensitivity and cell detection exist. In this study we aimed to overcome these limitations by manipulating the aminophospholipid composition of 19F nanoparticles in order to promote their uptake by dendritic cells (DCs). As critical components of biological membranes, phosphatidylethanolamines (PE) were studied. Both microscopy and MR spectroscopy methods revealed a striking (at least one order of magnitude) increase in cytoplasmic uptake of 19F nanoparticles in DCs following enrichment with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). The impact of enriching 19F nanoparticles with PE on DC migration was also investigated. By manipulating the nanoparticle composition and as a result the cellular uptake we provide here one way of boosting 19F signal per cell in order to overcome some of the limitations related to 19F MR signal sensitivity. The boost in signal is ultimately necessary to detect and track cells in vivo.

  14. 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

  15. 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. PMID:26517371

  16. 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).

  17. Interaction of peptidomimetics with bilayer membranes: biophysical characterization and cellular uptake.

    PubMed

    Jing, Xiaona; Kasimova, Marina R; Simonsen, Anders H; Jorgensen, Lene; Malmsten, Martin; Franzyk, Henrik; Foged, Camilla; Nielsen, Hanne M

    2012-03-20

    Enzymatically stable cell-penetrating α-peptide/β-peptoid peptidomimetics constitute promising drug delivery vehicles for the transport of therapeutic biomacromolecules across membrane barriers. The aim of the present study was to elucidate the mechanism of peptidomimetic-lipid bilayer interactions. A series of peptidomimetics consisting of alternating cationic and hydrophobic residues displaying variation in length and N-terminal end group were applied to fluid-phase, anionic lipid bilayers, and their interaction was investigated using isothermal titration calorimetry (ITC) and ellipsometry. Titration of lipid vesicles into solutions of peptidomimetics resulted in exothermic adsorption processes, and the interaction of all studied peptidomimetics with anionic lipid membranes was found to be enthalpy-driven. The enthalpy and Gibbs free energy (ΔG) proved more favorable with increasing chain length. However, not all charges contribute equally to the interaction, as evidenced by the charge-normalized ΔG being inversely correlated to the sequence length. Ellipsometry data suggested that the hydrophobic residues also played an important role in the interaction process. Furthermore, ΔG extracted from ellipsometry data showed good agreement with that obtained with ITC. To further elucidate their interaction with biological membranes, quantitative uptake and cellular distribution were studied in proliferating HeLa cells by flow cytometry and confocal microscopy. The cellular uptake of carboxyfluorescein-labeled peptidomimetics showed a similar ranking as that obtained from the adsorbed amount, and binding energy to model membranes demonstrated that the initial interaction with the membrane is of key importance for the cellular uptake.

  18. Accelerated Cellular Uptake and Metabolism of L-Thyroxine during Acute Salmonella typhimurium Sepsis

    PubMed Central

    DeRubertis, Frederick R.; Woeber, Kenneth A.

    1973-01-01

    The effects of acute Salmonella typhimurium sepsis on the kinetics of peripheral L-thyroxine (T4) distribution and metabolism and on serum total and free T4 concentrations were studied in rhesus monkeys inoculated i.v. with either heat-killed or viable organisms. The rate of disappearance of labeled T4 from serum was increased within 8 h after inoculation of monkeys with either heat-killed or viable Salmonella. The effects of the heat-killed organisms were transient and no longer evident by 16 h postinoculation. The monkeys inoculated with the viable Salmonella experienced a 2-3 day febrile, septic illness that was accompanied by an increase in the absolute rate of T4 disposal. In the infected monkeys, serum total T4 and endogenously labeled protein-bound iodine concentrations fell significantly during the period of acute sepsis and then rose during convalescence to values that exceeded the preinoculation values, suggesting that thyroidal secretion of hormone had increased in response to a primary depletion of the peripheral hormonal pool. Total cellular and hepatic uptakes of T4 were enhanced by 4 h after inoculation of monkeys with either heat-killed or viable Salmonella, but the increase in total cellular uptake persisted for 24 h only in the monkeys inoculated with the viable organisms. These alterations in T4 kinetics could neither be correlated with changes in the binding of T4 in plasma nor attributed to an increase in vascular permeability. Moreover, they could not be ascribed to an in vitro product of bacterial growth, suggesting that the presence of the organisms themselves was required. An acceleration of T4 disappearance was also observed during Escherichia coli and Diplococcus pucumoniae bacteremias. Our findings are consistent with a primary increase in the cellular uptake and metabolism of T4 during bacterial sepsis, possibly related to phagocytic cell function in the host. PMID:4629910

  19. Naringenin-loaded solid lipid nanoparticles: preparation, controlled delivery, cellular uptake, and pulmonary pharmacokinetics

    PubMed Central

    Ji, Peng; Yu, Tong; Liu, Ying; Jiang, Jie; Xu, Jie; Zhao, Ying; Hao, Yanna; Qiu, Yang; Zhao, Wenming; Wu, Chao

    2016-01-01

    Naringenin (NRG), a flavonoid compound, had been reported to exhibit extensive pharmacological effects, but its water solubility and oral bioavailability are only~46±6 µg/mL and 5.8%, respectively. The purpose of this study is to design and develop NRG-loaded solid lipid nanoparticles (NRG-SLNs) to provide prolonged and sustained drug release, with improved stability, involving nontoxic nanocarriers, and increase the bioavailability by means of pulmonary administration. Initially, a group contribution method was used to screen the best solid lipid matrix for the preparation of SLNs. NRG-SLNs were prepared by an emulsification and low-temperature solidification method and optimized using an orthogonal experiment approach. The morphology was examined by transmission electron microscopy, and the particle size and zeta potential were determined by photon correlation spectroscopy. The total drug content of NRG-SLNs was measured by high-performance liquid chromatography, and the encapsulation efficiency (EE) was determined by Sephadex gel-50 chromatography and high-performance liquid chromatography. The in vitro NRG release studies were carried out using a dialysis bag. The best cryoprotectant to prepare NRG-SLN lyophilized powder for future structural characterization was selected using differential scanning calorimetry, powder X-ray diffraction, and Fourier transform infrared spectroscopy. The short-term stability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, cellular uptake, and pharmacokinetics in rats were studied after pulmonary administration of NRG-SLN lyophilized powder. Glycerol monostearate was selected to prepare SLNs, and the optimal formulation of NRG-SLNs was spherical in shape, with a particle size of 98 nm, a polydispersity index of 0.258, a zeta potential of −31.4 mV, a total drug content of 9.76 mg, an EE of 79.11%, and a cumulative drug release of 80% in 48 hours with a sustained profile. In addition, 5% mannitol (w

  20. Naringenin-loaded solid lipid nanoparticles: preparation, controlled delivery, cellular uptake, and pulmonary pharmacokinetics.

    PubMed

    Ji, Peng; Yu, Tong; Liu, Ying; Jiang, Jie; Xu, Jie; Zhao, Ying; Hao, Yanna; Qiu, Yang; Zhao, Wenming; Wu, Chao

    2016-01-01

    Naringenin (NRG), a flavonoid compound, had been reported to exhibit extensive pharmacological effects, but its water solubility and oral bioavailability are only~46±6 µg/mL and 5.8%, respectively. The purpose of this study is to design and develop NRG-loaded solid lipid nanoparticles (NRG-SLNs) to provide prolonged and sustained drug release, with improved stability, involving nontoxic nanocarriers, and increase the bioavailability by means of pulmonary administration. Initially, a group contribution method was used to screen the best solid lipid matrix for the preparation of SLNs. NRG-SLNs were prepared by an emulsification and low-temperature solidification method and optimized using an orthogonal experiment approach. The morphology was examined by transmission electron microscopy, and the particle size and zeta potential were determined by photon correlation spectroscopy. The total drug content of NRG-SLNs was measured by high-performance liquid chromatography, and the encapsulation efficiency (EE) was determined by Sephadex gel-50 chromatography and high-performance liquid chromatography. The in vitro NRG release studies were carried out using a dialysis bag. The best cryoprotectant to prepare NRG-SLN lyophilized powder for future structural characterization was selected using differential scanning calorimetry, powder X-ray diffraction, and Fourier transform infrared spectroscopy. The short-term stability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, cellular uptake, and pharmacokinetics in rats were studied after pulmonary administration of NRG-SLN lyophilized powder. Glycerol monostearate was selected to prepare SLNs, and the optimal formulation of NRG-SLNs was spherical in shape, with a particle size of 98 nm, a polydispersity index of 0.258, a zeta potential of -31.4 mV, a total drug content of 9.76 mg, an EE of 79.11%, and a cumulative drug release of 80% in 48 hours with a sustained profile. In addition, 5% mannitol (w

  1. Plant nitrogen uptake drives responses of productivity to nitrogen and water addition in a grassland

    PubMed Central

    Lü, Xiao-Tao; Dijkstra, Feike A.; Kong, De-Liang; Wang, Zheng-Wen; Han, Xing-Guo

    2014-01-01

    Increased atmospheric nitrogen (N) deposition and altered precipitation regimes have profound impacts on ecosystem functioning in semiarid grasslands. The interactions between those two factors remain largely unknown. A field experiment with N and water additions was conducted in a semiarid grassland in northern China. We examined the responses of aboveground net primary production (ANPP) and plant N use during two contrasting hydrological growing seasons. Nitrogen addition had no impact on ANPP, which may be accounted for by the offset between enhanced plant N uptake and decreased plant nitrogen use efficiency (NUE). Water addition significantly enhanced ANPP, which was largely due to enhanced plant aboveground N uptake. Nitrogen and water additions significantly interacted to affect ANPP, plant N uptake and N concentrations at the community level. Our observations highlight the important role of plant N uptake and use in mediating the effects of N and water addition on ANPP. PMID:24769508

  2. Cellular Uptake and Intracellular Trafficking of Oligonucleotides: Implications for Oligonucleotide Pharmacology

    PubMed Central

    Ming, Xin; Carver, Kyle; Laing, Brian

    2014-01-01

    One of the major constraints on the therapeutic use of oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed. PMID:24383421

  3. Protein source and choice of anticoagulant decisively affect nanoparticle protein corona and cellular uptake

    NASA Astrophysics Data System (ADS)

    Schöttler, S.; Klein, Katja; Landfester, K.; Mailänder, V.

    2016-03-01

    Protein adsorption on nanoparticles has been a focus of the field of nanocarrier research in the past few years and more and more papers are dealing with increasingly detailed lists of proteins adsorbed to a plethora of nanocarriers. While there is an urgent need to understand the influence of this protein corona on nanocarriers' interactions with cells the strong impact of the protein source on corona formation and the consequence for interaction with different cell types are factors that are regularly neglected, but should be taken into account for a meaningful analysis. In this study, the importance of the choice of protein source used for in vitro protein corona analysis is concisely investigated. Major and decisive differences in cellular uptake of a polystyrene nanoparticle incubated in fetal bovine serum, human serum, human citrate and heparin plasma are reported. Furthermore, the protein compositions are determined for coronas formed in the respective incubation media. A strong influence of heparin, which is used as an anticoagulant for plasma generation, on cell interaction is demonstrated. While heparin enhances the uptake into macrophages, it prevents internalization into HeLa cells. Taken together we can give the recommendation that human plasma anticoagulated with citrate seems to give the most relevant results for in vitro studies of nanoparticle uptake.Protein adsorption on nanoparticles has been a focus of the field of nanocarrier research in the past few years and more and more papers are dealing with increasingly detailed lists of proteins adsorbed to a plethora of nanocarriers. While there is an urgent need to understand the influence of this protein corona on nanocarriers' interactions with cells the strong impact of the protein source on corona formation and the consequence for interaction with different cell types are factors that are regularly neglected, but should be taken into account for a meaningful analysis. In this study, the importance

  4. Mitochondria-targeting nanoplatform with fluorescent carbon dots for long time imaging and magnetic field-enhanced cellular uptake.

    PubMed

    Zhang, Ye; Shen, Yajing; Teng, Xiyao; Yan, Manqing; Bi, Hong; Morais, Paulo Cesar

    2015-05-20

    In this study, a biocompatible nanoplatform has been constructed on the basis of magnetic mesoporous silica nanoparticles (Fe3O4@mSiO2) via surface modification of triphenylphospine (TPP) and then conjugation with fluorescent carbon dots (CDs). The as-prepared Fe3O4@mSiO2-TPP/CDs nanoplatform shows a very low cytotoxicity and apoptosis rate in various cell lines such as A549, CHO, HeLa, SH-SY5Y, HFF, and HMEC-1. More importantly, this nanoplatform integrates long time cell imaging, mitochondria-targeting, and magnetic field-enhanced cellular uptake functionalities into an all-in-one system. Time-dependent mitochondrial colocalization in all of the cell lines has been proved by using confocal laser scanning microscopy and flow cytometry, while the multicolored fluorescence of the Fe3O4@mSiO2-TPP/CDs could remain bright and stable after coincubation for 24 h. In addition, the cellular uptake efficiency could be enhanced in a short time as a static magnetic field of 0.30 T was applied to the coincubation system of A549 and HFF cell lines. This bionanoplatform may have potential applications in targeted drug delivery for mitochondria diseases as well as early cancer diagnosis and treatment.

  5. 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. PMID:27400242

  6. Effects of macronutrient additions on nickel uptake and distribution in the dinoflagellate Prorocentrum donghaiense Lu.

    PubMed

    Hong, Hua-Sheng; Wang, Ming-Hua; Huang, Xu-Guang; Wang, Da-Zhi

    2009-06-01

    The influences of macronutrient additions on nickel (Ni) uptake and distribution in the subcellular structures and macromolecular components of the dinoflagellate Prorocentrum donghaiense Lu were examined using a radioisotope tracer method. The results showed that nitrate addition enhanced the uptake of Ni by P. donghaiense, whereas phosphate addition inhibited Ni uptake at high-Ni concentration. Nitrate or phosphate addition significantly affected Ni distribution in the subcellular structures and components. The majority of Ni was found in the soluble substances (>70%) and in the proteins (55.0-79.6%) of the algal cells. Urea reduced the Ni content in the amino acid-carbohydrate but elevated its content in proteins, and shown significantly correlated with the protein content of the algal cells. Thus, nutrient enrichment could influence both metal uptake and its distribution in the subcellular structures and components of the phytoplankton, as well as its subsequent transfer in marine food chains. PMID:19217194

  7. 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

  8. Enhanced cellular uptake and cytotoxicity of folate decorated doxorubicin loaded PLA-TPGS nanoparticles

    NASA Astrophysics Data System (ADS)

    Nguyen, Hoai Nam; Nhung Hoang, Thi My; Thu Trang Mai, Thi; Quynh Trang Nguyen, Thi; Doan Do, Hai; Hien Pham, Thi; Lap Nguyen, Thi; Thu Ha, Phuong

    2015-01-01

    Doxorubicin (DOX) is one of the most effective anticancer drugs for treating many types of cancer. However, the clinical applications of DOX were hindered because of serious side-effects resulting from the unselective delivery to cancer cell including congestive heart failure, chronic cardiomyopathy and drug resistance. Recently, it has been demonstrated that loading anti-cancer drugs onto drug delivery nanosystems helps to maximize therapeutic efficiency and minimize unwanted side-effects via passive and active targeting mechanisms. In this study we prepared folate decorated DOX loaded PLA-TPGS nanoparticles with the aim of improving the potential as well as reducing the side-effects of DOX. Characteristics of nanoparticles were investigated by field emission scanning electron microscopy (FESEM), dynamic light scattering (DLS) method and Fourier transform infrared spectroscopy (FTIR). Anticancer activity of the nanoparticles was evaluated through cytotoxicity and cellular uptake assays on HeLa and HT29 cancer cell lines. The results showed that prepared drug delivery system had size around 100 nm and exhibited higher cytotoxicity and cellular uptake on both tested HeLa and HT29 cells.

  9. Enhancement of cellular uptake, transport and oral absorption of protease inhibitor saquinavir by nanocrystal formulation

    PubMed Central

    He, Yuan; Xia, Deng-ning; Li, Qiu-xia; Tao, Jin-song; Gan, Yong; Wang, Chi

    2015-01-01

    Aim: Saquinavir (SQV) is the first protease inhibitor for the treatment of HIV infection, but with poor solubility. The aim of this study was to prepare a colloidal nanocrystal suspension for improving the oral absorption of SQV. Methods: SQV nanocrystals were prepared using anti-solvent precipitation–high pressure homogenization method. The nanocrystals were characterized by a Zetasizer and transmission electron microscopy (TEM). Their dissolution, cellular uptake and transport across the human colorectal adenocarcinoma cell line (Caco-2) monolayer were investigated. Bioimaging of ex vivo intestinal sections of rats was conducted with confocal laser scanning microscopy. Pharmacokinetic analysis was performed in rats administered nanocrystal SQV suspension (50 mg/kg, ig), and the plasma SQV concentrations were measured with HPLC. Results: The SQV nanocrystals were approximately 200 nm in diameter, with a uniform size distribution. The nanocrystals had a rod-like shape under TEM. The dissolution, cellular uptake, and transport across a Caco-2 monolayer of the nanocrystal formulation were significantly improved compared to those of the coarse crystals. The ex vivo intestinal section study revealed that the fluorescently labeled nanocrystals were located in the lamina propria and the epithelium of the duodenum and jejunum. Pharmacokinetic study showed that the maximal plasma concentration (Cmax) was 2.16-fold of that for coarse crystalline SQV suspension, whereas the area under the curve (AUC) of nanocrystal SQV suspension was 1.95-fold of that for coarse crystalline SQV suspension. Conclusion: The nanocrystal drug delivery system significantly improves the oral absorption of saquinavir. PMID:26256404

  10. Cellular response to empty and palladium-conjugated amino-polystyrene nanospheres uptake: a proteomic study.

    PubMed

    Pietrovito, Laura; Cano-Cortés, Victoria; Gamberi, Tania; Magherini, Francesca; Bianchi, Laura; Bini, Luca; Sánchez-Martín, Rosario M; Fasano, Mauro; Modesti, Alessandra

    2015-01-01

    Amino polystyrene nanospheres are shown to be efficient and controllable delivery devices, capable of transporting several bioactive cargoes. Recently, the design of a new device for prodrug activation, using these nanospheres with palladium encapsulated onto them, has been developed successfully. To study the influence of the cellular uptake of these nanodevices, we investigated the cellular response of human embryonic kidney cells (HEK-293T) and murine fibroblasts (L929) treated with empty or palladium-conjugated amino polystyrene nanospheres. To identify differentially expressed proteins, we performed an exhaustive proteomic analysis. In accordance with genomic data previously obtained, the uptake of the empty nanospheres did not induce significant variation in protein expression levels. Following the treatment with palladium-conjugated nanospheres, some changes in protein profiles in both cell lines were observed; these alterations affect proteins involved in cell metabolism and intracellular transport. No key regulator of the cell cycle result was differentially expressed after the treatment, confirming that these innovative drug delivery systems are harmless and well tolerated by the cells.

  11. Stable fluorescence conjugation of ZnO nanoparticles and their size dependent cellular uptake.

    PubMed

    Kim, Kyoung-Min; Kim, Min-Kyu; Paek, Hee-Jeong; Choi, Soo-Jin; Oh, Jae-Min

    2016-09-01

    We evaluated size dependent cellular uptake of ZnO nanoparticles utilizing stably introduced Cy5.5, which emits long-wavelength fluorescence. Through (3-aminopropyl)triethoxysilane modification, ZnO nanoparticles of different sizes (20 and 70nm) were functionalized with amine moiety, which was further reacted with Cy5.5-N-hydroxylsuccinimide ester to make covalently conjugated Cy5.5 dye on ZnO nanoparticles. Field emission-scanning electron microscopic images revealed that average particle size as well as particle morphology of ZnO nanoparticles were not altered by Cy5.5 conjugation. Zeta potential measurement confirmed that the positive surface charge of ZnO nanoparticles was well preserved after successive conjugation reactions. Based on infrared, ultraviolet-visible light and photoluminescence spectroscopies, we verify that the Cy5.5 was stably introduced to ZnO nanoparticles without serious aggregation. Surface conjugated Cy5.5 showed high stability in deionized water, phosphate buffered saline and cell culture medium, showing less than 2% of release during 85h. Confocal microscopy and fluorescence-activated cell sorting analysis demonstrated that smaller ZnO nanoparticles were more taken up in greater quantities by HaCaT cells. Moreover, systematic study on cellular uptake pathway showed that smaller ZnO nanoparticles were internalized into cells mainly by clathrin-mediated endocytosis, while larger ZnO nanoparticles entered cells via several pathways. PMID:27323344

  12. De novo synthesis and cellular uptake of organic nanocapsules with tunable surface chemistry.

    PubMed

    Huang, Kun; Jacobs, Amy; Rzayev, Javid

    2011-06-13

    Water-soluble organic nanocapsules were prepared from bottlebrush copolymers with triblock terpolymer side chains composed of a degradable inner block (polylactide), a cross-linkable middle block (poly(4-butenylstyrene)), and a functional outer block (poly(styrene-co-maleic anhydride)). Bottlebrush copolymers are macromolecules with a long linear backbone and shorter polymeric side chains densely grafted onto the backbone. Hollow cylindrical nanoparticles were prepared by peripheral cross-linking of the bottlebrush copolymers and subsequent selective removal of the core. Reactive anhydride groups of the outer functional layer allowed for the preparation of nanocapsules with tunable surface characteristics. Cellular uptake of negatively charged organic nanocapsules showed strong surface chemistry dependence. The presence of hydrophobic groups on the nanocapsule surface was necessary for their nonspecific association with the cell membrane and subsequent internalization by endocytosis. The length of surface grafted oligoethylene glycol chains also had a dramatic influence on the intracellular accumulation of nanocapsules. Macropinocytosis was shown to be the predominant pathway for the cellular uptake of organic nanocapsules.

  13. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

    PubMed Central

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-01-01

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems. PMID:26036864

  14. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.

    PubMed

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-06-03

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

  15. Shape effect in cellular uptake of PEGylated nanoparticles: comparison between sphere, rod, cube and disk

    NASA Astrophysics Data System (ADS)

    Li, Ying; Kröger, Martin; Liu, Wing Kam

    2015-10-01

    The size, shape, surface property and material composition of polymer-coated nanoparticles (NPs) are four important parameters in designing efficient NP-based carriers for targeted drug delivery. However, due to the complex interplay between size, shape and surface property, most studies lead to ambiguous descriptions of the relevance of shape. To clarify its influence on the cellular uptake of PEGylated NPs, large scale molecular simulations have been performed to study differently shaped convex NPs, such as sphere, rod, cube and disk. Comparing systems with identical NP surface area, ligand-receptor interaction strength, and grafting density of the polyethylene glycol, we find that the spherical NPs exhibit the fastest internalization rate, followed by the cubic NPs, then rod- and disk-like NPs. The spherical NPs thus demonstrate the highest uptake among these differently shaped NPs. Based on a detailed free energy analysis, the NP shape effect is found to be mainly induced by the different membrane bending energies during endocytosis. The spherical NPs need to overcome a minimal membrane bending energy barrier, compared with the non-spherical counterparts, while the internalization of disk-like NPs involves a strong membrane deformation, responsible for a large free energy barrier. Besides, the free energy change per tethered chain is about a single kBT regardless of NP shape, as revealed by our self-consistent field theory calculations, where kB and T denote Boltzmann constant and temperature, respectively. Thus, the NP shape only plays the secondary role in the free energy change of grafted PEG polymers during internalization. We also find that star-shaped NPs can be quickly wrapped by the cell membrane, similar to their spherical counterparts, indicating star-shaped NPs can be used for drug delivery with high efficacy. Our findings seem to provide useful guidance in the molecular design of PEGylated NPs for controllable cellular uptake and help establish

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

    SciTech Connect

    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. We 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.

  17. Cellular Uptake and Cytotoxicity of β-Lactoglobulin Nanoparticles: The Effects of Particle Size and Surface Charge

    PubMed Central

    Ha, Ho-Kyung; Kim, Jin Wook; Lee, Mee-Ryung; Jun, Woojin; Lee, Won-Jae

    2015-01-01

    It is necessary to understand the cellular uptake and cytotoxicity of food-grade delivery systems, such as β-lactoglobulin (β-lg) nanoparticles, for the application of bioactive compounds to functional foods. The objectives of this study were to investigate the relationships between the physicochemical properties of β-lg nanoparticles, such as particle size and zeta-potential value, and their cellular uptakes and cytotoxicity in Caco-2 cells. Physicochemical properties of β-lg nanoparticles were evaluated using particle size analyzer. Flow cytometry and confocal laser scanning microscopy were used to investigate cellular uptake and cytotoxicity of β-lg nanoparticles. The β-lg nanoparticles with various particle sizes (98 to 192 nm) and zeta-potential values (−14.8 to −17.6 mV) were successfully formed. A decrease in heating temperature from 70°C to 60°C resulted in a decrease in the particle size and an increase in the zeta-potential value of β-lg nanoparticles. Non-cytotoxicity was observed in Caco-2 cells treated with β-lg nanoparticles. There was an increase in cellular uptake of β-lg nanoparticles with a decrease in particle size and an increase in zeta-potential value. Cellular uptake β-lg nanoparticles was negatively correlated with particle size and positively correlated with zeta-potential value. Therefore, these results suggest that the particle size and zeta-potential value of β-lg nanoparticles play an important role in the cellular uptake. The β-lg nanoparticles can be used as a delivery system in foods due to its high cellular uptake and non-cytotoxicity. PMID:25656189

  18. Cellular Uptake of Two Fluoroketolides, HMR 3562 and HMR 3787, by Human Polymorphonuclear Neutrophils In Vitro

    PubMed Central

    Abdelghaffar, H.; Vazifeh, D.; Labro, M. T.

    2001-01-01

    We analyzed the cellular accumulation of two new fluoroketolides, HMR 3562 and HMR 3787, by human polymorphonuclear neutrophils (PMN) in vitro. Both compounds were rapidly taken up by PMN, with a cellular-to-extracellular concentration ratio (C/E) of about 141 (HMR 3562) and 117 (HMR 3787) at 5 min, and this was followed by a plateau at 60 to 180 min, with a C/E of >300 at 180 min. Both ketolides were mainly located in PMN granules (about 75%) and egressed slowly from loaded cells (about 40% at 60 min), owing to avid reuptake. Uptake was moderately sensitive to external pH, and activation energy was also moderate (about 70 kJ/mol). As with other macrolides and ketolides, the existence of an active transport system was suggested by (i) the strong interindividual variability in uptake kinetics, suggesting variability in the number or activity of a transport protein; (ii) the saturation kinetics characteristic of a carrier-mediated transport system (Vmax, about 2,300 ng/2.5 × 106 PMN/5 min; Km, about 50 μg/ml); (iii) the inhibitory effects of Ni2+ (a blocker of the Na+-Ca2+ exchanger), phorbol myristate acetate (a protein kinase C activator), and H89 (a protein kinase A inhibitor). Although these two ketolides are more related to HMR 3647 (telithromycin), it is interesting that the presence of a fluoride gave these molecules a cellular pharmacokinetics more like those of HMR 3004 than those of HMR 3647. The macrolide transport system has not been yet elucidated, but our data confirm that, despite variations in chemical structure, all erythromycin A derivatives share a transmembrane transport system. PMID:11557472

  19. Cellular uptake and toxicity effects of silver nanoparticles in mammalian kidney cells.

    PubMed

    Milić, Mirta; Leitinger, Gerd; Pavičić, Ivan; Zebić Avdičević, Maja; Dobrović, Slaven; Goessler, Walter; Vinković Vrček, Ivana

    2015-06-01

    The rapid progress and early commercial acceptance of silver-based nanomaterials is owed to their biocidal activity. Besides embracing the antimicrobial potential of silver nanoparticles (AgNPs), it is imperative to give special attention to the potential adverse health effects of nanoparticles owing to prolonged exposure. Here, we report a detailed study on the in vitro interactions of citrate-coated AgNPs with porcine kidney (Pk15) cells. As uncertainty remains whether biological/cellular responses to AgNPs are solely as a result of the release of silver ions or whether the AgNPs themselves have toxic effects, we investigated the effects of Ag(+) on Pk15 cells for comparison. Next, we investigated the cellular uptake of both AgNPs and Ag(+) in Pk15 cells at various concentrations applied. The detected Ag contents in cells exposed to 50 mg l(-1) AgNPs and 50 mg l(-1) Ag(+) were 209 and 25 µg of Ag per 10(6) cells, respectively. Transmission electron microscopy (TEM) images indicated that the Pk15 cells internalized AgNPs by endocytosis. Both forms of silver, nano and ionic, decreased the number of viable Pk15 cells after 24 h in a dose-dependent manner. In spite of a significant uptake into the cells, AgNPs had only insignificant toxicity at concentrations lower than 25 mg l(-1) , whereas Ag(+) exhibited a significant decrease in cell viability at one-fifth of this concentration. The Comet assay suggested that a rather high concentration of AgNP (above 25 mg l(-1) ) is able to induce genotoxicity in Pk15 cells. Further studies must seek deeper understanding of AgNP behavior in biological media and their interactions with cellular membranes. PMID:25352480

  20. Glycosaminoglycan-functionalized poly-lactide-co-glycolide nanoparticles: synthesis, characterization, cytocompatibility, and cellular uptake.

    PubMed

    Lamichhane, Surya P; Arya, Neha; Ojha, Nirdesh; Kohler, Esther; Shastri, V Prasad

    2015-01-01

    The efficient delivery of chemotherapeutics to the tumor via nanoparticle (NP)-based delivery systems remains a significant challenge. This is compounded by the fact that the tumor is highly dynamic and complex environment composed of a plurality of cell types and extracellular matrix. Since glycosaminoglycan (GAG) production is altered in many diseases (or pathologies), NPs bearing GAG moieties on the surface may confer some unique advantages in interrogating the tumor microenvironment. In order to explore this premise, in the study reported here poly-lactide-co-glycolide (PLGA) NPs in the range of 100-150 nm bearing various proteoglycans were synthesized by a single-step nanoprecipitation and characterized. The surface functionalization of the NPs with GAG moieties was verified using zeta potential measurements and X-ray photoelectron spectroscopy. To establish these GAG-bearing NPs as carriers of therapeutics, cellular toxicity assays were undertaken in lung epithelial adenocarcinoma (A549) cells, human pulmonary microvascular endothelial cells (HPMEC), and renal proximal tubular epithelial cells. In general NPs were well tolerated over a wide concentration range (100-600 μg/mL) by all cell types and were taken up to appreciable extents without any adverse cell response in A549 cells and HPMEC. Further, GAG-functionalized PLGA NPs were taken up to different extents in A459 cells and HPMEC. In both cell systems, the uptake of heparin-modified NPs was diminished by 50%-65% in comparison to that of unmodified PLGA. Interestingly, the uptake of chondroitin sulfate NPs was the highest in both cell systems with 40%-60% higher uptake when compared with that of PLGA, and this represented an almost twofold difference over heparin-modified NPs. These findings suggest that GAG modification can be explored as means of changing the uptake behavior of PLGA NPs and these NP systems have potential in cancer therapy.

  1. Glycosaminoglycan-functionalized poly-lactide-co-glycolide nanoparticles: synthesis, characterization, cytocompatibility, and cellular uptake

    PubMed Central

    Lamichhane, Surya P; Arya, Neha; Ojha, Nirdesh; Kohler, Esther; Shastri, V Prasad

    2015-01-01

    The efficient delivery of chemotherapeutics to the tumor via nanoparticle (NP)-based delivery systems remains a significant challenge. This is compounded by the fact that the tumor is highly dynamic and complex environment composed of a plurality of cell types and extracellular matrix. Since glycosaminoglycan (GAG) production is altered in many diseases (or pathologies), NPs bearing GAG moieties on the surface may confer some unique advantages in interrogating the tumor microenvironment. In order to explore this premise, in the study reported here poly-lactide-co-glycolide (PLGA) NPs in the range of 100–150 nm bearing various proteoglycans were synthesized by a single-step nanoprecipitation and characterized. The surface functionalization of the NPs with GAG moieties was verified using zeta potential measurements and X-ray photoelectron spectroscopy. To establish these GAG-bearing NPs as carriers of therapeutics, cellular toxicity assays were undertaken in lung epithelial adenocarcinoma (A549) cells, human pulmonary microvascular endothelial cells (HPMEC), and renal proximal tubular epithelial cells. In general NPs were well tolerated over a wide concentration range (100–600 μg/mL) by all cell types and were taken up to appreciable extents without any adverse cell response in A549 cells and HPMEC. Further, GAG-functionalized PLGA NPs were taken up to different extents in A459 cells and HPMEC. In both cell systems, the uptake of heparin-modified NPs was diminished by 50%–65% in comparison to that of unmodified PLGA. Interestingly, the uptake of chondroitin sulfate NPs was the highest in both cell systems with 40%–60% higher uptake when compared with that of PLGA, and this represented an almost twofold difference over heparin-modified NPs. These findings suggest that GAG modification can be explored as means of changing the uptake behavior of PLGA NPs and these NP systems have potential in cancer therapy. PMID:25632234

  2. Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking

    PubMed Central

    Gao, Xinxin; Stanger, Karen; Kaluarachchi, Harini; Maurer, Till; Ciepla, Paulina; Chalouni, Cecile; Franke, Yvonne; Hannoush, Rami N.

    2016-01-01

    Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery. PMID:27734922

  3. Residual CTAB Ligands as Mass Spectrometry Labels to Monitor Cellular Uptake of Au Nanorods.

    PubMed

    García, Isabel; Henriksen-Lacey, Malou; Sánchez-Iglesias, Ana; Grzelczak, Marek; Penadés, Soledad; Liz-Marzán, Luis M

    2015-06-01

    Gold nanorods have numerous applications in biomedical research, including diagnostics, bioimaging, and photothermal therapy. Even though surfactant removal and surface conjugation with antifouling molecules such as polyethylene glycol (PEG) are required to minimize nonspecific protein binding and cell uptake, the reliable characterization of these processes remains challenging. We propose here the use of laser desorption/ionization mass spectrometry (LDI-MS) to study the ligand exchange efficiency of cetyltrimethylammonium bromide (CTAB)-coated nanorods with different PEG grafting densities and to characterize nanorod internalization in cells. Application of LDI-MS analysis shows that residual CTAB consistently remains adsorbed on PEG-capped Au nanorods. Interestingly, such residual CTAB can be exploited as a mass barcode to discern the presence of nanorods in complex fluids and in vitro cellular systems, even at very low concentrations. PMID:26266492

  4. Secondary conformational conversion is involved in glycosaminoglycans-mediated cellular uptake of the cationic cell-penetrating peptide PACAP.

    PubMed

    Tchoumi Neree, Armelle; Nguyen, Phuong Trang; Chatenet, David; Fournier, Alain; Bourgault, Steve

    2014-12-20

    Glycosaminoglycans (GAGs) contribute to the cellular uptake of cationic cell-penetrating peptides (CPPs). However, molecular details about the contributions of GAGs in CPP internalization remain unclear. In this study, we examined the cellular uptake mechanism of the arginine-rich CPP pituitary adenylate-cyclase-activating polypeptide (PACAP). We observed that the uptake efficacy of PACAP is dependent on the expression of cell surface GAGs. As the binding of PACAP to sulfated GAGs induced a random coil-to-α-helix conformational conversion, we investigated the role of the helical formation in PACAP internalization. Whereas this secondary structure was not crucial for efficient internalization in GAGs-deficient cells, PACAP α-helix was essential for GAGs-dependent uptake.

  5. Enhanced cellular uptake and intracellular drug controlled release of VESylated gemcitabine prodrug nanocapsules.

    PubMed

    Fang, Yanfen; Du, Fang; Xu, Yanyun; Meng, Haijing; Huang, Jin; Zhang, Xiongwen; Lu, Wei; Liu, Shiyuan; Yu, Jiahui

    2015-04-01

    Gemcitabine, 2',2'-difluoro-2'-deoxycytidine (dFdC), is the first-line antitumor agent in the treatment of pancreatic tumors. However, it possesses certain drawbacks, such as poor biological half-life resulted from rapid metabolism and the induction of resistance, leading to its restricted therapeutic potential. With the purpose of overcoming the above drawbacks, we developed a novel VESylated gemcitabine (VES-dFdC) prodrug by coupling the N4-amino group of the pyrimidine ring of dFdC to the carboxylic group of vitamin E succinate (VES). The resulting amphiphilic compound could protect the N4-amino group of the pyrimidine ring of dFdC from being degraded by cytidine deaminase. What is more, the prodrug was able to form nanocapsules in aqueous media (similar to the structure of cytomembrane), confirmed by transmission electron microscope (TEM). Their average particle size is about 107 nm with zeta potential of -33.4 mV measured by dynamic light scattering (DLS). VES-dFdC nanocapsules showed accelerated accumulative drug release profile in simulated lysosome environment (sodium acetate buffer pH 5+cathepsin B, an enzyme in lysosome), due to the easily hydrolyzed property of amide bond by cathepsin B, while rather stable in PBS (pH 7.4) or sodium acetate buffer (pH 5.0) without cathepsin B, indicating their enhanced intracellular drug controlled release manner. Besides, VES-dFdC prodrug nanocapsules showed enhanced cellular uptake ability, and the amount of cellular uptake of the nanocapsules by the pancreatic cancer cell line BxPC-3 is seventy times higher than that of native gemcitabine in the first 1.5 h. Compared with free gemcitabine, VES-dFdC nanocapsules showed essentially increased growth inhibition activity against BxPC-3 cells, indicating its great potential as prodrug for pancreatic tumor therapy with improved antitumor activity.

  6. Real time observation and kinetic modeling of the cellular uptake and removal of silicon quantum dots.

    PubMed

    Ohta, Seiichi; Inasawa, Susumu; Yamaguchi, Yukio

    2012-06-01

    The time courses of uptake and removal of silicon quantum dots (Si-QDs) by human umbilical endothelial cells (HUVECs) were observed via confocal laser scanning microscope. Si-QDs were internalized via endocytosis and transported to late endosomes/lysosomes. The number of internalized Si-QDs increased with time and gradually reached a plateau value. When Si-QD-internalized HUVECs were subsequently washed and exposed to fresh culture medium, HUVECs removed internalized Si-QDs via exocytosis. The number of internalized Si-QDs decreased with time and gradually reached a plateau value. Not all internalized Si-QDs were removed from the cell interior but large numbers of internalized Si-QDs remained accumulated inside cells. A kinetic model based on the mass balance of Si-QDs and receptors in a cell was proposed to describe the cellular uptake and removal of Si-QDs. Model calculation fitted well with experimental results. Using this model, the dissociation constant between receptors and Si-QDs in the endosome, K(d,in), was found to be a determinant factor for Si-QD accumulation in cells after the removal process.

  7. Integrated multiplatform method for in vitro quantitative assessment of cellular uptake for fluorescent polymer nanoparticles

    NASA Astrophysics Data System (ADS)

    Ferrari, Raffaele; Lupi, Monica; Falcetta, Francesca; Bigini, Paolo; Paolella, Katia; Fiordaliso, Fabio; Bisighini, Cinzia; Salmona, Mario; D'Incalci, Maurizio; Morbidelli, Massimo; Moscatelli, Davide; Ubezio, Paolo

    2014-01-01

    Studies of cellular internalization of nanoparticles (NPs) play a paramount role for the design of efficient drug delivery systems, but so far they lack a robust experimental technique able to quantify the NP uptake in terms of number of NPs internalized in each cell. In this work we propose a novel method which provides a quantitative evaluation of fluorescent NP uptake by combining flow cytometry and plate fluorimetry with measurements of number of cells. Single cell fluorescence signals measured by flow cytometry were associated with the number of internalized NPs, exploiting the observed linearity between average flow cytometric fluorescence and overall plate fluorimeter measures, and previous calibration of the microplate reader with serial dilutions of NPs. This precise calibration has been made possible by using biocompatible fluorescent NPs in the range of 20-300 nm with a narrow particle size distribution, functionalized with a covalently bonded dye, Rhodamine B, and synthesized via emulsion free-radical polymerization. We report the absolute number of NPs internalized in mouse mammary tumor cells (4T1) as a function of time for different NP dimensions and surface charges and at several exposure concentrations. The obtained results indicate that 4T1 cells incorporated 103-104 polymer NPs in a short time, reaching an intracellular concentration 15 times higher than the external one.

  8. Magnetic field-enhanced cellular uptake of doxorubicin loaded magnetic nanoparticles for tumor treatment

    NASA Astrophysics Data System (ADS)

    Venugopal, Indu; Pernal, Sebastian; Duproz, Alexandra; Bentley, Jeromy; Engelhard, Herbert; Linninger, Andreas

    2016-09-01

    Cancer remains the second most common cause of death in the US, accounting for nearly 1 out of every 4 deaths. In recent years, several varieties of nanoparticles (NPs) have been synthesized with the intent of being utilized as tumor drug delivery vehicles. We have produced superparamagnetic, gold-coated magnetite (Fe3O4@Au) NPs and loaded them with the chemotherapeutic drug doxorubicin (DOX) for magnetic drug targeting (MDT) of tumors. The synthetic strategy uses the food thickening agent gellan gum (Phytagel) as a negatively charged shell around the Fe3O4@Au NP onto which the positively charged DOX molecules are loaded via electrostatic attraction. The resulting DOX-loaded magnetic nanoparticles (DOX-MNPs) were characterized using transmission electron microscopy, energy dispersive x-ray spectroscopy, superconducting quantum interference device magnetometry, surface area electron diffraction, zeta potential measurements, fourier transform infrared spectroscopy as well as UV/Vis and fluorescence spectroscopy. Cytotoxicity of the DOX-MNPs was demonstrated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay on C6 glioma cells. Cellular uptake of DOX-MNPs was enhanced with magnetic fields, which was quantitatively determined using flow cytometry. This improved uptake also led to greater tumor cell death, which was measured using MTT assay. These MDT results are promising for a new therapy for cancer.

  9. MATE1 regulates cellular uptake and sensitivity to imatinib in CML patients

    PubMed Central

    Harrach, S; Schmidt-Lauber, C; Pap, T; Pavenstädt, H; Schlatter, E; Schmidt, E; Berdel, W E; Schulze, U; Edemir, B; Jeromin, S; Haferlach, T; Ciarimboli, G; Bertrand, J

    2016-01-01

    Although imatinib is highly effective in the treatment of chronic myeloid leukemia (CML), 25–30% patients do not respond or relapse after initial response. Imatinib uptake into targeted cells is crucial for its molecular response and clinical effectiveness. The organic cation transporter 1 (OCT1) has been proposed to be responsible for this process, but its relevance has been discussed controversially in recent times. Here we found that the multidrug and toxin extrusion protein 1 (MATE1) transports imatinib with a manifold higher affinity. MATE1 mainly mediates the cellular uptake of imatinib into targeted cells and thereby controls the intracellular effectiveness of imatinib. Importantly, MATE1 but not OCT1 expression is reduced in total bone marrow cells of imatinib-non-responding CML patients compared with imatinib-responding patients, indicating that MATE1 but not OCT1 determines the therapeutic success of imatinib. We thus propose that imatinib non-responders could be identified early before starting therapy by measuring MATE1 expression levels. PMID:27635733

  10. STRA6 is critical for cellular vitamin A uptake and homeostasis

    PubMed Central

    Amengual, Jaume; Zhang, Ning; Kemerer, Mary; Maeda, Tadao; Palczewski, Krzysztof; Von Lintig, Johannes

    2014-01-01

    Vitamin A must be adequately distributed within the body to maintain the functions of retinoids in the periphery and chromophore production in the eyes. Blood transport of the lipophilic vitamin is mediated by the retinol-binding protein, RBP4. Biochemical evidence suggests that cellular uptake of vitamin A from RBP4 is facilitated by a membrane receptor. This receptor, identified as the Stimulated by retinoic acid gene 6 (Stra6) gene product, is highly expressed in epithelia that constitute blood–tissue barriers. Here we established a Stra6 knockout mouse model to analyze the metabolic basis of vitamin A homeostasis in peripheral tissues. These mice were viable when bred on diets replete in vitamin A, but evidenced markedly reduced levels of ocular retinoids. Ophthalmic imaging and histology revealed malformations in the choroid and retinal pigmented epithelium, early cone photoreceptor cell death, and reduced lengths of rod outer segments. Similar to the blood–retina barrier in the RPE, vitamin A transport through the blood–cerebrospinal fluid barrier in the brain's choroid plexus was impaired. Notably, treatment with pharmacological doses of vitamin A restored vitamin A transport across these barriers and rescued the vision of Stra6−/− mice. Furthermore, under conditions mimicking vitamin A excess and deficiency, our analyses revealed that STRA6-mediated vitamin A uptake is a regulated process mandatory for ocular vitamin A uptake when RBP4 constitutes the only transport mode in vitamin A deficiency. These findings identifying STRA6 as a bona fide vitamin A transporter have important implications for disease states associated with impaired blood vitamin A homeostasis. PMID:24852372

  11. Sequence-selective DNA recognition and enhanced cellular up-take by peptide-steroid conjugates.

    PubMed

    Ruiz García, Yara; Iyer, Abhishek; Van Lysebetten, Dorien; Pabon, Y Vladimir; Louage, Benoit; Honcharenko, Malgorzata; De Geest, Bruno G; Smith, C I Edvard; Strömberg, Roger; Madder, Annemieke

    2015-12-25

    Several GCN4 bZIP TF models have previously been designed and synthesized. However, the synthetic routes towards these constructs are typically tedious and difficult. We here describe the substitution of the Leucine zipper domain of the protein by a deoxycholic acid derivative appending the two GCN4 binding region peptides through an optimized double azide-alkyne cycloaddition click reaction. In addition to achieving sequence specific dsDNA binding, we have investigated the potential of these compounds to enter cells. Confocal microscopy and flow cytometry show the beneficial influence of the steroid on cell uptake. This unique synthetic model of the bZIP TF thus combines sequence specific dsDNA binding properties with enhanced cell-uptake. Given the unique properties of deoxycholic acid and the convergent nature of the synthesis, we believe this work represents a key achievement in the field of TF mimicry.

  12. Enhanced Cellular Uptake and Pharmacokinetic Characteristics of Doxorubicin-Valine Amide Prodrug.

    PubMed

    Park, Yohan; Park, Ju-Hwan; Park, Suryeon; Lee, Song Yi; Cho, Kwan Hyung; Kim, Dae-Duk; Shim, Won-Sik; Yoon, In-Soo; Cho, Hyun-Jong; Maeng, Han-Joo

    2016-09-22

    In this study, we synthesized the valine (Val)-conjugated amide prodrug of doxorubicin (DOX) by the formation of amide bonds between DOX and Val. The synthesis of the DOX-Val prodrug was identified by a proton nuclear magnetic resonance (¹H-NMR) assay. In the MCF-7 cells (human breast adenocarcinoma cell; amino acid transporter-positive cell), the cellular accumulation efficiency of DOX-Val was higher than that of DOX according to the flow cytometry analysis data. Using confocal laser scanning microscopy (CLSM) imaging, it was confirmed that DOX-Val as well as DOX was mainly distributed in the nucleus of cancer cells. DOX-Val was intravenously administered to rats at a dose of 4 mg/kg, and the plasma concentrations of DOX-Val (prodrug) and DOX (formed metabolite) were quantitatively determined. Based on the systemic exposure (represented as area under the curve (AUC) values) of DOX-Val (prodrug) and DOX (formed metabolite), approximately half of DOX-Val seemed to be metabolized into DOX. However, it is expected that the remaining DOX-Val may exert improved cellular uptake efficiency in cancer cells after its delivery to the cancer region.

  13. Enhanced Cellular Uptake and Pharmacokinetic Characteristics of Doxorubicin-Valine Amide Prodrug.

    PubMed

    Park, Yohan; Park, Ju-Hwan; Park, Suryeon; Lee, Song Yi; Cho, Kwan Hyung; Kim, Dae-Duk; Shim, Won-Sik; Yoon, In-Soo; Cho, Hyun-Jong; Maeng, Han-Joo

    2016-01-01

    In this study, we synthesized the valine (Val)-conjugated amide prodrug of doxorubicin (DOX) by the formation of amide bonds between DOX and Val. The synthesis of the DOX-Val prodrug was identified by a proton nuclear magnetic resonance (¹H-NMR) assay. In the MCF-7 cells (human breast adenocarcinoma cell; amino acid transporter-positive cell), the cellular accumulation efficiency of DOX-Val was higher than that of DOX according to the flow cytometry analysis data. Using confocal laser scanning microscopy (CLSM) imaging, it was confirmed that DOX-Val as well as DOX was mainly distributed in the nucleus of cancer cells. DOX-Val was intravenously administered to rats at a dose of 4 mg/kg, and the plasma concentrations of DOX-Val (prodrug) and DOX (formed metabolite) were quantitatively determined. Based on the systemic exposure (represented as area under the curve (AUC) values) of DOX-Val (prodrug) and DOX (formed metabolite), approximately half of DOX-Val seemed to be metabolized into DOX. However, it is expected that the remaining DOX-Val may exert improved cellular uptake efficiency in cancer cells after its delivery to the cancer region. PMID:27669201

  14. Nitrate removal in stream ecosystems measured by 15N addition experiments: Total uptake

    USGS Publications Warehouse

    Hall, R.O.; Tank, J.L.; Sobota, D.J.; Mulholland, P.J.; O'Brien, J. M.; Dodds, W.K.; Webster, J.R.; Valett, H.M.; Poole, G.C.; Peterson, B.J.; Meyer, J.L.; McDowell, W.H.; Johnson, S.L.; Hamilton, S.K.; Grimm, N. B.; Gregory, S.V.; Dahm, Clifford N.; Cooper, L.W.; Ashkenas, L.R.; Thomas, S.M.; Sheibley, R.W.; Potter, J.D.; Niederlehner, B.R.; Johnson, L.T.; Helton, A.M.; Crenshaw, C.M.; Burgin, A.J.; Bernot, M.J.; Beaulieu, J.J.; Arangob, C.P.

    2009-01-01

    We measured uptake length of 15NO-3 in 72 streams in eight regions across the United States and Puerto Rico to develop quantitative predictive models on controls of NO-3 uptake length. As part of the Lotic Intersite Nitrogen eXperiment II project, we chose nine streams in each region corresponding to natural (reference), suburban-urban, and agricultural land uses. Study streams spanned a range of human land use to maximize variation in NO-3 concentration, geomorphology, and metabolism. We tested a causal model predicting controls on NO-3 uptake length using structural equation modeling. The model included concomitant measurements of ecosystem metabolism, hydraulic parameters, and nitrogen concentration. We compared this structural equation model to multiple regression models which included additional biotic, catchment, and riparian variables. The structural equation model explained 79% of the variation in log uptake length (S Wtot). Uptake length increased with specific discharge (Q/w) and increasing NO-3 concentrations, showing a loss in removal efficiency in streams with high NO-3 concentration. Uptake lengths shortened with increasing gross primary production, suggesting autotrophic assimilation dominated NO-3 removal. The fraction of catchment area as agriculture and suburban-urban land use weakly predicted NO-3 uptake in bivariate regression, and did improve prediction in a set of multiple regression models. Adding land use to the structural equation model showed that land use indirectly affected NO-3 uptake lengths via directly increasing both gross primary production and NO-3 concentration. Gross primary production shortened SWtot, while increasing NO-3 lengthened SWtot resulting in no net effect of land use on NO- 3 removal. ?? 2009.

  15. Nitrate removal in stream ecosystems measured by 15N addition experiments: Total uptake

    SciTech Connect

    Mulholland, Patrick J; Hall, Robert; Tank, Jennifer; Sobota, Daniel; O'Brien, Jon; Webster, Jackson; Valett, H. Maurice; Dodds, Walter; Poole, Geoff; Peterson, Chris G.; Meyer, Judy; McDowell, William; Johnson, Sherri; Hamilton, Stephen; Gregory, Stanley; Grimm, Nancy; Dahm, Cliff; Cooper, Lee W; Ashkenas, Linda; Thomas, Suzanne; Sheibley, Rich; Potter, Jody; Niederlehner, Bobbie; Johnson, Laura; Helton, Ashley; Crenshaw, Chelsea; Burgin, Amy; Bernot, Melody; Beaulieu, Jake; Arango, Clay

    2009-01-01

    We measured uptake length of {sup 15}NO{sub 3}{sup -} in 72 streams in eight regions across the United States and Puerto Rico to develop quantitative predictive models on controls of NO{sub 3}{sup -} uptake length. As part of the Lotic Intersite Nitrogen Experiment II project, we chose nine streams in each region corresponding to natural (reference), suburban-urban, and agricultural land uses. Study streams spanned a range of human land use to maximize variation in NO{sub 3}{sup -} concentration, geomorphology, and metabolism. We tested a causal model predicting controls on NO{sub 3}{sup -} uptake length using structural equation modeling. The model included concomitant measurements of ecosystem metabolism, hydraulic parameters, and nitrogen concentration. We compared this structural equation model to multiple regression models which included additional biotic, catchment, and riparian variables. The structural equation model explained 79% of the variation in log uptake length (S{sub Wtot}). Uptake length increased with specific discharge (Q/w) and increasing NO{sub 3}{sup -} concentrations, showing a loss in removal efficiency in streams with high NO{sub 3}{sup -} concentration. Uptake lengths shortened with increasing gross primary production, suggesting autotrophic assimilation dominated NO{sub 3}{sup -} removal. The fraction of catchment area as agriculture and suburban-urban land use weakly predicted NO{sub 3}{sup -} uptake in bivariate regression, and did improve prediction in a set of multiple regression models. Adding land use to the structural equation model showed that land use indirectly affected NO{sub 3}{sup -} uptake lengths via directly increasing both gross primary production and NO{sub 3}{sup -} concentration. Gross primary production shortened S{sub Wtot}, while increasing NO{sub 3}{sup -} lengthened S{sub Wtot} resulting in no net effect of land use on NO{sub 3}{sup -} removal.

  16. High frequency application of nanosecond pulsed electric fields alters cellular membrane disruption and fluorescent dye uptake

    NASA Astrophysics Data System (ADS)

    Steelman, Zachary A.; Tolstykh, Gleb P.; Beier, Hope T.; Ibey, Bennett L.

    2016-03-01

    Cells exposed to nanosecond-pulsed electric fields (nsPEF) exhibit a wide variety of nonspecific effects, including blebbing, swelling, intracellular calcium bursts, apoptotic and necrotic cell death, formation of nanopores, and depletion of phosphatidylinositol 4,5-biphosphate (PIP2) to induce activation of the inositol trisphosphate/diacylglycerol pathway. While several studies have taken place in which multiple pulses were delivered to cells, the effect of pulse repetition rate (PRR) is not well understood. To better understand the effects of PRR, a laser scanning confocal microscope was used to observe CHO-K1 cells exposed to ten 600ns, 200V pulses at varying repetition rates (5Hz up to 500KHz) in the presence of either FM 1-43, YO-PRO-1, or Propidium Iodide (PI) fluorescent dyes, probes frequently used to indicate nanoporation or permeabilization of the plasma membrane. Dye uptake was monitored for 30 seconds after pulse application at a rate of 1 image/second. In addition, a single long pulse of equivalent energy (200V, 6 μs duration) was applied to test the hypothesis that very fast PRR will approximate the biological effects of a single long pulse of equal energy. Upon examination of the data, we found strong variation in the relationship between PRR and uptake in each of the three dyes. In particular, PI uptake showed little frequency dependence, FM 1-43 showed a strong inverse relationship between frequency and internal cell fluorescence, and YO-PRO-1 exhibited a "threshold" point of around 50 KHz, after which the inverse trend observed in FM 1-43 was seen to reverse itself. Further, a very high PRR of 500 KHz only approximated the biological effects of a single 6 μs pulse in cells stained with YO-PRO-1, suggesting that uptake of different dyes may proceed by different physical mechanisms.

  17. Neurotoxic potential and cellular uptake of T-2 toxin in human astrocytes in primary culture.

    PubMed

    Weidner, Maria; Lenczyk, Marlies; Schwerdt, Gerald; Gekle, Michael; Humpf, Hans-Ulrich

    2013-03-18

    The trichothecene mycotoxin T-2 toxin, which is produced by fungi of the Fusarium species, is a worldwide occurring contaminant of cereal based food and feed. The cytotoxic properties of T-2 toxin are already well described with apoptosis being a major mechanism of action in various cell lines as well as in primary cells of different origin. However, only few data on neurotoxic properties of T-2 toxin are reported so far, but in vivo studies showed different effects of T-2 toxin on behavior as well as on levels of brain amines in animals. To further investigate the cytotoxic properties of T-2 toxin on cells derived from brain tissue, normal human astrocytes in primary culture (NHA) were used in this study. Besides studies of cytotoxicity, apoptosis (caspase-3-activation, Annexin V) and necrosis (LDH-release), the cellular uptake and metabolism of T-2 toxin in NHA was analyzed and compared to the uptake in an established human cell line (HT-29). The results show that human astrocytes were highly sensitive to the cytotoxic properties of T-2 toxin, and apoptosis, induced at low concentrations, was identified for the first time as the mechanism of toxic action in NHA. Furthermore, a strong accumulation of T-2 toxin in NHA and HT-29 cells was detected, and T-2 toxin was subjected to metabolism leading to HT-2 toxin, a commonly found metabolite after T-2 toxin incubation in both cell types. This formation seems to occur within the cells since incubations of T-2 toxin with cell depleted culture medium did not lead to any degradation of the parent toxin. The results of this study emphasize the neurotoxic potential of T-2 toxin in human astrocytes at low concentrations after short incubation times. PMID:23363530

  18. Does cytotoxicity of metallointercalators correlate with cellular uptake or DNA affinity?

    PubMed

    Davis, Kimberley J; Carrall, Judith A; Lai, Barry; Aldrich-Wright, Janice R; Ralph, Stephen F; Dillon, Carolyn T

    2012-08-21

    The cytotoxicity of the metallointercalators, [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1R,2R-diaminocyclohexane)](2+) ([56MERR]) and [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1S,2S-diaminocyclohexane)](2+) ([56MESS]), towards A549 human lung cancer cells was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) value obtained following exposure of A549 cells to [56MESS] for 4 h was approximately three times smaller than that obtained when [56MERR] was administered under the same conditions, indicating that the former complex displayed greater cytotoxicity. Both IC(50) values were greater than that obtained after exposure of A549 cells to cisplatin, demonstrating that the latter compound was the most cytotoxic of the three examined. Microprobe synchrotron radiation X-ray fluorescence (SR-XRF) analyses of metallointercalator-treated A549 cells showed that platinum became localised in DNA-rich regions of the nucleus. In contrast, when the same cells were treated with cisplatin the metal became distributed throughout the cell. Determination of the maximum concentration of platinum present inside the cells using graphite furnace atomic absorption spectrophotometry (GFAAS) of platinum-treated cells suggested that there was greater uptake of [56MERR] compared to [56MESS] by the A549 cells, and that platinum uptake did not account for the greater toxicity of [56MESS], as assessed by the MTT assay. Electrospray ionization mass spectrometric (ESI-MS) and circular dichroism (CD) spectroscopic studies of solutions containing either [56MERR] or [56MESS], and a duplex hexadecamer molecule, showed the two metallointercalators displayed very similar affinity towards the nucleic acid. Overall these results indicate that the difference in cytotoxicity of the two platinum metallointercalators is probably the result of variations in their interactions with other cellular components. PMID:22740039

  19. Cellular uptake and transcytosis of lipid-based nanoparticles across the intestinal barrier: Relevance for oral drug delivery.

    PubMed

    Neves, Ana Rute; Queiroz, Joana Fontes; Costa Lima, Sofia A; Figueiredo, Francisco; Fernandes, Rui; Reis, Salette

    2016-02-01

    Oral administration is the preferred route for drug delivery and nanosystems represent a promising tool for protection and transport of hardly soluble, chemically unstable and poorly permeable drugs through the intestinal barrier. In the present work, we have studied lipid nanoparticles cellular uptake, internalization pathways and transcytosis routes through Caco-2 cell monolayers. Both lipid nanosystems presented similar size (∼180nm) and surface charge (-30mV). Nanostructured lipid carriers showed a higher cellular uptake and permeability across the barrier, but solid lipid nanoparticles could enter cells faster than the former. The internalization of lipid nanoparticles occurs mainly through a clathrin-mediated endocytosis mechanism, although caveolae-mediated endocytosis is also involved in the uptake. Both lipid nanoparticles were able to cross the intestinal barrier by a preferential transcellular route. This work contributed to a better knowledge of the developed nanosystems for the oral delivery of a wide spectrum of drugs.

  20. Cellular uptake mechanisms of functionalised multi-walled carbon nanotubes by 3D electron tomography imaging

    NASA Astrophysics Data System (ADS)

    Al-Jamal, Khuloud T.; Nerl, Hannah; Müller, Karin H.; Ali-Boucetta, Hanene; Li, Shouping; Haynes, Peter D.; Jinschek, Joerg R.; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas; Porter, Alexandra E.

    2011-06-01

    Carbon nanotubes (CNTs) are being investigated for a variety of biomedical applications. Despite numerous studies, the pathways by which carbon nanotubes enter cells and their subsequent intracellular trafficking and distribution remain poorly determined. Here, we use 3-D electron tomography techniques that offer optimum enhancement of contrast between carbon nanotubes and the plasma membrane to investigate the mechanisms involved in the cellular uptake of shortened, functionalised multi-walled carbon nanotubes (MWNT-NH3+). Both human lung epithelial (A549) cells, that are almost incapable of phagocytosis and primary macrophages, capable of extremely efficient phagocytosis, were used. We observed that MWNT-NH3+ were internalised in both phagocytic and non-phagocytic cells by any one of three mechanisms: (a) individually via membrane wrapping; (b) individually by direct membrane translocation; and (c) in clusters within vesicular compartments. At early time points following intracellular translocation, we noticed accumulation of nanotube material within various intracellular compartments, while a long-term (14-day) study using primary human macrophages revealed that MWNT-NH3+ were able to escape vesicular (phagosome) entrapment by translocating directly into the cytoplasm.Carbon nanotubes (CNTs) are being investigated for a variety of biomedical applications. Despite numerous studies, the pathways by which carbon nanotubes enter cells and their subsequent intracellular trafficking and distribution remain poorly determined. Here, we use 3-D electron tomography techniques that offer optimum enhancement of contrast between carbon nanotubes and the plasma membrane to investigate the mechanisms involved in the cellular uptake of shortened, functionalised multi-walled carbon nanotubes (MWNT-NH3+). Both human lung epithelial (A549) cells, that are almost incapable of phagocytosis and primary macrophages, capable of extremely efficient phagocytosis, were used. We observed

  1. Biomechanics and thermodynamics of nanoparticle interactions with plasma and endosomal membrane lipids in cellular uptake and endosomal escape.

    PubMed

    Peetla, Chiranjeevi; Jin, Shihua; Weimer, Jonathan; Elegbede, Adekunle; Labhasetwar, Vinod

    2014-07-01

    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

  2. Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes

    PubMed Central

    Yun, Bo; Azad, Mohammad A. K.; Nowell, Cameron J.; Nation, Roger L.; Thompson, Philip E.; Roberts, Kade D.

    2015-01-01

    Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity. PMID:26392495

  3. Preparation of bufalin-loaded pluronic polyetherimide nanoparticles, cellular uptake, distribution, and effect on colorectal cancer.

    PubMed

    Hu, Qiang; Liang, Bo; Sun, Ying; Guo, Xiao-Ling; Bao, Yi-Jie; Xie, Dong-Hao; Zhou, Ming; Duan, You-Rong; Yin, Pei-Hao; Peng, Zhi-Hai

    2014-01-01

    A large number of studies have shown that bufalin can have a significant antitumor effect in a variety of tumors. However, because of toxicity, insolubility in water, fast metabolism, short half-life, and other shortcomings, its application is limited in cancer therapy. In this study, we explored the anti-metastatic role of bufalin-loaded pluronic polyetherimide nanoparticles on HCT116 colon cancer-bearing mice. Nanoparticle size, shape, drug loading, encapsulation efficiency, and in vitro drug release were studied. Also, cellular uptake of nanoparticles, in vivo tumor targeting, and tumor metastasis were studied. The nanoparticles had a particle size of about 60 nm and an encapsulation efficiency of 75.71%, by weight. The in vitro release data showed that free bufalin was released faster than bufalin-loaded pluronic polyetherimide nanoparticles, and almost 80% of free bufalin was released after 32 hours. Nanoparticles had an even size distribution, were stable, and had a slow release and a tumor-targeting effect. Bufalin-loaded pluronic polyetherimide nanoparticles can significantly inhibit the growth and metastasis of colorectal cancer.

  4. Synthesis of Carbohydrate Capped Silicon Nanoparticles and their Reduced Cytotoxicity, In Vivo Toxicity, and Cellular Uptake.

    PubMed

    Ahire, Jayshree H; Behray, Mehrnaz; Webster, Carl A; Wang, Qi; Sherwood, Victoria; Saengkrit, Nattika; Ruktanonchai, Uracha; Woramongkolchai, Noppawan; Chao, Yimin

    2015-08-26

    The development of smart targeted nanoparticles (NPs) that can identify and deliver drugs at a sustained rate directly to cancer cells may provide better efficacy and lower toxicity for treating primary and advanced metastatic tumors. Obtaining knowledge of the diseases at the molecular level can facilitate the identification of biological targets. In particular, carbohydrate-mediated molecular recognitions using nano-vehicles are likely to increasingly affect cancer treatment methods, opening a new area in biomedical applications. Here, silicon NPs (SiNPs) capped with carbohydrates including galactose, glucose, mannose, and lactose are successfully synthesized from amine terminated SiNPs. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] analysis shows an extensive reduction in toxicity of SiNPs by functionalizing with carbohydrate moiety both in vitro and in vivo. Cellular uptake is investigated with flow cytometry and confocal fluorescence microscope. The results show the carbohydrate capped SiNPs can be internalized in the cells within 24 h of incubation, and can be taken up more readily by cancer cells than noncancerous cells. Moreover, these results reinforce the use of carbohydrates for the internalization of a variety of similar compounds into cancer cells. PMID:26121084

  5. Kinetics of cellular uptake of viruses and nanoparticles via clathrin-mediated endocytosis.

    PubMed

    Banerjee, Anand; Berezhkovskii, Alexander; Nossal, Ralph

    2016-02-01

    Several viruses exploit clathrin-mediated endocytosis to gain entry into host cells. This process is also used extensively in biomedical applications to deliver nanoparticles (NPs) to diseased cells. The internalization of these nano-objects is controlled by the assembly of a clathrin-containing protein coat on the cytoplasmic side of the plasma membrane, which drives the invagination of the membrane and the formation of a cargo-containing endocytic vesicle. Current theoretical models of receptor-mediated endocytosis of viruses and NPs do not explicitly take coat assembly into consideration. In this paper we study cellular uptake of viruses and NPs with a focus on coat assembly. We characterize the internalization process by the mean time between the binding of a particle to the membrane and its entry into the cell. Using a coarse-grained model which maps the stochastic dynamics of coat formation onto a one-dimensional random walk, we derive an analytical formula for this quantity. A study of the dependence of the mean internalization time on NP size shows that there is an upper bound above which this time becomes extremely large, and an optimal size at which it attains a minimum. Our estimates of these sizes compare well with experimental data. We also study the sensitivity of the obtained results on coat parameters to identify factors which significantly affect the internalization kinetics. PMID:26871680

  6. Synthesis of Carbohydrate Capped Silicon Nanoparticles and their Reduced Cytotoxicity, In Vivo Toxicity, and Cellular Uptake.

    PubMed

    Ahire, Jayshree H; Behray, Mehrnaz; Webster, Carl A; Wang, Qi; Sherwood, Victoria; Saengkrit, Nattika; Ruktanonchai, Uracha; Woramongkolchai, Noppawan; Chao, Yimin

    2015-08-26

    The development of smart targeted nanoparticles (NPs) that can identify and deliver drugs at a sustained rate directly to cancer cells may provide better efficacy and lower toxicity for treating primary and advanced metastatic tumors. Obtaining knowledge of the diseases at the molecular level can facilitate the identification of biological targets. In particular, carbohydrate-mediated molecular recognitions using nano-vehicles are likely to increasingly affect cancer treatment methods, opening a new area in biomedical applications. Here, silicon NPs (SiNPs) capped with carbohydrates including galactose, glucose, mannose, and lactose are successfully synthesized from amine terminated SiNPs. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] analysis shows an extensive reduction in toxicity of SiNPs by functionalizing with carbohydrate moiety both in vitro and in vivo. Cellular uptake is investigated with flow cytometry and confocal fluorescence microscope. The results show the carbohydrate capped SiNPs can be internalized in the cells within 24 h of incubation, and can be taken up more readily by cancer cells than noncancerous cells. Moreover, these results reinforce the use of carbohydrates for the internalization of a variety of similar compounds into cancer cells.

  7. An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium.

    SciTech Connect

    Jensen, M. P.; Gorman-Lewis, D.; Aryal, B. P.; Paunesku, T.; Vogt, S.; Rickert, P. G.; Seifert, S.; Lai, B.; Woloschak, G. E.; Soderholm, L.

    2011-08-01

    Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway -- receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small-angle scattering show that only transferrin with plutonium bound in the protein's C-terminal lobe (C-lobe) and iron bound in the N-terminal lobe (N-lobe) (Pu{sub c}Fe{sub N}Tf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal-binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin's two lobes act to restrict, but not eliminate, cellular Pu uptake.

  8. Kinetics of cellular uptake of viruses and nanoparticles via clathrin-mediated endocytosis

    NASA Astrophysics Data System (ADS)

    Banerjee, Anand; Berezhkovskii, Alexander; Nossal, Ralph

    2016-02-01

    Several viruses exploit clathrin-mediated endocytosis to gain entry into host cells. This process is also used extensively in biomedical applications to deliver nanoparticles (NPs) to diseased cells. The internalization of these nano-objects is controlled by the assembly of a clathrin-containing protein coat on the cytoplasmic side of the plasma membrane, which drives the invagination of the membrane and the formation of a cargo-containing endocytic vesicle. Current theoretical models of receptor-mediated endocytosis of viruses and NPs do not explicitly take coat assembly into consideration. In this paper we study cellular uptake of viruses and NPs with a focus on coat assembly. We characterize the internalization process by the mean time between the binding of a particle to the membrane and its entry into the cell. Using a coarse-grained model which maps the stochastic dynamics of coat formation onto a one-dimensional random walk, we derive an analytical formula for this quantity. A study of the dependence of the mean internalization time on NP size shows that there is an upper bound above which this time becomes extremely large, and an optimal size at which it attains a minimum. Our estimates of these sizes compare well with experimental data. We also study the sensitivity of the obtained results on coat parameters to identify factors which significantly affect the internalization kinetics.

  9. An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium

    PubMed Central

    Jensen, Mark P.; Gorman-Lewis, Drew; Aryal, Baikuntha; Paunesku, Tatjana; Vogt, Stefan; Rickert, Paul G.; Seifert, Soenke; Lai, Barry; Woloschak, Gayle E.; Soderholm, L.

    2012-01-01

    Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small angle X-ray scattering, receptor binding assays, and synchrotron X-ray fluorescence microscopy we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway, receptor-mediated endocytosis of the iron transport protein serum transferrin; however only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small angle scattering demonstrate that only transferrin with plutonium bound in the protein’s C-terminal lobe and iron bound in the N-lobe (PuCFeNTf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin’s two lobes act to restrict, but not eliminate, cellular Pu uptake. PMID:21706034

  10. Shape Effect of Glyco-Nanoparticles on Macrophage Cellular Uptake and Immune Response

    PubMed Central

    2016-01-01

    The shells of various poly(dl-lactide)-b-poly(acrylic acid) (PDLLA-b-PAA) spherical micelles and poly(l-lactide)-b-poly(acrylic acid) (PLLA-b-PAA) cylindrical micelles were functionalized with mannose to yield glyco-nanoparticles (GNPs) with different shapes and dimensions. All of these GNPs were shown to have good biocompatibility (up to 1 mg/mL). Cellular uptake experiments using RAW 264.7 have shown that the spherical GNPs were internalized to a much greater extent than the cylindrical GNPs and such a phenomenon was attributed to their different endocytosis pathways. It was demonstrated that spherical GNPs were internalized based on clathrin- and caveolin-mediated endocytosis while cylindrical GNPs mainly depended on clathrin-mediated endocytosis. We also found that longer cylindrical GNPs (Ln × Wn = 215 × 47 nm) can induce an inflammatory response (specifically interleukin 6) more efficiently than shorter cylindrical GNPs (Ln × Wn = 99 × 50 nm) and spherical GNPs (Dn = 46 nm). PMID:27695648

  11. An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium.

    PubMed

    Jensen, Mark P; Gorman-Lewis, Drew; Aryal, Baikuntha; Paunesku, Tatjana; Vogt, Stefan; Rickert, Paul G; Seifert, Soenke; Lai, Barry; Woloschak, Gayle E; Soderholm, L

    2011-06-26

    Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway--receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small-angle scattering show that only transferrin with plutonium bound in the protein's C-terminal lobe (C-lobe) and iron bound in the N-terminal lobe (N-lobe) (Pu(C)Fe(N)Tf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal-binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin's two lobes act to restrict, but not eliminate, cellular Pu uptake.

  12. Enhancing Cellular Uptake and Doxorubicin Delivery of Mesoporous Silica Nanoparticles via Surface Functionalization: Effects of Serum.

    PubMed

    Shahabi, Shakiba; Döscher, Svea; Bollhorst, Tobias; Treccani, Laura; Maas, Michael; Dringen, Ralf; Rezwan, Kurosch

    2015-12-01

    In this study, we demonstrate how functional groups on the surface of mesoporous silica nanoparticles (MSNPs) can influence the encapsulation and release of the anticancer drug doxorubicin, as well as cancer cell response in the absence or presence of serum proteins. To this end, we synthesized four differently functionalized MSNPs with amine, sulfonate, polyethylene glycol, or polyethylene imine functional surface groups, as well as one type of antibody-conjugated MSNP for specific cellular targeting, and we characterized these MSNPs regarding their physicochemical properties, colloidal stability in physiological media, and uptake and release of doxorubicin in vitro. Then, the MSNPs were investigated for their cytotoxic potential on cancer cells. Cationic MSNPs could not be loaded with doxorubicin and did therefore not show any cytotoxic and antiproliferative potential on osteosarcoma cells, although they were efficiently taken up into the cells in the presence or absence of serum. In contrast, substantial amounts of doxorubicin were loaded into negatively charged and unfunctionalized MSNPs. Especially, sulfonate-functionalized doxorubicin-loaded MSNPs were efficiently taken up into the cells in the presence of serum and showed an accelerated toxic and antiproliferative potential compared to unfunctionalized MSNPs, antibody-conjugated MSNPs, and even free doxorubicin. These findings stress the high importance of the surface charge as well as of the protein corona for designing and applying nanoparticles for targeted drug delivery.

  13. Cellular location of insulin-triggered signals and implications for glucose uptake.

    PubMed

    Patel, Nish; Huang, Carol; Klip, Amira

    2006-01-01

    Insulin stimulation of glucose uptake into muscle and fat cells requires movement of GLUT4-containing vesicles from intracellular compartments to the plasma membrane. Accordingly, insulin-derived signals must arrive at and be recognized by the appropriate intracellular GLUT4 pools. We describe the insulin signals participating in GLUT4 translocation, and review evidence that they are recruited to intracellular membranes in conjunction with cytoskeletal elements. Such segregation may facilitate the encounter between signals and target vesicles. In most animal and cellular models of insulin resistance, insulin-stimulated GLUT4 translocation to the plasma membrane is reduced. Insulin resistance caused by oxidative stress does not affect early insulin signals, rather their intracellular localization is altered. In this and several other insulin-resistant states, insulin-induced actin remodelling is concomitantly diminished. We summarize evidence suggesting that spatial localization of signals is critical for efficient insulin action, and that the cytoskeleton may act as a scaffold to promote efficient translocation of GLUT4 to the cell surface.

  14. Shape Effect of Glyco-Nanoparticles on Macrophage Cellular Uptake and Immune Response

    PubMed Central

    2016-01-01

    The shells of various poly(dl-lactide)-b-poly(acrylic acid) (PDLLA-b-PAA) spherical micelles and poly(l-lactide)-b-poly(acrylic acid) (PLLA-b-PAA) cylindrical micelles were functionalized with mannose to yield glyco-nanoparticles (GNPs) with different shapes and dimensions. All of these GNPs were shown to have good biocompatibility (up to 1 mg/mL). Cellular uptake experiments using RAW 264.7 have shown that the spherical GNPs were internalized to a much greater extent than the cylindrical GNPs and such a phenomenon was attributed to their different endocytosis pathways. It was demonstrated that spherical GNPs were internalized based on clathrin- and caveolin-mediated endocytosis while cylindrical GNPs mainly depended on clathrin-mediated endocytosis. We also found that longer cylindrical GNPs (Ln × Wn = 215 × 47 nm) can induce an inflammatory response (specifically interleukin 6) more efficiently than shorter cylindrical GNPs (Ln × Wn = 99 × 50 nm) and spherical GNPs (Dn = 46 nm).

  15. Isotopic discrimination of zinc during root-uptake and cellular incorporation in higher plants

    NASA Astrophysics Data System (ADS)

    Mason, T. F.; Weiss, D. J.; Coles, B. J.; Horstwood, M.; Parrish, R. R.; Zhao, F. J.; Kirk, G. J.

    2003-04-01

    solutions, the depletion from root to shoot is significantly larger with the former (at -0.15 to -0.25 ppm pamu compared with -0.13 to -0.18 ppm pamu). For rice plants cultivated on zinc-sufficient soils, isotopic enrichment from soil to root (+0.25 ppm pamu), and depletion from root to shoot (-0.11 ppm pamu) were observed. However, under zinc-deficient conditions no significant isotopic shifts between soil, root and shoot were found. From these results it is apparent that two or more processes are controlling the zinc isotopic composition of the plant materials: one that favours isotopically heavy zinc (which we tentatively link to isotopic partitioning between species within the nutrient/soil-solutions), and one that favours isotopically light zinc (which is consistent with biologically-mediated uptake and cellular incorporation by plants). The lack of isotopic variability in the zinc-deficient soil system may indicate the predominance of a high-affinity zinc uptake pathway that is not isotopically selective.

  16. pH effect on cellular uptake of Sn(IV) chlorine e6 dichloride trisodium salt by cancer cells in vitro.

    PubMed

    Al-Khaza'leh, Khaled A; Omar, Khalid; Jaafar, M S

    2011-01-01

    The effects of pH value and presence of serum in an incubation medium on photosensitizer drug cellular uptake in MCF7 cancer cells have been investigated. The results showed that the presence of serum in an incubation medium reduced the drug cellular uptake at all pH values. It has been found that decreasing on pH values of the incubation medium increased the cellular uptake of the drug, demonstrating selective uptake of the sensitizer. The HepG2 liver cancer cells exhibited more drug cellular uptake than CCD-18CO normal colon cells, which assessed the selectivity uptake of photosensitizer on cancerous cells. The concentration of photosensitizer measured in 10(6) cells showed a good correlation to the incubation time. Fluorescence and absorption spectroscopy been have used to examine the cells. PMID:22210969

  17. Glycan structure determinants for cation-independent mannose 6-phosphate receptor binding and cellular uptake of a recombinant protein.

    PubMed

    Zhou, Qun; Avila, Luis Z; Konowicz, Paul A; Harrahy, John; Finn, Patrick; Kim, Jennifer; Reardon, Michael R; Kyazike, Josephine; Brunyak, Elizabeth; Zheng, Xiaoyang; Patten, Scott M Van; Miller, Robert J; Pan, Clark Q

    2013-12-18

    The cation-independent mannose 6-phosphate receptor (CI-MPR) plays a critical role in intracellular transport of lysosomal enzymes as well as the uptake of recombinant proteins. To define the minimal glycan structure determinants necessary for receptor binding and cellular uptake, we synthesized a series of glycans containing mono-, di-, tri-, tetra-, and hexamannoses terminated with either one or two phosphates for conjugating to a model protein, recombinant human acid α-glucosidase. A high affinity interaction with the CI-MPR can be achieved for the enzyme conjugated to a dimannose glycan with a single phosphate. However, tightest binding to a CI-MPR affinity column was observed with a hexamannose structure containing two phosphates. Moreover, maximal cellular uptake and a 5-fold improvement in in vivo potency were achieved when the bisphosphorylated hexamannose glycan is conjugated to the protein by a β linker. Nevertheless, even a monophosphorylated dimannose glycan conjugate showed stronger binding to the receptor affinity column, higher cellular uptake, and significantly greater in vivo efficacy compared to the unconjugated protein which contains a low level of high affinity glycan structure. These results demonstrate that the phosphorylated dimannose moiety appears to be the minimal structure determinant for enhanced CI-MPR binding and that the orientation of the glycan is critical for maximum receptor interaction. In summary, we have improved the understanding of the mechanism of CI-MPR binding and developed a simple alternative for CI-MPR targeting.

  18. 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.

  19. Both FA- and mPEG-conjugated chitosan nanoparticles for targeted cellular uptake and enhanced tumor tissue distribution

    NASA Astrophysics Data System (ADS)

    Hou, Zhenqing; Zhan, Chuanming; Jiang, Qiwei; Hu, Quan; Li, Le; Chang, Di; Yang, Xiangrui; Wang, Yixiao; Li, Yang; Ye, Shefang; Xie, Liya; Yi, Yunfeng; Zhang, Qiqing

    2011-10-01

    Both folic acid (FA)- and methoxypoly(ethylene glycol) (mPEG)-conjugated chitosan nanoparticles (NPs) had been designed for targeted and prolong anticancer drug delivery system. The chitosan NPs were prepared with combination of ionic gelation and chemical cross-linking method, followed by conjugation with both FA and mPEG, respectively. FA-mPEG-NPs were compared with either NPs or mPEG-/FA-NPs in terms of their size, targeting cellular efficiency and tumor tissue distribution. The specificity of the mPEG-FA-NPs targeting cancerous cells was demonstrated by comparative intracellular uptake of NPs and mPEG-/FA-NPs by human adenocarcinoma HeLa cells. Mitomycin C (MMC), as a model drug, was loaded to the mPEG-FA-NPs. Results show that the chitosan NPs presented a narrow-size distribution with an average diameter about 200 nm regardless of the type of functional group. In addition, MMC was easily loaded to the mPEG-FA-NPs with drug-loading content of 9.1%, and the drug releases were biphasic with an initial burst release, followed by a subsequent slower release. Laser confocal scanning imaging proved that both mPEG-FA-NPs and FA-NPs could greatly enhance uptake by HeLa cells. In vivo animal experiments, using a nude mice xenograft model, demonstrated that an increased amount of mPEG-FA-NPs or FA-NPs were accumulated in the tumor tissue relative to the mPEG-NPs or NPs alone. These results suggest that both FA- and mPEG-conjugated chitosan NPs are potentially prolonged drug delivery system for tumor cell-selective targeting treatments.

  20. Scaling plant nitrogen use and uptake efficiencies in response to nutrient addition in peatlands

    SciTech Connect

    Iversen, Colleen M; Bridgham, Scott; Kellogg, Laurie E.

    2010-01-01

    Nitrogen (N) is the primary growth-limiting nutrient in many terrestrial ecosystems, and therefore plant production per unit N taken up (i.e., N use efficiency, NUE) is a fundamentally important component of ecosystem function. Nitrogen use efficiency comprises two components: N productivity (AN, plant production per peak biomass N content) and the mean residence time of N in plant biomass (MRTN). We utilized a five-year fertilization experiment to examine the manner in which increases in N and phosphorus (P) availability affected plant NUE at multiple biological scales (i.e., from leaf to community level). We fertilized a natural gradient of nutrient-limited peatland ecosystems in the Upper Peninsula of Michigan, USA, with 6 g Nm2yr1, 2 g Pm2yr1, or a combination of N and P. Our objectives were to determine how changes in carbon and N allocation within a plant to leaf and woody tissue and changes in species composition within a community, both above- and belowground, would affect (1) NUE; (2) the adaptive trade-off between the components of NUE; (3) the efficiency with which plants acquired N from the soil (N uptake efficiency); and (4) plant community production per unit soil N availability (N response efficiency, NRE). As expected, N and P addition generally increased aboveground production and N uptake. In particular, P availability strongly affected the way in which plants took up and used N. Nitrogen use efficiency response to nutrient addition was not straightforward. Nitrogen use efficiency differed between leaf and woody tissue, among species, and across the ombrotrophic minerotrophic gradient because plants and communities were adapted to maximize either AN or MRTN, but not both concurrently. Increased N availability strongly decreased plant and community N uptake efficiency, while increased P availability increased N uptake efficiency, particularly in a nitrogen-fixing shrub. Nitrogen uptake efficiency was more important in controlling overall plant

  1. Scaling plant nitrogen use and uptake efficiencies in response to nutrient addition in peatlands.

    PubMed

    Iversen, Colleen M; Bridgham, Scott D; Kellogg, Laurie E

    2010-03-01

    Nitrogen (N) is the primary growth-limiting nutrient in many terrestrial ecosystems, and therefore plant production per unit N taken up (i.e., N use efficiency, NUE) is a fundamentally important component of ecosystem function. Nitrogen use efficiency comprises two components: N productivity (A(N), plant production per peak biomass N content) and the mean residence time of N in plant biomass (MRT(N)). We utilized a five-year fertilization experiment to examine the manner in which increases in N and phosphorus (P) availability affected plant NUE at multiple biological scales (i.e., from leaf to community level). We fertilized a natural gradient of nutrient-limited peatland ecosystems in the Upper Peninsula of Michigan, USA, with 6 g N x m(-2) x yr(-1), 2 g P x m(-2) x yr(-1), or a combination of N and P. Our objectives were to determine how changes in carbon and N allocation within a plant to leaf and woody tissue and changes in species composition within a community, both above- and belowground, would affect (1) NUE; (2) the adaptive trade-off between the components of NUE; (3) the efficiency with which plants acquired N from the soil (N uptake efficiency); and (4) plant community production per unit soil N availability (N response efficiency, NRE). As expected, N and P addition generally increased aboveground production and N uptake. In particular, P availability strongly affected the way in which plants took up and used N. Nitrogen use efficiency response to nutrient addition was not straightforward. Nitrogen use efficiency differed between leaf and woody tissue, among species, and across the ombrotrophic-minerotrophic gradient because plants and communities were adapted to maximize either A(N) or MRT(N), but not both concurrently. Increased N availability strongly decreased plant and community N uptake efficiency, while increased P availability increased N uptake efficiency, particularly in a nitrogen-fixing shrub. Nitrogen uptake efficiency was more important

  2. Cellular uptake, intracellular trafficking, and antitumor efficacy of doxorubicin-loaded reduction-sensitive micelles.

    PubMed

    Cui, Can; Xue, Ya-Nan; Wu, Ming; Zhang, Yang; Yu, Ping; Liu, Lei; Zhuo, Ren-Xi; Huang, Shi-Wen

    2013-05-01

    Reduction-sensitive micelles were prepared from monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16), an amphiphilic poly(ethylene glycol) derivative containing a disulfide bond. The micelles were then used for the intracellular delivery of the anticancer drug doxorubicin (DOX) into tumor cells, and the cellular uptake mechanisms of the micelles were determined. To serve as a control, monomethoxy-poly(ethylene glycol)-C-C-hexadecyl (mPEG-C-C-C16) with an analogous structure but without a disulfide bond was also prepared. The polymer could self-assemble into micelles in an aqueous solution and be loaded with high-content DOX. In vitro release studies revealed that DOX-loaded mPEG-S-S-C16 micelles released DOX faster than DOX-loaded mPEG-C-C-C16 micelles in the presence of dithiothreitol (DTT), but showed similar release rates in the absence of DTT. MTT assay demonstrated significantly enhanced cytotoxicity of DOX-loaded mPEG-S-S-C16 micelles against the human cervical cancer cells (HeLa) compared with DOX-loaded mPEG-C-C-C16 micelles, but there was no significant difference in the cytotoxicity between the two DOX-loaded micelles against the african green monkey SV40-transformed kidney fibroblast cells (COS-7). Confocal laser scanning microscopy observation and flow cytometry analyses indicated that DOX-loaded mPEG-S-S-C16 micelles were efficiently internalized into HeLa cells, released DOX into the cytoplasm, and entered the nuclei. By contrast, in the case of DOX-loaded mPEG-C-C-C16 micelles, little DOX was found in the nuclei. Endocytosis inhibition results proved that both mPEG-S-S-C16 and mPEG-C-C-C16 micelles entered the HeLa cells mainly through the clathrin-mediated endocytosis pathway, and caveolae-mediated endocytosis was involved to a small extent. These results indicated that the different behaviors of cell uptake between reduction-sensitive and -insensitive micelles may occur after the micelles were internalized into the cells, but not during

  3. The comparison of protein-entrapped liposomes and lipoparticles: preparation, characterization, and efficacy of cellular uptake

    PubMed Central

    Chang, Wei-Kuo; Tai, Yu-Ju; Chiang, Chiao-Hsi; Hu, Chieh-Shen; Hong, Po-Da; Yeh, Ming-Kung

    2011-01-01

    Fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA)-loaded polyethylene glycol (PEG)-modified liposomes and lipoparticles with high protein entrapment were developed. The lipid formula of the liposomes contained PEGylated lipids and unsaturated fatty acids for enhancing membrane fluidity and effective delivery into cells. The preparation techniques, lipid content, and PEG-modified lipoparticle ratios were evaluated. The PEG-modified lipoparticles prepared by ethanol injection extrusion (100 nm pore size) achieve a population of blank liposomes with a mean size of 125 ± 2.3 nm and a zeta potential of −12.4 ± 1.5 mV. The average particle size of the PEG-modified lipoparticles was 133.7 ± 8.6 nm with a zeta potential of +13.3 mV. Lipoparticle conformation was determined using transmission electron microscopy and field-emission scanning electron microscopy. The FITC-BSA encapsulation efficiency was dramatically increased from 19.0% for liposomes to 59.7% for lipoparticles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results confirmed the preparation process, and an 8-hour leaching test did not harm the protein structure. Once prepared, the physical and chemical stability of the PEG-modified lipoparticle formulations was satisfactory over 90 days. In vitro retention tests indicated that the 50% retention time for the protein-containing lipoparticles was 7.9 hours, substantially longer than the liposomes at 3.3 hours. A Caco-2 cell model was used for evaluating the cytotoxicity and cell uptake efficiency of the PEG-modified lipoparticles. At a lipid content below 0.25 mM, neither the liposomes nor the lipoparticles caused significant cellular cytotoxicity (P < 0.01) and FITC-BSA was significantly taken up into cells within 60 minutes (P < 0.01). PMID:22072876

  4. Cellular senescence induced by prolonged subculture adversely affects glutamate uptake in C6 lineage.

    PubMed

    Pereira, Mery Stéfani Leivas; Zenki, Kamila; Cavalheiro, Marcela Mendonça; Thomé, Chairini Cássia; Filippi-Chiela, Eduardo Cremonese; Lenz, Guido; de Souza, Diogo Onofre Gomes; de Oliveira, Diogo Losch

    2014-05-01

    Several researchers have recently used C6 cells to evaluate functional properties of high-affinity glutamate transporters. However, it has been demonstrated that this lineage suffers several morphological and biochemical alterations according to the number of passages in culture. Currently, there are no reports showing whether functional properties of high-affinity glutamate transporters comply with these sub culturing-dependent modifications. The present study aimed to compare the functional properties of high-affinity glutamate transporters expressed in early (EPC6) and late (LPC6) passage C6 cells through a detailed pharmacological and biochemical characterization. Between 60-180 min of L-[(3)H]glu incubation, LPC6 presented an intracellular [(3)H] 55% lower than EPC6. Both cultures showed a time-dependent increase of intracellular [(3)H] reaching maximal levels at 120 min. Cultures incubated with D-[(3)H]asp showed a time-dependent increase of [(3)H] until 180 min. Moreover, LPC6 have a D-[(3)H]asp-derived intracellular [(3)H] 30-45% lower than EPC6 until 120 min. Only EAAT3 was immunodetected in cultures and its total content was equal between them. PMA-stimulated EAAT3 trafficking to membrane increased 50% of L-[(3)H]glu-derived intracellular [(3)H] in EPC6 and had no effect in LPC6. LPC6 displayed characteristics that resemble senescence, such as high β-Gal staining, cell enlargement and increase of large and regular nuclei. Our results demonstrated that LPC6 exhibited glutamate uptake impairment, which may have occurred due to its inability to mobilize EAAT3 to cell membrane. This profile might be related to senescent process observed in this culture. Our results suggest that LPC6 cells are an inappropriate glial cellular model to investigate the functional properties of high-affinity glutamate transporters.

  5. Role of toll-like receptors 3, 4 and 7 in cellular uptake and response to titanium dioxide nanoparticles

    NASA Astrophysics Data System (ADS)

    Chen, Peng; Kanehira, Koki; Taniguchi, Akiyoshi

    2013-02-01

    Innate immune response is believed to be among the earliest provisional cellular responses, and mediates the interactions between microbes and cells. Toll-like receptors (TLRs) are critical to these interactions. We hypothesize that TLRs also play an important role in interactions between nanoparticles (NPs) and cells, although little information has been reported concerning such an interaction. In this study, we investigated the role of TLR3, TLR4 and TLR7 in cellular uptake of titanium dioxide NP (TiO2 NP) agglomerates and the resulting inflammatory responses to these NPs. Our data indicate that TLR4 is involved in the uptake of TiO2 NPs and promotes the associated inflammatory responses. The data also suggest that TLR3, which has a subcellular location distinct from that of TLR4, inhibits the denaturation of cellular protein caused by TiO2 NPs. In contrast, the unique cellular localization of TLR7 has middle-ground functional roles in cellular response after TiO2 NP exposure. These findings are important for understanding the molecular interaction mechanisms between NPs and cells.

  6. Effect of low-intensity pulsed ultrasound on biocompatibility and cellular uptake of chitosan-tripolyphosphate nanoparticles

    PubMed Central

    Wu, Junyi; Liu, Gaojun; Qin, Yi-Xian; Meng, Yizhi

    2014-01-01

    Using low molecular weight chitosan nanoparticles (CNPs) prepared by an ionic gelation method, the authors report the effect of low-intensity pulsed ultrasound (US) on cell viability and nanoparticle uptake in cultured murine preosteoblasts. Particle size and zeta potential are measured using dynamic light scattering, and cell viability is evaluated using the of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] assay. Results show that 30 min delivery of CNPs at 0.5 mg/mL is able to prevent loss of cell viability due to either serum starvation or subsequent exposure to US (1 W/cm2 or 2 W/cm2, up to 1 min). Additionally, flow cytometry data suggest that there is a close association between cellular membrane integrity and the presence of CNPs when US at 2 W/cm2 is administered. PMID:25280857

  7. 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

  8. Increased cellular uptake of biocompatible superparamagnetic iron oxide nanoparticles into malignant cells by an external magnetic field.

    PubMed

    Prijic, Sara; Scancar, Janez; Romih, Rok; Cemazar, Maja; Bregar, Vladimir B; Znidarsic, Andrej; Sersa, Gregor

    2010-07-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) are used as delivery systems for different therapeutics including nucleic acids for magnetofection-mediated gene therapy. The aim of our study was to evaluate physicochemical properties, biocompatibility, cellular uptake and trafficking pathways of the custom-synthesized SPIONs for their potential use in magnetofection. Custom-synthesized SPIONs were tested for size, shape, crystalline composition and magnetic behavior using a transmission electron microscope, X-ray diffractometer and magnetometer. SPIONs were dispersed in different aqueous media to obtain ferrofluids, which were tested for pH and stability using a pH meter and zetameter. Cytotoxicity was determined using the MTS and clonogenic assays. Cellular uptake and trafficking pathways were qualitatively evaluated by transmission electron microscopy and quantitatively by inductively coupled plasma atomic emission spectrometry. SPIONs were composed of an iron oxide core with a diameter of 8-9 nm, coated with a 2-nm-thick layer of silica. SPIONs, dispersed in 0.9% NaCl solution, resulted in a stable ferrofluid at physiological pH for several months. SPIONs were not cytotoxic in a broad range of concentrations and were readily internalized into different cells by endocytosis. Exposure to neodymium-iron-boron magnets significantly increased the cellular uptake of SPIONs, predominantly into malignant cells. The prepared SPIONs displayed adequate physicochemical and biomedical properties for potential use in magnetofection. Their cellular uptake was dependent on the cell type, and their accumulation within the cells was dependent on the duration of exposure to an external magnetic field. PMID:20602230

  9. Increased Cellular Uptake of Biocompatible Superparamagnetic Iron Oxide Nanoparticles into Malignant Cells by an External Magnetic Field

    PubMed Central

    Prijic, Sara; Scancar, Janez; Romih, Rok; Cemazar, Maja; Bregar, Vladimir B.; Znidarsic, Andrej

    2010-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) are used as delivery systems for different therapeutics including nucleic acids for magnetofection-mediated gene therapy. The aim of our study was to evaluate physicochemical properties, biocompatibility, cellular uptake and trafficking pathways of the custom-synthesized SPIONs for their potential use in magnetofection. Custom-synthesized SPIONs were tested for size, shape, crystalline composition and magnetic behavior using a transmission electron microscope, X-ray diffractometer and magnetometer. SPIONs were dispersed in different aqueous media to obtain ferrofluids, which were tested for pH and stability using a pH meter and zetameter. Cytotoxicity was determined using the MTS and clonogenic assays. Cellular uptake and trafficking pathways were qualitatively evaluated by transmission electron microscopy and quantitatively by inductively coupled plasma atomic emission spectrometry. SPIONs were composed of an iron oxide core with a diameter of 8–9 nm, coated with a 2-nm-thick layer of silica. SPIONs, dispersed in 0.9% NaCl solution, resulted in a stable ferrofluid at physiological pH for several months. SPIONs were not cytotoxic in a broad range of concentrations and were readily internalized into different cells by endocytosis. Exposure to neodymium-iron-boron magnets significantly increased the cellular uptake of SPIONs, predominantly into malignant cells. The prepared SPIONs displayed adequate physicochemical and biomedical properties for potential use in magnetofection. Their cellular uptake was dependent on the cell type, and their accumulation within the cells was dependent on the duration of exposure to an external magnetic field. PMID:20602230

  10. Enhanced Cellular Uptake of Silica-Coated Magnetite Nanoparticles Compared with PEG-Coated Ones in Stem Cells.

    PubMed

    Lee, Dong Heon; Kang, Myunggoo; Lee, Hong Jai; Kim, Jeong Ah; Choi, Yun-Kyong; Cho, Hyunjin; Park, Jung-Keug; Park, Tai Hyun; Jung, Hyun

    2015-08-01

    Monodispersed magnetite (Fe3O4) nanoparticles (NPs) were prepared through the thermal decomposition method. The obtained NPs were surface modified with silica (SiO2) and polyethylene glycol (PEG), to enhance their stability in aqueous environment and their cellular uptake efficiency for biomedical applications. The NPs were characterized by X-ray diffraction (XRD), high-resolution transmission electron microscopy (HR-TEM), Fourier transform infrared (FT-IR) spectroscopy, and dynamic light scattering (DLS). The cytotoxicity of these NPs on bone marrow mesenchymal stem cells (BM-MSCs) was measured by MTT assay (cell viability test) at various concentrations (2, 5, 12.5, 25, and 50 µg/mL). The cells remained more than 90% viable at concentrations as high as 50 µg/mL. To compare the cellular uptake efficiency, these NPs were treated in BM-MSCs and the Fe concentration within the cells was measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES) analysis. The uptake process displayed a time- and dose-dependency. The uptake amount of SiO2-coated Fe3O4 (Fe3O4@SiO2) NPs was about 10 times higher than that of the PEG-coated ones (Fe3O4@PEG). PMID:26369110

  11. Taurine Boosts Cellular Uptake of Small d-Peptides for Enzyme-Instructed Intracellular Molecular Self-Assembly

    PubMed Central

    2016-01-01

    Due to their biostability, d-peptides are emerging as an important molecular platform for biomedical applications. Being proteolytically resistant, d-peptides lack interactions with endogenous transporters and hardly enter cells. Here we show that taurine, a natural amino acid, drastically boosts the cellular uptake of small d-peptides in mammalian cells by >10-fold, from 118 μM (without conjugating taurine) to >1.6 mM (after conjugating taurine). The uptake of a large amount of the ester conjugate of taurine and d-peptide allows intracellular esterase to trigger intracellular self-assembly of the d-peptide derivative, further enhancing their cellular accumulation. The study on the mechanism of the uptake reveals that the conjugates enter cells via both dynamin-dependent endocytosis and macropinocytosis, but likely not relying on taurine transporters. Differing fundamentally from the positively charged cell-penetrating peptides, the biocompatibility, stability, and simplicity of the enzyme-cleavable taurine motif promise new ways to promote the uptake of bioactive molecules for countering the action of efflux pump and contributing to intracellular molecular self-assembly. PMID:26235707

  12. Taurine Boosts Cellular Uptake of Small D-Peptides for Enzyme-Instructed Intracellular Molecular Self-Assembly.

    PubMed

    Zhou, Jie; Du, Xuewen; Li, Jie; Yamagata, Natsuko; Xu, Bing

    2015-08-19

    Due to their biostability, D-peptides are emerging as an important molecular platform for biomedical applications. Being proteolytically resistant, D-peptides lack interactions with endogenous transporters and hardly enter cells. Here we show that taurine, a natural amino acid, drastically boosts the cellular uptake of small D-peptides in mammalian cells by >10-fold, from 118 μM (without conjugating taurine) to >1.6 mM (after conjugating taurine). The uptake of a large amount of the ester conjugate of taurine and D-peptide allows intracellular esterase to trigger intracellular self-assembly of the D-peptide derivative, further enhancing their cellular accumulation. The study on the mechanism of the uptake reveals that the conjugates enter cells via both dynamin-dependent endocytosis and macropinocytosis, but likely not relying on taurine transporters. Differing fundamentally from the positively charged cell-penetrating peptides, the biocompatibility, stability, and simplicity of the enzyme-cleavable taurine motif promise new ways to promote the uptake of bioactive molecules for countering the action of efflux pump and contributing to intracellular molecular self-assembly.

  13. Taurine Boosts Cellular Uptake of Small D-Peptides for Enzyme-Instructed Intracellular Molecular Self-Assembly.

    PubMed

    Zhou, Jie; Du, Xuewen; Li, Jie; Yamagata, Natsuko; Xu, Bing

    2015-08-19

    Due to their biostability, D-peptides are emerging as an important molecular platform for biomedical applications. Being proteolytically resistant, D-peptides lack interactions with endogenous transporters and hardly enter cells. Here we show that taurine, a natural amino acid, drastically boosts the cellular uptake of small D-peptides in mammalian cells by >10-fold, from 118 μM (without conjugating taurine) to >1.6 mM (after conjugating taurine). The uptake of a large amount of the ester conjugate of taurine and D-peptide allows intracellular esterase to trigger intracellular self-assembly of the D-peptide derivative, further enhancing their cellular accumulation. The study on the mechanism of the uptake reveals that the conjugates enter cells via both dynamin-dependent endocytosis and macropinocytosis, but likely not relying on taurine transporters. Differing fundamentally from the positively charged cell-penetrating peptides, the biocompatibility, stability, and simplicity of the enzyme-cleavable taurine motif promise new ways to promote the uptake of bioactive molecules for countering the action of efflux pump and contributing to intracellular molecular self-assembly. PMID:26235707

  14. Improving solubility, stability, and cellular uptake of resveratrol by nanoencapsulation with chitosan and γ-poly (glutamic acid).

    PubMed

    Jeon, Young Ok; Lee, Ji-Soo; Lee, Hyeon Gyu

    2016-11-01

    Resveratrol (RES), a polyphenolic compound found in grape skins, is a potent antioxidant with broad health benefits. However, its utilization in food has been limited by its poor water solubility, instability, and low bioavailability. The purpose of this study is to improve the solubility, stability, and cellular uptake of RES by nanoencapsulation using chitosan (CS) and γ-poly (glutamic acid) (γ-PGA). The size of nanoparticles significantly decreases with a decrease in the CS/γ-PGA ratio (p<0.05). The nanoparticle size with CS/γ-PGA ratio of 5 was 100-150nm. The entrapment efficiency and UV-light protection effect significantly increases (p<0.05), with an increase in the CS and γ-PGA concentration. The solubility of RES increases 3.2 and 4.2 times before and after lyophilization by nanoencapsulation, respectively. Compared with non-nanoencapsulated RES, the nanoencapsulated RES tends to maintain its solubility and antioxidant activity during storage. CS/γ-PGA nanoencapsulation was able to significantly enhance the transport of RES across a Caco-2 cell monolayer (p<0.05). The highest cellular uptake was found for nanoparticles prepared with 0.5mg/mL CS and 0.1mg/mL γ-PGA, which showed the highest solubility and antioxidant activity during storage. Therefore, CS/γ-PGA nanoencapsulation is found to be a potentially valuable technique for improving the solubility, stability, and cellular uptake of RES. PMID:27518454

  15. Increasing cellular uptake of mesoporous silica nanoparticles in human embryonic kidney cell line 293T cells by using Lipofectamine 2000.

    PubMed

    Wang, Jiandong; Teng, Zhaogang; Tian, Ying; Fang, Tian; Ma, Jie; Sun, Jin; Zhu, Feipeng; Wu, Jinrong; Wang, Xin; Yang, Nannan; Zhou, Xiaojun; Yun, Shifeng; Lu, Guangming

    2013-11-01

    Mesoporous silica nanoparticles (MSNs) are ideal nanocarriers that have recently gained attention in important bioapplications such as drug, gene, and protein delivery. The efficacy of endocytosis greatly affects the biological functions of MSNs. In the present study, we investigated the effect of cationic liposomes of Lipofectamine 2000 on cellular uptake of MSNs and the cytotoxicity of cationic liposomes combining with MSNs both in vitro and in vivo. Therefore, mesoporous silica nanoparticles with an average diameter of 130 nm and negative surface charge were synthesized and characterized. The possible role of Lipofectamine 2000 in cellular uptake of MSNs was evaluated in human embryonic kidney cell line 293T cells by transmission electron microscopy (TEM) and with inductively coupled plasma (ICP) analysis. The toxicities of liposomes combining with MSNs were tested in vitro via cell apoptosis assay and MTT cell viability assay, and in vivo by histological examination of six organs of mice after intravenous injection. The endocytosis efficiency of MSNs in human embryonic kidney 293T cells was greatly increased using Lipofectamine 2000 compared with controls (P < 0.001). No apparent in vitro or in vivo cytotoxicity was found for Lipofectamine 2000 combining with MSNs. Our data indicate that cationic liposomes of Lipofectamine 2000 has the potential to greatly increase cellular uptake of MSNs with negative surface charge in human renal 293T cells without apparent toxicity. PMID:24059087

  16. Evaluation of Chitosan-Tripolyphosphate Nanoparticles as a p-shRNA Delivery Vector: Formulation, Optimization and Cellular Uptake Study

    PubMed Central

    Karimi, Mahdi; Avci, Pinar; Ahi, Mohsen; Gazori, Tarane; Hamblin, Michael R.; Naderi-Manesh, Hossein

    2015-01-01

    Polysaccharides (especially chitosan) have recently attracted much attention as gene therapy delivery vehicles for their unique properties such as biocompatibility, biodegradability, low toxicity, and controlled release. Nanoparticles have strong potential as a carrier of plasmid short hairpin RNA (p-shRNA). This study aimed to find the optimum conditions for obtaining Chitosan/triphosphate (TPP)/p-shRNA nanoparticles by the ionic gelation method, and investigating the cellular uptake of the optimized nanoparticles. After applying the central composite design of response surface methodology (RSM), the optimum conditions for preparation of nanoparticles with small size and high loading efficiency were: chitosan/TPP ratio = 10, pH = 5.5 and N/P ratio = 11. The resulting nanoparticles had an average size of 172.8 ± 7 nm and loading efficiency of 71.5 ± 5%. SEM images showed spherical and smooth nanoparticles. The nanoparticles complexed with p-shRNA and may protect it against nuclease digestion. Cytotoxicity studies with HeLa and PC3 human cancer cells demonstrated that chitosan/TPP nanoparticles had low toxicity. Cellular uptake studies using HeLa cells showed that the nanoparticles entered the cells (cellular uptake) and delivered DNA, probably due to their favorable Zeta potential (approximately +28 mV) and small size. PMID:26989641

  17. Cellular uptake of Nigella sativa oil-PLGA microparticle by PC-12 cell line.

    PubMed

    Doolaanea, Abd Almonem; Mansor, Nur 'Izzati; Mohd Nor, Nurul Hafizah; Mohamed, Farahidah

    2014-01-01

    The aim of this study is to investigate the cell uptake of Nigella sativa oil (NSO)-PLGA microparticle by neuron-like PC-12 cells in comparison to surfactants; hydrophilic (Tween 80 & Triton X100) and hydrophobic (Span 80). Solvent evaporation was used to precisely control the size, zeta potential and morphology of the particle. The results revealed varying efficiencies of the cell uptake by PC-12 cells, which may be partially attributed to the surface hydrophobicity of the microparticles. Interestingly, the uptake efficiency of PC-12 cells was higher with the more hydrophilic microparticle. NSO microparticle showed evidence of being preferably internalised by mitotic cells. Tween 80 microparticle showed the highest cell uptake efficiency with a concentration-dependent pattern suggesting its use as uptake enhancer for non-scavenging cells. In conclusion, PC-12 cells can take up NSO-PLGA microparticle which may have potential in the treatment of neurodegenerative disease. PMID:24697178

  18. 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.

  19. 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

  20. Synthesis, cellular uptake and structure-activity relationships for potent cytotoxic trichloridoiridium(III) polypyridyl complexes.

    PubMed

    Scharwitz, Michael A; Ott, Ingo; Gust, Ronald; Kromm, Anna; Sheldrick, William S

    2008-08-01

    The complexes fac-[IrCl(3)(DMSO)(pp)] 1a-5a may be prepared by stepwise reaction of IrCl(3) x 3H(2)O with the appropriate polypyridyl ligand (pp=bpy, phen, dpq, dppz, dppn) and DMSO in CH(3)OH solution in the dark. The fac isomers of 1a-5a are stable in light-protected CD(2)Cl(2) solution but, with the exception of 5a, isomerize rapidly to a mixture of the fac and mer isomers in the presence of light. In contrast, solutions of the fac isomers in the polar solvents D(2)O and CD(3)OD are stable under such conditions. The isomer mer-[IrCl(3)(DMSO-kappa S)(phen)] 2b was, however, isolated by slow evaporation of an H(2)O/CH(3)OH solution of 2a and characterized by X-ray structural analysis. UV/Vis and CD studies of the interaction of 1a-5a with calf thymus DNA are in accordance with an effective absence of intercalation. (1)H NMR studies indicate that the complexes react slowly with compounds containing soft S donor atoms (e. g. N-acetylmethionine) but do not react with the guanine base of 5'-GMP(2-). The complexes 2a-5a are potent in vitro cytotoxic agents toward the human cell lines MCF-7 and HT-29 and their IC(50) values are dependent on the size of the polypyridyl ligand in the order phen, dpq>dppz>dppn. For instance IC(50) values of 5.5 (0.9), 0.8 (0.3) and 0.21 (0.11)microM were established for 3a-5a against MCF-7 cells and 6.1 (0.7), 1.5 (0.2) and 1.3 (0.4)microM against HT-29 cells. These values correlate with the cellular uptake efficiency which, on exposure to 10 microM solutions, reaches its highest levels (19.3(0.8) and 37.4(8.9) ng Ir/mg protein for MCF-7 and HT-29, respectively) for the dppn compound 5a. PMID:18472166

  1. 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

  2. The inactive-active phase transition in the noisy additive (exclusive-or) probabilistic cellular automaton

    NASA Astrophysics Data System (ADS)

    Mendonça, J. Ricardo G.

    2016-07-01

    We investigate the inactive-active phase transition in an array of additive (exclusive-or) cellular automata (CA) under noise. The model is closely related with the Domany-Kinzel (DK) probabilistic cellular automaton (PCA), for which there are rigorous as well as numerical estimates on the transition probabilities. Here, we characterize the critical behavior of the noisy additive cellular automaton by mean field analysis and finite-size scaling and show that its phase transition belongs to the directed percolation universality class of critical behavior. As a by-product of our analysis, we argue that the critical behavior of the noisy elementary CA 90 and 102 (in Wolfram’s enumeration scheme) must be the same. We also perform an empirical investigation of the mean field equations to assess their quality and find that away from the critical point (but not necessarily very far away) the mean field approximations provide a reasonably good description of the dynamics of the PCA.

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

    PubMed

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

    2015-12-18

    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. PMID:26584818

  4. 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.

  5. 47 CFR 1.20007 - Additional assistance capability requirements for wireline, cellular, and PCS telecommunications...

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 1 2014-10-01 2014-10-01 false Additional assistance capability requirements for wireline, cellular, and PCS telecommunications carriers. 1.20007 Section 1.20007 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE Grants by Random Selection Communications Assistance for Law Enforcement Act...

  6. 47 CFR 1.20007 - Additional assistance capability requirements for wireline, cellular, and PCS telecommunications...

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 1 2013-10-01 2013-10-01 false Additional assistance capability requirements for wireline, cellular, and PCS telecommunications carriers. 1.20007 Section 1.20007 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE Grants by Random Selection Communications Assistance for Law Enforcement Act...

  7. Comparison of In vitro Nanoparticles Uptake in Various Cell Lines and In vivo Pulmonary Cellular Transport in Intratracheally Dosed Rat Model

    NASA Astrophysics Data System (ADS)

    Lai, Yurong; Chiang, Po-Chang; Blom, Jason D.; Li, Na; Shevlin, Kimberly; Brayman, Timothy G.; Hu, Yiding; Selbo, Jon G.; Hu, Liangbiao George

    2008-09-01

    In present study, the potential drug delivery of nanoformulations was validated via the comparison of cellular uptake of nanoparticles in various cell lines and in vivo pulmonary cellular uptake in intratracheally (IT) dosed rat model. Nanoparticles were prepared by a bench scale wet milling device and incubated with a series of cell lines, including Caco-2, RAW, MDCK and MDCK transfected MDR1 cells. IT dosed rats were examined for the pulmonary cellular uptake of nanoparticles. The processes of nanoparticle preparation did not alter the crystalline state of the material. The uptake of nanoparticles was observed most extensively in RAW cells and the least in Caco-2 cells. Efflux transporter P-gp did not prevent cell from nanoparticles uptake. The cellular uptake of nanoparticles was also confirmed in bronchoalveolar lavage (BAL) fluid cells and in bronchiolar epithelial cells, type II alveolar epithelial cells in the intratracheally administrated rats. The nanoparticles uptake in MDCK, RAW cells and in vivo lung epithelial cells indicated the potential applications of nanoformulation for poorly soluble compounds. The observed limited direct uptake of nanoparticles in Caco-2 cells suggests that the improvement in oral bioavailability by particle size reduction is via increased dissolution rate rather than direct uptake.

  8. Adaptation of the pore diffusion model to describe multi-addition batch uptake high-throughput screening experiments.

    PubMed

    Traylor, Steven J; Xu, Xuankuo; Li, Yi; Jin, Mi; Li, Zheng Jian

    2014-11-14

    Equilibrium isotherm and kinetic mass transfer measurements are critical to mechanistic modeling of binding and elution behavior within a chromatographic column. However, traditional methods of measuring these parameters are impractically time- and labor-intensive. While advances in high-throughput robotic liquid handling systems have created time and labor-saving methods of performing kinetic and equilibrium measurements of proteins on chromatographic resins in a 96-well plate format, these techniques continue to be limited by physical constraints on protein addition, incubation and separation times; the available concentration of protein stocks and process pools; and practical constraints on resin and fluid volumes in the 96-well format. In this study, a novel technique for measuring protein uptake kinetics (multi-addition batch uptake) has been developed to address some of these limitations during high-throughput batch uptake kinetic measurements. This technique uses sequential additions of protein stock to chromatographic resin in a 96-well plate and the subsequent removal of each addition by centrifugation or vacuum separation. The pore diffusion model was adapted here to model multi-addition batch uptake and was tested and compared with traditional batch uptake measurements of uptake of an Fc-fusion protein on an anion exchange resin. Acceptable agreement between the two techniques is achieved for the two solution conditions investigated here. In addition, a sensitivity analysis of the model to the physical inputs is presented and the advantages and limitations of the multi-addition batch uptake technique are explored.

  9. 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. PMID:26481477

  10. Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.

    PubMed

    Rezgui, Rachid; Blumer, Katy; Yeoh-Tan, Gilbert; Trexler, Adam J; Magzoub, Mazin

    2016-07-01

    Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane. PMID:27033412

  11. Selenium addition alters mercury uptake, bioavailability in the rhizosphere and root anatomy of rice (Oryza sativa)

    PubMed Central

    Wang, Xun; Tam, Nora Fung-Yee; Fu, Shi; Ametkhan, Aray; Ouyang, Yun; Ye, Zhihong

    2014-01-01

    Background and Aims Mercury (Hg) is an extremely toxic pollutant, especially in the form of methylmercury (MeHg), whereas selenium (Se) is an essential trace element in the human diet. This study aimed to ascertain whether addition of Se can produce rice with enriched Se and lowered Hg content when growing in Hg-contaminated paddy fields and, if so, to determine the possible mechanisms behind these effects. Methods Two cultivars of rice (Oryza sativa, japonica and indica) were grown in either hydroponic solutions or soil rhizobags with different Se and Hg treatments. Concentrations of total Hg, MeHg and Se were determined in the roots, shoots and brown rice, together with Hg uptake kinetics and Hg bioavailability in the soil. Root anatonmy was also studied. Key Results The high Se treatment (5 μg g–1) significantly increased brown rice yield by 48 % and total Se content by 2·8-fold, and decreased total Hg and MeHg by 47 and 55 %, respectively, compared with the control treatments. The high Se treatment also markedly reduced ‘water-soluble’ Hg and MeHg concentrations in the rhizosphere soil, decreased the uptake capacity of Hg by roots and enhanced the development of apoplastic barriers in the root endodermis. Conclusions Addition of Se to Hg-contaminated soil can help produce brown rice that is simultaneously enriched in Se and contains less total Hg and MeHg. The lowered accumulation of total Hg and MeHg appears to be the result of reduced bioavailability of Hg and production of MeHg in the rhizosphere, suppression of uptake of Hg into the root cells and an enhancement of the development of apoplastic barriers in the endodermis of the roots. PMID:24948669

  12. 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.

  13. Cellular uptake of lead in the blood-cerebrospinal fluid barrier: Novel roles of Connexin 43 hemichannel and its down-regulations via Erk phosphorylation.

    PubMed

    Song, Han; Zheng, Gang; Liu, Yang; Shen, Xue-Feng; Zhao, Zai-Hua; Aschner, Michael; Luo, Wen-Jing; Chen, Jing-Yuan

    2016-04-15

    As the structural basis of blood-cerebrospinal fluid barrier (BCB), epithelial cells in the choroid plexus (CP) are targets for lead (Pb). Pb is known to accumulate in the CP; however, the mechanism of Pb uptake in the choroidal epithelial cells remains unknown. Recently, hemichannels of Connexin 43 (Cx43), the most ubiquitously expressed gap junction proteins in the CP, were found to be important pathways for many substances. This study was designed to investigate the roles of Cx43 in Pb uptake in the epithelial cells. Autometallography was used to outline Pb's subcellular location, and the characteristics of Pb transport into CP cells, including concentration- and time-dependence were analyzed by atomic absorption spectroscopy. Knockdown/overexpression of Cx43 with transient siRNA/plasmids transfections before Pb exposure diminished/increased the Pb accumulation. In the Z310 cell-based doxycycline-inducible Cx43 expression cell line (iZCx43), doxycycline induced a significant increase (3-fold) in Pb uptake, corresponding to the increased Cx43 levels. Activation of Cx43 hemichannels by reduced serum conditions caused an increase of Pb concentrations. Cx43-induced Pb uptake was attenuated after blockage of Cx43 hemichannels with its inhibitor, carbenoxolone. Additionally, down-regulation of Cx43 protein levels by Pb exposure paralleled cellular Pb concentrations in the time study. Concomitantly, expressions of phosphor-Src and phosphor-Erk were both significantly increased by Pb. However, inactivation of Erk, not Src pathway, reversed Pb-induced downregulation of Cx43. Taken together, these data establish that Pb can accumulate in the BCB and validate the role of Cx43 hemichannel in Pb uptake and its regulations through Erk phosphorylation.

  14. Improvement of Cellular Uptake and Transfection Ability of pDNA Using α-Cyclodextrin-Polyamidoamine Conjugates as Gene Delivery System.

    PubMed

    Qin, Linghao; Cao, Duanwen; Huang, Huan; Ji, Gangjian; Feng, Min; Chen, Jianhai; Pan, Shirong

    2016-02-01

    Polyamidoamine (PAMAM) dendrimers are a class of unique nanomaterials which attracted attention because of their extraordinary properties, such as highly branched structure and types of terminal primary groups. In addition, development in PAMAM chemical modification has broadened its biological application especially for drug and gene delivery. In this study, PAMAMs are covalently conjugated onto α-Cyclodextrin (α-CD) via amide bonds obtaining the starburst cationic polymers (CD-PG2). The chemical structure and composition of CD-PG2 was characterized by IH NMR. Physicochemical and biological properties of CD-PG2/pDNA polyplex were evaluated by agarose gel retardation, stability test against DNasecñ, MTT assay, DLS measurement, CLSM observation, LDH leakage test, cellular uptake route analysis and in-vitro cell transfection. Results showed that CD-PG2 can efficiently condense pDNA into nanoscale particles with a narrow size distribution, and protect pDNA form DNase I degradation. Compared with free PEI-25K and commercial product Lipofectamine2000, CD-PG2 shows excellent gene transfection efficiency without serum interference as well as relatively low cytotoxicity. Cellular uptake of CD-PG2/pDNA polyplex is mainly through CME and CvME route and further investigations demonstrate that α-CD can regulate CvME pathway to improve polyplex transfection behavior. In conclusion, CD-PG2 can be considered as a versatile tool for gene delivery, especially for gene transfer in-vivo. PMID:27305760

  15. 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.

  16. Evaluation of in-vitro cytotoxicity and cellular uptake efficiency of zidovudine-loaded solid lipid nanoparticles modified with Aloe Vera in glioma cells.

    PubMed

    K S, Joshy; Sharma, Chandra P; Kalarikkal, Nandakumar; Sandeep, K; Thomas, Sabu; Pothen, Laly A

    2016-09-01

    Zidovudine loaded solid lipid nanoparticles of stearic acid modified with Aloe Vera (AV) have been prepared via simple emulsion solvent evaporation method which showed excellent stability at room temperature and refrigerated condition. The nanoparticles were examined by Fourier transform infrared spectroscopy (FT-IR), which revealed the overlap of the AV absorption peak with the absorption peak of modified stearic acid nanoparticles. The inclusion of AV to stearic acid decreased the crystallinity and improved the hydrophilicity of lipid nanoparticles and thereby improved the drug loading efficacy of lipid nanoparticles. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) imaging revealed that, the average particle size of unmodified (bare) nanoparticles was 45.66±12.22nm and modified solid lipid nanoparticles showed an average size of 265.61±80.44nm. Solid lipid nanoparticles with well-defined morphology were tested in vitro for their possible application in drug delivery. Cell culture studies using C6 glioma cells on the nanoparticles showed enhanced growth and proliferation of cells without exhibiting any toxicity. In addition, normal cell morphology and improved uptake were observed by fluorescence microscopy images of rhodamine labeled modified solid lipid nanoparticles compared with unmodified nanoparticles. The cellular uptake study suggested that these nanoparticles could be a promising drug delivery system to enhance the uptake of antiviral drug by brain cells and it could be a suitable drug carrier system for the treatment of HIV. PMID:27207037

  17. Positive newborn screen for methylmalonic aciduria identifies the first mutation in TCblR/CD320, the gene for cellular uptake of transcobalamin-bound vitamin B(12).

    PubMed

    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

    2010-08-01

    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.

  18. Vitamin E TPGS modified liposomes enhance cellular uptake and targeted delivery of luteolin: An in vivo/in vitro evaluation.

    PubMed

    Li, Jinli; Cheng, Xudong; Chen, Yan; He, Weiming; Ni, Li; Xiong, Peihua; Wei, Minggang

    2016-10-15

    Luteolin (LUT) is an active agent in cancer prevention and a potential candidate for clinical chemotherapy. However, poor water solubility and extensive excretion have limited the clinical use of luteolin. Herein, we attempted to develop vitamin E d-α-tocopherol acid polyethylene glycol 1000 succinate (TPGS) coated liposomes to improve the accumulation of luteolin in lung cancer. Luteolin or coumarin-6 loaded liposomes were prepared using film-dispersion methods and characterized according to their particle size, polydispersity, zeta potential, and drug encapsulation efficiency. Cellular uptake properties and cytotoxicities of luteolin or coumarin-6 loaded liposomes were determined in A549 cells. Optical in vivo imaging was employed as a method of assessing tumor targeting. The antitumor effect was evaluated in mice xenotransplanted with A549 cancer cells. The results showed that TPGS coated liposomes are spherical, and are ∼176.2nm in size. TPGS coated liposomes enhanced cellular uptake and apoptosis by upregulating the Bax/Bcl-2 ratio, resulting in improved cytotoxicity in A549 cells. TPGS coated liposomes significantly accumulated in tumor tissue and enhanced tumor inhibition without altering the structures of other organs. Thereby, TPGS coated liposomes provide an effective strategy in cancer chemotherapy for model drugs with poor water solubility such as luteolin. PMID:27545748

  19. In vitro control release, cytotoxicity assessment and cellular uptake of methotrexate loaded liquid-crystalline folate nanocarrier.

    PubMed

    Misra, Rahul; Upadhyay, Mohita; Perumal, Vivekanandan; Mohanty, Sanat

    2015-02-01

    Folate molecules self-assemble in the form of stacks to form liquid-crystalline solutions. Nanocarriers from self-assembled folates are composed of highly ordered structures, which offer high encapsulation of drug (95-98%), controlled drug release rates, active cellular uptake and biocompatibility. Recently, we have shown that the release rates of methotrexate can be controlled by varying the size of nanoparticles, cross-linking cation and cross-linking concentration. The present study reports the in vitro cytotoxic behavior of methotrexate loaded liquid-crystalline folate nanoparticles on cultured HeLa cells. Changing drug release rates can influence cytotoxicity of cancer cells. Therefore, to study the correlation of release rate and cytotoxic behavior, the effect of release controlling parameters on HeLa cells was studied through MTT assay. It is reported that by controlling the methotrexate release, the survival rates of HeLa cells can be controlled. Released methotrexate kills HeLa cells as effectively as free methotrexate solution. The co-culture based in vitro cellular uptake study through fluorescence microscopy on folate receptor positive and negative cancer cells shows that the present nanocarrier has the potential to distinguish cancer cells from normal cells. Overall, the present study reports the in vitro performance of self-assembled liquid-crystalline folate nanoparticles, which will be a platform for further in vivo studies and clinical trials. PMID:25661345

  20. Tuning the Surface of Nanoparticles: Impact of Poly(2-ethyl-2-oxazoline) on Protein Adsorption in Serum and Cellular Uptake.

    PubMed

    Koshkina, Olga; Westmeier, Dana; Lang, Thomas; Bantz, Christoph; Hahlbrock, Angelina; Würth, Christian; Resch-Genger, Ute; Braun, Ulrike; Thiermann, Raphael; Weise, Christoph; Eravci, Murat; Mohr, Benjamin; Schlaad, Helmut; Stauber, Roland H; Docter, Dominic; Bertin, Annabelle; Maskos, Michael

    2016-09-01

    Due to the adsorption of biomolecules, the control of the biodistribution of nanoparticles is still one of the major challenges of nanomedicine. Poly(2-ethyl-2-oxazoline) (PEtOx) for surface modification of nanoparticles is applied and both protein adsorption and cellular uptake of PEtOxylated nanoparticles versus nanoparticles coated with poly(ethylene glycol) (PEG) and non-coated positively and negatively charged nanoparticles are compared. Therefore, fluorescent poly(organosiloxane) nanoparticles of 15 nm radius are synthesized, which are used as a scaffold for surface modification in a grafting onto approach. With multi-angle dynamic light scattering, asymmetrical flow field-flow fractionation, gel electrophoresis, and liquid chromatography-mass spectrometry, it is demonstrated that protein adsorption on PEtOxylated nanoparticles is extremely low, similar as on PEGylated nanoparticles. Moreover, quantitative microscopy reveals that PEtOxylation significantly reduces the non-specific cellular uptake, particularly by macrophage-like cells. Collectively, studies demonstrate that PEtOx is a very effective alternative to PEG for stealth modification of the surface of nanoparticles.

  1. 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

  2. Effect of chirality on cellular uptake, imaging and photodynamic therapy of photosensitizers derived from chlorophyll-a.

    PubMed

    Srivatsan, Avinash; Pera, Paula; Joshi, Penny; Wang, Yanfang; Missert, Joseph R; Tracy, Erin C; Tabaczynski, Walter A; Yao, Rutao; Sajjad, Munawwar; Baumann, Heinz; Pandey, Ravindra K

    2015-07-01

    We have previously shown that the (124)I-analog of methyl 3-(1'-m-iodobenzyloxy) ethyl-3-devinyl-pyropheophorbide-a derived as racemic mixture from chlorophyll-a can be used for PET (positron emission tomography)-imaging in animal tumor models. On the other hand, as a non-radioactive analog, it showed excellent fluorescence and photodynamic therapy (PDT) efficacy. Thus, a single agent in a mixture of radioactive ((124)I-) and non-radioactive ((127)I) material can be used for both dual-imaging and PDT of cancer. Before advancing to Phase I human clinical trials, we evaluated the activity of the individual isomers as well as the impact of a chiral center at position-3(1) in directing in vitro/in vivo cellular uptake, intracellular localization, epithelial tumor cell-specific retention, fluorescence/PET imaging, and photosensitizing ability. The results indicate that both isomers (racemates), either as methyl ester or carboxylic acid, were equally effective. However, the methyl ester analogs, due to subcellular deposition into vesicular structures, were preferentially retained. All derivatives containing carboxylic acid at the position-17(2) were noted to be substrate for the ABCG2 (a member of the ATP binding cassette transporters) protein explaining their low retention in lung tumor cells expressing this transporter. The compounds in which the chirality at position-3 has been substituted by a non-chiral functionality showed reduced cellular uptake, retention and lower PDT efficacy in mice bearing murine Colon26 tumors.

  3. Managing magnetic nanoparticle aggregation and cellular uptake: a precondition for efficient stem-cell differentiation and MRI tracking.

    PubMed

    Fayol, Delphine; Luciani, Nathalie; Lartigue, Lenaic; Gazeau, Florence; Wilhelm, Claire

    2013-02-01

    The labeling of stem cells with iron oxide nanoparticles is increasingly used to enable MRI cell tracking and magnetic cell manipulation, stimulating the fields of tissue engineering and cell therapy. However, the impact of magnetic labeling on stem-cell differentiation is still controversial. One compromising factor for successful differentiation may arise from early interactions of nanoparticles with cells during the labeling procedure. It is hypothesized that the lack of control over nanoparticle colloidal stability in biological media may lead to undesirable nanoparticle localization, overestimation of cellular uptake, misleading MRI cell tracking, and further impairment of differentiation. Herein a method is described for labeling mesenchymal stem cells (MSC), in which the physical state of citrate-coated nanoparticles (dispersed versus aggregated) can be kinetically tuned through electrostatic and magnetic triggers, as monitored by diffusion light scattering in the extracellular medium and by optical and electronic microscopy in cells. A set of statistical cell-by-cell measurements (flow cytometry, single-cell magnetophoresis, and high-resolution MRI cellular detection) is used to independently quantify the nanoparticle cell uptake and the effects of nanoparticle aggregation. Such aggregation confounds MRI cell detection as well as global iron quantification and has adverse effects on chondrogenetic differentiation. Magnetic labeling conditions with perfectly stable nanoparticles-suitable for obtaining differentiation-capable magnetic stem cells for use in cell therapy-are subsequently identified. PMID:23184893

  4. Tuning the Surface of Nanoparticles: Impact of Poly(2-ethyl-2-oxazoline) on Protein Adsorption in Serum and Cellular Uptake.

    PubMed

    Koshkina, Olga; Westmeier, Dana; Lang, Thomas; Bantz, Christoph; Hahlbrock, Angelina; Würth, Christian; Resch-Genger, Ute; Braun, Ulrike; Thiermann, Raphael; Weise, Christoph; Eravci, Murat; Mohr, Benjamin; Schlaad, Helmut; Stauber, Roland H; Docter, Dominic; Bertin, Annabelle; Maskos, Michael

    2016-09-01

    Due to the adsorption of biomolecules, the control of the biodistribution of nanoparticles is still one of the major challenges of nanomedicine. Poly(2-ethyl-2-oxazoline) (PEtOx) for surface modification of nanoparticles is applied and both protein adsorption and cellular uptake of PEtOxylated nanoparticles versus nanoparticles coated with poly(ethylene glycol) (PEG) and non-coated positively and negatively charged nanoparticles are compared. Therefore, fluorescent poly(organosiloxane) nanoparticles of 15 nm radius are synthesized, which are used as a scaffold for surface modification in a grafting onto approach. With multi-angle dynamic light scattering, asymmetrical flow field-flow fractionation, gel electrophoresis, and liquid chromatography-mass spectrometry, it is demonstrated that protein adsorption on PEtOxylated nanoparticles is extremely low, similar as on PEGylated nanoparticles. Moreover, quantitative microscopy reveals that PEtOxylation significantly reduces the non-specific cellular uptake, particularly by macrophage-like cells. Collectively, studies demonstrate that PEtOx is a very effective alternative to PEG for stealth modification of the surface of nanoparticles. PMID:27281039

  5. In vitro control release, cytotoxicity assessment and cellular uptake of methotrexate loaded liquid-crystalline folate nanocarrier.

    PubMed

    Misra, Rahul; Upadhyay, Mohita; Perumal, Vivekanandan; Mohanty, Sanat

    2015-02-01

    Folate molecules self-assemble in the form of stacks to form liquid-crystalline solutions. Nanocarriers from self-assembled folates are composed of highly ordered structures, which offer high encapsulation of drug (95-98%), controlled drug release rates, active cellular uptake and biocompatibility. Recently, we have shown that the release rates of methotrexate can be controlled by varying the size of nanoparticles, cross-linking cation and cross-linking concentration. The present study reports the in vitro cytotoxic behavior of methotrexate loaded liquid-crystalline folate nanoparticles on cultured HeLa cells. Changing drug release rates can influence cytotoxicity of cancer cells. Therefore, to study the correlation of release rate and cytotoxic behavior, the effect of release controlling parameters on HeLa cells was studied through MTT assay. It is reported that by controlling the methotrexate release, the survival rates of HeLa cells can be controlled. Released methotrexate kills HeLa cells as effectively as free methotrexate solution. The co-culture based in vitro cellular uptake study through fluorescence microscopy on folate receptor positive and negative cancer cells shows that the present nanocarrier has the potential to distinguish cancer cells from normal cells. Overall, the present study reports the in vitro performance of self-assembled liquid-crystalline folate nanoparticles, which will be a platform for further in vivo studies and clinical trials.

  6. Effects of nanoparticle size on cellular uptake and liver MRI with polyvinylpyrrolidone-coated iron oxide nanoparticles.

    PubMed

    Huang, Jing; Bu, Lihong; Xie, Jin; Chen, Kai; Cheng, Zhen; Li, Xingguo; Chen, Xiaoyuan

    2010-12-28

    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 T(2) 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.

  7. Evidence for Enhanced Cellular Uptake and Binding of Thyroxine In Vivo during Acute Infection with Diplococcus pneumoniae

    PubMed Central

    DeRubertis, Frederick R.; Woeber, Kenneth A.

    1972-01-01

    Previous work has demonstrated that acute pneumococcal infections in man and in the rhesus monkey are accompanied by accelerated metabolic disposal of L-thyroxine (T4). In order to study the influence of acute pneumococcal infection on the kinetics of hormone distribution, the early cellular uptake of T4 (CT4), reflecting the net effect of plasma and cellular binding factors, was assessed in rhesus monkeys from the differences in instantaneous distribution volumes of T4-131I and albumin-125I during the first 60 min after their simultaneous injection. Hepatic and renal uptakes of 131I were also determined. Plasma binding of T4 was assessed by measuring the per cent of free T4 (% FT4) in serum. Six monkeys were studied 12 hr (INF-12) and seven 24 hr (INF-24) after intravenous inoculation with Diplococcus pneumoniae; seven controls were inoculated with a heat-killed culture. CT4 at 60 min as per cent administered dose was 31.5 ±2.0 (mean ±SE) in INF-12 and 33.0±0.8 in INF-24, values significantly greater than control (22.4±1.3). By contrast, mean% FT4 was identical in control and INF-12 (0.028 ±0.002 and 0.028 ±0.001) and variably increased in INF-24 (0.034 ±0.003). Thus, in the infected monkeys CT4 and% FT4 were not significantly correlated. The increased CT4 in the infected monkeys could not be ascribed to an increase in vascular permeability and did not correlate with the magnitude of fever. Although the increased CT4 could not be accounted for by increased hepatic or renal uptake of hormone, hepatic and renal T4 spaces were increased, results consistent with increased binding by these tissues. Our data indicate that the cellular uptake of T4 is increased early in acute pneumococcal infection and suggest that this results from a primary enhancement of cell-associated binding factors for T4. PMID:5014612

  8. Stimulation of polyunsaturated fatty acid oxidation in myocytes by regulating its cellular uptake

    SciTech Connect

    Abdel-aleem, S.; Frangakis, C. ); Badr, M. )

    1991-01-01

    In order to investigate the regulation of polyunsaturated fatty acid oxidation in the heart, the effect of the phosphodiesterase inhibitor enoximone on the oxidation of (1-{sup 14}C) arachidonic acid, and (1-{sup 14}C) arachidonyl-CoA, were studied in adult rat myocytes, and isolated rat heart mitochondria. Enoximone stimulated arachidonate oxidation by 94%, at a concentration of 0.25 mM. The apparent Vmax value of archidonate oxidation in the presence of enoximone was approximately 75% higher than the value observed with the control in isolated myocytes. Also, enoximone stimulated arachidonate uptake by 27% at a concentration of 0.25 mM. On the other hand, enoximone had no effect on the oxidation of (1-{sup 14}C) arachidonyl-CoA in isolated rat heart mitochondria. These results suggest that the oxidation of polyunsaturated fatty acids in myocytes is regulated by the rate of uptake of these acids across sarcolemmal membranes.

  9. The cellular uptake mechanism, intracellular transportation, and exocytosis of polyamidoamine dendrimers in multidrug-resistant breast cancer cells

    PubMed Central

    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. PMID:27536106

  10. 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. PMID:27536106

  11. Cellular uptake of lipoproteins and persistent organic compounds-An update and new data

    SciTech Connect

    Hjelmborg, Philip Sebastian; Andreassen, Thomas Kjaergaard; Bonefeld-Jorgensen, Eva Cecilie

    2008-10-15

    There are a number of interactions related to the transport of lipophilic xenobiotic compounds in the blood stream of mammals. This paper will focus on the interactions between lipoproteins and persistent organic pollutants (POPs) and how these particles are taken up by cells. A number of POPs including the pesticide p,p'-dichlorodiphenyltrichloroethane (DDT), and especially its metabolite p,p'-dichlorodiphenyldichloroethene (DDE), interacts with nuclear hormone receptors causing these to malfunction, which in turn results in a range of deleterious health effects in humans. The aim of the present study was to determine the role of lipoprotein receptors in mouse embryonic fibroblast (MEF) cells in conjunction with uptake of DDT-lipoprotein complexes from supplemented media in vitro. Uptake of DDT by MEF cells was investigated using MEF1 cells carrying the receptors low-density lipoprotein receptor-related protein (LRP) and low-density lipoprotein receptor (LDLR) present and MEF4 cells with no LRP and LDLR expression. Cells were incubated together with the complex of low-density lipoproteins (LDL) and [{sup 14}C]DDT. The receptor function was further evaluated by adding the 40 kDa receptor-associated protein (RAP) which blocks receptor activity. The results showed that [{sup 14}C]DDT uptake was decreasing when the LDL concentration was increasing. There was no strong evidence for a receptor-mediated uptake of the [{sup 14}C]DDT-lipoprotein complex. To conclude, DDT travels in the blood stream and can cross cell membranes while being transported as a DDT-lipoprotein complex. The lipoproteins do not need receptors to cross cell membranes since passive diffusion constitutes a major passageway.

  12. Yersinia enterocolitica inhibits Salmonella enterica serovar Typhimurium and Listeria monocytogenes cellular uptake.

    PubMed

    Habyarimana, Fabien; Swearingen, Matthew C; Young, Glenn M; Seveau, Stephanie; Ahmer, Brian M M

    2014-01-01

    Yersinia enterocolitica biovar 1B employs two type three secretion systems (T3SS), Ysa and Ysc, which inject effector proteins into macrophages to prevent phagocytosis. Conversely, Salmonella enterica serovar Typhimurium uses a T3SS encoded by Salmonella pathogenicity island 1 (SPI1) to actively invade cells that are normally nonphagocytic and a second T3SS encoded by SPI2 to survive within macrophages. Given the distinctly different outcomes that occur with regard to host cell uptake of S. Typhimurium and Y. enterocolitica, we investigated how each pathogen influences the internalization outcome of the other. Y. enterocolitica reduces S. Typhimurium invasion of HeLa and Caco-2 cells to a level similar to that observed using an S. Typhimurium SPI1 mutant alone. However, Y. enterocolitica had no effect on S. Typhimurium uptake by J774.1 or RAW264.7 macrophage-like cells. Y. enterocolitica was also able to inhibit the invasion of epithelial and macrophage-like cells by Listeria monocytogenes. Y. enterocolitica mutants lacking either the Ysa or Ysc T3SS were partially defective, while double mutants were completely defective, in blocking S. Typhimurium uptake by epithelial cells. S. Typhimurium encodes a LuxR homolog, SdiA, which detects N-acylhomoserine lactones (AHLs) produced by Y. enterocolitica and upregulates the expression of an invasin (Rck) and a putative T3SS effector (SrgE). Two different methods of constitutively activating the S. Typhimurium SdiA regulon failed to reverse the uptake blockade imposed by Y. enterocolitica.

  13. Sulfur and nitrogen uptake by loblolly pine seedlings as influenced by nitrogen and sulfur addition

    SciTech Connect

    Kelly, J.M.; Johnson, D.W.

    1982-12-01

    The influence of increasing levels of nitrogen addition at several levels of sulfur input on nitrogen and sulfur uptake by loblolly pine seedlings was evaluated in a greenhouse study. All possible combinations on nitrogen and sulfur were incorporated into soil collected from the A horizon of a southeastern forest soil at rates of 0, 200, 500, and 1,000 ..mu..g/g of N, and 0, 14, 35, and 70 ..mu..g/g of S. Soil samples collected at the end of the study indicated that a similar amount of soil SO/sub 4/-S had been mineralized in all treatment combinations, compared to a general pattern of increasing soil nitrogen mineralization with increasing nitrogen input. Most mineralized sulfate appeared to come from nonprotein organic compounds as there was not a significant concomitant release of nitrogen. Both shoot and root biomass responded significantly to nitrogen addition, but there was no sulfur or nitrogen-sulfur interaction response. Nitrogen treatment generally increased shoot nitrogen concentration compared to a general decrease in shoot total-, sulfate-, and organically bound-sulfur. Organically bound-sulfur concentrations were 26 to 60 percent below sulfur values calculated from an S/N ratio of 0.03 on a gram atom basis. The results show that increased nitrogen addition affected both growth and sulfur status of loblolly pine seedlings, but not entirely in the manner predicted by theoretical considerations.

  14. Sulfur and nitrogen uptake by loblolly pine seedlings as influenced by nitrogen and sulfur addition

    SciTech Connect

    Kelly, J.M.; Johnson, D.W.

    1982-12-01

    The influence of increasing levels of nitrogen addition at several levels of sulfur input on nitrogen and sulfur uptake by loblolly pine seedlings was evaluated in a greenhouse study. All possible combinations of nitrogen and sulfur were incorporated into soil collected from the A horizon of a southeastern forest soil at rates of 0, 200, 500, and 1,000 ..mu..g/g of N, and 0, 14, 35, and 70 ..mu..g/g of S. Soil samples collected at the end of the study indicated that a similar amount of soil SO/sub 4/-S had been mineralized in all treatment combinations, compared to a general pattern of increasing soil nitrogen mineralization with increasing nitrogen input. Most mineralized sulfate appeared to come from nonprotein organic compounds as there was not a significant concomitant release of nitrogen. Both shoot and root biomass responded significantly to nitrogen addition, but there was no sulfur or nitrogen-sulfur interaction response. Nitrogen treatment generally increased shoot nitrogen concentration compared to a general decrease in shoot total-, sulfate-, and organically bound-sulfur. Organically bound-sulfur concentrations were 26 to 60 percent below sulfur values calculated from an S/N ratio of 0.03 on a gram atom basis. The results show that increased nitrogen addition affected both growth and sulfur status of loblolly pine seedlings, but not entirely in the manner predicted by theoretical considerations.

  15. PDR-type ABC transporter mediates cellular uptake of the phytohormone abscisic acid

    PubMed Central

    Kang, Joohyun; Hwang, Jae-Ung; Kim, Yu-Young; Assmann, Sarah M.; Martinoia, Enrico; Lee, Youngsook

    2010-01-01

    Abscisic acid (ABA) is a ubiquitous phytohormone involved in many developmental processes and stress responses of plants. ABA moves within the plant, and intracellular receptors for ABA have been recently identified; however, no ABA transporter has been described to date. Here, we report the identification of the ATP-binding cassette (ABC) transporter Arabidopsis thaliana Pleiotropic drug resistance transporter PDR12 (AtPDR12)/ABCG40 as a plasma membrane ABA uptake transporter. Uptake of ABA into yeast and BY2 cells expressing AtABCG40 was increased, whereas ABA uptake into protoplasts of atabcg40 plants was decreased compared with control cells. In response to exogenous ABA, the up-regulation of ABA responsive genes was strongly delayed in atabcg40 plants, indicating that ABCG40 is necessary for timely responses to ABA. Stomata of loss-of-function atabcg40 mutants closed more slowly in response to ABA, resulting in reduced drought tolerance. Our results integrate ABA-dependent signaling and transport processes and open another avenue for the engineering of drought-tolerant plants. PMID:20133880

  16. BRAF inhibition decreases cellular glucose uptake in melanoma in association with reduction in cell volume

    PubMed Central

    Theodosakis, Nicholas; Held, Matthew A.; Marzuka-Alcala, Alexander; Meeth, Katrina M.; Micevic, Goran; Long, Georgina V.; Scolyer, Richard A.; Stern, David F.; Bosenberg, Marcus W.

    2015-01-01

    BRAF kinase inhibitors have dramatically impacted treatment of BRAFV600E/K-driven metastatic melanoma. Early responses assessed using [18F]fluorodeoxyglucose uptake-positron emission tomography (FDG-PET) have shown dramatic reduction of radiotracer signal within two weeks of treatment. Despite high response rates, relapse occurs in nearly all cases, frequently at sites of treated metastatic disease. It remains unclear whether initial loss of 18FDG uptake is due to tumor cell death or other reasons. Here we provide evidence of melanoma cell volume reduction in a patient cohort treated with BRAF inhibitors. We present data demonstrating that BRAF inhibition reduces melanoma glucose uptake per cell, but that this change is no longer significant following normalization for cell volume changes. We also demonstrate that volume normalization greatly reduces differences in transmembrane glucose transport and hexokinase-mediated phosphorylation. Mechanistic studies suggest that this loss of cell volume is due in large part to decreases in new protein translation as a consequence of vemurafenib treatment. Ultimately, our findings suggest that cell volume regulation constitutes an important physiologic parameter that may significantly contribute to radiographic changes observed in clinic. PMID:25948295

  17. Mapping the functional domains of TCblR/CD320, the receptor for cellular uptake of transcobalamin-bound cobalamin

    PubMed Central

    Jiang, Wenxia; Nakayama, Yasumi; Sequeira, Jeffrey M.; Quadros, Edward V.

    2013-01-01

    The membrane receptor TCblR/CD320 binds transcobalamin (TC) saturated with vitamin B12 [cobalamin (Cbl)] and mediates cellular uptake of the vitamin. The specificity of TC for Cbl and of the receptor for TC-Cbl ensures efficient uptake of Cbl into cells. The high-affinity interaction of TCblR with TC-Cbl (Ka=10 nM−1) was investigated using deletions and mutations of amino acid sequences in TCblR. Only the extracellular region (aa 32–229) is needed for TC-Cbl binding, but the N-glycosylation sites (N126, N195, and N213) are of no importance for this function. Deleting the cysteine-rich region (aa 95–141) that separates the two low-density lipoprotein receptor type A (LDLR-A) domains does not affect TC-Cbl binding (Ka = 19–24 nM−1). The two LDLR-A domains (aa 54–89 and 132–167) with the negatively charged acidic residues involved in Ca2+ binding are critical determinants of ligand binding. The cytoplasmic tail is apparently crucial for internalization of the ligand. Within this region, the RPLGLL motif and the PDZ binding motifs (QERL/KESL) appear to be involved in initiating and completing the process of ligand internalization. Mutations and deletions of these regions involved in binding and internalization of TC-Cbl are likely to produce the biochemical and clinical phenotype of Cbl deficiency.—Jiang, W., Nakayama, Y., Sequeira, J. M., Quadros, E. V. Mapping the functional domains of TCblR/CD320, the receptor for cellular uptake of transcobalamin-bound cobalamin. PMID:23603833

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. Multifunctional Hyaluronic Acid and Chondroitin Sulfate Nanoparticles: Impact of Glycosaminoglycan Presentation on Receptor Mediated Cellular Uptake and Immune Activation.

    PubMed

    Oommen, Oommen P; Duehrkop, Claudia; Nilsson, Bo; Hilborn, Jöns; Varghese, Oommen P

    2016-08-17

    Hyaluronic acid (HA) and chondroitin sulfate (CS) polymers are extensively used for various biomedical applications, such as for tissue engineering, drug delivery, and gene delivery. Although both these biopolymers are known to target cell surface CD44 receptors, their relative cellular targeting properties and immune activation potential have never been evaluated. In this article, we present the synthesis and characterization of novel self-assembled supramolecular HA and CS nanoparticles (NPs). These NPs were developed using fluorescein as a hydrophobic component that induced amphiphilicity in biopolymers and also efficiently stabilized anticancer drug doxorubicin (DOX) promoting a near zero-order drug release. The cellular uptake and cytotoxicity studies of these NPs in different human cancer lines, namely, human colorectal carcinoma cell line HCT116 and human breast cancer cell line MCF-7 demonstrated dose dependent cytotoxicity. Interestingly, both NPs showed CD44 dependent cellular uptake with the CS-DOX NP displaying higher dose-dependent cytotoxicity than the HA-DOX NP in different mammalian cells tested. Immunological evaluation of these nanocarriers in an ex vivo human whole blood model revealed that unlike unmodified polymers, the HA NP and CS NP surprisingly showed platelet aggregation and thrombin-antithrombin complex formation at high concentrations (0.8 mg/mL). We also observed a clear difference in early- and late-stage complement activation (C3a and sC5b-9) with CS and CS NP triggering significant complement activation at high concentrations (0.08-0.8 mg/mL), unlike HA and HA NP. These results offer new insight into designing glycosaminoglycan-based NPs and understanding their hematological responses and targeting ability. PMID:27468113

  20. Chiral Ruthenium(II) Polypyridyl Complexes: Stabilization of G-Quadruplex DNA, Inhibition of Telomerase Activity and Cellular Uptake

    PubMed Central

    Yu, Qianqian; Liu, Yanan; Wang, Chuan; Sun, Dongdong; Yang, Xingcheng; Liu, Yanyu; Liu, Jie

    2012-01-01

    Two ruthenium(II) complexes, Λ-[Ru(phen)2(p-HPIP)]2+ and Δ-[Ru(phen)2(p-HPIP)]2+, were synthesized and characterized via proton nuclear magnetic resonance spectroscopy, electrospray ionization-mass spectrometry, and circular dichroism spectroscopy. This study aims to clarify the anticancer effect of metal complexes as novel and potent telomerase inhibitors and cellular nucleus target drug. First, the chiral selectivity of the compounds and their ability to stabilize quadruplex DNA were studied via absorption and emission analyses, circular dichroism spectroscopy, fluorescence-resonance energy transfer melting assay, electrophoretic mobility shift assay, and polymerase chain reaction stop assay. The two chiral compounds selectively induced and stabilized the G-quadruplex of telomeric DNA with or without metal cations. These results provide new insights into the development of chiral anticancer agents for G-quadruplex DNA targeting. Telomerase repeat amplification protocol reveals the higher inhibitory activity of Λ-[Ru(phen)2(p-HPIP)]2+ against telomerase, suggesting that Λ-[Ru(phen)2(p-HPIP)]2+ may be a potential telomerase inhibitor for cancer chemotherapy. MTT assay results show that these chiral complexes have significant antitumor activities in HepG2 cells. More interestingly, cellular uptake and laser-scanning confocal microscopic studies reveal the efficient uptake of Λ-[Ru(phen)2(p-HPIP)]2+ by HepG2 cells. This complex then enters the cytoplasm and tends to accumulate in the nucleus. This nuclear penetration of the ruthenium complexes and their subsequent accumulation are associated with the chirality of the isomers as well as with the subtle environment of the ruthenium complexes. Therefore, the nucleus can be the cellular target of chiral ruthenium complexes for anticancer therapy. PMID:23236402

  1. Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

    PubMed

    Poutiainen, Pekka K; Rönkkö, Teemu; Hinkkanen, Ari E; Palvimo, Jorma J; Närvänen, Ale; Turhanen, Petri; Laatikainen, Reino; Weisell, Janne; Pulkkinen, Juha T

    2014-01-15

    In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 μM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

  2. A novel method for measuring cellular antibody uptake using imaging flow cytometry reveals distinct uptake rates for two different monoclonal antibodies targeting L1.

    PubMed

    Hazin, John; Moldenhauer, Gerhard; Altevogt, Peter; Brady, Nathan R

    2015-08-01

    Monoclonal antibodies (mAbs) have emerged as a promising tool for cancer therapy. Differing approaches utilize mAbs to either deliver a drug to the tumor cells or to modulate the host's immune system to mediate tumor kill. The rate by which a therapeutic antibody is being internalized by tumor cells is a decisive feature for choosing the appropriate treatment strategy. We herein present a novel method to effectively quantitate antibody uptake of tumor cells by using image-based flow cytometry, which combines image analysis with high throughput of sample numbers and sample size. The use of this method is established by determining uptake rate of an anti-EpCAM antibody (HEA125), from single cell measurements of plasma membrane versus internalized antibody, in conjunction with inhibitors of endocytosis. The method is then applied to two mAbs (L1-9.3, L1-OV52.24) targeting the neural cell adhesion molecule L1 (L1CAM) at two different epitopes. Based on median cell population responses, we find that mAb L1-OV52.24 is rapidly internalized by the ovarian carcinoma cell line SKOV3ip while L1 mAb 9.3 is mainly retained at the cell surface. These findings suggest the L1 mAb OV52.24 as a candidate to be further developed for drug-delivery to cancer cells, while L1-9.3 may be optimized to tag the tumor cells and stimulate immunogenic cancer cell killing. Furthermore, when analyzing cell-to-cell variability, we observed L1 mAb OV52.24 rapidly transition into a subpopulation with high-internalization capacity. In summary, this novel high-content method for measuring antibody internalization rate provides a high level of accuracy and sensitivity for cell population measurements and reveals further biologically relevant information when taking into account cellular heterogeneity.

  3. A novel method for measuring cellular antibody uptake using imaging flow cytometry reveals distinct uptake rates for two different monoclonal antibodies targeting L1.

    PubMed

    Hazin, John; Moldenhauer, Gerhard; Altevogt, Peter; Brady, Nathan R

    2015-08-01

    Monoclonal antibodies (mAbs) have emerged as a promising tool for cancer therapy. Differing approaches utilize mAbs to either deliver a drug to the tumor cells or to modulate the host's immune system to mediate tumor kill. The rate by which a therapeutic antibody is being internalized by tumor cells is a decisive feature for choosing the appropriate treatment strategy. We herein present a novel method to effectively quantitate antibody uptake of tumor cells by using image-based flow cytometry, which combines image analysis with high throughput of sample numbers and sample size. The use of this method is established by determining uptake rate of an anti-EpCAM antibody (HEA125), from single cell measurements of plasma membrane versus internalized antibody, in conjunction with inhibitors of endocytosis. The method is then applied to two mAbs (L1-9.3, L1-OV52.24) targeting the neural cell adhesion molecule L1 (L1CAM) at two different epitopes. Based on median cell population responses, we find that mAb L1-OV52.24 is rapidly internalized by the ovarian carcinoma cell line SKOV3ip while L1 mAb 9.3 is mainly retained at the cell surface. These findings suggest the L1 mAb OV52.24 as a candidate to be further developed for drug-delivery to cancer cells, while L1-9.3 may be optimized to tag the tumor cells and stimulate immunogenic cancer cell killing. Furthermore, when analyzing cell-to-cell variability, we observed L1 mAb OV52.24 rapidly transition into a subpopulation with high-internalization capacity. In summary, this novel high-content method for measuring antibody internalization rate provides a high level of accuracy and sensitivity for cell population measurements and reveals further biologically relevant information when taking into account cellular heterogeneity. PMID:25967950

  4. Versatile cellular uptake mediated by catanionic vesicles: simultaneous spontaneous membrane fusion and endocytosis.

    PubMed

    Mauroy, Chloé; Castagnos, Pauline; Orio, Julie; Blache, Marie-Claire; Rico-Lattes, Isabelle; Teissié, Justin; Rols, Marie-Pierre; Blanzat, Muriel

    2015-01-01

    Lactose-derived catanionic vesicles offer unique opportunities to overcome cellular barriers. These potential nanovectors, very easy to formulate as drug delivery systems, are able to encapsulate drugs of various hydrophilicity. This article highlights versatile interaction mechanisms between these catanionic vesicles, labeled with hydrophilic and amphiphilic fluorescent probes, and a mammalian cell line, Chinese Hamster Ovary. Confocal microscopy and flow cytometry techniques show that these vesicles are internalized by cells through cellular energy dependent processes, as endocytosis, but are simultaneously able to spontaneously fuse with cell plasma membranes and release their hydrophilic content directly inside the cytosol. Such innovative and polyvalent nanovectors, able to deliver their content via different internalization pathways, would positively be a great progress for the coadministration of drugs of complementary efficiency. PMID:25310849

  5. Slight temperature changes affect protein affinity and cellular uptake/toxicity of nanoparticles.

    PubMed

    Mahmoudi, Morteza; Shokrgozar, Mohammad A; Behzadi, Shahed

    2013-04-21

    It is known that what the cell actually "sees" at the nanoscale is an outer shell formed of 'protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature.

  6. Inhibition of Glutathione Production Induces Macrophage CD36 Expression and Enhances Cellular-oxidized Low Density Lipoprotein (oxLDL) Uptake.

    PubMed

    Yang, Xiaoxiao; Yao, Hui; Chen, Yuanli; Sun, Lei; Li, Yan; Ma, Xingzhe; Duan, Shengzhong; Li, Xiaoju; Xiang, Rong; Han, Jihong; Duan, Yajun

    2015-09-01

    The glutathione (GSH)-dependent antioxidant system has been demonstrated to inhibit atherosclerosis. Macrophage CD36 uptakes oxidized low density lipoprotein (oxLDL) thereby facilitating foam cell formation and development of atherosclerosis. It remains unknown if GSH can influence macrophage CD36 expression and cellular oxLDL uptake directly. Herein we report that treatment of macrophages with l-buthionine-S,R-sulfoximine (BSO) decreased cellular GSH production and ratios of GSH to glutathione disulfide (GSH/GSSG) while increasing production of reactive oxygen species. Associated with decreased GSH levels, macrophage CD36 expression was increased, which resulted in enhanced cellular oxLDL uptake. In contrast, N-acetyl cysteine and antioxidant enzyme (catalase or superoxide dismutase) blocked BSO-induced CD36 expression as well as oxLDL uptake. In vivo, administration of mice with BSO increased CD36 expression in peritoneal macrophages and kidneys. BSO had no effect on CD36 mRNA expression and promoter activity but still induced CD36 protein expression in macrophages lacking peroxisome proliferator-activated receptor γ expression, suggesting it induced CD36 expression at the translational level. Indeed, we determined that BSO enhanced CD36 translational efficiency. Taken together, our study demonstrates that cellular GSH levels and GSH/GSSG status can regulate macrophage CD36 expression and cellular oxLDL uptake and demonstrate an important anti-atherogenic function of the GSH-dependent antioxidant system by providing a novel molecular mechanism.

  7. Inhibition of Glutathione Production Induces Macrophage CD36 Expression and Enhances Cellular-oxidized Low Density Lipoprotein (oxLDL) Uptake*

    PubMed Central

    Yang, Xiaoxiao; Yao, Hui; Chen, Yuanli; Sun, Lei; Li, Yan; Ma, Xingzhe; Duan, Shengzhong; Li, Xiaoju; Xiang, Rong; Han, Jihong; Duan, Yajun

    2015-01-01

    The glutathione (GSH)-dependent antioxidant system has been demonstrated to inhibit atherosclerosis. Macrophage CD36 uptakes oxidized low density lipoprotein (oxLDL) thereby facilitating foam cell formation and development of atherosclerosis. It remains unknown if GSH can influence macrophage CD36 expression and cellular oxLDL uptake directly. Herein we report that treatment of macrophages with l-buthionine-S,R-sulfoximine (BSO) decreased cellular GSH production and ratios of GSH to glutathione disulfide (GSH/GSSG) while increasing production of reactive oxygen species. Associated with decreased GSH levels, macrophage CD36 expression was increased, which resulted in enhanced cellular oxLDL uptake. In contrast, N-acetyl cysteine and antioxidant enzyme (catalase or superoxide dismutase) blocked BSO-induced CD36 expression as well as oxLDL uptake. In vivo, administration of mice with BSO increased CD36 expression in peritoneal macrophages and kidneys. BSO had no effect on CD36 mRNA expression and promoter activity but still induced CD36 protein expression in macrophages lacking peroxisome proliferator-activated receptor γ expression, suggesting it induced CD36 expression at the translational level. Indeed, we determined that BSO enhanced CD36 translational efficiency. Taken together, our study demonstrates that cellular GSH levels and GSH/GSSG status can regulate macrophage CD36 expression and cellular oxLDL uptake and demonstrate an important anti-atherogenic function of the GSH-dependent antioxidant system by providing a novel molecular mechanism. PMID:26187465

  8. The effect of neutral-surface iron oxide nanoparticles on cellular uptake and signaling pathways

    PubMed Central

    Kim, Eunjoo; Kim, Joon Mee; Kim, Lucia; Choi, Suk Jin; Park, In Suh; Han, Jee Young; Chu, Young Chae; Choi, Eun Sook; Na, Kun; Hong, Soon-Sun

    2016-01-01

    In recent years, iron oxide nanoparticles (IONPs) have been applied widely to biomedical fields. However, the relationship between the physicochemical properties of IONPs and their biological behavior is not fully understood yet. We prepared 3-methacryloxypropyltrimethoxysilane (MPS)-coated IONPs, which have a neutral hydrophobic surface, and compared their biological behavior to that of Resovist (ferucarbotran), a commercialized IONP formulation modified with carboxymethyl dextran. The rate of MPS-IONP uptake by human aortic endothelial cells (HAoECs) was higher than ferucarbotran uptake, indicating that the neutral hydrophobic nature of MPS-IONPs allowed them to be absorbed more readily through the plasma membrane. However, the signaling pathways activated by MPS-IONPs and ferucarbotran were comparable, suggesting that surface charge is not a key factor for inducing changes in HAoECs. In vivo fate analysis showed that MPS-IONPs accumulated for longer periods in tissues than hydrophilic ferucarbotran. These findings could enlarge our understanding of NP behavior for advanced applications in the biomedical field. PMID:27695320

  9. The effect of neutral-surface iron oxide nanoparticles on cellular uptake and signaling pathways

    PubMed Central

    Kim, Eunjoo; Kim, Joon Mee; Kim, Lucia; Choi, Suk Jin; Park, In Suh; Han, Jee Young; Chu, Young Chae; Choi, Eun Sook; Na, Kun; Hong, Soon-Sun

    2016-01-01

    In recent years, iron oxide nanoparticles (IONPs) have been applied widely to biomedical fields. However, the relationship between the physicochemical properties of IONPs and their biological behavior is not fully understood yet. We prepared 3-methacryloxypropyltrimethoxysilane (MPS)-coated IONPs, which have a neutral hydrophobic surface, and compared their biological behavior to that of Resovist (ferucarbotran), a commercialized IONP formulation modified with carboxymethyl dextran. The rate of MPS-IONP uptake by human aortic endothelial cells (HAoECs) was higher than ferucarbotran uptake, indicating that the neutral hydrophobic nature of MPS-IONPs allowed them to be absorbed more readily through the plasma membrane. However, the signaling pathways activated by MPS-IONPs and ferucarbotran were comparable, suggesting that surface charge is not a key factor for inducing changes in HAoECs. In vivo fate analysis showed that MPS-IONPs accumulated for longer periods in tissues than hydrophilic ferucarbotran. These findings could enlarge our understanding of NP behavior for advanced applications in the biomedical field.

  10. Cellular uptake behavior of unfunctionalized and functionalized PBCA particles prepared in a miniemulsion.

    PubMed

    Weiss, Clemens K; Lorenz, Myriam R; Landfester, Katharina; Mailänder, Volker

    2007-07-01

    Fluorescent dye labeled unfunctionalized and functionalized poly(n-butylcyanoacrylate) nanoparticles were prepared using a miniemulsion technique. Amino acid and methoxyPEG functionalization could be introduced by using aqueous solutions as an initiator for the anionic polymerization in the heterophase. All the particles prepared had sizes smaller than 250 nm and negative zeta-potentials. The molar mass distribution of the polymer was dependent on the acid used as the continuous phase and the initiator solution applied. Cells of three lines (HeLa, Jurkat and mesenchymal stem cells) were incubated with the particles. The molar mass of the polymer determined the onset and extent of apoptosis, and the total uptake was determined by the size and functionalization of the particles. Different uptake kinetics were obtained with HeLa and Jurkat cells after incubation with the same particle batch. The intracellular particle distribution, visualized by confocal laser scanning microscopy, did not show significant differences for either of the cell lines or particle batches.

  11. A five-year study of the impact of nitrogen addition on methane uptake in alpine grassland.

    PubMed

    Yue, Ping; Li, Kaihui; Gong, Yanming; Hu, Yukun; Mohammat, Anwar; Christie, Peter; Liu, Xuejun

    2016-01-01

    It remains unclear how nitrogen (N) deposition affects soil methane (CH4) uptake in semiarid and arid zones. An in situ field experiment was conducted from 2010 to 2014 to systematically study the effect of various N application rates (0, 10, 30, and 90 kg N ha(-1) yr(-1)) on CH4 flux in alpine grassland in the Tianshan Mountains. No significant influence of N addition on CH4 uptake was found. Initially the CH4 uptake rate increased with increasing N application rate by up to 11.5% in 2011 and then there was gradual inhibition by 2014. However, the between-year variability in CH4 uptake was very highly significant with average uptake ranging from 52.9 to 106.6 μg C m(-2) h(-1) and the rate depended largely on seasonal variability in precipitation and temperature. CH4 uptake was positively correlated with soil temperature, air temperature and to a lesser extent with precipitation, and was negatively correlated with soil moisture and NO3(-)-N content. The results indicate that between-year variability in CH4 uptake was impacted by precipitation and temperature and was not sensitive to elevated N deposition in alpine grassland. PMID:27571892

  12. A five-year study of the impact of nitrogen addition on methane uptake in alpine grassland

    PubMed Central

    Yue, Ping; Li, Kaihui; Gong, Yanming; Hu, Yukun; Mohammat, Anwar; Christie, Peter; Liu, Xuejun

    2016-01-01

    It remains unclear how nitrogen (N) deposition affects soil methane (CH4) uptake in semiarid and arid zones. An in situ field experiment was conducted from 2010 to 2014 to systematically study the effect of various N application rates (0, 10, 30, and 90 kg N ha−1 yr−1) on CH4 flux in alpine grassland in the Tianshan Mountains. No significant influence of N addition on CH4 uptake was found. Initially the CH4 uptake rate increased with increasing N application rate by up to 11.5% in 2011 and then there was gradual inhibition by 2014. However, the between-year variability in CH4 uptake was very highly significant with average uptake ranging from 52.9 to 106.6 μg C m−2 h−1 and the rate depended largely on seasonal variability in precipitation and temperature. CH4 uptake was positively correlated with soil temperature, air temperature and to a lesser extent with precipitation, and was negatively correlated with soil moisture and NO3−-N content. The results indicate that between-year variability in CH4 uptake was impacted by precipitation and temperature and was not sensitive to elevated N deposition in alpine grassland. PMID:27571892

  13. Localizing Organomercury Uptake And Accumulation in Zebrafish Larvae at the Tissue And Cellular Level

    SciTech Connect

    Korbas, M.; Blechinger, S.R.; Krone, P.H.; Pickering, I.J.; George, G.N.

    2009-05-20

    Using synchrotron x-ray fluorescence mapping, we have examined the uptake and localization of organic mercury in zebrafish larvae. Strikingly, the greatest accumulation of methyl and ethyl mercury compounds was highly localized in the rapidly dividing lens epithelium, with lower levels going to brain, optic nerve, and various other organs. The data suggest that the reported impairment of visual processes by mercury may arise not only from previously reported neurological effects, but also from direct effects on the ocular tissue. This novel approach is a powerful tool for directly investigating the molecular toxicology of heavy metals, and should be equally applicable to the study of a wide range of elements in developing embryos.

  14. Slight temperature changes affect protein affinity and cellular uptake/toxicity of nanoparticles

    NASA Astrophysics Data System (ADS)

    Mahmoudi, Morteza; Shokrgozar, Mohammad A.; Behzadi, Shahed

    2013-03-01

    It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature.It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular

  15. Recent advances in interactions of designed nanoparticles and cells with respect to cellular uptake, intracellular fate, degradation and cytotoxicity.

    PubMed

    Deng, Jun; Gao, Changyou

    2016-10-14

    The unique features of nanomaterials have led to their rapid development in the biomedical field. In particular, functionalized nanoparticles (NPs) are extensively used in the delivery of drugs and genes, bio-imaging and diagnosis. Hence, the interaction between NPs and cells is one of the most important issues towards understanding the true nature of the NP-mediated biological effects. Moreover, the intracellular safety concern of the NPs as a result of intracellular NP degradation remains to be clarified in detail. This review presents recent advances in the interactions of designed NPs and cells. The focus includes the governing factors on cellular uptake and the intracellular fate of NPs, and the degradation of NPs and its influence on nanotoxicity. Some basic consideration is proposed for optimizing the NP-cell interaction and designing NPs of better biocompatiblity for biomedical application. PMID:27609340

  16. 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.

  17. Recent advances in interactions of designed nanoparticles and cells with respect to cellular uptake, intracellular fate, degradation and cytotoxicity

    NASA Astrophysics Data System (ADS)

    Deng, Jun; Gao, Changyou

    2016-10-01

    The unique features of nanomaterials have led to their rapid development in the biomedical field. In particular, functionalized nanoparticles (NPs) are extensively used in the delivery of drugs and genes, bio-imaging and diagnosis. Hence, the interaction between NPs and cells is one of the most important issues towards understanding the true nature of the NP-mediated biological effects. Moreover, the intracellular safety concern of the NPs as a result of intracellular NP degradation remains to be clarified in detail. This review presents recent advances in the interactions of designed NPs and cells. The focus includes the governing factors on cellular uptake and the intracellular fate of NPs, and the degradation of NPs and its influence on nanotoxicity. Some basic consideration is proposed for optimizing the NP-cell interaction and designing NPs of better biocompatiblity for biomedical application.

  18. Recent advances in interactions of designed nanoparticles and cells with respect to cellular uptake, intracellular fate, degradation and cytotoxicity.

    PubMed

    Deng, Jun; Gao, Changyou

    2016-10-14

    The unique features of nanomaterials have led to their rapid development in the biomedical field. In particular, functionalized nanoparticles (NPs) are extensively used in the delivery of drugs and genes, bio-imaging and diagnosis. Hence, the interaction between NPs and cells is one of the most important issues towards understanding the true nature of the NP-mediated biological effects. Moreover, the intracellular safety concern of the NPs as a result of intracellular NP degradation remains to be clarified in detail. This review presents recent advances in the interactions of designed NPs and cells. The focus includes the governing factors on cellular uptake and the intracellular fate of NPs, and the degradation of NPs and its influence on nanotoxicity. Some basic consideration is proposed for optimizing the NP-cell interaction and designing NPs of better biocompatiblity for biomedical application.

  19. Measuring in vitro cellular uptake of nanoparticles by transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Brown, A. P.; Brydson, R. M. D.; Hondow, N. S.

    2014-06-01

    Biomedical application of engineered nanoparticles (NPs) is a growing area of research and development. Uncertainty remains as to the mode of action of many NP types and TEM is a tool capable of addressing this if used in conjunction with standard cellular response assays. We will demonstrate imaging of thin sections of fixed, plastic embedded cells by analytical TEM to identify: superparamagnetic iron oxide NP translocation into cell compartments such as endosomes; amorphous silica NP penetration through a cell membrane without membrane encapsulation and zinc oxide NP degradation in cell compartments. We will then discuss how the in vitro cellular responses to a dose of NPs exposed to cell lines can be correlated to the internalized dose per cell section noting however that quantification of the latter requires random sampling procedures or correlation to higher throughout techniques to measure a population of whole cells. Similarly, analytical TEM measures of NP degradation within intracellular compartments will require a more appropriate sample preparation such as cryo-fixation.

  20. Preparation, cellular uptake and angiogenic suppression of shikonin-containing liposomes in vitro and in vivo

    PubMed Central

    Xia, Hongmei; Tang, Chengyi; Gui, Heng; Wang, Xiaoming; Qi, Jinliang; Wang, Xiuqiang; Yang, Yonghua

    2012-01-01

    Shikonin has anticancer activity, but it has not yet been applied into clinical use. In the present study, shikonin was prepared using liposomes. We aimed to examine several aspects of sh-L (shikonin-containing liposomes): preparation, angiogenic suppression and cellular uptake through self-fluorescence. Sh-L were prepared using soybean phospholipid and cholesterol to form the membrane and shikonin was encapsulated into the phospholipid membrane. Three liposomes were prepared with shikonin. They had red fluorescence and were analysed using a flow cytometer. Angiogenic suppression of sh-L was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], Transwell tests, chick CAM (chorioallantoic membrane) and Matrigel™ plug assay. MTT assay showed the median IC50 (inhibitory concentrations) as follows: shikonin, sh-L1 and sh-L2 were 4.99±0.23, 5.81±0.57 and 7.17±0.69 μM, respectively. The inhibition rates of migration were 53.58±7.05, 46.56±4.36 and 41.19±3.59% for 3.15 μM shikonin, sh-L1 and sh-L2, respectively. The results of CAM and Matrigel plug assay demonstrated that shikonin and sh-L can decrease neovascularization. Effect of shikonin was more obvious than sh-L at the same concentration. The results showed that sh-L decreased the toxicity, the rate of inhibition of migration and angiogenic suppression. The cellular uptake of the sh-L could be pictured because of the self-fluorescence. The self-fluorescence will be useful for conducting further research. Sh-L might be an excellent preparation for future clinical application to cancer patients. PMID:23176403

  1. Protein Transduction Domains Fused to Virus Receptors Improve Cellular Virus Uptake and Enhance Oncolysis by Tumor-Specific Replicating Vectors

    PubMed Central

    Kühnel, Florian; Schulte, Bernd; Wirth, Thomas; Woller, Norman; Schäfers, Sonja; Zender, Lars; Manns, Michael; Kubicka, Stefan

    2004-01-01

    Expression of cellular receptors determines viral tropism and limits gene delivery by viral vectors. Protein transduction domains (PTDs) have been shown to deliver proteins, antisense oligonucleotides, liposomes, or plasmid DNA into cells. In our study, we investigated the role of several PTD motifs in adenoviral infection. When physiologically expressed, a PTD from human immunodeficiency virus transactivator of transcription (Tat) did not improve adenoviral infection. We therefore fused PTDs to the ectodomain of the coxsackievirus-adenovirus receptor (CARex) to attach PTDs to adenoviral fiber knobs. CARex-Tat and CARex-VP22 allowed efficient adenoviral infection in nonpermissive cells and significantly improved viral uptake rates in permissive cells. Dose-dependent competition of CARex-PTD-mediated infection using CARex and inhibition experiments with heparin showed that binding of CARex-PTD to both adenoviral fiber and cellular glycosaminoglycans is essential for the improvement of infection. CARex-PTD-treated adenoviruses retained their properties after density gradient ultracentrifugation, indicating stable binding of CARex-PTD to adenoviral particles. Consequently, the mechanism of CARex-PTD-mediated infection involves coating of the viral fiber knobs by CARex-PTD, rather than placement of CARex domains on cell surfaces. Expression of CARex-PTDs led to enhanced lysis of permissive and nonpermissive tumor cells by replicating adenoviruses, indicating that CARex-PTDs are valuable tools to improve the efficacy of oncolytic therapy. Together, our study shows that CARex-PTDs facilitate gene transfer in nonpermissive cells and improve viral uptake at reduced titers and infection times. The data suggest that PTDs fused to virus binding receptors may be a valuable tool to overcome natural tropism of vectors and could be of great interest for gene therapeutic approaches. PMID:15564483

  2. Encapsulated plant extract (Gelsemium sempervirens) poly (lactide-co-glycolide) nanoparticles enhance cellular uptake and increase bioactivity in vitro.

    PubMed

    Bhattacharyya, Soumya Sundar; Paul, Saili; Khuda-Bukhsh, Anisur Rahman

    2010-06-01

    Ethanolic extract of Gelsemium sempervirens (family: Loganiaceae), henceforth to be called EEGS, is used in various traditional systems of medicine. In homeopathy, EEGS is known as mother tincture of G. sempervirens, which is generally used to treat pain and respiratory ailments. We demonstrated earlier anticancer activity of crude EEGS by in vitro studies on human HeLa cells. To test the hypothesis if nanoparticle-encapsulated extract (now onwards to be called NEEGS) could enhance cellular uptake and thereby improve bioactivity, we formulated nanoparticle encapsulation based on poly (lactide-co-glycolide) (PLGA) and confirmed encapsulation by scanning electron microscopy (SEM) and atomic force microscopy. EEGS was encapsulated with 81.6% efficiency in PLGA biodegradable nanoparticle formulation and F68 (polyoxyethylene-polyoxypropylene) was used as a stabilizer. Dynamic laser light scattering and SEM indicated a particle diameter of 122.6 nm. The zeta potential of the drug-loaded nanoparticles was -14.8 mV. NEEGS was characterized for their biological activities in a skin cancer cell line A375 in vitro. NEEGS exhibited relatively rapid (30 min) and more efficient cellular uptake than their un-encapsulated counterpart (45 min). Analysis of data of apoptosis study using Annexin V-FITC, terminal transferase dUTP nick end labeling assay and DNA ladder revealed that encapsulated EEGS was more potent than their un-encapsulated counterpart in inducing apoptosis of A375 cells. Reverse transcriptase-polymerase chain reaction data of survivin, cyclin-D1, caspase-3, PCNA and p53 also corroborated well to suggest greater potentials of NEEGS as anticancer agents.

  3. Combinations of Cocaine with Other Dopamine Uptake Inhibitors: Assessment of Additivity

    PubMed Central

    Tanda, Gianluigi; Newman, Amy Hauck; Ebbs, Aaron L.; Tronci, Valeria; Green, Jennifer L.; Tallarida, Ronald J.; Katz, Jonathan L.

    2009-01-01

    Drugs that inhibit dopamine (DA) reuptake through actions at the dopamine transporter (DAT) have been proposed as candidates for development as pharmacotherapies for cocaine abuse. Accordingly, it is important to understand the potential pharmacological interactions of cocaine with other drugs acting at the DAT. Effects of combinations of cocaine with a cocaine analog, 2β-carbomethoxy-3β-(4-fluorophenyl)tropane (WIN 35,428), were compared quantitatively with the combinations of cocaine with the N-butyl,4′,4″-diF benztropine analog, 3-(bis(4-fluorophenyl)methoxy)-8-butyl-8-azabicyclo[3.2.1]octane (JHW 007), to determine whether their effects on DA levels in the shell of the nucleus accumbens (NAC) in mice differed. Each of the drugs alone produced dose-related elevations in NAC DA levels. In contrast to the other drugs, JHW 007 was less effective, producing maximal effects that approached 400% of control versus ∼700% with the other drugs. In addition, the JHW 007 dose-effect curve was not as steep as those for cocaine and WIN 35,428. Combinations of cocaine with its analog, WIN 35,428, were most often greater than those predicted based on dose additivity. In contrast, combinations of cocaine with JHW 007 were most often subadditive. This outcome is consistent with recent studies suggesting that structurally divergent DA uptake inhibitors bind to different domains of the DAT, which can result in different DAT conformations. The conformational changes occurring with JHW 007 binding may result in functional outcomes that alter its abuse liability and its effects in combination with cocaine. PMID:19483071

  4. Cellular uptake and degradation behaviour of biodegradable poly(ethylene glycol-graft-methyl methacrylate) nanoparticles crosslinked with dimethacryloyl hydroxylamine.

    PubMed

    Scheler, Stefan; Kitzan, Martina; Fahr, Alfred

    2011-01-17

    Crosslinked polymers with hydrolytically cleavable linkages are highly interesting materials for the design of biodegradable drug carriers. The aim of this study was to investigate if nanoparticles made of such polymers have the potential to be used also for intracellular drug delivery. PEGylated nanoparticles were prepared by copolymerization of methacrylic acid esters and N,O-dimethacryloylhydroxylamine (DMHA). The particles were stable at pH 5.0. At pH 7.4 and 9.0 the degradation covered a time span of about 14 days, following first-order kinetics with higher crosslinked particles degrading slower. Cellular particle uptake and cytotoxicity were tested with L929 mouse fibroblasts. The particle uptake rate was found to correlate linearly with the surface charge and to increase as the zeta potential becomes less negative. Coating of the particle surface with polysorbate 80 drops the internalization rate close to zero and the charge dependence disappears. This indicates the existence of a second effect apart from surface charge. A similar pattern of correlation with zeta potential and coating was also found for the degree of membrane damage while there was no effect of polysorbate on the cell metabolism which increased as the negative charge decreased. It is discussed whether exocytotic processes may explain this behaviour.

  5. Synthesis of diethylenetriaminepentaacetic acid conjugated inulin and utility for cellular uptake of liposomes

    SciTech Connect

    Essien, H.; Lai, J.Y.; Hwang, K.J.

    1988-05-01

    The synthesis, binding of radioactive cations, liposomal encapsulation, and biodistribution of the oxidized-inulin reaction product with ethylenediamine and diethylenetriaminepentaacetic acid (4) are described. The four-step synthesis of the inulin derivative proceeded in a good overall yield of 72%. The complex of the inulin derivative with either /sup 67/Ga3+ or /sup 111/In3+ was stable in vivo and did not readily distribute into tissues, being excreted primarily in urine after intravenous administration to mice. The liposome-entrapped inulin derivative can be loaded with radioactive heavy metal cations by mobile ionophores in high radiochemical yields of 80-91%. Following the intravenous administration of the liposomal encapsulation of the indium-111-labeled inulin derivative, the entrapped compound had a biodistribution characteristic of liposomes and allowed an estimation of the extent of the intracellular uptake of liposomes. The ability of the inulin derivative to chelate many different types of metals will allow the use of this probe for studying subtle differences in tissue distribution resulting from different drug targeting or delivery protocols in the same animal by multiple labeling techniques. Moreover, the chelate-conjugated inulin permits studies of the applications of drug delivery systems in primates or human subjects by noninvasive techniques such as gamma-scintigraphic or nuclear magnetic resonance imaging methods.

  6. Lipid Cross-Linking of Nanolipoprotein Particles Substantially Enhances Serum Stability and Cellular Uptake.

    PubMed

    Gilmore, Sean F; Blanchette, Craig D; Scharadin, Tiffany M; Hura, Greg L; Rasley, Amy; Corzett, Michele; Pan, Chong-Xian; Fischer, Nicholas O; Henderson, Paul T

    2016-08-17

    Nanolipoprotein particles (NLPs) consist of a discoidal phospholipid lipid bilayer confined by an apolipoprotein belt. NLPs are a promising platform for a variety of biomedical applications due to their biocompatibility, size, definable composition, and amphipathic characteristics. However, poor serum stability hampers the use of NLPs for in vivo applications such as drug formulation. In this study, NLP stability was enhanced upon the incorporation and subsequent UV-mediated intermolecular cross-linking of photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC in the bilayer and UV exposure time significantly affected the resulting X-NLP stability in 100% serum, as assessed by size exclusion chromatography (SEC) of fluorescently labeled particles. Cross-linking did not significantly impact the size of X-NLPs as determined by dynamic light scattering and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum, which is a drastic improvement compared to non-cross-linked NLPs (50% degradation by ∼10 min). X-NLPs had greater uptake into the human ATCC 5637 bladder cancer cell line compared to non-cross-linked particles, indicating their potential utility for targeted drug delivery. X-NLPs also exhibited enhanced stability following intravenous administration in mice. These results collectively support the potential utility of X-NLPs for a variety of in vivo applications. PMID:27411034

  7. Enhanced cellular uptake of a glutathione selective fluorogenic probe encapsulated in nanoparticles

    NASA Astrophysics Data System (ADS)

    Glówka, Eliza; Lamprecht, Alf; Ubrich, Nathalie; Maincent, Philippe; Lulek, Janina; Coulon, Joël; Leroy, Pierre

    2006-05-01

    Selective fluorogenic probes for the labelling of intracellular reduced glutathione (GSH), i.e. ortho-phthaldialdehyde (OPA) and naphthalene-2,3-dicarboxaldehyde (NDA), have been encapsulated in polymeric nanoparticles (NPs) and the ability of the NPs to enhance uptake of the probe by microbial cells has been evaluated. Preparation of the probe-loaded NPs composed of Eudragit® E was based on an oil-in-water emulsification solvent evaporation method using an ultrasonic probe and polyvinyl alcohol as the surfactant. The encapsulation efficiency of the probes in lyophilized NPs was determined using high performance liquid chromatography (HPLC). A higher encapsulation rate of NDA than OPA was found: 47.6 ± 9.9 (n = 6) and 2.1 ± 0.2% (n = 3), respectively. The NDA-loaded particle diameter and zeta potential were 224.6 ± 14.7 nm and +40.9 ± 6.5 mV, respectively. After 20 min incubation of cultured Candida albicans yeast cells with either free NDA or NDA-loaded NPs (final NDA concentration 100 µM), cells were harvested and corresponding lysates were analysed using HPLC coupled with spectrofluorimetric detection. Incubation of cells with NDA-loaded NPs increased intracellular levels of NDA-GSH adduct by about nine-fold in comparison with the free probe. Adhesion on the cells and the penetration behaviour of NPs loaded with either NDA or fluorescent label (Nile Red) were characterized qualitatively by confocal laser scanning microscopy.

  8. Size-dependent cytotoxicity of silver nanoparticles in human lung cells: the role of cellular uptake, agglomeration and Ag release

    PubMed Central

    2014-01-01

    Background Silver nanoparticles (AgNPs) are currently one of the most manufactured nanomaterials. A wide range of toxicity studies have been performed on various AgNPs, but these studies report a high variation in toxicity and often lack proper particle characterization. The aim of this study was to investigate size- and coating-dependent toxicity of thoroughly characterized AgNPs following exposure of human lung cells and to explore the mechanisms of toxicity. Methods BEAS-2B cells were exposed to citrate coated AgNPs of different primary particle sizes (10, 40 and 75 nm) as well as to 10 nm PVP coated and 50 nm uncoated AgNPs. The particle agglomeration in cell medium was investigated by photon cross correlation spectroscopy (PCCS); cell viability by LDH and Alamar Blue assay; ROS induction by DCFH-DA assay; genotoxicity by alkaline comet assay and γH2AX foci formation; uptake and intracellular localization by transmission electron microscopy (TEM); and cellular dose as well as Ag release by atomic absorption spectroscopy (AAS). Results The results showed cytotoxicity only of the 10 nm particles independent of surface coating. In contrast, all AgNPs tested caused an increase in overall DNA damage after 24 h assessed by the comet assay, suggesting independent mechanisms for cytotoxicity and DNA damage. However, there was no γH2AX foci formation and no increased production of intracellular reactive oxygen species (ROS). The reasons for the higher toxicity of the 10 nm particles were explored by investigating particle agglomeration in cell medium, cellular uptake, intracellular localization and Ag release. Despite different agglomeration patterns, there was no evident difference in the uptake or intracellular localization of the citrate and PVP coated AgNPs. However, the 10 nm particles released significantly more Ag compared with all other AgNPs (approx. 24 wt% vs. 4–7 wt%) following 24 h in cell medium. The released fraction in cell medium did not induce any

  9. 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

    2016-01-01

    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 (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. PMID:27405011

  10. 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

  11. Enhanced cellular uptake and gene silencing activity of siRNA molecules mediated by chitosan-derivative nanocomplexes.

    PubMed

    Guzman-Villanueva, Diana; El-Sherbiny, Ibrahim M; Vlassov, Alexander V; Herrera-Ruiz, Dea; Smyth, Hugh D C

    2014-10-01

    The RNA interference (RNAi) constitutes a conservative mechanism in eukaryotic cells that induces silencing of target genes. In mammalians, the RNAi is triggered by siRNA (small interfering RNA) molecules. Due to its potential in silencing specific genes, the siRNA has been considered a potential alternative for the treatment of genetic and acquired diseases. However, the siRNA therapy has been limited by its low stability and rapid degradation in presence of nucleases, low cellular uptake, and immune response activation. In order to overcome these drawbacks, we propose the synthesis and characterization of non-viral delivery systems using chitosan derivatives to obtain siRNA complexes (polyplexes). The non-viral delivery systems synthesized included PEG-g-OCs (oligochitosan) and PEG-g-Cs (chitosan medium molecular weight). Both systems allowed the formation of siRNA polyplexes, increased the stability of siRNA in the presence of nucleases, enhanced cellular internalization, and showed low toxicity in the A549 cell line. Finally, the complexes obtained with the PEG-g-OCs system showed silencing activity in a GFP model in the cell line A549 in comparison with naked siRNA. PMID:25063077

  12. 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.

  13. 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.

  14. Intracellular trafficking and cellular uptake mechanism of mPEG-PLGA-PLL and mPEG-PLGA-PLL-Gal nanoparticles for targeted delivery to hepatomas.

    PubMed

    Liu, Peifeng; Sun, Yanming; Wang, Qi; Sun, Ying; Li, He; Duan, Yourong

    2014-01-01

    The lysosomal escape of nanoparticles is crucial to enhancing their delivery and therapeutic efficiency. Here, we report the cellular uptake mechanism, lysosomal escape, and organelle morphology effect of monomethoxy (polyethylene glycol)-poly (D,L-lactide-co-glycolide)-poly (L-lysine) (mPEG-PLGA-PLL, PEAL) and 4-O-beta-D-Galactopyranosyl-D-gluconic acid (Gal)-modified PEAL (PEAL-Gal) for intracellular delivery to HepG2, Huh7, and PLC hepatoma cells. These results indicate that PEAL is taken up by clathrin-mediated endocytosis of HepG2, Huh7 and PLC cells. For PEAL-Gal, sialic acid receptor-mediated endocytosis and clathrin-mediated endocytosis are the primary uptake pathways in HepG2 cells, respectively, whereas PEAL-Gal is internalized by sag vesicle- and clathrin-mediated endocytosis in Huh7 cells. In the case of PLC cells, clathrin-mediated endocytosis and sialic acid receptor play a primary role in the uptake of PEAL-Gal. TEM results verify that PEAL and PEAL-Gal lead to a different influence on organelle morphology of HepG2, Huh7 and PLC cells. In addition, the results of intracellular distribution reveal that PEAL and PEAL-Gal are less entrapped in the lysosomes of HepG2 and Huh7 cells, demonstrating that they effectively escape from lysosomes and contribute to enhance the efficiency of intracellular delivery and tumor therapy. In vivo tumor targeting image results demonstrate that PEAL-Gal specifically delivers Rhodamine B (Rb) to the tumor tissue of mice with HepG2, Huh7, and PLC hepatomas and remains at a high concentration in tumor tissue until 48 h, properties that will greatly contribute to enhanced antitumor efficiency.

  15. Biocompatible multilayer capsules engineered with a graphene oxide derivative: synthesis, characterization and cellular uptake

    NASA Astrophysics Data System (ADS)

    Del Mercato, Loretta L.; Guerra, Flora; Lazzari, Gianpiero; Nobile, Concetta; Bucci, Cecilia; Rinaldi, Rosaria

    2016-03-01

    Graphene-based capsules have strong potential for a number of applications, including drug/gene delivery, tissue engineering, sensors, catalysis and reactors. The ability to integrate graphene into carrier systems with three-dimensional (3D) geometry may open new perspectives both for fundamental tests of graphene mechanics and for novel (bio)technological applications. However, the assembly of 3D complexes from graphene or its derivatives is challenging because of its poor stability under biological conditions. In this work, we attempted to integrate a layer of graphene oxide derivative into the shell of biodegradable capsules by exploiting a facile layer-by-layer (LbL) protocol. As a first step we optimized the LbL protocol to obtain colloidal suspensions of isolated capsules embedding the graphene oxide derivative. As a following step, we investigated in detail the morphological properties of the hybrid capsules, and how the graphene oxide derivative layer influences the porosity and the robustness of the multilayer composite shells. Finally, we verified the uptake of the capsules modified with the GO derivative by two cell lines and studied their intracellular localization and biocompatibility. As compared to pristine capsules, the graphene-modified capsules possess reduced porosity, reduced shell thickness and a higher stability against osmotic pressure. They show remarkable biocompatibility towards the tested cells and long-term colloidal stability and dispersion. By combining the excellent mechanical properties of a graphene oxide derivative with the high versatility of the LbL method, robust and flexible biocompatible polymeric capsules with novel characteristics have been fabricated.Graphene-based capsules have strong potential for a number of applications, including drug/gene delivery, tissue engineering, sensors, catalysis and reactors. The ability to integrate graphene into carrier systems with three-dimensional (3D) geometry may open new perspectives

  16. Iron(III) complexes of a pyridoxal Schiff base for enhanced cellular uptake with selectivity and remarkable photocytotoxicity.

    PubMed

    Basu, Uttara; Pant, Ila; Hussain, Akhtar; Kondaiah, Paturu; Chakravarty, Akhil R

    2015-04-20

    Iron(III) complexes of pyridoxal (vitamin B6, VB6) or salicylaldehyde Schiff bases and modified dipicolylamines, namely, [Fe(B)(L)](NO3) (1-5), where B is phenyl-N,N-bis((pyridin-2-yl)methyl)methanamine (phbpa in 1), (anthracen-9-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (anbpa in 2, 4) and (pyren-1-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (pybpa in 3, 5) (H2L(1) is 3-hydroxy-5-(hydroxymethyl)-4-(((2-hydroxyphenyl)imino)methyl)-2-methylpyridine (1-3) and H2L(2) is 2-[(2-hydroxyphenyl-imino)methyl]phenol), were prepared and their uptake in cancer cells and photocytotoxicity were studied. Complexes 4 and 5, having a non-pyridoxal Schiff base, were prepared to probe the role of the pyridoxal group in tumor targeting and cellular uptake. The PF6 salt (1a) of complex 1 is structurally characterized. The complexes have a distorted six-coordinate FeN4O2 core where the metal is in the +3 oxidation state with five unpaired electrons. The complexes display a ligand to metal charge transfer band near 520 and 420 nm from phenolate to the iron(III) center. The photophysical properties of the complexes are explained from the time dependent density functional theory calculations. The redox active complexes show a quasi-reversible Fe(III)/Fe(II) response near -0.3 V vs saturated calomel electrode. Complexes 2 and 3 exhibit remarkable photocytotoxicity in various cancer cells with IC50 values ranging from 0.4 to 5 μM with 10-fold lower dark toxicity. The cell death proceeded by the apoptotic pathway due to generation of reactive oxygen species upon light exposure. The nonvitamin complexes 4 and 5 display 3-fold lower photocytotoxicity compared to their VB6 analogues, possibly due to preferential and faster uptake of the vitamin complexes in the cancer cells. Complexes 2 and 3 show significant uptake in the endoplasmic reticulum, while complexes 4 and 5 are distributed throughout the cells without any specific localization pattern.

  17. Cytotoxicity and cellular uptake of ZnS:Mn nanocrystals biofunctionalized with chitosan and aminoacids

    NASA Astrophysics Data System (ADS)

    Sajimol Augustine, M.; Anas, Abdulaziz; Das, Ani V.; Sreekanth, S.; Jayalekshmi, S.

    2015-02-01

    Highly luminescent, manganese doped, zinc sulphide (ZnS:Mn) nanocrystals biofunctionalized with chitosan and various aminoacids such as L-citrulline, L-lysine, L-arginine, L-serine, L-histidine and glycine were synthesized by chemical capping co-precipitation method at room temperature, which is a simple and cost effective technique. The synthesized nanocrystals were structurally characterized by TEM, XRD, EDXS and FT-IR spectroscopy techniques. They possess high colloidal stability with strong orange red photoluminescence emission at 598 nm. The intensity of orange red emission has been observed to be maximum in L-citrulline capped ZnS:Mn nanocrystals in which the emission at 420 nm is effectively quenched by surface passivation due to capping. Taking into consideration the prospects of these highly luminescent, bio-compatible ZnS:Mn nanocrystals in bio-imaging applications, cytotoxicity studies were conducted to identify the capping combination which would accomplish minimum toxic effects. ZnS:Mn nanocrystals biofunctionalized with chitosan, L-citrulline, glycine, L-artginine, L-serine and L-histidine showed least toxicity up to 10 nM concentrations in mouse fibroblast L929 cells, which further confirms their cytocompatibility. Also the ZnS:Mn nanocrystals biofunctionalized with L-arginine showed maximum uptake in in vitro studies carried out in human embryonic kidney cells, HEK-293T, which shows the significant role of this particular amino acid in fetoplacental nutrition. The present study highlights the suitability of aminoacid conjugated ZnS:Mn nanocrystals, as promising candidates for biomedical applications.

  18. Molybdate uptake by Agrobacterium tumefaciens correlates with the cellular molybdenum cofactor status.

    PubMed

    Hoffmann, Marie-Christine; Ali, Koral; Sonnenschein, Marleen; Robrahn, Laura; Strauss, Daria; Narberhaus, Franz; Masepohl, Bernd

    2016-09-01

    Many enzymes require the molybdenum cofactor, Moco. Under Mo-limiting conditions, the high-affinity ABC transporter ModABC permits molybdate uptake and Moco biosynthesis in bacteria. Under Mo-replete conditions, Escherichia coli represses modABC transcription by the one-component regulator, ModE, consisting of a DNA-binding and a molybdate-sensing domain. Instead of a full-length ModE protein, many bacteria have a shorter ModE protein, ModE(S) , consisting of a DNA-binding domain only. Here, we asked how such proteins sense the intracellular molybdenum status. We show that the Agrobacterium tumefaciens ModE(S) protein Atu2564 is essential for modABC repression. ModE(S) binds two Mo-boxes in the modA promoter as shown by electrophoretic mobility shift assays. Northern analysis revealed cotranscription of modE(S) with the upstream gene, atu2565, which was dispensable for ModE(S) activity. To identify genes controlling ModE(S) function, we performed transposon mutagenesis. Tn5 insertions resulting in derepressed modA transcription mapped to the atu2565-modE(S) operon and several Moco biosynthesis genes. We conclude that A. tumefaciens ModE(S) activity responds to Moco availability rather than to molybdate concentration directly, as is the case for E. coli ModE. Similar results in Sinorhizobium meliloti suggest that Moco dependence is a common feature of ModE(S) regulators.

  19. Molybdate uptake by Agrobacterium tumefaciens correlates with the cellular molybdenum cofactor status.

    PubMed

    Hoffmann, Marie-Christine; Ali, Koral; Sonnenschein, Marleen; Robrahn, Laura; Strauss, Daria; Narberhaus, Franz; Masepohl, Bernd

    2016-09-01

    Many enzymes require the molybdenum cofactor, Moco. Under Mo-limiting conditions, the high-affinity ABC transporter ModABC permits molybdate uptake and Moco biosynthesis in bacteria. Under Mo-replete conditions, Escherichia coli represses modABC transcription by the one-component regulator, ModE, consisting of a DNA-binding and a molybdate-sensing domain. Instead of a full-length ModE protein, many bacteria have a shorter ModE protein, ModE(S) , consisting of a DNA-binding domain only. Here, we asked how such proteins sense the intracellular molybdenum status. We show that the Agrobacterium tumefaciens ModE(S) protein Atu2564 is essential for modABC repression. ModE(S) binds two Mo-boxes in the modA promoter as shown by electrophoretic mobility shift assays. Northern analysis revealed cotranscription of modE(S) with the upstream gene, atu2565, which was dispensable for ModE(S) activity. To identify genes controlling ModE(S) function, we performed transposon mutagenesis. Tn5 insertions resulting in derepressed modA transcription mapped to the atu2565-modE(S) operon and several Moco biosynthesis genes. We conclude that A. tumefaciens ModE(S) activity responds to Moco availability rather than to molybdate concentration directly, as is the case for E. coli ModE. Similar results in Sinorhizobium meliloti suggest that Moco dependence is a common feature of ModE(S) regulators. PMID:27196733

  20. Enhanced cellular uptake of a glutathione selective fluorogenic probe encapsulated in nanoparticles.

    PubMed

    Główka, Eliza; Lamprecht, Alf; Ubrich, Nathalie; Maincent, Philippe; Lulek, Janina; Coulon, Joël; Leroy, Pierre

    2006-05-28

    Selective fluorogenic probes for the labelling of intracellular reduced glutathione (GSH), i.e. ortho-phthaldialdehyde (OPA) and naphthalene-2,3-dicarboxaldehyde (NDA), have been encapsulated in polymeric nanoparticles (NPs) and the ability of the NPs to enhance uptake of the probe by microbial cells has been evaluated. Preparation of the probe-loaded NPs composed of Eudragit(®) E was based on an oil-in-water emulsification solvent evaporation method using an ultrasonic probe and polyvinyl alcohol as the surfactant. The encapsulation efficiency of the probes in lyophilized NPs was determined using high performance liquid chromatography (HPLC). A higher encapsulation rate of NDA than OPA was found: 47.6 ± 9.9 (n = 6) and 2.1 ± 0.2% (n = 3), respectively. The NDA-loaded particle diameter and zeta potential were 224.6 ± 14.7 nm and +40.9 ± 6.5 mV, respectively. After 20 min incubation of cultured Candida albicans yeast cells with either free NDA or NDA-loaded NPs (final NDA concentration 100 µM), cells were harvested and corresponding lysates were analysed using HPLC coupled with spectrofluorimetric detection. Incubation of cells with NDA-loaded NPs increased intracellular levels of NDA-GSH adduct by about nine-fold in comparison with the free probe. Adhesion on the cells and the penetration behaviour of NPs loaded with either NDA or fluorescent label (Nile Red) were characterized qualitatively by confocal laser scanning microscopy. PMID:21727503

  1. PCSK9 and LDLR The Yin-Yang in the Cellular Uptake of Cholesterol.

    PubMed

    Morales-Villegas, E

    2014-07-01

    The intracellular concentration of cholesterol is a vital constant influenced by the uptake, metabolism and excretion of cholesterol. The synthesis and expression of the PCSK9-LDLR duo is one of the most important mechanisms to regulate this constant; in a physiological state, the yin-yang balance between PCSK9 and LDLR regulates the entry of cholesterol into the cell to keep the intracellular cholesterol concentration stable. The mapping of the human gene encoding the serine protease PCSK9, located at 1p32-3, has allowed the identification of mutations with "gain" and "loss" of protease functions. Gain of function mutations causes decreased LDLR resulting in increased LDL-C and increased incidence of cardiovascular events. Loss of function mutations have opposite effect, increased LDLR, decreased LDL-C and decreased incidence of cardiovascular events. The identification of human mutations with PCSK9 "loss" of function demostrated the benefit of decreased PCSK9 and opened the door to developing new anti-PCSK9 therapies. The goal of this research area is to reduce the incidence of cardiovascular events beyond statins; the strategy is to mimic the state of PCSK9 "loss" of function by tactics as oligonucleotide therapies targeting PCSK9 mRNA and/or biological therapies with human monoclonal antibodies targeting PCSK9. This chapter reviews, the characteristics of the PCSK9, the physiological significance of the PCSK9-LDLR duo, and the therapeutic implications of the human genetic models of PCSK9 "loss" of function. The phase I-II clinical trial data of two promising monoclonal antibodies to PCSK9, Alirocumab formerly SAR236553/REGN727and AMG145 will be presented. PMID:24993279

  2. Biocompatible multilayer capsules engineered with a graphene oxide derivative: synthesis, characterization and cellular uptake.

    PubMed

    del Mercato, Loretta L; Guerra, Flora; Lazzari, Gianpiero; Nobile, Concetta; Bucci, Cecilia; Rinaldi, Rosaria

    2016-04-14

    Graphene-based capsules have strong potential for a number of applications, including drug/gene delivery, tissue engineering, sensors, catalysis and reactors. The ability to integrate graphene into carrier systems with three-dimensional (3D) geometry may open new perspectives both for fundamental tests of graphene mechanics and for novel (bio)technological applications. However, the assembly of 3D complexes from graphene or its derivatives is challenging because of its poor stability under biological conditions. In this work, we attempted to integrate a layer of graphene oxide derivative into the shell of biodegradable capsules by exploiting a facile layer-by-layer (LbL) protocol. As a first step we optimized the LbL protocol to obtain colloidal suspensions of isolated capsules embedding the graphene oxide derivative. As a following step, we investigated in detail the morphological properties of the hybrid capsules, and how the graphene oxide derivative layer influences the porosity and the robustness of the multilayer composite shells. Finally, we verified the uptake of the capsules modified with the GO derivative by two cell lines and studied their intracellular localization and biocompatibility. As compared to pristine capsules, the graphene-modified capsules possess reduced porosity, reduced shell thickness and a higher stability against osmotic pressure. They show remarkable biocompatibility towards the tested cells and long-term colloidal stability and dispersion. By combining the excellent mechanical properties of a graphene oxide derivative with the high versatility of the LbL method, robust and flexible biocompatible polymeric capsules with novel characteristics have been fabricated. PMID:26892453

  3. Branched amphiphilic peptide capsules: cellular uptake and retention of encapsulated solutes.

    PubMed

    Sukthankar, Pinakin; Avila, L Adriana; Whitaker, Susan K; Iwamoto, Takeo; Morgenstern, Alfred; Apostolidis, Christos; Liu, Ke; Hanzlik, Robert P; Dadachova, Ekaterina; Tomich, John M

    2014-09-01

    Branched amphiphilic peptide capsules (BAPCs) are peptide nano-spheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble to form bilayer delimited capsules. In two recent publications we described the lipid analogous characteristics of our BAPCs, examined their initial assembly, mode of fusion, solute encapsulation, and resizing and delineated their capability to be maintained at a specific size by storing them at 4°C. In this report we describe the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs in vivo. The ability of our constructs to retain alpha particle emitting radionuclides without any apparent leakage and their persistence in the peri-nuclear region of the cell for extended periods of time, coupled with their ease of preparation and potential tune-ability, makes them attractive as biocompatible carriers for targeted cancer therapy using particle emitting radioisotopes. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

  4. Branched amphiphilic peptide capsules: Cellular uptake and retention of encapsulated solutes☆

    PubMed Central

    Sukthankar, Pinakin; Avila, L. Adriana; Whitaker, Susan K.; Iwamoto, Takeo; Morgenstern, Alfred; Apostolidis, Christos; Liu, Ke; Hanzlik, Robert P.; Dadachova, Ekaterina; Tomich, John M.

    2014-01-01

    Branched amphiphilic peptide capsules (BAPCs) are peptide nanospheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble to form bi-layer delimited capsules. In two recent publications we described the lipid analogous characteristics of our BAPCs, examined their initial assembly, mode of fusion, solute encapsulation, and resizing and delineated their capability to be maintained at a specific size by storing them at 4 °C. In this report we describe the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs in vivo. The ability of our constructs to retain alpha particle emitting radionuclides without any apparent leakage and their persistence in the peri-nuclear region of the cell for extended periods of time, coupled with their ease of preparation and potential tune-ability, makes them attractive as biocompatible carriers for targeted cancer therapy using particle emitting radioisotopes. This article is part of a Special Issue entitled: Interfacially active peptides and proteins. PMID:24565797

  5. Branched amphiphilic peptide capsules: cellular uptake and retention of encapsulated solutes.

    PubMed

    Sukthankar, Pinakin; Avila, L Adriana; Whitaker, Susan K; Iwamoto, Takeo; Morgenstern, Alfred; Apostolidis, Christos; Liu, Ke; Hanzlik, Robert P; Dadachova, Ekaterina; Tomich, John M

    2014-09-01

    Branched amphiphilic peptide capsules (BAPCs) are peptide nano-spheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble to form bilayer delimited capsules. In two recent publications we described the lipid analogous characteristics of our BAPCs, examined their initial assembly, mode of fusion, solute encapsulation, and resizing and delineated their capability to be maintained at a specific size by storing them at 4°C. In this report we describe the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs in vivo. The ability of our constructs to retain alpha particle emitting radionuclides without any apparent leakage and their persistence in the peri-nuclear region of the cell for extended periods of time, coupled with their ease of preparation and potential tune-ability, makes them attractive as biocompatible carriers for targeted cancer therapy using particle emitting radioisotopes. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. PMID:24565797

  6. Enhanced Cellular Uptake of Albumin-Based Lyophilisomes when Functionalized with Cell-Penetrating Peptide TAT in HeLa Cells

    PubMed Central

    van Bracht, Etienne; Versteegden, Luuk R. M.; Stolle, Sarah; Verdurmen, Wouter P. R.; Woestenenk, Rob; Raavé, René; Hafmans, Theo; Oosterwijk, Egbert; Brock, Roland; van Kuppevelt, Toin H.; Daamen, Willeke F.

    2014-01-01

    Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells. PMID:25369131

  7. Membrane Surface-Associated Helices Promote Lipid Interactions and Cellular Uptake of Human Calcitonin-Derived Cell Penetrating Peptides

    PubMed Central

    Herbig, Michael E.; Weller, Kathrin; Krauss, Ulrike; Beck-Sickinger, Annette G.; Merkle, Hans P.; Zerbe, Oliver

    2005-01-01

    hCT(9-32) is a human calcitonin (hCT)-derived cell-penetrating peptide that has been shown to translocate the plasma membrane of mammalian cells. It has been suggested as a cellular carrier for drugs, green fluorescent protein, and plasmid DNA. Because of its temperature-dependent cellular translocation resulting in punctuated cytoplasmatic distribution, its uptake is likely to follow an endocytic pathway. To gain insight into the molecular orientation of hCT(9-32) when interacting with lipid models, and to learn more about its mode of action, various biophysical techniques from liposome partitioning to high-resolution NMR spectroscopy were utilized. Moreover, to establish the role of individual residues for the topology of its association with the lipid membrane, two mutants of hCT(9-32), i.e., W30-hCT(9-32) and A23-hCT(9-32), were also investigated. Although unstructured in aqueous solution, hCT(9-32) adopted two short helical stretches when bound to dodecylphosphocholine micelles, extending from Thr10 to Asn17 and from Gln24 to Val29. A23-hCT(9-32), in which the helix-breaking Pro23 was replaced by Ala, displayed a continuous α-helix extending from residue 12 to 26. Probing with the spin label 5-doxylstearate revealed that association with dodecylphosphocholine micelles was such that the helix engaged in parallel orientation to the micelle surface. Moreover, the Gly to Trp exchange in W30-hCT(9-32) resulted in a more stable anchoring of the C-terminal segment close to the interface, as reflected by a twofold increase in the partition coefficient in liposomes. Interestingly, tighter binding to model membranes was associated with an increase in the in vitro uptake in human cervix epithelial andenocarcinoma cell line cells. Liposome leakage studies excluded pore formation, and the punctuated fluorescence pattern of internalized peptide indicated vesicular localization and, in conclusion, strongly suggested an endocytic pathway of translocation. PMID:16183886

  8. The aspect ratio effect of drug nanocrystals on cellular internalization efficiency, uptake mechanisms, and in vitro and in vivo anticancer efficiencies

    NASA Astrophysics Data System (ADS)

    Tian, Baishun; Zhang, Xiujuan; Yu, Caitong; Zhou, Mengjiao; Zhang, Xiaohong

    2015-02-01

    In this paper, we investigated the aspect ratio (AR) effect of anticancer drug nanocrystals (NCs) on their cellular internalization efficiency, uptake mechanisms, biodistributions as well as in vitro and in vivo antitumor efficiencies. Both confocal imaging and flow cytometry show that shorter NCs with AR = 1.3 have a much faster cellular uptake rate and a much higher anticancer efficacy than longer NCs. All NCs with different ARs were found to enter the cells via an energy-dependent clathrin-mediated pathway. In vivo experiments indicate that NCs with higher ARs have a shorter half-life and are more easily captured by the liver, while the corresponding tumor uptake decreased. We also observed that NCs with the smallest AR have the highest therapeutic efficacy with appreciably less weight loss. These results would assist in the future design of drug NCs and may lead to the development of new drug nanostructures for biomedical applications.In this paper, we investigated the aspect ratio (AR) effect of anticancer drug nanocrystals (NCs) on their cellular internalization efficiency, uptake mechanisms, biodistributions as well as in vitro and in vivo antitumor efficiencies. Both confocal imaging and flow cytometry show that shorter NCs with AR = 1.3 have a much faster cellular uptake rate and a much higher anticancer efficacy than longer NCs. All NCs with different ARs were found to enter the cells via an energy-dependent clathrin-mediated pathway. In vivo experiments indicate that NCs with higher ARs have a shorter half-life and are more easily captured by the liver, while the corresponding tumor uptake decreased. We also observed that NCs with the smallest AR have the highest therapeutic efficacy with appreciably less weight loss. These results would assist in the future design of drug NCs and may lead to the development of new drug nanostructures for biomedical applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06743f

  9. Multi-functional bio-synthetic hybrid nanostructures for enhanced cellular uptake, endosomal escape and targeted delivery toward diagnostics and therapeutics

    NASA Astrophysics Data System (ADS)

    Shrestha, Ritu

    Applications of nanotechnology in medicine, also known as nanomedicine, is a rapidly growing field as it holds great potential in the development of novel therapeutics toward treatment of various diseases. Shell crosslinked knedel-like nanoparticles (SCKs) that are self assembled from amphiphilic block copolymers into polymeric micelles followed by crosslinking selectively throughout the shell domain have been investigated as theranostic agents for the delivery of nucleic acids and incorporation of imaging probes. The main focus of this dissertation is to design and develop unique multifunctional bio-synthetic hybrid nanoparticles that can carry agents for radiolabeling, moieties for inducing stealth properties to minimize protein adsorption in vivo, ligands for site-specific targeting, therapeutic payloads, and are optimized for efficient delivery of cargoes intracellularly and to the target sites toward constructing novel nanoscopic objects for therapy and diagnosis. Alteration of polymeric building blocks of the nanoparticles provides opportunities for precise control over the sizes, shapes, compositions, structures and properties of the nanoparticles. To ensure ideal performance of nanoparticles as theranostic agents, it is critical to ensure high intracellular bioavailability of the therapeutic payload conjugated to nanoparticles. Special efforts were made by employing well-defined multi-step polymerization and polymer modification reactions that involved conjugation of peptide nucleic acids (PNAs) to chain terminus of poly(ethylene glycol) (PEG) chain grafts such that they were presented at the outermost surface of SCKs. Additionally, chemical modification reactions were performed on the polymer backbone to integrate positive charges onto the shell of the nanoparticles to afford cationic SCKs (cSCKs) for facilitating cellular entry and electrostatic interactions with negatively charged nucleic acids. Covalent conjugation of F3, a tumor homing peptide, post

  10. Protease-Triggered, Integrin-Targeted Cellular Uptake of Recombinant Protein Micelles.

    PubMed

    Gao, Chen; Vargo, Kevin B; Hammer, Daniel A

    2016-09-01

    Targeting nanoparticles for drug delivery has great potential for improving efficacy and reducing side effects from systemic toxicity. New developments in the assembly of materials afford the opportunity to expose cryptic targeting domains in tissue-specific microenvironments in which certain proteases are expressed. Here, recombinant proteins are designed to combine the responsiveness to environmental proteases with specific targeting. Materials made recombinantly allow complete control over amino acid sequence, in which each molecule is identically functionalized. Previously, oleosin, a naturally occurring plant protein that acts as a surfactant, has been engineered to self-assemble into spherical micelles-a useful structure for drug delivery. To make oleosins that are locally activated to bind receptors, oleosin is genetically modified to incorporate the integrin-binding motif RGDS just behind a domain cleavable by thrombin. The resulting modified oleosin self-assembles into spherical micelles in aqueous environments, with the RGDS motif protected by the thrombin-cleavable domain. Upon the addition of thrombin, the RGDS is exposed and the binding of the spherical micelles to breast cancer cells is increased fourfold. PMID:27284959

  11. Intradermal Gene Immunization: The Possible Role of DNA Uptake in the Induction of Cellular Immunity to Viruses

    NASA Astrophysics Data System (ADS)

    Raz, Eyal; Carson, Dennis A.; Parker, Suezanne E.; Parr, Tyler B.; Abai, Anna M.; Aichinger, Gerald; Gromkowski, Stanislaw H.; Singh, Malini; Lew, Denise; Yankauckas, Michelle A.; Baird, Stephen M.; Rhodes, Gary H.

    1994-09-01

    The skin and mucous membranes are the anatomical sites where most viruses are first encountered by the immune system. Previous experiments have suggested that striated muscle cells are unique among mammalian cell types in their capacity to take up and express free DNA in the absence of a viral vector or physical carrier. However, we have found that mice injected into the superficial skin with free (naked) plasmid DNA encoding the influenza nucleoprotein gene had discrete foci of epidermal and dermal cells, including cells with dendritic morphology, that contained immunoreactive nucleoprotein antigen. A single intradermal administration of 0.3-15 μ g of free plasmid DNA induced anti-nucleoprotein-specific antibody and cytotoxic T lymphocytes that persisted for at least 68-70 weeks after vaccination. Intradermal gene administration induced higher antibody titers than did direct gene injection into skeletal muscle and did not cause local inflammation or necrosis. Compared with control animals, the gene-injected mice were resistant to challenge with a heterologous strain of influenza virus. These results indicate that the cells of the skin can take up and express free foreign DNA and induce cellular and humoral immune responses against the encoded protein. We suggest that DNA uptake by the skin-associated lymphoid tissues may play a role in the induction of cytotoxic T cells against viruses and other intracellular pathogens.

  12. Real-time and label-free monitoring of nanoparticle cellular uptake using capacitance-based assays.

    PubMed

    Lee, Rimi; Jo, Dong Hyun; Chung, Sang J; Na, Hee-Kyung; Kim, Jeong Hun; Lee, Tae Geol

    2016-01-01

    Nanoparticles have shown great potential as vehicles for the delivery of drugs, nucleic acids, and therapeutic proteins; an efficient, high-throughput screening method to analyze nanoparticle interaction with the cytomembrane would substantially improve the efficiency and accuracy of the delivery. Here, we developed a capacitance sensor array that monitored the capacitance values of nanoparticle-treated cells in a real-time manner, without the need for labeling. Upon cellular uptake of the nanoparticles, a capacitance peak was observed at a low frequency (e.g., 100 Hz) as a function of time based on zeta potential changes. In the high frequency region (e.g., 15-20 kHz), the rate of decreasing capacitance slowed as a function of time compared to the cell growth control group, due to increased cytoplasm resistance and decreased membrane capacitance and resistance. The information provided by our capacitance sensor array will be a powerful tool for scientists designing nanoparticles for specific purposes. PMID:27641838

  13. Effects of Graphene Oxide and Oxidized Carbon Nanotubes on the Cellular Division, Microstructure, Uptake, Oxidative Stress, and Metabolic Profiles.

    PubMed

    Hu, Xiangang; Ouyang, Shaohu; Mu, Li; An, Jing; Zhou, Qixing

    2015-09-15

    Nanomaterial oxides are common formations of nanomaterials in the natural environment. Herein, the nanotoxicology of typical graphene oxide (GO) and carboxyl single-walled carbon nanotubes (C-SWCNT) was compared. The results showed that cell division of Chlorella vulgaris was promoted at 24 h and then inhibited at 96 h after nanomaterial exposure. At 96 h, GO and C-SWCNT inhibited the rates of cell division by 0.08-15% and 0.8-28.3%, respectively. Both GO and C-SWCNT covered the cell surface, but the uptake percentage of C-SWCNT was 2-fold higher than that of GO. C-SWCNT induced stronger plasmolysis and mitochondrial membrane potential loss and decreased the cell viability to a greater extent than GO. Moreover, C-SWCNT-exposed cells exhibited more starch grains and lysosome formation and higher reactive oxygen species (ROS) levels than GO-exposed cells. Metabolomics analysis revealed significant differences in the metabolic profiles among the control, C-SWCNT and GO groups. The metabolisms of alkanes, lysine, octadecadienoic acid and valine was associated with ROS and could be considered as new biomarkers of ROS. The nanotoxicological mechanisms involved the inhibition of fatty acid, amino acid and small molecule acid metabolisms. These findings provide new insights into the effects of GO and C-SWCNT on cellular responses. PMID:26295980

  14. Dual-drug delivery by porous silicon nanoparticles for improved cellular uptake, sustained release, and combination therapy.

    PubMed

    Wang, Chang-Fang; Mäkilä, Ermei M; Kaasalainen, Martti H; Hagström, Marja V; Salonen, Jarno J; Hirvonen, Jouni T; Santos, Hélder A

    2015-04-01

    Dual-drug delivery of antiangiogenic and chemotherapeutic drugs can enhance the therapeutic effect for cancer therapy. Conjugation of methotrexate (MTX) to porous silicon (PSi) nanoparticles (MTX-PSi) with positively charged surface can improve the cellular uptake of MTX and inhibit the proliferation of cancer cells. Herein, MTX-PSi conjugates sustained the release of MTX up to 96 h, and the released fragments including MTX were confirmed by mass spectrometry. The intracellular distribution of the MTX-PSi nanoparticles was confirmed by transmission electron microscopy. Compared to pure MTX, the MTX-PSi achieved similar inhibition of cell proliferation in folate receptor (FR) over-expressing U87 MG cancer cells, and a higher effect in low FR-expressing EA.hy926 cells. Nuclear fragmentation analysis demonstrated programmed cell apoptosis of MTX-PSi in the high/low FR-expressing cancer cells, whereas PSi alone at the same dose had a minor effect on cell apoptosis. Finally, the porous structure of MTX-PSi enabled a successful concomitant loading of another anti-angiogenic hydrophobic drug, sorafenib, and considerably enhanced the dissolution rate of sorafenib. Overall, the MTX-PSi nanoparticles can be used as a platform for combination chemotherapy by simultaneously enhancing the dissolution rate of a hydrophobic drug and sustaining the release of a conjugated chemotherapeutic drug.

  15. Effects of Graphene Oxide and Oxidized Carbon Nanotubes on the Cellular Division, Microstructure, Uptake, Oxidative Stress, and Metabolic Profiles.

    PubMed

    Hu, Xiangang; Ouyang, Shaohu; Mu, Li; An, Jing; Zhou, Qixing

    2015-09-15

    Nanomaterial oxides are common formations of nanomaterials in the natural environment. Herein, the nanotoxicology of typical graphene oxide (GO) and carboxyl single-walled carbon nanotubes (C-SWCNT) was compared. The results showed that cell division of Chlorella vulgaris was promoted at 24 h and then inhibited at 96 h after nanomaterial exposure. At 96 h, GO and C-SWCNT inhibited the rates of cell division by 0.08-15% and 0.8-28.3%, respectively. Both GO and C-SWCNT covered the cell surface, but the uptake percentage of C-SWCNT was 2-fold higher than that of GO. C-SWCNT induced stronger plasmolysis and mitochondrial membrane potential loss and decreased the cell viability to a greater extent than GO. Moreover, C-SWCNT-exposed cells exhibited more starch grains and lysosome formation and higher reactive oxygen species (ROS) levels than GO-exposed cells. Metabolomics analysis revealed significant differences in the metabolic profiles among the control, C-SWCNT and GO groups. The metabolisms of alkanes, lysine, octadecadienoic acid and valine was associated with ROS and could be considered as new biomarkers of ROS. The nanotoxicological mechanisms involved the inhibition of fatty acid, amino acid and small molecule acid metabolisms. These findings provide new insights into the effects of GO and C-SWCNT on cellular responses.

  16. Magnetic nanoparticles to recover cellular organelles and study the time resolved nanoparticle-cell interactome throughout uptake.

    PubMed

    Bertoli, Filippo; Davies, Gemma-Louise; Monopoli, Marco P; Moloney, Micheal; Gun'ko, Yurii K; Salvati, Anna; Dawson, Kenneth A

    2014-08-27

    Nanoparticles in contact with cells and living organisms generate quite novel interactions at the interface between the nanoparticle surface and the surrounding biological environment. However, a detailed time resolved molecular level description of the evolving interactions as nanoparticles are internalized and trafficked within the cellular environment is still missing and will certainly be required for the emerging arena of nanoparticle-cell interactions to mature. In this paper promising methodologies to map out the time resolved nanoparticle-cell interactome for nanoparticle uptake are discussed. Thus silica coated magnetite nanoparticles are presented to cells and their magnetic properties used to isolate, in a time resolved manner, the organelles containing the nanoparticles. Characterization of the recovered fractions shows that different cell compartments are isolated at different times, in agreement with imaging results on nanoparticle intracellular location. Subsequently the internalized nanoparticles can be further isolated from the recovered organelles, allowing the study of the most tightly nanoparticle-bound biomolecules, analogous to the 'hard corona' that so far has mostly been characterized in extracellular environments. Preliminary data on the recovered nanoparticles suggest that significant portion of the original corona (derived from the serum in which particles are presented to the cells) is preserved as nanoparticles are trafficked through the cells.

  17. Real-time and label-free monitoring of nanoparticle cellular uptake using capacitance-based assays

    PubMed Central

    Lee, Rimi; Jo, Dong hyun; Chung, Sang J.; Na, Hee-Kyung; Kim, Jeong Hun; Lee, Tae Geol

    2016-01-01

    Nanoparticles have shown great potential as vehicles for the delivery of drugs, nucleic acids, and therapeutic proteins; an efficient, high-throughput screening method to analyze nanoparticle interaction with the cytomembrane would substantially improve the efficiency and accuracy of the delivery. Here, we developed a capacitance sensor array that monitored the capacitance values of nanoparticle-treated cells in a real-time manner, without the need for labeling. Upon cellular uptake of the nanoparticles, a capacitance peak was observed at a low frequency (e.g., 100 Hz) as a function of time based on zeta potential changes. In the high frequency region (e.g., 15–20 kHz), the rate of decreasing capacitance slowed as a function of time compared to the cell growth control group, due to increased cytoplasm resistance and decreased membrane capacitance and resistance. The information provided by our capacitance sensor array will be a powerful tool for scientists designing nanoparticles for specific purposes. PMID:27641838

  18. Prevention of DNA photodamage by vitamin E compounds and sunscreens: roles of ultraviolet absorbance and cellular uptake.

    PubMed

    McVean, M; Liebler, D C

    1999-03-01

    Topical application of alpha-tocopherol (alphaTH), the most prominent naturally occurring form of vitamin E, inhibits ultraviolet (UV) B-induced photocarcinogenesis and DNA photodamage in C3H mice in vivo. In this study, we compared alphaTH with other vitamin E compounds and with three commercial sunscreen compounds for their ability to inhibit DNA photodamage in C3H mouse skin in vivo. When applied in a 5% dispersion in a neutral cream vehicle, alpha-tocopherol (alphaTH), gamma-tocopherol (gammaTH), and delta-tocopherol (deltaTH) each produced a statistically significant inhibition of thymine dimer formation, whereas alpha-tocopherol acetate (alphaTAc) and alpha-tocopherol methyl ether (alphaTOMe) did not. Application of 5% dispersions of the commercial sunscreen agent octylmethoxycinnamate also inhibited dimer formation, whereas ethylhexyl salicylate and oxybenzone did not, despite their considerably greater UVB absorbances than alphaTH. To test the hypothesis that cellular uptake and distribution are necessary for optimal photoprotection by tocopherols, photoprotection was studied in mouse 308 keratinocyte cells in vitro. Preincubation of 308 cells with 1 microM alphaTH for at least 2 h before exposure to 2.5 J/m2/s UVB for 10 min significantly (P < 0.05) attenuated thymine dimer formation. Pre-incubation with 1 microM gammaTH, deltaTH, alphaTAc, or alphaTOMe for 2 h did not inhibit thymine dimer formation significantly. Uptake of alphaTH was measured after incubation with 1 microM [2H3]alphaTH (d3-alphaTH) and resulted in a time-dependent increase in alphaTH levels. Use of d3-alphaTH allowed separate, simultaneous measurement of added d3-alphaTH and unlabeled endogenous alphaTH by gas chromatography-mass spectrometry. Accumulation of 167 +/- 62 pmol d3-alphaTH/mg protein was measured within 1 h in whole-cell fractions. d3-AlphaTH in the nuclear fraction reached levels of 15 +/- 4 pmol d3-alphaTH/mg protein at 2 h. Accumulation of alphaTH in the whole cell and

  19. Prevention of DNA photodamage by vitamin E compounds and sunscreens: roles of ultraviolet absorbance and cellular uptake.

    PubMed

    McVean, M; Liebler, D C

    1999-03-01

    Topical application of alpha-tocopherol (alphaTH), the most prominent naturally occurring form of vitamin E, inhibits ultraviolet (UV) B-induced photocarcinogenesis and DNA photodamage in C3H mice in vivo. In this study, we compared alphaTH with other vitamin E compounds and with three commercial sunscreen compounds for their ability to inhibit DNA photodamage in C3H mouse skin in vivo. When applied in a 5% dispersion in a neutral cream vehicle, alpha-tocopherol (alphaTH), gamma-tocopherol (gammaTH), and delta-tocopherol (deltaTH) each produced a statistically significant inhibition of thymine dimer formation, whereas alpha-tocopherol acetate (alphaTAc) and alpha-tocopherol methyl ether (alphaTOMe) did not. Application of 5% dispersions of the commercial sunscreen agent octylmethoxycinnamate also inhibited dimer formation, whereas ethylhexyl salicylate and oxybenzone did not, despite their considerably greater UVB absorbances than alphaTH. To test the hypothesis that cellular uptake and distribution are necessary for optimal photoprotection by tocopherols, photoprotection was studied in mouse 308 keratinocyte cells in vitro. Preincubation of 308 cells with 1 microM alphaTH for at least 2 h before exposure to 2.5 J/m2/s UVB for 10 min significantly (P < 0.05) attenuated thymine dimer formation. Pre-incubation with 1 microM gammaTH, deltaTH, alphaTAc, or alphaTOMe for 2 h did not inhibit thymine dimer formation significantly. Uptake of alphaTH was measured after incubation with 1 microM [2H3]alphaTH (d3-alphaTH) and resulted in a time-dependent increase in alphaTH levels. Use of d3-alphaTH allowed separate, simultaneous measurement of added d3-alphaTH and unlabeled endogenous alphaTH by gas chromatography-mass spectrometry. Accumulation of 167 +/- 62 pmol d3-alphaTH/mg protein was measured within 1 h in whole-cell fractions. d3-AlphaTH in the nuclear fraction reached levels of 15 +/- 4 pmol d3-alphaTH/mg protein at 2 h. Accumulation of alphaTH in the whole cell and

  20. Effect of crystals and fibrous network polymer additives on cellular morphology of microcellular foams

    NASA Astrophysics Data System (ADS)

    Miyamoto, Ryoma; Utano, Tatsumi; Yasuhara, Shunya; Ishihara, Shota; Ohshima, Masahiro

    2015-05-01

    In this study, the core-back foam injection molding was used for preparing microcelluar polypropylene (PP) foam with either a 1,3:2,4 bis-O-(4-methylbenzylidene)-D-sorbitol gelling agent (Gel-all MD) or a fibros network polymer additive (Metablen 3000). Both agent and addiive could effectively control the celluar morphology in foams but somehow different ways. In course of cooling the polymer with Gel-all MD in the mold caity, the agent enhanced the crystal nucleation and resulted in the large number of small crystals. The crystals acted as effective bubble nucleation agent in foaming process. Thus, the agent reduced the cell size and increased the cell density, drastically. Furthermore, the small crystals provided an inhomogenuity to the expanding cell wall and produced the high open cell content with nano-scale fibril structure. Gell-all as well as Metablene 3000 formed a gel-like fibrous network in melt. The network increased the elongational viscosity and tended to prevent the cell wall from breaking up. The foaming temperature window was widened by the presence of the network. Especially, the temperature window where the macro-fibrous structure was formed was expanded to the higher temperature. The effects of crystal nucleating agent and PTFE on crystals' size and number, viscoelsticity, rheological propreties of PP and cellular morphology were compared and thorougly investigated.

  1. BREFELDIN A INHIBITS CHOLESTEROL EFFLUX WITHOUT AFFECTING THE RATE OF CELLULAR UPTAKE AND RESECRETION OF APOLIPOPROTEIN A-I IN ADIPOCYTES

    PubMed Central

    Verghese, Philip B; Arrese, Estela L; Howard, Alisha D; Soulages, Jose L

    2008-01-01

    A possible role of cellular uptake and re-secretion of apoA-I in the mechanism of cholesterol efflux induced by apoA-I was investigated using a novel experimental approach. Incubation of adipocytes with a recombinant human apoA-I containing a consensus PKA phosphorylation site, pka-ApoA-I, leads to the appearance of phosphorylated protein in the cell culture medium unambiguously proving cellular uptake and re-secretion of pka-ApoA-I. Phosphorylation of apoA-I is abolished by PKA inhibitors and enhanced by PKA activators demonstrating the specific involvement of PKA. Studies on the concentration dependence of pka-apoA-I phosphorylation and competition experiments with human apoA-I suggest that apolipoprotein uptake is a receptor mediated process. A possible role of apoA-I recycling in the mechanism of cholesterol efflux was investigated by determining the rates of apoA-I induced cholesterol efflux and apoA-I recycling in the presence and in the absence of Brefeldin A (BFA). The studies showed that BFA strongly inhibits cholesterol efflux without affecting the rate of apoA-I recycling. Since BFA affects vesicular trafficking of ABCA1, this study suggests that the interaction of apoA-I with ABCA1 does not mediate apolipoprotein uptake and re-secretion. This result suggests that lipidation of apoA-I and apolipoprotein uptake/re-secretion are independent processes. PMID:18708026

  2. Influence of the side-chain length on the cellular uptake and the cytotoxicity of rhenium triscarbonyl derivatives: a bimodal infrared and luminescence quantitative study.

    PubMed

    Clède, Sylvain; Lambert, François; Saint-Fort, Rénette; Plamont, Marie-Aude; Bertrand, Hélène; Vessières, Anne; Policar, Clotilde

    2014-07-01

    Rhenium triscarbonyl complexes fac-[Re(CO)3 (N^N)] with appropriate ancillary N^N ligands are relevant for fluorescent bio-imaging. Recently, we have shown that [Re(CO)3 ] cores can also be efficiently mapped inside cells using their IR signature and that they can thus be used in a bimodal approach. To describe them we have coined the term SCoMPIs for single-core multimodal probes for imaging. In the context of the use of these SCoMPIs in bio-imaging, the questions of their cellular uptake and cytotoxicity are critical. We report here a series of compounds derived from the [Re(CO)3 Cl(pyta)] core (pyta=4-(2-pyridyl)-1,2,3-triazole). The pyta ligand is of interest because it can be easily functionalized. Aliphatic side chains (C4 , C8 , and C12 ) were appended to this core. A correlative study involving IR and luminescence was performed to monitor and quantify their cellular internalization. We studied the relationship between lipophilicity (log P(o/w)), cytotoxicity (IC50 ), and cellular uptake, and we showed that both uptake and cytotoxicity increase with the length of the side chain, with a higher uptake for the C12 derivative. This study stresses the distinction that has to be made between apparent toxicity, determined as an incubation concentration IC50 , and intrinsic toxicity. Indeed, the intrinsic toxicity of a compound can remain hidden if it is not cell permeable. Therefore it must be kept in mind that IC50 values are composite values, reflecting both cellular uptake and intrinsic toxicity.

  3. Stealth CD44-targeted hyaluronic acid supramolecular nanoassemblies for doxorubicin delivery: probing the effect of uncovalent pegylation degree on cellular uptake and blood long circulation.

    PubMed

    Han, Xiaopeng; Li, Zhenbao; Sun, Jin; Luo, Cong; Li, Lin; Liu, Yuhai; Du, Yuqian; Qiu, Shuhong; Ai, Xiaoyu; Wu, Chunnuan; Lian, He; He, Zhonggui

    2015-01-10

    Stealth active targeting nanoparticles (NPs) usually include two types of ligand sites: ligand anchored on distal ends of the polyethylene glycol (PEG) and ligand buried under pegylated layer. The latter typical case is hyaluronic acid (HA)-based NPs; however, there is little information available for the latter NPs about effect of the optimal density of surface PEG coating on the blood circulation time, cellular uptake and in vivo anticancer activity. Thus, in this study, in order to optimize the anticancer effects of HA-based NPs, we focus on how uncovalent pegylation degree modulates blood circulation time and cellular uptake of HA-based NPs. We firstly designed a new double-hydrophilic copolymer by conjugating HP-β-cyclodextrin with HA, and this carrier was further pegylated with adamantyl-peg (ADA-PEG) to form inclusion complex HA-HPCD/ADA-PEG, termed as HCPs. The supramolecular nanoassemblies were fabricated by host-guest and polar interactions between HCPs and doxorubicin (Dox), with vitamin E succinate (VES) being a nanobridge. Despite the active recognition between HA and CD44 receptor, the cellular uptake and targeting efficiency of HA-NPs decreased with the increasing peg density, demonstrating HA was partly buried by high density peg coating. However, the high density of peg coating was beneficial to long circulation time, tumor biodistribution and anticancer activity in vivo. NPs with 5% peg coating had the optimal cellular targeting efficiency in vitro and anticancer effects in vivo. The findings suggest that balancing long circulation property and cellular uptake is important to achieve the optimal antitumor efficacy for pegylated HA-based NPs, and that PEG coating densities cannot be extended beyond a certain density for shielding effect without compromising the efficacy of hyaluronic acid targeted delivery.

  4. Cellular Uptake of Aminoglycosides

    ERIC Educational Resources Information Center

    Steyger, Peter S.

    2005-01-01

    Aminoglycosides exert their cytotoxic effect at three different locations: at the cell surface, in the cytosol, or in the nucleus. At the cell surface, aminoglycoside binding can cause temporary hearing loss, motor paralysis at the neuromuscular junction, ion wasting in kidneys, or analgesia in mechano- and nocioreceptors (touch and pain sensory…

  5. Monocomponent hexa- and dodecaethylene glycol succinyl-tocopherol esters: self-assembly structures, cellular uptake and sensitivity to enzyme hydrolysis.

    PubMed

    Folmer, Britta M; Barron, Denis; Hughes, Eric; Miguet, Laurence; Sanchez, Belén; Heudi, Olivier; Rouvet, Martine; Sagalowicz, Laurent; Callier, Philippe; Michel, Martin; Williamson, Gary

    2009-12-15

    We have chemically synthesized two water-soluble forms of tocopherol succinate linked via an ester bond to hexaethylene glycol and dodecaethylene glycol. The self-assembly structure of the former in water is vesicular, whereas the latter forms elongated micelles. We treated Caco-2 cells with these compounds in these physical forms, in addition to a mixed micelle form. The intact compounds were taken up into the cells, influenced by both the chain length and the physical structure. In addition, the tocopherol derivatives were also metabolized into tocopherol succinate and tocopherol inside the cell. The total hydrolysis and uptake into the cells was two-fold higher from tocopherol hexaethylene glycol succinate in the form of mixed micelles than in vesicular form as assessed by analyzing intracellular tocopherol and tocopherol succinate. The longer polyethylene glycol chain gave a higher intracellular tocopherol succinate/tocopherol ratio. The major intracellular esterase in Caco-2 cells is carboxyl esterase 1 (EC 3.1.1.1), and in silico modelling studies show that the position of docking and hence the site of hydrolysis is influenced by the chain length. The in silico prediction is consistent with the in vitro data, since a longer chain length is predicted to favour hydrolysis of the ester bond between the succinate and polyethylene glycol moieties. PMID:19631613

  6. Structural parameters modulating the cellular uptake of disulfide-rich cyclic cell-penetrating peptides: MCoTI-II and SFTI-1.

    PubMed

    D'Souza, Charlotte; Henriques, Sónia Troeira; Wang, Conan K; Craik, David J

    2014-12-17

    Peptides are emerging as a new class of therapeutics due to their high potency and specificity for a range of targets, including the inhibition of protein-protein interactions. Disulfide-rich cyclic peptides, in particular, have attracted much attention in drug design due to their ultra-stable structure. Moreover, some of them have been shown to internalize into cells, which makes them potential scaffolds to deliver pharmaceutically bioactive sequences to intracellular targets. Here we examined the effects of structural modifications on the cell-penetrating properties of two disulfide-rich cyclic cell-penetrating peptides, Momordica cochinchinensis trypsin inhibitor II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1). We found that the cellular uptake of MCoTI-II can be improved by increasing the overall positive charge of the native sequence. On the other hand, mutations to SFTI-1 did not significantly influence its cellular uptake, suggesting a non-specific endocytosis-dependent mechanism of cellular uptake. This study provides an understanding of the structural features affecting the internalization of MCoTI-II and SFTI-1, and hence provides a guide for the development of these disulfide-rich cyclic scaffolds into potential drug leads. PMID:24985034

  7. Structural parameters modulating the cellular uptake of disulfide-rich cyclic cell-penetrating peptides: MCoTI-II and SFTI-1.

    PubMed

    D'Souza, Charlotte; Henriques, Sónia Troeira; Wang, Conan K; Craik, David J

    2014-12-17

    Peptides are emerging as a new class of therapeutics due to their high potency and specificity for a range of targets, including the inhibition of protein-protein interactions. Disulfide-rich cyclic peptides, in particular, have attracted much attention in drug design due to their ultra-stable structure. Moreover, some of them have been shown to internalize into cells, which makes them potential scaffolds to deliver pharmaceutically bioactive sequences to intracellular targets. Here we examined the effects of structural modifications on the cell-penetrating properties of two disulfide-rich cyclic cell-penetrating peptides, Momordica cochinchinensis trypsin inhibitor II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1). We found that the cellular uptake of MCoTI-II can be improved by increasing the overall positive charge of the native sequence. On the other hand, mutations to SFTI-1 did not significantly influence its cellular uptake, suggesting a non-specific endocytosis-dependent mechanism of cellular uptake. This study provides an understanding of the structural features affecting the internalization of MCoTI-II and SFTI-1, and hence provides a guide for the development of these disulfide-rich cyclic scaffolds into potential drug leads.

  8. Impacts of carbon source addition on denitrification and phosphorus uptake in enhanced biological phosphorus removal systems.

    PubMed

    Begum, Shamim A; Batista, Jacimaria R

    2013-01-01

    In this study, simultaneous denitrification and phosphorus (P) removal were investigated in batch tests using nitrified mixed liquor and secondary wastewater influent from a full-scale treatment plant and different levels of acetate and propionate as supplemental carbon sources. Without supplemental carbon source, denitrification occurred at low rate and P release and P uptake was negatively affected (i.e., P removal of only 59.7%). When acetate and propionate were supplied, denitrification and P release occurred simultaneously under anoxic conditions. For acetate and propionate at a C/N stoichiometric ratio of 7.6, P release was negatively affected by denitrification. For acetate, the percent P removal and denitrification were very similar for C/N ratios of 22 (5X stoichiometric) and 59 (10X stoichiometric). For propionate, both percent P removal and denitrification deteriorated for C/N ratios of 22 (5X stoichiometric) and 45 (10X stoichiometric). It was observed that carbon source added in excess to stoichiometric ratio was consumed in the aerobic zone, but P was not taken up. This implies that PAO bacteria may utilize the excess carbon source in the aerobic zone rather than their polyhydroxyalkanoate (PHA) reserves, thereby promoting deterioration of the system.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Surface-anchored poly(acryloyl-L(D)-valine) with enhanced chirality-selective effect on cellular uptake of gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Deng, Jun; Wu, Sai; Yao, Mengyun; Gao, Changyou

    2016-08-01

    Chirality is one of the ubiquitous phenomena in biological systems. The left handed (L-) amino acids and right handed (D-) sugars are normally found in proteins, and in RNAs and DNAs, respectively. The effect of chiral surfaces at the nanoscale on cellular uptake has, however, not been explored. This study reveals for the first time the molecular chirality on gold nanoparticles (AuNPs) functions as a direct regulator for cellular uptake. Monolayers of 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) and poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were formed on AuNPs surface, respectively. The internalized amount of PAV-AuNPs was several times larger than that of MAV-AuNPs by A549 and HepG2 cells, regardless of the chirality difference. However, the D-PAV-AuNPs were internalized with significantly larger amount than the L-PAV-AuNPs. This chirality-dependent uptake effect is likely attributed to the preferable interaction between the L-phospholipid-based cell membrane and the D-enantiomers.

  11. Surface-anchored poly(acryloyl-L(D)-valine) with enhanced chirality-selective effect on cellular uptake of gold nanoparticles

    PubMed Central

    Deng, Jun; Wu, Sai; Yao, Mengyun; Gao, Changyou

    2016-01-01

    Chirality is one of the ubiquitous phenomena in biological systems. The left handed (L-) amino acids and right handed (D-) sugars are normally found in proteins, and in RNAs and DNAs, respectively. The effect of chiral surfaces at the nanoscale on cellular uptake has, however, not been explored. This study reveals for the first time the molecular chirality on gold nanoparticles (AuNPs) functions as a direct regulator for cellular uptake. Monolayers of 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) and poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were formed on AuNPs surface, respectively. The internalized amount of PAV-AuNPs was several times larger than that of MAV-AuNPs by A549 and HepG2 cells, regardless of the chirality difference. However, the D-PAV-AuNPs were internalized with significantly larger amount than the L-PAV-AuNPs. This chirality-dependent uptake effect is likely attributed to the preferable interaction between the L-phospholipid-based cell membrane and the D-enantiomers. PMID:27531648

  12. Surface-anchored poly(acryloyl-L(D)-valine) with enhanced chirality-selective effect on cellular uptake of gold nanoparticles.

    PubMed

    Deng, Jun; Wu, Sai; Yao, Mengyun; Gao, Changyou

    2016-01-01

    Chirality is one of the ubiquitous phenomena in biological systems. The left handed (L-) amino acids and right handed (D-) sugars are normally found in proteins, and in RNAs and DNAs, respectively. The effect of chiral surfaces at the nanoscale on cellular uptake has, however, not been explored. This study reveals for the first time the molecular chirality on gold nanoparticles (AuNPs) functions as a direct regulator for cellular uptake. Monolayers of 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) and poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were formed on AuNPs surface, respectively. The internalized amount of PAV-AuNPs was several times larger than that of MAV-AuNPs by A549 and HepG2 cells, regardless of the chirality difference. However, the D-PAV-AuNPs were internalized with significantly larger amount than the L-PAV-AuNPs. This chirality-dependent uptake effect is likely attributed to the preferable interaction between the L-phospholipid-based cell membrane and the D-enantiomers. PMID:27531648

  13. 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.

  14. Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression.

    PubMed

    Sakurai, F; Inoue, R; Nishino, Y; Okuda, A; Matsumoto, O; Taga, T; Yamashita, F; Takakura, Y; Hashida, M

    2000-05-15

    In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MBT-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [32P]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA

  15. Cellular uptake mechanism and comparative evaluation of antineoplastic effects of paclitaxel-cholesterol lipid emulsion on triple-negative and non-triple-negative breast cancer cell lines.

    PubMed

    Ye, Jun; Xia, Xuejun; Dong, Wujun; Hao, Huazhen; Meng, Luhua; Yang, Yanfang; Wang, Renyun; Lyu, Yuanfeng; Liu, Yuling

    2016-01-01

    There is no effective clinical therapy for triple-negative breast cancers (TNBCs), which have high low-density lipoprotein (LDL) requirements and express relatively high levels of LDL receptors (LDLRs) on their membranes. In our previous study, a novel lipid emulsion based on a paclitaxel-cholesterol complex (PTX-CH Emul) was developed, which exhibited improved safety and efficacy for the treatment of TNBC. To date, however, the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul have not been investigated. In order to offer powerful proof for the therapeutic effects of PTX-CH Emul, we systematically studied the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul and made a comparative evaluation of antineoplastic effects on TNBC (MDA-MB-231) and non-TNBC (MCF7) cell lines through in vitro and in vivo experiments. The in vitro antineoplastic effects and in vivo tumor-targeting efficiency of PTX-CH Emul were significantly more enhanced in MDA-MB-231-based models than those in MCF7-based models, which was associated with the more abundant expression profile of LDLR in MDA-MB-231 cells. The results of the cellular uptake mechanism indicated that PTX-CH Emul was internalized into breast cancer cells through the LDLR-mediated internalization pathway via clathrin-coated pits, localized in lysosomes, and then released into the cytoplasm, which was consistent with the internalization pathway and intracellular trafficking of native LDL. The findings of this paper further confirm the therapeutic potential of PTX-CH Emul in clinical applications involving TNBC therapy. PMID:27601899

  16. Clathrin-dependent endocytosis plays a predominant role in cellular uptake of double-stranded RNA in the red flour beetle.

    PubMed

    Xiao, Da; Gao, Xiwu; Xu, Jiaping; Liang, Xiao; Li, Qingqing; Yao, Jianxiu; Zhu, Kun Yan

    2015-05-01

    RNA interference (RNAi) is a highly conserved gene regulatory mechanism in eukaryotic organisms; however, an understanding of mechanisms of cellular uptake of double-stranded RNA (dsRNA) in different organisms remains elusive. By using pharmacological inhibitors of different endocytic pathways in conjunction with RNAi of a marker gene (lethal giant larvae, TcLgl) in the red flour beetle (Tribolium castaneum), we demonstrated that two inhibitors (chlorpromazine and bafilomycin-A1) of clathrin-dependent endocytosis can nearly abolish or significantly diminish RNAi of TcLgl, whereas methyl-β-cyclodextrin and cytochalasin-D, known to inhibit other endocytic pathways, showed no effect on RNAi of TcLgl. By using Cy3-labeled TcLgl dsRNA, we observed significantly reduced cellular uptake of TcLgl dsRNA in midgut cells after larvae were injected with each of the two clathrin-dependent endocytosis inhibitors. By using an "RNAi of RNAi" strategy, we further demonstrated that suppression of each transcript of the four key genes encoding clathrin heavy chain (TcChc), clathrin coat assembly protein AP50 (TcAP50), vacuolar (H(+))-ATPase subunit H (TcVhaSFD) and a ras-related protein (TcRab7) in clathrin-dependent endocytosis by RNAi can significantly impair RNAi of TcLgl. These results support our conclusion that clathrin-dependent endocytosis is a major mechanism in cellular uptake of dsRNA in T. castaneum. Our study also provides new insights into improving RNAi efficiency by enhancing dsRNA endosomal release.

  17. Cellular uptake mechanism and comparative evaluation of antineoplastic effects of paclitaxel–cholesterol lipid emulsion on triple-negative and non-triple-negative breast cancer cell lines

    PubMed Central

    Ye, Jun; Xia, Xuejun; Dong, Wujun; Hao, Huazhen; Meng, Luhua; Yang, Yanfang; Wang, Renyun; Lyu, Yuanfeng; Liu, Yuling

    2016-01-01

    There is no effective clinical therapy for triple-negative breast cancers (TNBCs), which have high low-density lipoprotein (LDL) requirements and express relatively high levels of LDL receptors (LDLRs) on their membranes. In our previous study, a novel lipid emulsion based on a paclitaxel–cholesterol complex (PTX-CH Emul) was developed, which exhibited improved safety and efficacy for the treatment of TNBC. To date, however, the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul have not been investigated. In order to offer powerful proof for the therapeutic effects of PTX-CH Emul, we systematically studied the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul and made a comparative evaluation of antineoplastic effects on TNBC (MDA-MB-231) and non-TNBC (MCF7) cell lines through in vitro and in vivo experiments. The in vitro antineoplastic effects and in vivo tumor-targeting efficiency of PTX-CH Emul were significantly more enhanced in MDA-MB-231-based models than those in MCF7-based models, which was associated with the more abundant expression profile of LDLR in MDA-MB-231 cells. The results of the cellular uptake mechanism indicated that PTX-CH Emul was internalized into breast cancer cells through the LDLR-mediated internalization pathway via clathrin-coated pits, localized in lysosomes, and then released into the cytoplasm, which was consistent with the internalization pathway and intracellular trafficking of native LDL. The findings of this paper further confirm the therapeutic potential of PTX-CH Emul in clinical applications involving TNBC therapy. PMID:27601899

  18. Cellular uptake mechanism and comparative evaluation of antineoplastic effects of paclitaxel–cholesterol lipid emulsion on triple-negative and non-triple-negative breast cancer cell lines

    PubMed Central

    Ye, Jun; Xia, Xuejun; Dong, Wujun; Hao, Huazhen; Meng, Luhua; Yang, Yanfang; Wang, Renyun; Lyu, Yuanfeng; Liu, Yuling

    2016-01-01

    There is no effective clinical therapy for triple-negative breast cancers (TNBCs), which have high low-density lipoprotein (LDL) requirements and express relatively high levels of LDL receptors (LDLRs) on their membranes. In our previous study, a novel lipid emulsion based on a paclitaxel–cholesterol complex (PTX-CH Emul) was developed, which exhibited improved safety and efficacy for the treatment of TNBC. To date, however, the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul have not been investigated. In order to offer powerful proof for the therapeutic effects of PTX-CH Emul, we systematically studied the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul and made a comparative evaluation of antineoplastic effects on TNBC (MDA-MB-231) and non-TNBC (MCF7) cell lines through in vitro and in vivo experiments. The in vitro antineoplastic effects and in vivo tumor-targeting efficiency of PTX-CH Emul were significantly more enhanced in MDA-MB-231-based models than those in MCF7-based models, which was associated with the more abundant expression profile of LDLR in MDA-MB-231 cells. The results of the cellular uptake mechanism indicated that PTX-CH Emul was internalized into breast cancer cells through the LDLR-mediated internalization pathway via clathrin-coated pits, localized in lysosomes, and then released into the cytoplasm, which was consistent with the internalization pathway and intracellular trafficking of native LDL. The findings of this paper further confirm the therapeutic potential of PTX-CH Emul in clinical applications involving TNBC therapy.

  19. pH luminescence switching, dihydrogen phosphate sensing, and cellular uptake of a heterobimetallic ruthenium(II)-rhenium(I) complex.

    PubMed

    Zheng, Ze-Bao; Wu, Yong-Quan; Wang, Ke-Zhi; Li, Fuyou

    2014-02-28

    A new heterobimetallic ruthenium(II)-rhenium(I) complex of [Ru(bpy)2(HL)Re(CO)3Cl](ClO4)2·6H2O (RuHLRe) {bpy = 2,2'-bipyridine and HL = 2-(4-(2,6-di(pyridin-2-yl)pyridin-4-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} was synthesised and characterised by elemental analysis, proton nuclear magnetic resonance spectroscopy, and mass spectrometry. The ground- and excited-state acid-base properties of RuHLRe were studied using UV-Vis absorption spectrophotometric and spectrofluorimetric titrations in a 100 : 1 (v/v) Britton-Robinson buffer-CH3CN solution combined with luminescence lifetime measurements. The complex exhibited two-step separate protonation-deprotonation processes in both the ground and excited states. The complex acted as pH-induced "off-on-off" luminescence switches (I(on)/I(off) = 31.0 and 14.6), with one of the switching actions being driven by pH variations over the physiological pH range (5.3-8.0). Importantly, cellular imaging and cytotoxicity experiments demonstrated that RuHLRe rapidly and selectively illuminated the membrane of HeLa cells over fixed cells and exhibited reduced cytotoxicity at the imaging concentration compared to the Re(I)-free parent Ru(II) complex. In addition, RuHLRe acted as an efficient "turn on" emission sensor for H2PO4(-) and "turn off" emission sensor for F(-) and OAc(-).

  20. [Effects of applying nitrogen fertilizer and fertilizer additive on rice yield and rice plant nitrogen uptake, translocation, and utilization].

    PubMed

    Li, Wen-jun; Xia, Yong-qiu; Yang, Xiao-yun; Guo, Miao; Yan, Xiao-yuan

    2011-09-01

    A field experiment was conducted in the Taihu Lake region of southern Jiangsu to study the effects of applying nitrogen (N) fertilizer and fertilizer additive on the rice yield and the rice plant N uptake, translocation, and utilization. Applying N fertilizer had significant positive effects on the rice yield, accumulative absorbed N at all growth stages and at each growth stage, and N translocation rate after anthesis (P < 0.01). However, when the N application rate exceeded 200 kg x hm(-2), its yield-increasing effect was not significant (P > 0.05). The N translocation rate after anthesis and the N fertilizer use efficiency decreased with increasing N application rate. Applying fertilizer additive further improved the rice yield, accumulative absorbed N, N translocation rate after anthesis, and N fertilizer use efficiency, and this effect was more evident when the N application rate was equal to or greater than 200 kg x hm(-2). Relatively high rice yield and N use efficiency were achieved when applying 150 kg x hm(-2) of N fertilizer without the application of fertilizer additive.

  1. Flow cytometric assessment of reactive oxygen species generations that are directly related to cellular ZnO nanoparticle uptake.

    PubMed

    Yoo, Hyun Ju; Yoon, Tae Hyun

    2014-07-01

    In this study, a simple flow cytometry protocol to evaluate nanoparticle associated biological response was proposed. Particularly, we have evaluated the effect of surface charge on the cellular nanoparticle associations and nanoparticle-induced apoptosis. Significant enhancement in side scattering intensity was observed for the HeLa cells treated with positively charged (PLL)ZnO nanoparticles, suggesting that the (PLL)ZnO nanoparticles may induce cell death via adsorption and endocytosis of the nanoparticles. On the other hand, the negatively charged (PAA)ZnO nanoparticle seems to cause cell death process indirectly via the released Zn ions, with less contribution from cellular association of nanoparticles. Time- and dose-dependent studies on cellular association of ZnO nanoparticles, and ZnO associated reactive oxygen species generation were also performed for the HeLa cells exposed to the (PLL)ZnO nanoparticle. For those cells associated with (PLL)ZnO nanoparticle, a significant enhancement in reactive oxygen species generation was observed even at a lower concentration (10 ppm), which was not observable for the results with the whole cell population. By using this approach, we are able to distinguish biological responses (e.g., reactive oxygen species (ROS) generation) directly related to the cellular associations of NPs from those indirectly related to the cellular associations of NPs, such as the cytotoxicity caused by the NP released metal ions.

  2. An Amphiphilic BODIPY-Porphyrin Conjugate: Intense Two-Photon Absorption and Rapid Cellular Uptake for Two-Photon-Induced Imaging and Photodynamic Therapy.

    PubMed

    Zhang, Tao; Lan, Rongfeng; Gong, Longlong; Wu, Baoyan; Wang, Yuzhi; Kwong, Daniel W J; Wong, Wai-Kwok; Wong, Ka-Leung; Xing, Da

    2015-11-01

    The new amphiphilic BODPY-porphyrin conjugate BZnPP and its precursor BZnPH were synthesised, and their linear and two-photon photophysical properties, together with their cellular uptake and photo-cytotoxicity, were studied. This amphiphilic conjugate consists of a hydrophobic BODIPY moiety and a hydrophilic tetra(ethylene glycol) chain bridging a cationic triphenylphosphonium group to an amphiphilic porphyrin ZnP through acetylide linkers at its meso positions. A large two-photon absorption cross-section (σ=1725 GM) and a high singlet oxygen quantum yield (0.52) were recorded. Intense linear- and two-photon-induced red emissions were also observed for both BZnPP and BZnPH. Further in vitro studies showed that BZnPP exhibited very efficient cellular uptake and strong photocytotoxic but weak dark cytotoxic properties towards human breast carcinoma MCF-7 cells. In summary, the two-photon-induced emission and the potent photo-cytotoxicity of BZnPP make it an efficacious dual-purpose tumour-imaging and photodynamic therapeutic agent in the tissue-transparent spectral windows.

  3. Spectroscopic characterization of protein-wrapped single-wall carbon nanotubes and quantification of their cellular uptake in multiple cell generations

    NASA Astrophysics Data System (ADS)

    Bertulli, Cristina; Beeson, Harry J.; Hasan, Tawfique; Huang, Yan Yan S.

    2013-07-01

    We study the spectral characteristics of bovine serum albumin (BSA) protein conjugated single-wall carbon nanotubes (SWNTs), and quantify their uptake by macrophages. The binding of BSA onto the SWNT surface is found to change the protein structure and to increase the doping of the nanotubes. The G-band Raman intensity follows a well-defined power law for SWNT concentrations of up to 33 μg ml-1 in aqueous solutions. Subsequently, in vitro experiments demonstrate that incubation of BSA-SWNT complexes with macrophages affects neither the cellular growth nor the cellular viability over multiple cell generations. Using wide spot Raman spectroscopy as a fast, non-destructive method for statistical quantification, we observe that macrophages effectively uptake BSA-SWNT complexes, with the average number of nanotubes internalized per cell remaining relatively constant over consecutive cell generations. The number of internalized SWNTs is found to be ˜30 × 106 SWNTs/cell for a 60 mm-2 seeding density and ˜100 × 106 SWNTs/cell for a 200 mm-2 seeding density. Our results show that BSA-functionalized SWNTs are an efficient molecular transport system with low cytotoxicity maintained over multiple cell generations.

  4. Identification of a Pro-Angiogenic Potential and Cellular Uptake Mechanism of a LMW Highly Sulfated Fraction of Fucoidan from Ascophyllum nodosum

    PubMed Central

    Marinval, Nicolas; Saboural, Pierre; Haddad, Oualid; Maire, Murielle; Bassand, Kevin; Geinguenaud, Frederic; Djaker, Nadia; Ben Akrout, Khadija; Lamy de la Chapelle, Marc; Robert, Romain; Oudar, Olivier; Guyot, Erwan; Laguillier-Morizot, Christelle; Sutton, Angela; Chauvierre, Cedric; Chaubet, Frederic; Charnaux, Nathalie; Hlawaty, Hanna

    2016-01-01

    Herein we investigate the structure/function relationships of fucoidans from Ascophyllum nodosum to analyze their pro-angiogenic effect and cellular uptake in native and glycosaminoglycan-free (GAG-free) human endothelial cells (HUVECs). Fucoidans are marine sulfated polysaccharides, which act as glycosaminoglycans mimetics. We hypothesized that the size and sulfation rate of fucoidans influence their ability to induce pro-angiogenic processes independently of GAGs. We collected two fractions of fucoidans, Low and Medium Molecular Weight Fucoidan (LMWF and MMWF, respectively) by size exclusion chromatography and characterized their composition (sulfate, fucose and uronic acid) by colorimetric measurement and Raman and FT-IR spectroscopy. The high affinities of fractionated fucoidans to heparin binding proteins were confirmed by Surface Plasmon Resonance. We evidenced that LMWF has a higher pro-angiogenic (2D-angiogenesis on Matrigel) and pro-migratory (Boyden chamber) potential on HUVECs, compared to MMWF. Interestingly, in a GAG-free HUVECs model, LMWF kept a pro-angiogenic potential. Finally, to evaluate the association of LMWF-induced biological effects and its cellular uptake, we analyzed by confocal microscopy the GAGs involvement in the internalization of a fluorescent LMWF. The fluorescent LMWF was mainly internalized through HUVEC clathrin-dependent endocytosis in which GAGs were partially involved. In conclusion, a better characterization of the relationships between the fucoidan structure and its pro-angiogenic potential in GAG-free endothelial cells was required to identify an adapted fucoidan to enhance vascular repair in ischemia. PMID:27763505

  5. Effect of hydrophobic scaffold on the cellular uptake and gene transfection activities of DNA-encapsulating liposomal nanoparticles via intracerebroventricular administration.

    PubMed

    Akita, Hidetaka; Nakatani, Taichi; Kuroki, Kimiko; Maenaka, Katsumi; Tange, Kota; Nakai, Yuta; Harashima, Hideyoshi

    2015-07-25

    Efficient DNA carriers are needed as a gene medication for curing brain disorders. In the present study, the function of a neutral lipid envelope-type nanoparticle (LNP) encapsulating pDNA was evaluated after intracerebroventricular administration. The lipid envelope was composed of a series of SS-cleavable and pH-activated lipid like materials (ssPalm) including myristic acid, vitamin A and vitamin E in the hydrophobic scaffold (LNPssPalmM, LNPssPalmA, LNPssPalmE, respectively). The LNPssPalmA and LNPssPalmE were extensively distributed in the corpus callosum, and then gene expression occurred mainly astrocytes in this region, while not in LNPssPalmM. The recombinant human ApoE3-dependent enhancement of the uptake into an astrocyte-derived cell line (KT-5) was observed in LNPssPalmA and LNPssPalmE. Thus, ApoE in the brain plays a key role in the cellular uptake of these particles by astrocytes, and this uptake is dependent on the structure of the hydrophobic scaffold.

  6. Cellular sites of estrogen and antiestrogen uptake, retention and action: comparative autoradiographic studies in the immature rat uterus

    SciTech Connect

    Ennis, B.W.

    1987-01-01

    This purpose of this study is to clarify the mechanism of action of antiestrogens: agents used for treating breast cancer and as probes for studying the mechanisms of action of estrogen. Nuclear uptake and retention of estrogen and antiestrogen were determined in the different cell types of the immature rat uterus, by quantitative autoradiography, after an injection of tritiated hydroxytamoxifen ((/sup 3/H)TAM(OH)) or tritiated estradiol ((/sup 3/H)E/sub 2/). The effect of TAM(OH) and E/sub 2/ on progesterone receptor content was assessed in the different cell types by determining nuclear uptake of the synthetic progestin (/sup 3/H)Org 2058. The results indicate that antiestrogen and estrogen localize to nuclei of the same uterine cell types, but that this nuclear uptake differs among the uterine tissue compartments, that antiestrogen is taken up considerably slower and retained longer than estrogen and that antiestrogen and estrogen differentially affect progesterone receptor content in the different cell types. The results further suggest that antiestrogen-specific binding sites exist in the cytoplasm of uterine luminal epithelium.

  7. Modeling of time dependent localized flow shear stress and its impact on cellular growth within additive manufactured titanium implants.

    PubMed

    Zhang, Ziyu; Yuan, Lang; Lee, Peter D; Jones, Eric; Jones, Julian R

    2014-11-01

    Bone augmentation implants are porous to allow cellular growth, bone formation and fixation. However, the design of the pores is currently based on simple empirical rules, such as minimum pore and interconnects sizes. We present a three-dimensional (3D) transient model of cellular growth based on the Navier-Stokes equations that simulates the body fluid flow and stimulation of bone precursor cellular growth, attachment, and proliferation as a function of local flow shear stress. The model's effectiveness is demonstrated for two additive manufactured (AM) titanium scaffold architectures. The results demonstrate that there is a complex interaction of flow rate and strut architecture, resulting in partially randomized structures having a preferential impact on stimulating cell migration in 3D porous structures for higher flow rates. This novel result demonstrates the potential new insights that can be gained via the modeling tool developed, and how the model can be used to perform what-if simulations to design AM structures to specific functional requirements.

  8. Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells.

    PubMed

    Gu, Jijin; Hao, Junguo; Fang, Xiaoling; Sha, Xianyi

    2016-04-01

    Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization. PMID:26741268

  9. Targeting Cells With MR Imaging Probes: Cellular Interaction And Intracellular Magnetic Iron Oxide Nanoparticles Uptake In Brain Capillary Endothelial and Choroidal Plexus Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Cambianica, I.; Bossi, M.; Gasco, P.; Gonzalez, W.; Idee, J. M.; Miserocchi, G.; Rigolio, R.; Chanana, M.; Morjan, I.; Wang, D.; Sancini, G.

    2010-10-01

    Magnetic iron oxide nanoparticles (NPs) are considered for various diagnostic and therapeutic applications in brain including their use as contrast agent for magnetic resonance imaging. In delivery application, the critical step is the transport across cell layers and the internalization of NPs into specific cells, a process often limited by poor targeting specificity and low internalization efficiency. The development of the models of brain endothelial cells and choroidal plexus epithelial cells in culture has allowed us to investigate into these mechanisms. Our strategy is aimed at exploring different routes to the entrapment of iron oxide NPs in these brain related cells. Here we demonstrated that not only cells endowed with a good phagocytic activity like activated macrophages but also endothelial brain capillary and choroidal plexus epithelial cells do internalize iron oxide NPs. Our study of the intracellular trafficking of NPs by TEM, and confocal microscopy revealed that NPs are mainly internalized by the endocytic pathway. Iron oxide NPs were dispersed in water and coated with 3,4-dihydroxyl-L-phenylalanine (L-DOPA) using standard procedures. Magnetic lipid NPs were prepared by NANOVECTOR: water in oil in water (W/O/W) microemulsion process has been applied to directly coat different iron based NPs by lipid layer or to encapsulate them into Solid Lipid Nanoparticles (SLNs). By these coating/loading the colloidal stability was improved without strong alteration of the particle size distribution. Magnetic lipid NPs could be reconstituted after freeze drying without appreciable changes in stability. L-DOPA coated NPs are stable in PBS and in MEM (Modified Eagle Medium) medium. The magnetic properties of these NPs were not altered by the coating processes. We investigated the cellular uptake, cytotoxicity, and interaction of these NPs with rat brain capillary endothelial (REB4) and choroidal plexus epithelial (Z310) cells. By means of widefield, confocal

  10. Assessment of the enhancement of PLGA nanoparticle uptake by dendritic cells through the addition of natural receptor ligands and monoclonal antibody.

    PubMed

    Walters, Adam A; Somavarapu, Satyanarayana; Riitho, Victor; Stewart, Graham R; Charleston, Bryan; Steinbach, Falko; Graham, Simon P

    2015-11-27

    Targeting of specific receptors on antigen-presenting cells is an appealing prospect in the production of novel nanoparticulate vaccines. In particular, the targeting of vaccines to dendritic cell (DC) subsets has been shown in models to significantly improve the induction of immune responses. This paper describes the evaluation of natural ligands, mannan and chitosan, and monoclonal antibodies as targeting motifs to enhance uptake of PLGA nanoparticle carriers by bovine DCs. To assess enhancement of uptake after the addition of natural ligands a bovine monocyte derived DC (MoDC) model was used. For the assessment of monoclonal antibody targeting, the model was expanded to include afferent lymph DCs (ALDCs) in a competitive uptake assay. Mannan, proved unsuccessful at enhancing uptake or targeting by MoDCs. Chitosan coated particle uptake could be impeded by the addition of mannan suggesting uptake may be mediated through sugar receptors. Inclusion of monoclonal antibodies specific for the DEC-205 (CD205) receptor increased the number of receptor expressing DCs associated with particles as well as the number of particles taken up by individual cells. These results support the further evaluation of active targeting of nanovaccines to DCs to enhance their immunogenicity in cattle and other large mammalian species including humans. PMID:26529067

  11. Assessment of the enhancement of PLGA nanoparticle uptake by dendritic cells through the addition of natural receptor ligands and monoclonal antibody.

    PubMed

    Walters, Adam A; Somavarapu, Satyanarayana; Riitho, Victor; Stewart, Graham R; Charleston, Bryan; Steinbach, Falko; Graham, Simon P

    2015-11-27

    Targeting of specific receptors on antigen-presenting cells is an appealing prospect in the production of novel nanoparticulate vaccines. In particular, the targeting of vaccines to dendritic cell (DC) subsets has been shown in models to significantly improve the induction of immune responses. This paper describes the evaluation of natural ligands, mannan and chitosan, and monoclonal antibodies as targeting motifs to enhance uptake of PLGA nanoparticle carriers by bovine DCs. To assess enhancement of uptake after the addition of natural ligands a bovine monocyte derived DC (MoDC) model was used. For the assessment of monoclonal antibody targeting, the model was expanded to include afferent lymph DCs (ALDCs) in a competitive uptake assay. Mannan, proved unsuccessful at enhancing uptake or targeting by MoDCs. Chitosan coated particle uptake could be impeded by the addition of mannan suggesting uptake may be mediated through sugar receptors. Inclusion of monoclonal antibodies specific for the DEC-205 (CD205) receptor increased the number of receptor expressing DCs associated with particles as well as the number of particles taken up by individual cells. These results support the further evaluation of active targeting of nanovaccines to DCs to enhance their immunogenicity in cattle and other large mammalian species including humans.

  12. Effect of organic matter additions on uptake of weathered DDT by Cucurbita pepo ssp. pepo cv. Howden.

    PubMed

    Lunney, Alissa I; Rutter, Allison; Zeeb, Barbara A

    2010-01-01

    Greenhouse studies were conducted to assess the impact of organic matter additions on plant uptake of DDT [2,2-bis(chlorophenyl)-1,1,1-trichloroethane] from weathered soil. Cucurbita pepo ssp. pepo cv. Howden pumpkins were grown in 100 g of DDT contaminated soil ([DDT] - 1100 ng/g) mixed with equal volumes of either clean soil, perlite, vermiculite, peat, potting soil, or granular activated carbon (GAC) to give total organic carbon contents of 2.4%, 2.5%, 2.6%, 11.5%, 12.2%, and 27.3%, respectively. As in other studies, root DDT concentrations were significantly lower in soils with high organic matter. Root bioaccumulation factors (BAF = [DDT]root/[DDT]soil) approximated this trend. Root concentrations correlated with organic matter concentrations and not with soil DDT concentrations. Conversely, shoot DDT concentrations, shoot BAFs and translocation factors (TLF = BAF(shoot)/BAF(root)) were not significantly different between treatment groups, except for plants grown in GAC/DDT soil. This suggests that amendments with a range of organic matter contents may be added to improve soil conditions at industrial sites without significant adverse effects on phytoextraction potential of C. pepo ssp. pepo. PMID:20734916

  13. Hybrid nanoparticle architecture for cellular uptake and bioimaging: direct crystallization of a polymer immobilized with magnetic nanoparticles on carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Depan, D.; Misra, R. D. K.

    2012-09-01

    We describe here the success of an innovative approach of direct immobilization of magnetic nanoparticles (MNPs) onto carbon nanotubes (CNTs). The approach involved functionalization of magnetic nanoparticles and consequent covalent linkage to a copolymer (PE-b-PEG). Next, the immobilized magnetic nanoparticles on the copolymer were directly crystallized on the long axis of CNTs, where the interfacial adhesion comes from electrostatic and van der Waals interaction. The intracellular trafficking of a hybrid nanoparticle system [(PE-b-PEG)-MNP-CNT-FITC] in HeLa cells was monitored using a fluorescent marker, FITC, conjugated to the nanoparticle system. The distribution of the nanoparticle system inside cells was studied by fluorescence microscopy in a time and dose dependent manner, and it was observed that the nanoparticles are located in the cytoplasm and no apparent cell death was observed at the concentration studied. Also, the effect of an externally applied magnetic field on actin cytoskeleton, cell morphology and intracellular uptake of iron was studied. The approach described here is promising for simultaneous imaging and monitoring intracellular uptake.

  14. Effects of Lipids on in Vitro Release and Cellular Uptake of β-Carotene in Nanoemulsion-Based Delivery Systems.

    PubMed

    Yi, Jiang; Zhong, Fang; Zhang, Yuzhu; Yokoyama, Wallace; Zhao, Liqing

    2015-12-23

    β-Carotene (BC) nanoemulsions were successfully prepared by microfluidization. BC micellarization was significantly affected by bile salts and pancreatin concentration. Positive and linear correlation was observed between BC release and bile salts concentration. Pancreatin facilitated BC's release in simulated digestion. Compared to the control (bulk oil) (4.6%), nanoemulsion delivery systems significantly improved the micellarization of BC (70.9%). The amount of BC partitioned into micelles was positively proportional to the length of carrier oils. Unsaturated fatty acid (UFA)-rich oils were better than saturated fatty acid (SFA)-rich oils in transferring BC (p < 0.05). No significant difference was observed between monounsaturated fatty acid (MUFA)-rich oils and polyunsaturated fatty acid (PUFA)-rich oils (p > 0.05). A positive and linear relationship between the degree of lipolysis and the release of BC in vitro digestion was observed. Bile salts showed cytotoxicity to Caco-2 cells below 20 times dilution. BC uptake by Caco-2 cells was not affected by fatty acid (FA) compositions in micelles, but BC uptake was proportional to its concentration in the diluted micelle fraction. The results obtained are beneficial to encapsulate and deliver BC or other bioactive lipophilic carotenoids in a wide range of commercial products. PMID:26629789

  15. Reduction-sensitive liposomes from a multifunctional lipid conjugate and natural phospholipids: reduction and release kinetics and cellular uptake.

    PubMed

    Goldenbogen, Björn; Brodersen, Nicolai; Gramatica, Andrea; Loew, Martin; Liebscher, Jürgen; Herrmann, Andreas; Egger, Holger; Budde, Bastian; Arbuzova, Anna

    2011-09-01

    The development of targeted and triggerable delivery systems is of high relevance for anticancer therapies. We report here on reduction-sensitive liposomes composed of a novel multifunctional lipidlike conjugate, containing a disulfide bond and a biotin moiety, and natural phospholipids. The incorporation of the disulfide conjugate into vesicles and the kinetics of their reduction were studied using dansyl-labeled conjugate 1 in using the dansyl fluorescence environmental sensitivity and the Förster resonance energy transfer from dansyl to rhodamine-labeled phospholipids. Cleavage of the disulfide bridge (e.g., by tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), l-cysteine, or glutathione (GSH)) removed the hydrophilic headgroup of the conjugate and thus changed the membrane organization leading to the release of entrapped molecules. Upon nonspecific uptake of vesicles by macrophages, calcein release from reduction-sensitive liposomes consisting of the disulfide conjugate and phospholipids was more efficient than from reduction-insensitive liposomes composed only of phospholipids. The binding of streptavidin to the conjugates did not interfere with either the subsequent reduction of the disulfide bond of the conjugate or the release of entrapped molecules. Breast cancer cell line BT-474, overexpressing the HER2 receptor, showed a high uptake of the reduction-sensitive doxorubicin-loaded liposomes functionalized with the biotin-tagged anti-HER2 antibody. The release of the entrapped cargo inside the cells was observed, implying the potential of using our system for active targeting and delivery.

  16. Hybrid nanoparticle architecture for cellular uptake and bioimaging: direct crystallization of a polymer immobilized with magnetic nanoparticles on carbon nanotubes.

    PubMed

    Depan, D; Misra, R D K

    2012-10-21

    We describe here the success of an innovative approach of direct immobilization of magnetic nanoparticles (MNPs) onto carbon nanotubes (CNTs). The approach involved functionalization of magnetic nanoparticles and consequent covalent linkage to a copolymer (PE-b-PEG). Next, the immobilized magnetic nanoparticles on the copolymer were directly crystallized on the long axis of CNTs, where the interfacial adhesion comes from electrostatic and van der Waals interaction. The intracellular trafficking of a hybrid nanoparticle system [(PE-b-PEG)-MNP-CNT-FITC] in HeLa cells was monitored using a fluorescent marker, FITC, conjugated to the nanoparticle system. The distribution of the nanoparticle system inside cells was studied by fluorescence microscopy in a time and dose dependent manner, and it was observed that the nanoparticles are located in the cytoplasm and no apparent cell death was observed at the concentration studied. Also, the effect of an externally applied magnetic field on actin cytoskeleton, cell morphology and intracellular uptake of iron was studied. The approach described here is promising for simultaneous imaging and monitoring intracellular uptake.

  17. Albumin and Uptake of Drugs in Cells: Additional Validation Exercises of a Recently Published Equation that Quantifies the Albumin-Facilitated Uptake Mechanism(s) in Physiologically Based Pharmacokinetic and Pharmacodynamic Modeling Research.

    PubMed

    Poulin, Patrick; Haddad, Sami

    2015-12-01

    The impact of albumin concentration on the uptake of drugs in cells might involve mechanisms going beyond the free drug concentration hypothesis. Proceeding from the assumption that both the unbound and protein-bound drug fractions can be available for uptake, several authors have argued that the uptake of highly bound drugs in cells might be driven mainly by the albumin-facilitated uptake mechanism(s). Hence, a novel approach quantifying the additional contribution of the protein-bound drug complex and pH gradient effect in diverse in vitro-to-in vivo extrapolation (IVIVE) procedures of drug uptake and clearance has been proposed and extensively validated by Poulin et al. (2015. J Pharm Sci. Epub ahead of print); this approach consisted of replacing the unbound fraction in plasma (fup ) with an adjusted fup value (fup-adjusted ). After a second review of literature, the objective of the present study was to perform further validation exercises of the concept of fup-adjusted by using additional case examples of IVIVEs that covered diverse drug properties and experimental settings with varied albumin concentrations (e.g., perfused liver, isolated and suspended hepatocytes, and cultured cells overexpressing transporters). Again, the novel IVIVE method based on fup-adjusted was the best-performing prediction method of the uptake rate (or clearance) as a function of protein binding compared with the conventional method based on the fup theory (absolute average fold error of 1.4 vs. 7.4). Therefore, the present study confirms the utility of fup-adjusted compared with fup in IVIVE procedures for drugs highly bound to albumin, and the improvement was observed particularly in the higher range of albumin concentrations. From these findings, we may conclude that uptake of these drugs in cells is primarily driven by the albumin-bound form. Consequently, it is suggested to estimate the uptake kinetic parameters with cell-based assays incubated in 100% human serum or to make a

  18. Addition of Alanyl-Glutamine to Dialysis Fluid Restores Peritoneal Cellular Stress Responses – A First-In-Man Trial

    PubMed Central

    Boehm, Michael; Herzog, Rebecca; Gruber, Katharina; Lichtenauer, Anton Michael; Kuster, Lilian; Csaicsich, Dagmar; Gleiss, Andreas; Alper, Seth L.; Aufricht, Christoph; Vychytil, Andreas

    2016-01-01

    Background Peritonitis and ultrafiltration failure remain serious complications of chronic peritoneal dialysis (PD). Dysfunctional cellular stress responses aggravate peritoneal injury associated with PD fluid exposure, potentially due to peritoneal glutamine depletion. In this randomized cross-over phase I/II trial we investigated cytoprotective effects of alanyl-glutamine (AlaGln) addition to glucose-based PDF. Methods In a prospective randomized cross-over design, 20 stable PD outpatients underwent paired peritoneal equilibration tests 4 weeks apart, using conventional acidic, single chamber 3.86% glucose PD fluid, with and without 8 mM supplemental AlaGln. Heat-shock protein 72 expression was assessed in peritoneal effluent cells as surrogate parameter of cellular stress responses, complemented by metabolomics and functional immunocompetence assays. Results AlaGln restored peritoneal glutamine levels and increased the primary outcome heat-shock protein expression (effect 1.51-fold, CI 1.07–2.14; p = 0.022), without changes in peritoneal ultrafiltration, small solute transport, or biomarkers reflecting cell mass and inflammation. Further effects were glutamine-like metabolomic changes and increased ex-vivo LPS-stimulated cytokine release from healthy donor peripheral blood monocytes. In patients with a history of peritonitis (5 of 20), AlaGln supplementation decreased dialysate interleukin-8 levels. Supplemented PD fluid also attenuated inflammation and enhanced stimulated cytokine release in a mouse model of PD-associated peritonitis. Conclusion We conclude that AlaGln-supplemented, glucose-based PD fluid can restore peritoneal cellular stress responses with attenuation of sterile inflammation, and may improve peritoneal host-defense in the setting of PD. PMID:27768727

  19. The perlecan heparan sulfate proteoglycan mediates cellular uptake of HIV-1 Tat through a pathway responsible for biological activity

    SciTech Connect

    Argyris, Elias G.; Kulkosky, Joseph; Meyer, Marie E.; Xu Yan; Mukhtar, Muhammad; Pomerantz, Roger J. . E-mail: roger.j.pomerantz@jefferson.edu; Williams, Kevin Jon . E-mail: K_Williams@mail.jci.tju.edu

    2004-12-20

    Cell surface heparan sulfate proteoglycans (HSPGs) mediate internalization of HIV-1 Tat. Herein, we report that human WiDr cells, which express perlecan but no other HSPGs, can internalize {sup 125}I-labeled Tat with minimal lysosomal degradation. Pre-treatment of cells with heparitinase almost completely abolished {sup 125}I-Tat surface binding, while the use of an HIV-1 long terminal repeat (LTR) promoter-reporter construct demonstrated that transactivation was potently blocked by pretreatment of cells with heparitinase, indicating an essential role for perlecan in the biologic effects of Tat. We conclude that the perlecan mediates Tat uptake and is required for HIV-1 LTR-directed transactivation in this human cell type.

  20. Interaction of Actinide Species with Microorganisms & Microbial Chelators: Cellular Uptake, Toxicity, & Implications for Bioremediation of Soil & Ground Water.

    SciTech Connect

    Hakim Boukhalfa Mary, P. Neu Alvin Crumbliss

    2006-03-28

    Microorganisms influence the natural cycle of major elements, including C, N, P, S, and transition metals such as Mn and Fe. Bacterial processes can also influence the behavior of actinides in soil and ground water. While radionuclides have no known biological utility, they have the potential to interact with microorganisms and to interfere with processes involving other elements such as Fe and Mn. These interactions can transform radionuclides and affect their fate and transport. Organic acids, extruded by-products of cell metabolism, can solubilize radionuclides and facilitate their transport. The soluble complexes formed can be taken up by the cells and incorporated into biofilm structures. We have examined the interactions of Pu species with bacterial metabolites, studied Pu uptake by microorganisms and examined the toxicity of Pu and other toxic metals to environmentally relevant bacteria. We have also studied the speciation of Pu(IV) in the presence of natural and synthetic chelators.

  1. Enhancement of indocyanine green stability and cellular uptake by incorporating cationic lipid into indocyanine green-loaded nanoemulsions.

    PubMed

    Lee, Eun-Hye; Kim, Jin-Ki; Lim, Joon-Seok; Lim, Soo-Jeong

    2015-12-01

    Indocyanine green (ICG) is a near-infrared optical dye approved by the Food and Drug Administration. ICG has been investigated as a simultaneous color and fluorescence-imaging tracer for the intraoperative identification of sentinel lymph nodes, but its use has recently expanded to include application as a photosensitizer for the local photodynamic/thermal treatment of identified lymph node metastases. The current study was designed to develop an ICG-loaded nanoemulsion as an effective agent for both the diagnosis and treatment of lymph node metastases. Incorporating the cationic lipid stearylamine (SA) together with ICG in the shell of nanoemulsions did not affect the loaded ICG concentration, but changed the surface charge of nanoemulsions from a negative to a positive value and improved the physical stability of nanoemulsions. Loading ICG into SA-incorporated nanoemulsions more effectively blocked the aggregation and degradation of ICG compared to loading in SA-free nanoemulsions. SA incorporation also enhanced tumor cell uptake of ICG-loaded nanoemulsions, resulting in greater cell killing upon light irradiation. After subcutaneous injection into the footpad of mice, SA-incorporated nanoemulsions increased the concentration of ICG accumulated in popliteal lymph nodes to a greater extent than SA-free nanoemulsions without affecting the kinetics of lymph node uptake of nanoemulsions. These multiple beneficial effects of incorporating SA in nanoemulsions are likely attributable to the electrostatic interaction between anionic ICG and cationic SA, as well as the change in the nanoemulsion surface charge from negative to positive. Our findings indicate that SA-incorporated nanoemulsions are promising ICG carriers for combined diagnosis and treatment of lymph node metastases.

  2. Positively charged and pH self-buffering quantum dots for efficient cellular uptake by charge mediation and monitoring cell membrane permeability

    NASA Astrophysics Data System (ADS)

    Wang, Suhua; Song, Haipeng; Ong, Wei Yi; Han, Ming Yong; Huang, Dejian

    2009-10-01

    Positively charged and pH self-buffering quantum dots (Tren-QDs) were achieved by surface functionalization with tris(2-aminoethyl)amine (Tren) derivatives, which are attached to the inorganic cores of QDs through bidentate chelating of dithiocarbamates. The Tren-QDs exhibit pH buffering capability by absorbing or releasing protons due to the surface polyamine groups as the surrounding pH fluctuates. Such self-buffering capability stabilizes the photoluminescence of the Tren-QDs against acid. The Tren-QDs bear positive charges through protonation of the surface polyamine groups under physiological conditions and the surface positive charges improve their cellular uptake efficiency by charge mediation, which has been demonstrated by BV-2 microglia cells. The photoluminescence of Tren-QDs shows a selective Stern-Volmer response to copper ions and this property has been preliminarily evaluated for investigating the BV-2 cell membrane structure by monitoring the photoluminescence of intracellular Tren-QDs.

  3. Monte Carlo simulation of irradiation and killing in three-dimensional cell populations with lognormal cellular uptake of radioactivity

    PubMed Central

    Howell, Roger W.; Rajon, Didier; Bolch, Wesley E.

    2013-01-01

    Purpose The biological response of tissue exposed to radiations emitted by internal radioactivity is often correlated with the mean absorbed dose to a tissue element. However, experimental studies show that even when the mean absorbed dose to the tissue element is constant, the response of the cell population within the tissue element can vary significantly depending on the distribution of radioactivity at the cellular and multicellular levels. The present work develops theoretical models to simulate these observations. Materials and methods Two theoretical models were created to simulate experimental three-dimensional cell culture models with homogeneous and inhomogeneous tissue environments. The cells were assigned activities according to lognormal distributions of an alpha particle emitter or a monoenergetic electron emitter. Absorbed doses to the cell nuclei were assessed with point-kernel geometric-factor and Electron Gamma Shower version nrc (EGSnrc) Monte Carlo radiation transport simulations, respectively. The self- and cross-dose to individual cell nuclei were calculated and a Monte Carlo method was used to determine their fate. Survival curves were produced after tallying the live and dead cells. Results Both percent cells labeled and breadth of lognormal distribution affected the dose distribution at the cellular level, which in turn, influenced the shape of the cell survival curves. Conclusions Multicellular Monte Carlo dosimetry-models offer improved capacity to predict response to radiopharmaceuticals compared to approaches based on mean absorbed dose to the tissue. PMID:21745001

  4. In vitro release characteristics and cellular uptake of poly(D,L-lactic-co-glycolic acid) nanoparticles for topical delivery of antisense oligodeoxynucleotides.

    PubMed

    Chen, Ying-Shan; Alany, Raid G; Young, Simon A; Green, Colin R; Rupenthal, Ilva D

    2011-01-01

    The efficacy of antisense oligodeoxynucleotides (AsODNs) is compromised by their poor stability in biological fluids and the inefficient cellular uptake due to their size and negative charge. Since chemical modifications of these molecules have resulted in a number of non-antisense activities, incorporation into particulate delivery systems has offered a promising alternative. The aim of this study was to evaluate various poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles for AsODN entrapment and delivery. PLGA nanoparticles were prepared using the double emulsion solvent evaporation method. The influence of formulation parameters such as PLGA concentration and volume ratio of internal aqueous phase volume (Va1) to organic phase volume (Vo) to external aqueous phase volume (Va2) on particle size, polydispersity index (PDI) and zeta potential (ZP) was investigated using a full factorial study. The particle size increased with increasing PLGA concentrations and volume ratios, with an interaction detectable between the two factors. AsODN entrapment efficiencies ranged between 49.97% and 54.95% with no significant difference between various formulations. By fitting the in vitro release profiles to a dual first order release model it was shown that the AsODN release occurred via two processes: a diffusion controlled process in the early phase (25 to 32% within one day) and a PLGA degradation process in the latter (39 to 70% after 14 days). Cellular uptake studies using primary corneal epithelial cells suggested active transport of nanoparticles via endocytosis. PLGA nanoparticles therefore show potential to successfully entrap AsODNs, transport them into cells and release them over time due to polymer erosion.

  5. Cellular Uptake and Cytotoxic Effect of Epidermal Growth Factor Receptor Targeted and Plitidepsin Loaded Co-Polymeric Polymersomes on Colorectal Cancer Cell Lines.

    PubMed

    Goñi-de-Cerio, Felipe; Thevenot, Julie; Oliveira, Hugo; Pérez-Andrés, Encarnación; Berra, Edurne; Masa, Marc; Suárez-Merino, Blanca; Lecommandoux, Sébastien; Heredia, Pedro

    2015-11-01

    Encapsulating chemotherapy drugs in targeted nanodelivery systems is one of the most promising approaches to tackle cancer disease, avoiding side effects of common treatment. In the last decade, several nanocarriers with different nature have been tested, but polypeptide-based copolymers have attracted considerable attention for their biocompatibility, controlled and slow biodegradability as well as their low toxicity. In this work, we synthesized, characterized and evaluated poly(trimethylene carbonate)-bock-poly(L-glutamic acid) derived polymersomes, targeted to epidermal growth factor receptor (EGFR), loaded with plitidepsin and ultimately tested in HT29 and LS174T colorectal cancer cell lines for specificity and efficacy. Furthermore, morphology, physico-chemical properties and plitidepsin loading were carefully investigated. A thorough in vitro cytotoxicity analysis of the unloaded polymersomes was carried out for biocompatibility check, studying viability, cell membrane asymmetry and reactive oxygen species levels. Those cytotoxicity assays showed good biocompatibility for plitidepsin-unloaded polymersomes. Cellular uptake and cytotoxic effect of EGFR targeted and plitidepsin loaded polymersome indicated that colorectal cancer cell lines were.more sensitive to anti-EGFR-drug-loaded than untargeted drug-loaded polymersomes. Also, in both cell lines, the use of untargeted polymersomes greatly reduced plitidepsin cytotoxicity as well as the cellular uptake, indicating that the use of this targeted nanocarrier is a promising approach to tackle colorectal cancer disease and avoid the undesired effects of the usual treatment. Furthermore, in vivo assays support the in vitro conclusions that EGFR targeted polymersomes could be a good drug delivery system. This work provides a proof of concept for the use of encapsulated targeted drugs as future therapeutic treatments for cancer.

  6. Factor VIII C1 domain spikes 2092-2093 and 2158-2159 comprise regions that modulate cofactor function and cellular uptake.

    PubMed

    Bloem, Esther; van den Biggelaar, Maartje; Wroblewska, Aleksandra; Voorberg, Jan; Faber, Johan H; Kjalke, Marianne; Stennicke, Henning R; Mertens, Koen; Meijer, Alexander B

    2013-10-11

    The C1 domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent "hot spot" on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091-2104 and 2157-2162 from hydrogen-deuterium exchange. These comprise the two C1 domain spikes 2092-2093 and 2158-2159. Spike 2092-2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158-2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 domain spikes 2092-2093 and 2158-2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 domain in FVIII biology. PMID:24009077

  7. Factor VIII C1 Domain Spikes 2092–2093 and 2158–2159 Comprise Regions That Modulate Cofactor Function and Cellular Uptake

    PubMed Central

    Bloem, Esther; van den Biggelaar, Maartje; Wroblewska, Aleksandra; Voorberg, Jan; Faber, Johan H.; Kjalke, Marianne; Stennicke, Henning R.; Mertens, Koen; Meijer, Alexander B.

    2013-01-01

    The C1 domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent “hot spot” on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091–2104 and 2157–2162 from hydrogen-deuterium exchange. These comprise the two C1 domain spikes 2092–2093 and 2158–2159. Spike 2092–2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158–2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 domain spikes 2092–2093 and 2158–2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 domain in FVIII biology. PMID:24009077

  8. Inhibition of MDR1 gene expression and enhancing cellular uptake for effective colon cancer treatment using dual-surface–functionalized nanoparticles

    PubMed Central

    Xiao, Bo; Zhang, Mingzhen; Viennois, Emilie; Zhang, Yuchen; Wei, Na; Baker, Mark T.; Jung, Yunjin; Merlin, Didier

    2015-01-01

    Nanomedicine options for colon cancer therapy have been limited by the lack of suitable carriers capable of delivering sufficient drug into tumors to cause lethal toxicity. To circumvent this limitation, we fabricated a camptothecin (CPT)-loaded poly(lactic-co-glycolic acid) nanoparticle (NP) with dual-surface functionalization—Pluronic F127 and chitosan—for inhibiting multi-drug resistant gene 1 (MDR1) expression and enhancing tumor uptake. The resultant spherical NPs-P/C had a desirable particle size (~268 nm), slightly positive zeta-potential, and the ability to efficiently down-regulate the expression of MDR1. In vitro cytotoxicity tests revealed that the 24 and 48 h IC50 values of NPs-P/C1 were 2.03 and 0.67 µM, respectively, which were much lower than those for free CPT and other NPs. Interestingly, NPs-P/C1 showed the highest cellular uptake efficiency (approximately 85.5%) among the different drug formulations. Most importantly, treatment of colon tumor-bearing mice with various drug formulations confirmed that the introduction of Pluronic F127 and chitosan to the NP surface significantly enhanced the therapeutic efficacy of CPT, induced tumor cell apoptosis, and reduced systemic toxicity. Collectively, these findings suggest that our one-step–fabricated, dual-surface–functionalized NPs may hold promise as a readily scalable and effective drug carrier with clinical potential in colon cancer therapy. PMID:25701040

  9. Dietary uptake of Cu sorbed to hydrous iron oxide is linked to cellular toxicity and feeding inhibition in a benthic grazer

    USGS Publications Warehouse

    Cain, Daniel J.; Croteau, Marie-Noele; Fuller, Christopher C.; Ringwood, Amy H.

    2016-01-01

    Whereas feeding inhibition caused by exposure to contaminants has been extensively documented, the underlying mechanism(s) are less well understood. For this study, the behavior of several key feeding processes, including ingestion rate and assimilation efficiency, that affect the dietary uptake of Cu were evaluated in the benthic grazer Lymnaea stagnalis following 4–5 h exposures to Cu adsorbed to synthetic hydrous ferric oxide (Cu–HFO). The particles were mixed with a cultured alga to create algal mats with Cu exposures spanning nearly 3 orders of magnitude at variable or constant Fe concentrations, thereby allowing first order and interactive effects of Cu and Fe to be evaluated. Results showed that Cu influx rates and ingestion rates decreased as Cu exposures of the algal mat mixture exceeded 104 nmol/g. Ingestion rate appeared to exert primary control on the Cu influx rate. Lysosomal destabilization rates increased directly with Cu influx rates. At the highest Cu exposure where the incidence of lysosomal membrane damage was greatest (51%), the ingestion rate was suppressed 80%. The findings suggested that feeding inhibition was a stress response emanating from excessive uptake of dietary Cu and cellular toxicity.

  10. A smart tumor targeting peptide-drug conjugate, pHLIP-SS-DOX: synthesis and cellular uptake on MCF-7 and MCF-7/Adr cells.

    PubMed

    Song, Qin; Chuan, Xingxing; Chen, Binlong; He, Bing; Zhang, Hua; Dai, Wenbing; Wang, Xueqing; Zhang, Qiang

    2016-06-01

    Doxorubicin (DOX) is a potent anticancer drug for the treatment of tumors, but the poor specificity and multi-drug resistance (MDR) on tumor cells have restricted its application. Here, a pH and reduction-responsive peptide-drug conjugate (PDC), pHLIP-SS-DOX, was synthesized to overcome these drawbacks. pH low insertion peptide (pHLIP) is a cell penetrating peptide (CPP) with pH-dependent transmembrane ability. And because of the unique cell membrane insertion pattern, it might reverse the MDR. The cellular uptake study showed that on both drug-sensitive MCF-7 and drug-resistant MCF-7/Adr cells, pHLIP-SS-DOX obviously facilitated the uptake of DOX at pH 6.0 and the uptake level on MCF-7/Adr cells was similar with that on MCF-7 cells, indicating that pHLIP-SS-DOX had the ability to target acidic tumor cells and reverse MDR. In vitro cytotoxicity study mediated by GSH-OEt demonstrated that the cytotoxic effect of pHLIP-SS-DOX was reduction responsive, with obvious cytotoxicity at pH 6.0; while it had poor cytotoxicity at pH 7.4, no matter with or without GSH-OEt pretreatment. This illustrated that pHLIP could deliver DOX into tumor cells with acidic microenvironment specifically and could not deliver drugs into normal cells with neutral microenvironment. In summary, pHLIP-SS-DOX is a promising strategy to target drugs to tumors and provides a possibility to overcome MDR.

  11. Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect

    PubMed Central

    Veldhoen, Sandra; Laufer, Sandra D.; Trampe, Alexander; Restle, Tobias

    2006-01-01

    Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGα, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGα-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC50 value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGα/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGα/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGα/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGα-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed. PMID:17135188

  12. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  13. Differential roles of the protein corona in the cellular uptake of nanoporous polymer particles by monocyte and macrophage cell lines.

    PubMed

    Yan, Yan; Gause, Katelyn T; Kamphuis, Marloes M J; Ang, Ching-Seng; O'Brien-Simpson, Neil M; Lenzo, Jason C; Reynolds, Eric C; Nice, Edouard C; Caruso, Frank

    2013-12-23

    Many biomolecules, mainly proteins, adsorb onto polymer particles to form a dynamic protein corona in biological environments. The protein corona can significantly influence particle-cell interactions, including internalization and pathway activation. In this work, we demonstrate the differential roles of a given protein corona formed in cell culture media in particle uptake by monocytes and macrophages. By exposing disulfide-stabilized poly(methacrylic acid) nanoporous polymer particles (PMASH NPPs) to complete cell growth media containing 10% fetal bovine serum, a protein corona, with the most abundant component being bovine serum albumin, was characterized. Upon adsorption onto the PMASH NPPs, native bovine serum albumin (BSA) was found to undergo conformational changes. The denatured BSA led to a significant decrease in internalization efficiency in human monocytic cells, THP-1, compared with the bare particles, due to reduced cell membrane adhesion. In contrast, the unfolded BSA on the NPPs triggered class A scavenger receptor-mediated phagocytosis in differentiated macrophage-like cells (dTHP-1) without a significant impact on the overall internalization efficiency. Taken together, this work demonstrates the disparate effects of a given protein corona on particle-cell interactions, highlighting the correlation between protein corona conformation in situ and relevant biological characteristics for biological functionalities.

  14. TAT and HA2 Facilitate Cellular Uptake of Gold Nanoparticles but Do Not Lead to Cytosolic Localisation

    PubMed Central

    Free, Paul; Lévy, Raphaël

    2015-01-01

    The methods currently available to deliver functional labels and drugs to the cell cytosol are inefficient and this constitutes a major obstacle to cell biology (delivery of sensors and imaging probes) and therapy (drug access to the cell internal machinery). As cell membranes are impermeable to most molecular cargos, viral peptides have been used to bolster their internalisation through endocytosis and help their release to the cytosol by bursting the endosomal vesicles. However, conflicting results have been reported on the extent of the cytosolic delivery achieved. To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery. We evaluated the number of gold nanoparticles present in cells after internalisation using photothermal microscopy and their subcellular localisation by electron microscopy. While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles. PMID:25836335

  15. Lysine-functionalized nanodiamonds as gene carriers: development of stable colloidal dispersion for in vitro cellular uptake studies and siRNA delivery application

    PubMed Central

    Alwani, Saniya; Kaur, Randeep; Michel, Deborah; Chitanda, Jackson M; Verrall, Ronald E; Karunakaran, Chithra; Badea, Ildiko

    2016-01-01

    Purpose Nanodiamonds (NDs) are emerging as an attractive tool for gene therapeutics. To reach their full potential for biological application, NDs should maintain their colloidal stability in biological milieu. This study describes the behavior of lysine-functionalized ND (lys-ND) in various dispersion media, with an aim to limit aggregation and improve the colloidal stability of ND-gene complexes called diamoplexes. Furthermore, cellular and macromolecular interactions of lys-NDs are also analyzed in vitro to establish the understanding of ND-mediated gene transfer in cells. Methods lys-NDs were synthesized earlier through covalent conjugation of lysine amino acid to carboxylated NDs surface generated through re-oxidation in strong oxidizing acids. In this study, dispersions of lys-NDs were prepared in various media, and the degree of sedimentation was monitored for 72 hours. Particle size distributions and zeta potential measurements were performed for a period of 25 days to characterize the physicochemical stability of lys-NDs in the medium. The interaction profile of lys-NDs with fetal bovine serum showed formation of a protein corona, which was evaluated by size and charge distribution measurements. Uptake of lys-NDs in cervical cancer cells was analyzed by scanning transmission X-ray microscopy, flow cytometry, and confocal microscopy. Cellular uptake of diamoplexes (complex of lys-NDs with small interfering RNA) was also analyzed using flow cytometry. Results Aqueous dispersion of lys-NDs showed minimum sedimentation and remained stable over a period of 25 days. Size distributions showed good stability, remaining under 100 nm throughout the testing period. A positive zeta potential of >+20 mV indicated a preservation of surface charges. Size distribution and zeta potential changed for lys-NDs after incubation with blood serum, suggesting an interaction with biomolecules, mainly proteins, and a possible formation of a protein corona. Cellular internalization

  16. Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase:RTB lectin fusion.

    PubMed

    Condori, Jose; Acosta, Walter; Ayala, Jorge; Katta, Varun; Flory, Ashley; Martin, Reid; Radin, Jonathan; Cramer, Carole L; Radin, David N

    2016-02-01

    cleaved and rapidly degraded. The activated β-gal was still detected at 48h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of β-gal:RTB supported significantly greater accumulation of β-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived β-gal. These data demonstrate that plant-made β-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis - delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments.

  17. Droplet aerodynamics, cellular uptake, and efficacy of a nebulizable corticosteroid nanosuspension are superior to a micronized dosage form.

    PubMed

    Britland, Stephen; Finter, Wayne; Chrystyn, Henry; Eagland, Donald; Abdelrahim, Mohamed E

    2012-01-01

    Inhaled corticosteroids are considered to be an effective prophylactic against the morbid symptoms of several lung diseases, but scope remains for improvement in drug delivery technology to benefit bioavailability and treatment compliance. To ascertain whether dosage form might influence bioavailability, the emission characteristics and efficacy of a nanoparticulate budesonide formulation (Nanagel®) were compared with those of a proprietary micronized suspension (Pulmicort®) when delivered as a nebulized aerosol to human airway epithelial cells in a culture model. Having the visual appearance of a clear solution, Nanagel® was delivered by both jet and vibrating mesh nebulizers as an increased fine particle fraction and with a smaller mass median aerodynamic diameter (MMAD) compared to the micronized suspension. Quantitative high performance liquid chromatography (HPLC) analysis of cultured epithelia one hour after treatment with Nanagel® revealed a significantly greater cellular accumulation of budesonide when compared with Pulmicort® for an equivalent dose, a differential which persisted 24 and 48 h later. A quantitative in vitro assay measuring the activity of enzymes involved in superoxide production revealed that stressed HaCaT cells (a long-lived, spontaneously immortalized human keratinocyte line) treated with Nanagel® continued to show significantly greater attenuation of inflammatory response compared with Pulmicort®-treated cells 24 h after the application of an equivalent budesonide dose. The present in vitro findings suggest that formulation of inhalable drugs such as budesonide as aerosolized nanoparticulate, rather than microparticulate, suspensions can enhance bioavailability with concomitant improvements in efficacy.

  18. Synthesis and characterization of Her2-NLP peptide conjugates targeting circulating breast cancer cells: cellular uptake and localization by fluorescent microscopic imaging.

    PubMed

    Cai, Huawei; Singh, Ajay N; Sun, Xiankai; Peng, Fangyu

    2015-01-01

    To synthesize a fluorescent Her2-NLP peptide conjugate consisting of Her2/neu targeting peptide and nuclear localization sequence peptide (NLP) and assess its cellular uptake and intracellular localization for radionuclide cancer therapy targeting Her2/neu-positive circulating breast cancer cells (CBCC). Fluorescent Cy5.5 Her2-NLP peptide conjugate was synthesized by coupling a bivalent peptide sequence, which consisted of a Her2-binding peptide (NH2-GSGKCCYSL) and an NLP peptide (CGYGPKKKRKVGG) linked by a polyethylene glycol (PEG) chain with 6 repeating units, with an activated Cy5.5 ester. The conjugate was separated and purified by HPLC and then characterized by Maldi-MS. The intracellular localization of fluorescent Cy5.5 Her2-NLP peptide conjugate was assessed by fluorescent microscopic imaging using a confocal microscope after incubation of Cy5.5-Her2-NLP with Her2/neu positive breast cancer cells and Her2/neu negative control breast cancer cells, respectively. Fluorescent signals were detected in cytoplasm of Her2/neu positive breast cancer cells (SKBR-3 and BT474 cell lines), but not or little in cytoplasm of Her2/neu negative breast cancer cells (MDA-MB-231), after incubation of the breast cancer cells with Cy5.5-Her2-NLP conjugates in vitro. No fluorescent signals were detected within the nuclei of Her2/neu positive SKBR-3 and BT474 breast cancer cells, neither Her2/neu negative MDA-MB-231 cells, incubated with the Cy5.5-Her2-NLP peptide conjugates, suggesting poor nuclear localization of the Cy5.5-Her2-NLP conjugates localized within the cytoplasm after their cellular uptake and internalization by the Her2/neu positive breast cancer cells. Her2-binding peptide (KCCYSL) is a promising agent for radionuclide therapy of Her2/neu positive breast cancer using a β(-) or α emitting radionuclide, but poor nuclear localization of the Her2-NLP peptide conjugates may limit its use for eradication of Her2/neu-positive CBCC using I-125 or other Auger electron

  19. Cationic lipid-nanoceria hybrids, a novel nonviral vector-mediated gene delivery into mammalian cells: investigation of the cellular uptake mechanism.

    PubMed

    Das, Joydeep; Han, Jae Woong; Choi, Yun-Jung; Song, Hyuk; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

    2016-07-06

    Gene therapy is a promising technique for the treatment of various diseases. The development of minimally toxic and highly efficient non-viral gene delivery vectors is the most challenging undertaking in the field of gene therapy. Here, we developed dimethyldioctadecylammonium bromide (DODAB)-nanoceria (CeO2) hybrids as a new class of non-viral gene delivery vectors. These DODAB-modified CeO2 nanoparticles (CeO2/DODAB) could effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines. The CeO2/DODAB nanovectors were also found to be non-toxic and did not induce ROS formation as well as any stress responsive and pro-survival signaling pathways. The overall vector performance of CeO2/DODAB nanohybrids was comparable with lipofectamine and DOTAP, and higher than calcium phosphate and DEAE-dextran for transfecting small plasmids. The increased cellular uptake of the nanovector/DNA complexes through clathrin- and caveolae-mediated endocytosis and subsequent release from the endosomes further support the increased gene transfection efficiency of the CeO2/DODAB vectors. Besides, CeO2/DODAB nanovectors could transfect genes in vivo without any sign of toxicity. Taken together, this new nano-vector has the potential to be used for gene delivery in biomedical applications.

  20. Supramolecular Nanotube of Chaperonin GroEL: Length Control for Cellular Uptake Using Single-Ring GroEL Mutant as End-Capper.

    PubMed

    Sim, Seunghyun; Niwa, Tatsuya; Taguchi, Hideki; Aida, Takuzo

    2016-09-01

    How to modulate supramolecular protein nanotubes without sacrificing their thermodynamic stability? This challenging issue emerged with an enhanced reality since our successful development of a protein nanotube of chaperonin GroELMC as a novel ATP-responsive 1D nanocarrier because the nanotube length may potentially affect the cellular uptake efficiency. Herein, we report a molecularly engineered protein end-capper (SRMC) that firmly binds to the nanotube termini since the end-capper originates from GroEL. According to the single-ring mutation of GroEL, we obtained a single-ring version of GroEL bearing cysteine mutations (GroELCys) and modified its 14 apical cysteine residues with merocyanine (MC). Whereas SRMC self-dimerizes upon treatment with Mg(2+), we confirmed that SRMC serves as the efficient end-capper for the Mg(2+)-mediated supramolecular polymerization of GroELMC and allows for modulating the average nanotube length over a wide range from 320 to 40 nm by increasing the feed molar ratio SRMC/GroELMC up to 5.4. We also found that the nanotubes shorter than 100 nm are efficiently taken up into HEP3B cells. PMID:27545864

  1. Portal vein glucose entry triggers a coordinated cellular response that potentiates hepatic glucose uptake and storage in normal but not high-fat/high-fructose-fed dogs.

    PubMed

    Coate, Katie C; Kraft, Guillaume; Irimia, Jose M; Smith, Marta S; Farmer, Ben; Neal, Doss W; Roach, Peter J; Shiota, Masakazu; Cherrington, Alan D

    2013-02-01

    The cellular events mediating the pleiotropic actions of portal vein glucose (PoG) delivery on hepatic glucose disposition have not been clearly defined. Likewise, the molecular defects associated with postprandial hyperglycemia and impaired hepatic glucose uptake (HGU) following consumption of a high-fat, high-fructose diet (HFFD) are unknown. Our goal was to identify hepatocellular changes elicited by hyperinsulinemia, hyperglycemia, and PoG signaling in normal chow-fed (CTR) and HFFD-fed dogs. In CTR dogs, we demonstrated that PoG infusion in the presence of hyperinsulinemia and hyperglycemia triggered an increase in the activity of hepatic glucokinase (GK) and glycogen synthase (GS), which occurred in association with further augmentation in HGU and glycogen synthesis (GSYN) in vivo. In contrast, 4 weeks of HFFD feeding markedly reduced GK protein content and impaired the activation of GS in association with diminished HGU and GSYN in vivo. Furthermore, the enzymatic changes associated with PoG sensing in chow-fed animals were abolished in HFFD-fed animals, consistent with loss of the stimulatory effects of PoG delivery. These data reveal new insight into the molecular physiology of the portal glucose signaling mechanism under normal conditions and to the pathophysiology of aberrant postprandial hepatic glucose disposition evident under a diet-induced glucose-intolerant condition.

  2. 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.

  3. DNA Binding and Photocleavage Properties, Cellular Uptake and Localization, and in-Vitro Cytotoxicity of Dinuclear Ruthenium(II) Complexes with Varying Lengths in Bridging Alkyl Linkers.

    PubMed

    Liu, Ping; Wu, Bao-Yan; Liu, Jin; Dai, Yong-Cheng; Wang, You-Jun; Wang, Ke-Zhi

    2016-02-15

    Two new dinuclear Ru(II) polypyridyl complexes containing three and ten methylene chains in their bridging linkers are synthesized and characterized. Their calf thymus DNA-binding and plasmid DNA photocleavage behaviors are comparatively studied with a previously reported, six-methylene-containing analog by absorption and luminescence spectroscopy, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, DNA viscosity measurements, DNA thermal denaturation, and agarose gel electrophoresis analyses. Theoretical calculations applying the density functional theory (DFT) method for the three complexes are also performed to understand experimentally observed DNA binding properties. The results show that the two complexes partially intercalate between the base pairs of DNA. Cellular uptake and colocalization studies have demonstrated that the complexes could enter HeLa cells efficiently and localize within lysosomes. The in-vitro antitumor activity against HeLa and MCF-7 tumor cells of the complexes are studied by MTT cytotoxic analysis. A new method, high-content analysis (HCA), is also used to assess cytotoxicity, apoptosis and cell cycle arrest of the three complexes. The results show that the lengths of the alkyl linkers could effectively tune their biological properties and that HCA is suitable for rapidly identifying cytotoxicity and can be substituted for MTT assays to evaluate the cell cytotoxicity of chemotherapeutic agents.

  4. Acidity-promoted cellular uptake and drug release mediated by amine-functionalized block polycarbonates prepared via one-shot ring-opening copolymerization.

    PubMed

    Wang, Hua-Fen; Jia, Hui-Zhen; Chu, Yan-Feng; Feng, Jun; Zhang, Xian-Zheng; Zhuo, Ren-Xi

    2014-04-01

    This paper reports a drug nanovehicle self-assembled from an amine-functionalized block copolymer poly(6,14-dimethyl-1,3,9,11-tetraoxa-6,14-diaza-cyclohexadecane-2,10-dione)-block-poly(1,3-dioxepan-2-one) (PADMC-b-PTeMC), which is prepared by controlable ring-opening block copolymerization attractively in a "one-shot feeding" pathway. The copolymers display high cell-biocompatibility with no apparent cytotoxicities detected in 293T and HeLa cells. Due to their amphiphilic nature, PADMC-b-PTeMC copolymers can self-assemble into nanosized micelles capable of loading anticancer drugs such as camptothecin (CPT) and doxorubicin (DOX). In particular, the outer PADMC shell endows the PADMC-b-PTeMC nanomicelles with pH-dependent control over the micellar morphology, cell uptake efficiency, and the drug release pattern. Confocal inspection reveals the remarkably enhanced cellular internalization of drug loaded micelles by cancerous HeLa cells at relatively lower pH 5.8 simulating the mildly acid microenvironment in tumors. Along with the acidity-triggered volume expansion of micelles, an accelerated CPT release in vitro occurs. The obtained results adumbrate the possibility of completely biodegradable PADMC-b-PTeMC as pH-sensitive drug carriers for tumor chemotherapy.

  5. Cationic lipid-nanoceria hybrids, a novel nonviral vector-mediated gene delivery into mammalian cells: investigation of the cellular uptake mechanism

    PubMed Central

    Das, Joydeep; Han, Jae Woong; Choi, Yun-Jung; Song, Hyuk; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

    2016-01-01

    Gene therapy is a promising technique for the treatment of various diseases. The development of minimally toxic and highly efficient non-viral gene delivery vectors is the most challenging undertaking in the field of gene therapy. Here, we developed dimethyldioctadecylammonium bromide (DODAB)–nanoceria (CeO2) hybrids as a new class of non-viral gene delivery vectors. These DODAB-modified CeO2 nanoparticles (CeO2/DODAB) could effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines. The CeO2/DODAB nanovectors were also found to be non-toxic and did not induce ROS formation as well as any stress responsive and pro-survival signaling pathways. The overall vector performance of CeO2/DODAB nanohybrids was comparable with lipofectamine and DOTAP, and higher than calcium phosphate and DEAE-dextran for transfecting small plasmids. The increased cellular uptake of the nanovector/DNA complexes through clathrin- and caveolae-mediated endocytosis and subsequent release from the endosomes further support the increased gene transfection efficiency of the CeO2/DODAB vectors. Besides, CeO2/DODAB nanovectors could transfect genes in vivo without any sign of toxicity. Taken together, this new nano-vector has the potential to be used for gene delivery in biomedical applications. PMID:27380727

  6. Cationic lipid-nanoceria hybrids, a novel nonviral vector-mediated gene delivery into mammalian cells: investigation of the cellular uptake mechanism.

    PubMed

    Das, Joydeep; Han, Jae Woong; Choi, Yun-Jung; Song, Hyuk; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

    2016-01-01

    Gene therapy is a promising technique for the treatment of various diseases. The development of minimally toxic and highly efficient non-viral gene delivery vectors is the most challenging undertaking in the field of gene therapy. Here, we developed dimethyldioctadecylammonium bromide (DODAB)-nanoceria (CeO2) hybrids as a new class of non-viral gene delivery vectors. These DODAB-modified CeO2 nanoparticles (CeO2/DODAB) could effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines. The CeO2/DODAB nanovectors were also found to be non-toxic and did not induce ROS formation as well as any stress responsive and pro-survival signaling pathways. The overall vector performance of CeO2/DODAB nanohybrids was comparable with lipofectamine and DOTAP, and higher than calcium phosphate and DEAE-dextran for transfecting small plasmids. The increased cellular uptake of the nanovector/DNA complexes through clathrin- and caveolae-mediated endocytosis and subsequent release from the endosomes further support the increased gene transfection efficiency of the CeO2/DODAB vectors. Besides, CeO2/DODAB nanovectors could transfect genes in vivo without any sign of toxicity. Taken together, this new nano-vector has the potential to be used for gene delivery in biomedical applications. PMID:27380727

  7. Supramolecular Nanotube of Chaperonin GroEL: Length Control for Cellular Uptake Using Single-Ring GroEL Mutant as End-Capper.

    PubMed

    Sim, Seunghyun; Niwa, Tatsuya; Taguchi, Hideki; Aida, Takuzo

    2016-09-01

    How to modulate supramolecular protein nanotubes without sacrificing their thermodynamic stability? This challenging issue emerged with an enhanced reality since our successful development of a protein nanotube of chaperonin GroELMC as a novel ATP-responsive 1D nanocarrier because the nanotube length may potentially affect the cellular uptake efficiency. Herein, we report a molecularly engineered protein end-capper (SRMC) that firmly binds to the nanotube termini since the end-capper originates from GroEL. According to the single-ring mutation of GroEL, we obtained a single-ring version of GroEL bearing cysteine mutations (GroELCys) and modified its 14 apical cysteine residues with merocyanine (MC). Whereas SRMC self-dimerizes upon treatment with Mg(2+), we confirmed that SRMC serves as the efficient end-capper for the Mg(2+)-mediated supramolecular polymerization of GroELMC and allows for modulating the average nanotube length over a wide range from 320 to 40 nm by increasing the feed molar ratio SRMC/GroELMC up to 5.4. We also found that the nanotubes shorter than 100 nm are efficiently taken up into HEP3B cells.

  8. Magnetic Particle Spectroscopy Reveals Dynamic Changes in the Magnetic Behavior of Very Small Superparamagnetic Iron Oxide Nanoparticles During Cellular Uptake and Enables Determination of Cell-Labeling Efficacy.

    PubMed

    Poller, Wolfram C; Löwa, Norbert; Wiekhorst, Frank; Taupitz, Matthias; Wagner, Susanne; Möller, Konstantin; Baumann, Gert; Stangl, Verena; Trahms, Lutz; Ludwig, Antje

    2016-02-01

    In vivo tracking of nanoparticle-labeled cells by magnetic resonance imaging (MRI) crucially depends on accurate determination of cell-labeling efficacy prior to transplantation. Here, we analyzed the feasibility and accuracy of magnetic particle spectroscopy (MPS) for estimation of cell-labeling efficacy in living THP-1 cells incubated with very small superparamagnetic iron oxide nanoparticles (VSOP). Cell viability and proliferation capacity were not affected by the MPS measurement procedure. In VSOP samples without cell contact, MPS enabled highly accurate quantification. In contrast, MPS constantly overestimated the amount of cell associated and internalized VSOP. Analyses of the MPS spectrum shape expressed as harmonic ratio A₅/A₃ revealed distinct changes in the magnetic behavior of VSOP in response to cellular uptake. These changes were proportional to the deviation between MPS and actual iron amount, therefore allowing for adjusted iron quantification. Transmission electron microscopy provided visual evidence that changes in the magnetic properties correlated with cell surface interaction of VSOP as well as with alterations of particle structure and arrangement during the phagocytic process. Altogether, A₅/A₃-adjusted MPS enables highly accurate, cell-preserving VSOP quantification and furthermore provides information on the magnetic characteristics of internalized VSOP. PMID:27305767

  9. Impaired NFKBIE gene function decreases cellular uptake of methotrexate by down-regulating SLC19A1 expression in a human rheumatoid arthritis cell line

    PubMed Central

    Imamura, Hitoshi; Yoshina, Sawako; Ikari, Katsunori; Miyazawa, Keiji; Momohara, Shigeki; Mitani, Shohei

    2016-01-01

    Abstract Objective: A non-synonymous single nucleotide polymorphism (nsSNP, rs2233434, Val194Ala) in the NFKBIE (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon) gene is known to be a rheumatoid arthritis (RA) susceptibility polymorphism in the Japanese RA population and could be closely associated with nuclear factor kappaB (NF-κB) activity. Inflammation caused by RA is sometimes associated with changes in expression levels of MTX (methotrexate) pathway-related genes. It is of interest to examine whether the NFKBIE gene had any influences on the mode of MTX action. Methods: Both knockdown of NFKBIE gene expression and overexpression of wild-type NFKBIE and Val194Ala mutation were performed. A transfected human RA synovial cell line was cultured and then gene expressions in the MTX pathway were measured. In addition, we measured the uptake and efflux of MTX derivatives under the NFKBIE knockdown condition. Results: Knockdown of NFKBIE reduced the mRNA for SLC19A1, a main MTX membrane transporter, and the intracellular accumulations of MTX derivatives. Moreover, our experiments also confirmed that overexpression of Val194Ala mutant NFKBIE decreased the SLC19A1 mRNA when compared to that of wild-type NFKBIE. Conclusions: We suggest that the impairment of NFKBIE gene function can reduce the uptake of MTX into cells, suggesting that the gene is an important factor for the RA outcome. PMID:26587663

  10. Short-term enhancement effect of nitrogen addition on microbial degradation and plant uptake of polybrominated diphenyl ethers (PBDEs) in contaminated mangrove soil.

    PubMed

    Chen, Juan; Zhou, Hai Chao; Wang, Chao; Zhu, Chun Quan; Tam, Nora Fung-Yee

    2015-12-30

    Effects of nitrogen (N) addition on the microbial degradation and uptake of a mixture of BDE-47 and -209 by Aegiceras corniculatum, a typical mangrove plant species were investigated. At the end of 3-month experiment, a significant dissipation of BDE-47 was observed in the planted soil, and this dissipation, particularly in rhizosphere soil, was significantly accelerated by the frequent addition of N in the form of ammonium chloride. The removal percentage of BDE-47 in the rhizosphere soil without N addition was 47.3% and increased to 58.2% with N. However, the unplanted soil only removed less than 25% BDE-47, irrespective to N supply. The N addition in planted treatments significantly increased soil N content, urease and dehydrogenase activities, and the abundances of total bacteria and dehalogenating bacteria, leading to more microbial degradation of BDE-47. The N addition also enhanced the root uptake and translocation of PBDEs to above-ground tissues of A. corniculatum. These results suggested that N addition could enhance the phytoremediation of BDE-47-contaminated soil within a short period of time. Different from BDE-47, BDE-209 in all contaminated soils was difficult to be removed due to its persistence and low bioavailability.

  11. Cellular Uptake of Elastic Nanoparticles

    NASA Astrophysics Data System (ADS)

    Yi, Xin; Shi, Xinghua; Gao, Huajian

    2011-08-01

    A fundamental understanding of cell-nanomaterial interaction is of essential importance to nanomedicine and safe applications of nanotechnology. Here we investigate the adhesive wrapping of a soft elastic vesicle by a lipid membrane. We show that there exist a maximum of five distinct wrapping phases based on the stability of full wrapping, partial wrapping, and no wrapping states. The wrapping phases depend on the vesicle size, adhesion energy, surface tension of membrane, and bending rigidity ratio between vesicle and membrane. These results are of immediate interest to the study of vesicular transport and endocytosis or phagocytosis of elastic particles into cells.

  12. Emissive behavior, cytotoxic activity, cellular uptake, and PEGylation properties of new luminescent rhenium(I) polypyridine poly(ethylene glycol) complexes.

    PubMed

    Choi, Alex Wing-Tat; Louie, Man-Wai; Li, Steve Po-Yam; Liu, Hua-Wei; Chan, Bruce Ting-Ngok; Lam, Tonlex Chun-Ying; Lin, Alex Chun-Chi; Cheng, Shuk-Han; Lo, Kenneth Kam-Wing

    2012-12-17

    We report here a new class of biological reagents derived from luminescent rhenium(I) polypyridine complexes modified with a poly(ethylene glycol) (PEG) pendant. The PEG-amine complexes [Re(N(^)N)(CO)(3)(py-PEG-NH(2))](PF(6)) (py-PEG-NH(2) = 3-amino-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine, MW(PEG) = 5000 Da, PDI(PEG) < 1.08; N(^)N = 1,10-phenanthroline (phen) (1-PEG-NH(2)), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me(4)-phen) (2-PEG-NH(2)), 4,7-diphenyl-1,10-phenanthroline (Ph(2)-phen) (3-PEG-NH(2))) and [Re(bpy-PEG)(CO)(3)(py-NH(2))](PF(6)) (bpy-PEG = 4-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine; py-NH(2) = 3-aminopyridine) (4-PEG-NH(2)) have been synthesized and characterized. The photophysical properties, lipophilicity, water solubility, cytotoxic activity, and cellular uptake properties of these complexes have been compared to those of their PEG-free counterparts [Re(N(^)N)(CO)(3)(py-Et-NH(2))](PF(6)) (py-Et-NH(2) = 3-amino-5-(N-(ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-Et-NH(2)), Me(4)-phen (2-Et-NH(2)), Ph(2)-phen (3-Et-NH(2))) and [Re(bpy-Et)(CO)(3)(py-NH(2))](PF(6)) (bpy-Et = 4-(N-(ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine) (4-Et-NH(2)). The PEG complexes exhibited significantly higher water solubility and lower cytotoxicity (IC(50) = 6.6 to 1152 μM) than their PEG-free counterparts (IC(50) = 3.6 to 159 μM), indicating that the covalent attachment of a PEG pendant to rhenium(I) polypyridine complexes is an effective way to increase their biocompatibility. The amine complexes 1-PEG-NH(2)-4-PEG-NH(2) have been activated with thiophosgene to yield the isothiocyanate complexes [Re(N(^)N)(CO)(3)(py-PEG-NCS)](PF(6)) (py-PEG-NCS = 3-isothiocyanato-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-PEG-NCS), Me(4)-phen (2-PEG-NCS), Ph(2)-phen (3-PEG-NCS)), and [Re(bpy-PEG)(CO)(3)(py-NCS)](PF(6)) (py-NCS = 3-isothiocyanatopyridine) (4-PEG-NCS) as a new class

  13. Does photocatalytic activity of TiO2 nanoparticles correspond to photo-cytotoxicity? Cellular uptake of TiO2 nanoparticles is important in their photo-cytotoxicity.

    PubMed

    Horie, Masanori; Sugino, Sakiko; Kato, Haruhisa; Tabei, Yosuke; Nakamura, Ayako; Yoshida, Yasukazu

    2016-05-01

    Titanium dioxide (TiO2) nanoparticles are important industrial nano-objects with wide applications, including as photocatalysts and sunscreen components. Recently, the phototoxicity of TiO2 nanoparticles has been a concern. However, phototoxicity caused by photocatalytic activity may differ between anatase and rutile nanoparticles. In the present study, we compared the phototoxicity of anatase and rutile nanoparticles. Human keratinocyte HaCaT cells were treated with stable TiO2 nanoparticle suspensions. Without UVA irradiation, TiO2 nanoparticles did not affect mitochondrial activity or cell membranes. However, exposure to rutile nanoparticle suspensions inhibited cell growth and induced HO-1 gene expression without UVA irradiation. These effects may be explained by the hydrophobic surface of rutile nanoparticles. Next, TiO2-exposed cells were irradiated with UVA for 4 h and effects of TiO2 nanoparticles on cells were examined. The rutile nanoparticles did not show any cellular effects after UVA irradiation. However, the anatase nanoparticles caused strong phototoxicity. Decreased mitochondrial activity, cell membrane damage and the induction of oxidative stress were observed in the cells exposed to anatase nanoparticles with UVA irradiation. Cellular uptake of the nanoparticles was observed in both anatase- and rutile-exposed cells. These results suggest that internalized anatase nanoparticles are important for phototoxicity. Additionally, the exposure of a 3D skin model to TiO2 nanoparticles did not result in significant toxicity. In conclusion, rutile nanoparticles used in sunscreen did not exhibit phototoxic activity. Despite the strong phototoxic activity of anatase nanoparticles in cell cultures, they demonstrated no phototoxicity using a 3D skin model. PMID:27142467

  14. Quantitative evaluation of cellular uptake, DNA incorporation and adduct formation in cisplatin sensitive and resistant cell lines: Comparison of different Pt-containing drugs.

    PubMed

    Corte-Rodríguez, M; Espina, M; Sierra, L M; Blanco, E; Ames, T; Montes-Bayón, M; Sanz-Medel, A

    2015-11-01

    The use of Pt-containing compounds as chemotherapeutic agents facilitates drug monitoring by using highly sensitive elemental techniques like inductively coupled plasma mass spectrometry (ICP-MS). However, methodological problems arise when trying to compare different experiments due to the high variability of biological parameters. In this work we have attempted to identify and correct such variations in order to compare the biological behavior of cisplatin, oxaliplatin and pyrodach-2 (a novel platinum-containing agent). A detailed study to address differential cellular uptake has been conducted in three different cell lines: lung adenocarcinoma (A549); cisplatin-sensitive ovarian carcinoma (A2780); and cisplatin-resistant ovarian carcinoma (A2780cis). The normalization of Pt results to cell mass, after freeze-drying, has been used to minimize the errors associated with cell counting. Similarly, Pt accumulation in DNA has been evaluated by referencing the Pt results to the DNA concentration, as measured by (31)P monitoring using flow-injection and ICP-MS detection. These strategies have permitted to address significantly lower Pt levels in the resistant cells when treated with cisplatin or oxaliplatin as well as an independent behaviour from the cell type (sensitive or resistant) for pyrodach-2. Similarly, different levels of incorporation in DNA have been found for the three drugs depending on the cell model revealing a different behavior regarding cell cisplatin resistance. Further speciation experiments (by using complementary HPLC-ICP-MS and HPLC-ESI-Q-TOF MS) have shown that the main target in DNA is still the N7 of the guanine but with different kinetics of the ligand exchange mechanism for each of the compounds under evaluation.

  15. Doxorubicin-loaded biodegradable self-assembly zein nanoparticle and its anti-cancer effect: Preparation, in vitro evaluation, and cellular uptake.

    PubMed

    Dong, Fangyuan; Dong, Xiaoli; Zhou, Liping; Xiao, Huihui; Ho, Pui-Yu; Wong, Man-Sau; Wang, Yi

    2016-04-01

    Cancer is one top leading cause of the deaths worldwide. Various anticancer drugs, which can effectively kill cancer cells, have been developed in the last decade. However, the problem is still about the low therapeutic index of the drugs, which means that the effective dose of drugs will cause cytotoxicity to normal cells. A strategy based on drug nano-encapsulation is applied to achieve an effective anti-cancer therapy. In this study, we use zein, which is an amphiphilic protein, to make the anti-cancer drug nano-encapsulation. Doxorubicin (DOX), a popular anti-cancer drug, is selected as the core drug. The results show that DOX could be successfully encapsulated into zein to form spherical nanoparticles. The encapsulation efficiency and loading efficiency could reach as high as 90.06% and 15.01 mg/g, respectively. The cumulative release result showed a desired pH-responsible release behavior: DOX could be released faster in acidic buffer solutions (pH 5.0 and 6.5) than neutral one (pH 7.4). The effects of the nano-encapsulation on the anti-proliferation of HeLa cells were also examined. It indicated that, compared with free DOX, the DOX-loaded zein nanoparticles (DOX-zein-NPs) had a better effect on cancer cell killing at low DOX concentrations. We also investigated the cellular uptake of DOX-zein-NPs using confocal laser scanning microscopy (CLSM), flow cytometry, and transmission electron microscopy (TEM). And the endocytosis mechanism of DOX-zein-NPs entering into HeLa cells was studied using various endocytosis pathway inhibitors. PMID:26764113

  16. Receptor-independent, vacuolar ATPase-mediated cellular uptake of histamine receptor-1 ligands: Possible origin of pharmacological distortions and side effects

    SciTech Connect

    Morissette, Guillaume |; Lodge, Robert; Bouthillier, Johanne |; Marceau, Francois |

    2008-06-15

    The aims of this study were to investigate whether several histamine receptor agonists and antagonists are subjected to receptor-independent ion trapping into acidic organelles, and whether this sequestration influences their pharmacological or toxicological properties. Vacuolar (V)-ATPase-dependent intracellular sequestration of agonists was recognized as morphological alterations (large fluid-filled vacuoles for betahistine and 1-methylhistamine, granular uptake for fluorescent BODIPY FL histamine) prevented by the specific V-ATPase inhibitor bafilomycin A1 in rabbit vascular smooth muscle cells. Lipophilicity was the major determinant of these cellular effects (order of potency: BODIPY FL histamine > betahistine > 1-methylhistamine > histamine) that occurred at high concentrations. This ranking was dissociable from the potency order for H{sub 1} receptor-mediated contraction of the rabbit aorta, a response uninfluenced by bafilomycin. Antihistamines are inherently more lipophilic and caused vacuolization of a proportion of cells at 5-500 {mu}M. Agonist or antagonist-induced vacuoles were of macroautophagic nature (labeled with GFP-conjugated LC3, Rab7 and CD63; detection of LC3 II). Further, the 2 most lipophilic antihistamines tested, astemizole and terfenadine, were potentiated by V-ATPase blockade in the aortic contractility assay (13- and 3.6-fold more potent, respectively, pA{sub 2} scale), suggesting that V-ATPase-mediated cation trapping sequesters these antagonists from the vicinity of H{sub 1} receptors in the therapeutic concentration range. This potentiation did not apply to less lipophilic antagonists (pyrilamine, diphenhydramine). While some agonists and all tested antagonists of the histamine H{sub 1} receptors induce the V-ATPase-dependent vacuolar and autophagic cytopathology, sequestration affects the pharmacology of only the most lipophilic antagonists, the ones prone to off-target arrhythmogenic side effects.

  17. Cellular uptake and anticancer activity of salvianolic acid B phospholipid complex loaded nanoparticles in head and neck cancer and precancer cells.

    PubMed

    Li, Hongquan; Shi, Linjun; Wei, Jie; Zhang, Chenping; Zhou, Zengtong; Wu, Lan; Liu, Wei

    2016-11-01

    Salvianolic acid B (SalB) was demonstrated to be a promising chemopreventive agent for head and neck squamous cell carcinoma (HNSCC) in the previous studies by our and other research institution, but the properties like low efficacy, poor systemic delivery, and low bioavailability has hampered its clinical applications. To continue our research program focused on the use of natural compounds on cancer chemoprevention, we propose a first example of phospholipid complex loaded nanoparticles (PLC-NPs) encapsulating SalB as a potential carrier for intervention of HNSCC (HN13, HN30) cells and precancer Leuk1 cells in this study. Qualitative and quantitive studies of cellular uptake showed that intracellular accumulation of SalB was significantly higher when HN13, HN30 and Leuk1 cells were incubated with SalB-PLC-NPs complex (nano-SalB) as against free-SalB. Cell viability assay revealed that the cell growth of HN13 and HN30 cells was significantly inhibited of 56.1% and 29.3%, respectively, for nano-SalB compared to an equivalent amount of free-SalB (P<0.001). Moreover, cell cycle and apoptosis assay showed that a clear trend of cell cycle arrest and induction of apoptosis was also observed within the HNSCC cells treated with nano-SalB. Collectively, this study demonstrated that nano-SalB was significantly more potent had an anticancer effect against HNSCC cells, which serves as the first step toward establishing SalB nano-formulations as promising cancer chemopreventive agents. The current study could pave a new way for the development of drugs that target HNSCC in the future.

  18. Miscellaneous additives can enhance plant uptake and affect geochemical fractions of copper in a heavily polluted riparian grassland soil.

    PubMed

    Rinklebe, Jörg; Shaheen, Sabry M

    2015-09-01

    The problem of copper (Cu) pollution in riverine ecosystems is world-wide and has significant environmental, eco-toxicological, and agricultural relevance. We assessed the suitability and effectiveness of application rate of 1% of activated charcoal, bentonite, biochar, cement kiln dust, chitosan, coal fly ash, limestone, nano-hydroxyapatite, organo-clay, sugar beet factory lime, and zeolite as soil amendments together with rapeseed as bioenergy crop as a possible remediation option for a heavily Cu polluted floodplain soil (total Cu=3041.9mgkg(-1)) that has a very high proportion of sorbed/carbonate fraction (484.6mgkg(-1)) and potential mobile fraction of Cu (1611.9mgkg(-1)). Application changed distribution of Cu among geochemical fractions: alkaline materials lead to increased carbonate bounded fraction and the acid rhizosphere zone might cause release of this Cu. Thus, mobilization of Cu and uptake of Cu by rapeseed were increased compared to the control (except for organo-clay) under the prevailing conditions. PMID:25968602

  19. Miscellaneous additives can enhance plant uptake and affect geochemical fractions of copper in a heavily polluted riparian grassland soil.

    PubMed

    Rinklebe, Jörg; Shaheen, Sabry M

    2015-09-01

    The problem of copper (Cu) pollution in riverine ecosystems is world-wide and has significant environmental, eco-toxicological, and agricultural relevance. We assessed the suitability and effectiveness of application rate of 1% of activated charcoal, bentonite, biochar, cement kiln dust, chitosan, coal fly ash, limestone, nano-hydroxyapatite, organo-clay, sugar beet factory lime, and zeolite as soil amendments together with rapeseed as bioenergy crop as a possible remediation option for a heavily Cu polluted floodplain soil (total Cu=3041.9mgkg(-1)) that has a very high proportion of sorbed/carbonate fraction (484.6mgkg(-1)) and potential mobile fraction of Cu (1611.9mgkg(-1)). Application changed distribution of Cu among geochemical fractions: alkaline materials lead to increased carbonate bounded fraction and the acid rhizosphere zone might cause release of this Cu. Thus, mobilization of Cu and uptake of Cu by rapeseed were increased compared to the control (except for organo-clay) under the prevailing conditions.

  20. New [(η(5)-C5H5)Ru(N-N)(PPh3)][PF6] compounds: colon anticancer activity and GLUT-mediated cellular uptake of carbohydrate-appended complexes.

    PubMed

    Florindo, Pedro R; Pereira, Diane M; Borralho, Pedro M; Costa, Paulo J; Piedade, M F M; Rodrigues, Cecília M P; Fernandes, Ana C

    2016-07-26

    Eight ruthenium(ii) compounds of the general formula [(η(5)-C5H5)Ru(N-N)(PPh3)][PF6] were rationally designed, exhibiting high cytotoxicity against HCT116 human colon cancer cells, with IC50 between 14.56 and 1.56 μM; importantly, compounds 5Ru and 6Ru are the first reported ruthenium glycoconjugates exploiting glucose transporters, widely overexpressed in cancer, for cellular uptake. PMID:27216868

  1. Cellular uptake, antioxidant and antiproliferative activity of entrapped α-tocopherol and γ-tocotrienol in poly (lactic-co-glycolic) acid (PLGA) and chitosan covered PLGA nanoparticles (PLGA-Chi).

    PubMed

    Alqahtani, Saeed; Simon, Lacey; Astete, Carlos E; Alayoubi, Alaadin; Sylvester, Paul W; Nazzal, Sami; Shen, Yixiao; Xu, Zhimin; Kaddoumi, Amal; Sabliov, Cristina M

    2015-05-01

    The aim of this study was to formulate and characterize α-tocopherol (α-T) and tocotrienol-rich fraction (TRF) entrapped in poly (lactide-co-glycolide) (PLGA) and chitosan covered PLGA (PLGA-Chi) based nanoparticles. The resultant nanoparticles were characterized and the effect of nanoparticles entrapment on the cellular uptake, antioxidant, and antiproliferative activity of α-T and TRF were tested. In vitro uptake studies in Caco2 cells showed that PLGA and PLGA-Chi nanoparticles displayed a greater enhancement in the cellular uptake of α-T and TRF when compared with the control without causing toxicity to the cells (p<0.0001). Furthermore, the cellular internalization of both PLGA and PLGA-Chi nanoparticles labeled with FITC was investigated by fluorescence microscopy; both types of nanoparticles were able to get internalized into the cells with reasonable amounts. However, PLGA-Chi nanoparticles showed significantly higher (3.5-fold) cellular uptake compared to PLGA nanoparticles. The antioxidant activity studies demonstrated that entrapment of α-T and TRF in PLGA and PLGA-Chi nanoparticles exhibited greater ability in inhibiting cholesterol oxidation at 48 h compared to the control. In vitro antiproliferative studies confirmed marked cytotoxicity of TRF on MCF-7 and MDA-MB-231 cell lines when delivered by PLGA and PLGA-Chi nanoparticles after 48 h incubation compared to control. In summary, PLGA and PLGA-Chi nanoparticles may be considered as an attractive and promising approach to enhance the bioavailability and activity of poorly water soluble compounds such as α-tocopherol and tocotrienols.

  2. A role for dZIP89B in Drosophila dietary zinc uptake reveals additional complexity in the zinc absorption process.

    PubMed

    Richards, Christopher D; Warr, Coral G; Burke, Richard

    2015-12-01

    Dietary zinc is the principal source of zinc in eukaryotes, with its uptake and distribution controlled by a complex network of numerous membrane-spanning transport proteins. Dietary absorption is achieved by members of the SLC39A (ZIP) gene family, which encode proteins that are generally responsible for the movement of zinc into the cytosol. ZIP4 is thought to be the primary mammalian zinc uptake gene in the small intestine, with mutations in this gene causing the zinc deficiency disease Acrodermatitis enteropathica. In Drosophila, dual knockdown of the major dietary zinc uptake genes dZIP42C.1 (dZIP1) and dZIP42C.2 (dZIP2) results in a severe sensitivity to zinc-deficient media. However, the symptoms associated with ZIP4 loss can be reversed by zinc supplementation and dZIP42C.1 and 2 knockdown has minimal effect under normal dietary conditions, suggesting that additional pathways for zinc absorption exist in both mammals and flies. This study provides evidence that dZIP89B is an ideal candidate for this role in Drosophila, encoding a low-affinity zinc uptake transporter active in the posterior midgut. Flies lacking dZIP89B, while viable and apparently healthy, show indications of low midgut zinc levels, including reduced metallothionein B expression and compensatory up-regulation of dZIP42C.1 and 2. Furthermore dZIP89B mutants display a dramatic resistance to toxic dietary zinc levels which is abrogated by midgut-specific restoration of dZIP89B activity. We postulate that dZIP89B works in concert with the closely related dZIP42C.1 and 2 to ensure optimal zinc absorption under a range of dietary conditions.

  3. Potentiating the cellular targeting and anti-tumor activity of Dp44mT via binding to human serum albumin: two saturable mechanisms of Dp44mT uptake by cells

    PubMed Central

    Merlot, Angelica M.; Sahni, Sumit; Lane, Darius J.R.; Fordham, Ashleigh M.; Pantarat, Namfon; Hibbs, David E.; Richardson, Vera; Doddareddy, Munikumar R.; Ong, Jennifer A.; Huang, Michael L.H.

    2015-01-01

    Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) demonstrates potent anti-cancer activity. We previously demonstrated that 14C-Dp44mT enters and targets cells through a carrier/receptor-mediated uptake process. Despite structural similarity, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and pyridoxal isonicotinoyl hydrazone (PIH) enter cells via passive diffusion. Considering albumin alters the uptake of many drugs, we examined the effect of human serum albumin (HSA) on the cellular uptake of Dp44mT, Bp4eT and PIH. Chelator-HSA binding studies demonstrated the following order of relative affinity: Bp4eT≈PIH>Dp44mT. Interestingly, HSA decreased Bp4eT and PIH uptake, potentially due to its high affinity for the ligands. In contrast, HSA markedly stimulated Dp44mT uptake by cells, with two saturable uptake mechanisms identified. The first mechanism saturated at 5-10 μM (Bmax:1.20±0.04 × 107 molecules/cell; Kd:33±3 μM) and was consistent with a previously identified Dp44mT receptor/carrier. The second mechanism was of lower affinity, but higher capacity (Bmax:2.90±0.12 × 107 molecules/cell; Kd:65±6 μM), becoming saturated at 100 μM and was only evident in the presence of HSA. This second saturable Dp44mT uptake process was inhibited by excess HSA and had characteristics suggesting it was mediated by a specific binding site. Significantly, the HSA-mediated increase in the targeting of Dp44mT to cancer cells potentiated apoptosis and could be important for enhancing efficacy. PMID:25848850

  4. Enhancing degradation of total petroleum hydrocarbons and uptake of heavy metals in a wetland microcosm planted with Phragmites communis by humic acids addition.

    PubMed

    Sung, Kijune; Kim, Ki Seob; Park, Soyoung

    2013-01-01

    The effects of humic acid (HA) on heavy-metal uptake by plants and degradation of total petroleum hydrocarbons (TPHs) in a wetland microcosm planted with Phragmites communis were evaluated by comparing waterlogged soils and water-drained upland soils. Experiments were conducted on soils artificially contaminated with heavy metals (Pb, Cu, Cd, Ni) and diesel fuel. HA showed a positive influence on biomass increase for all conditions, but more for belowground than aboveground biomass, and lower in contaminated than uncontaminated soil. The bioavailability and leachability factor (BLF) for all heavy metals except Ni increased with HA addition in both the control and the P. communis planted microcosms, suggesting that more heavy metals could be potentially phytoavailable for plant uptake. Microbial activities were not affected by both heavy metals and TPH contamination, and HA effects on stimulating microbial activities were much greater in the contaminated soil than under uncontaminated conditions. HA addition enhanced the degradation of TPH and n-alkane in waterlogged conditions. The results show that HA can increase the remedial performance in P. communis dominated wetlands simultaneously contaminated with heavy metals and petroleum hydrocarbons and thus prevent contamination of groundwater or other adjacent ecosystems.

  5. Liv.52 up-regulates cellular antioxidants and increase glucose uptake to circumvent oleic acid induced hepatic steatosis in HepG2 cells.

    PubMed

    Vidyashankar, Satyakumar; Sharath Kumar, L M; Barooah, Vandana; Sandeep Varma, R; Nandakumar, Krishna S; Patki, Pralhad Sadashiv

    2012-10-15

    HepG2 cells were rendered steatotic by supplementing 2.0mM oleic acid (OA) in the culture media for 24h. OA induced hepatic steatosis in HepG2 cells was marked by significant accumulation of lipid droplets as determined by Oil-Red-O (ORO) based colorimetric assay, increased triacylglycerol (TAG) and increased lipid peroxidation. It was also marked by increased inflammatory cytokines TNF-α and IL-8 with decreased enzymic and non-enzymic antioxidant molecules and decreased cell proliferation associated with insulin resistance and DNA fragmentation. Addition of Liv.52 hydro-alcoholic extract (LHAE) 50μg/mL to the steatotic cells was effective in increasing the insulin mediated glucose uptake by 3.13 folds and increased cell proliferation by 3.81 folds with decreased TAG content (55%) and cytokines. The intracellular glutathione content was increased by 8.9 folds without substantial increase in GSSG content. LHAE decreased TNF-α and IL-8 by 51% and 6.5% folds respectively, lipid peroxidation by 65% and inhibited DNA fragmentation by 69%. The superoxide dismutase, catalase and glutathione peroxidase activities were increased by 88%, 128% and 64% respectively. Albumin and urea content was increased while the alanine aminotransferase (ALAT) activity was significantly decreased by LHAE. Hence, LHAE effectively attenuate molecular perturbations associated with non-alcoholic fatty liver disease (NAFLD) indications in HepG2 cells. PMID:22940028

  6. Liv.52 up-regulates cellular antioxidants and increase glucose uptake to circumvent oleic acid induced hepatic steatosis in HepG2 cells.

    PubMed

    Vidyashankar, Satyakumar; Sharath Kumar, L M; Barooah, Vandana; Sandeep Varma, R; Nandakumar, Krishna S; Patki, Pralhad Sadashiv

    2012-10-15

    HepG2 cells were rendered steatotic by supplementing 2.0mM oleic acid (OA) in the culture media for 24h. OA induced hepatic steatosis in HepG2 cells was marked by significant accumulation of lipid droplets as determined by Oil-Red-O (ORO) based colorimetric assay, increased triacylglycerol (TAG) and increased lipid peroxidation. It was also marked by increased inflammatory cytokines TNF-α and IL-8 with decreased enzymic and non-enzymic antioxidant molecules and decreased cell proliferation associated with insulin resistance and DNA fragmentation. Addition of Liv.52 hydro-alcoholic extract (LHAE) 50μg/mL to the steatotic cells was effective in increasing the insulin mediated glucose uptake by 3.13 folds and increased cell proliferation by 3.81 folds with decreased TAG content (55%) and cytokines. The intracellular glutathione content was increased by 8.9 folds without substantial increase in GSSG content. LHAE decreased TNF-α and IL-8 by 51% and 6.5% folds respectively, lipid peroxidation by 65% and inhibited DNA fragmentation by 69%. The superoxide dismutase, catalase and glutathione peroxidase activities were increased by 88%, 128% and 64% respectively. Albumin and urea content was increased while the alanine aminotransferase (ALAT) activity was significantly decreased by LHAE. Hence, LHAE effectively attenuate molecular perturbations associated with non-alcoholic fatty liver disease (NAFLD) indications in HepG2 cells.

  7. 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

  8. 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.

    2016-01-01

    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

  9. Effect of additive oxygen gas on cellular response of lung cancer cells induced by atmospheric pressure helium plasma jet.

    PubMed

    Joh, Hea Min; Choi, Ji Ye; Kim, Sun Ja; Chung, T H; Kang, Tae-Hong

    2014-10-16

    The atmospheric pressure helium plasma jet driven by pulsed dc voltage was utilized to treat human lung cancer cells in vitro. The properties of plasma plume were adjusted by the injection type and flow rate of additive oxygen gas in atmospheric pressure helium plasma jet. The plasma characteristics such as plume length, electric current and optical emission spectra (OES) were measured at different flow rates of additive oxygen to helium. The plasma plume length and total current decreased with an increase in the additive oxygen flow rate. The electron excitation temperature estimated by the Boltzmann plot from several excited helium emission lines increased slightly with the additive oxygen flow. The oxygen atom density in the gas phase estimated by actinometry utilizing argon was observed to increase with the additive oxygen flow. The concentration of intracellular reactive oxygen species (ROS) measured by fluorescence assay was found to be not exactly proportional to that of extracellular ROS (measured by OES), but both correlated considerably. It was also observed that the expression levels of p53 and the phospho-p53 were enhanced in the presence of additive oxygen flow compared with those from the pure helium plasma treatment.

  10. Effect of additive oxygen gas on cellular response of lung cancer cells induced by atmospheric pressure helium plasma jet

    PubMed Central

    Joh, Hea Min; Choi, Ji Ye; Kim, Sun Ja; Chung, T. H.; Kang, Tae-Hong

    2014-01-01

    The atmospheric pressure helium plasma jet driven by pulsed dc voltage was utilized to treat human lung cancer cells in vitro. The properties of plasma plume were adjusted by the injection type and flow rate of additive oxygen gas in atmospheric pressure helium plasma jet. The plasma characteristics such as plume length, electric current and optical emission spectra (OES) were measured at different flow rates of additive oxygen to helium. The plasma plume length and total current decreased with an increase in the additive oxygen flow rate. The electron excitation temperature estimated by the Boltzmann plot from several excited helium emission lines increased slightly with the additive oxygen flow. The oxygen atom density in the gas phase estimated by actinometry utilizing argon was observed to increase with the additive oxygen flow. The concentration of intracellular reactive oxygen species (ROS) measured by fluorescence assay was found to be not exactly proportional to that of extracellular ROS (measured by OES), but both correlated considerably. It was also observed that the expression levels of p53 and the phospho-p53 were enhanced in the presence of additive oxygen flow compared with those from the pure helium plasma treatment. PMID:25319447

  11. Effect of additive oxygen gas on cellular response of lung cancer cells induced by atmospheric pressure helium plasma jet

    NASA Astrophysics Data System (ADS)

    Joh, Hea Min; Choi, Ji Ye; Kim, Sun Ja; Chung, T. H.; Kang, Tae-Hong

    2014-10-01

    The atmospheric pressure helium plasma jet driven by pulsed dc voltage was utilized to treat human lung cancer cells in vitro. The properties of plasma plume were adjusted by the injection type and flow rate of additive oxygen gas in atmospheric pressure helium plasma jet. The plasma characteristics such as plume length, electric current and optical emission spectra (OES) were measured at different flow rates of additive oxygen to helium. The plasma plume length and total current decreased with an increase in the additive oxygen flow rate. The electron excitation temperature estimated by the Boltzmann plot from several excited helium emission lines increased slightly with the additive oxygen flow. The oxygen atom density in the gas phase estimated by actinometry utilizing argon was observed to increase with the additive oxygen flow. The concentration of intracellular reactive oxygen species (ROS) measured by fluorescence assay was found to be not exactly proportional to that of extracellular ROS (measured by OES), but both correlated considerably. It was also observed that the expression levels of p53 and the phospho-p53 were enhanced in the presence of additive oxygen flow compared with those from the pure helium plasma treatment.

  12. Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4

    PubMed Central

    Yang, Jinju; Qin, Nannan; Zhang, Hongwei; Yang, Rui; Xiang, Benqiong; Wei, Qun

    2016-01-01

    Our previous research showed that recombinant calcineurin B (rhCnB) stimulates cytokine secretion by immune cells, probably through TLR4. Exogenous CnB can be incorporated into many different tumour cells in vitro, but the mode of uptake and receptors required remain unknown. Here, we report that exogenous CnB is taken up by cells in a time- and concentration-dependent manner via clathrin-dependent receptor-mediated internalization. Our findings further confirm that uptake is mediated by the TLR4/MD2 complex together with the co-receptor CD14. The MST results revealed a high affinity between CnB and the TLR4 receptor complex. No binding was detected between CnB and LPS. CnB inhibited the uptake of LPS, and LPS also inhibited the uptake of CnB. These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS. Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB. CnB could be a potential endogenous ligand of TLR4 and function as an agonist of TLR4. These properties of CnB support its potential for development as an anti-cancer drug. PMID:27090571

  13. Mitochondria-acting hexokinase II peptides carried by short-length carbon nanotubes with increased cellular uptake, endosomal evasion, and enhanced bioactivity against cancer cells

    NASA Astrophysics Data System (ADS)

    Yoong, Sia Lee; Lau, Wei Liang; Liu, Ang Yu; Prendergast, D'arcy; Ho, Han Kiat; Yu, Victor Chun Kong; Lee, Chengkuo; Ang, Wee Han; Pastorin, Giorgia

    2015-08-01

    Type II hexokinase (HKII) has emerged as a viable therapeutic target due to its involvement in metabolic reprogramming and also apoptosis prevention. The peptide derived from the fifteen amino acid sequence in the HKII N-terminal region [HKII(pep)] can compete with endogenous proteins for binding on mitochondria and trigger apoptosis. However, this peptide is not cell-permeable. In this study, multi-walled carbon nanotubes (MWCNTs) were used to effectively deliver HKII(pep) across cellular barriers without compromising their bioactivity. The peptide was conjugated on either oxidized MWCNTs or 2,2'-(ethylenedioxy)bis(ethylamine)-functionalized MWCNTs, yielding MWCNT-HKII(pep) and MWCNT-TEG-HKII(pep), respectively. Both conjugates were shown to be internalized by breast cancer MCF-7 cells using confocal microscopy. Moreover, these nanoconjugates seemed to have escaped from endosomes and be in the vicinity of mitochondria. The WST-1 cytotoxicity assay conducted on MCF-7 and colon carcinoma HCT116 cells revealed that MWCNT-peptide conjugates were significantly more effective in curbing cancer cell growth compared to a commercially available cell permeable HKII fusion peptide. In addition, both nanoconjugates displayed an enhanced ability in eliciting apoptosis and depleting the ATP level in HCT116 cells compared to the mere HKII peptide. Importantly, hexokinase II release from mitochondria was demonstrated in MWCNT-HKII(pep) and MWCNT-TEG-HKII(pep) treated cells, highlighting that the structure and bioactivity of HKII(pep) were not compromised after covalent conjugation to MWCNTs.Type II hexokinase (HKII) has emerged as a viable therapeutic target due to its involvement in metabolic reprogramming and also apoptosis prevention. The peptide derived from the fifteen amino acid sequence in the HKII N-terminal region [HKII(pep)] can compete with endogenous proteins for binding on mitochondria and trigger apoptosis. However, this peptide is not cell-permeable. In this study

  14. Curcumin and Its Analogue Induce Apoptosis in Leukemia Cells and Have Additive Effects with Bortezomib in Cellular and Xenograft Models

    PubMed Central

    Nagy, L. I.; Fehér, L. Z.; Szebeni, G. J.; Gyuris, M.; Sipos, P.; Alföldi, R.; Ózsvári, B.; Hackler, L.; Balázs, A.; Batár, P.; Kanizsai, I.; Puskás, L. G.

    2015-01-01

    Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination. PMID:26075279

  15. Biochar addition to an arsenic contaminated soil increases arsenic concentrations in the pore water but reduces uptake to tomato plants (Solanum lycopersicum L.).

    PubMed

    Beesley, Luke; Marmiroli, Marta; Pagano, Luca; Pigoni, Veronica; Fellet, Guido; Fresno, Teresa; Vamerali, Teofilo; Bandiera, Marianna; Marmiroli, Nelson

    2013-06-01

    Arsenic (As) concentrations in soil, soil pore water and plant tissues were evaluated in a pot experiment following the transplantation of tomato (Solanum lycopersicum L.) plantlets to a heavily As contaminated mine soil (~6000 mg kg(-1) pseudo-total As) receiving an orchard prune residue biochar amendment, with and without NPK fertiliser. An in-vitro test was also performed to establish if tomato seeds were able to germinate in various proportions of biochar added to nutrient solution (MS). Biochar significantly increased arsenic concentrations in pore water (500 μg L(-1)-2000 μg L(-1)) whilst root and shoot concentrations were significantly reduced compared to the control without biochar. Fruit As concentrations were very low (<3 μg kg(-1)), indicating minimal toxicity and transfer risk. Fertilisation was required to significantly increase plant biomass above the control after biochar addition whilst plants transplanted to biochar only were heavily stunted and chlorotic. Given that increasing the amount of biochar added to nutrient solution in-vitro reduced seed germination by up to 40%, a lack of balanced nutrient provision from biochar could be concluded. In summary, solubility and mobility of As were increased by biochar addition to this soil, but uptake to plant was reduced, and toxicity-transfer risk was negligible. Therefore leaching rather than food chain transfer appears the most probable immediate consequence of biochar addition to As contaminated soils. PMID:23583727

  16. Effects of past sewage sludge additions on heavy metal availability in light textured soils: implications for crop yields and metal uptakes.

    PubMed

    Bhogal, A; Nicholson, F A; Chambers, B J; Shepherd, M A

    2003-01-01

    The effect of heavy metal additions in past sewage sludge applications on soil metal availability and the growth and yield of crops was evaluated at two sites in the UK. At Gleadthorpe, sewage sludges enriched with salts of zinc (Zn), copper (Cu) and nickel (Ni) had been applied to a loamy sand in 1982 and additionally naturally contaminated Zn and Cu sludge cakes in 1986. At Rosemaund, sewage sludges naturally contaminated with Zn, Cu, Ni and chromium (Cr) had been applied in 1968-1971 to a sandy loam. From 1994 to 1997, the yields of both cereals and legumes at Gleadthorpe were up to 3 t/ha lower than the no-sludge control where total topsoil Zn and Cu concentrations exceeded 200 and 120 mg/kg, respectively, but only when topsoil ammonium nitrate extractable metal levels also exceeded 40 mg/kg Zn and 0.9 mg/kg Cu. At Rosemaund, yields were only decreased where total topsoil Cu concentrations exceeded 220 mg/kg or 0.7 mg/kg ammonium nitrate extractable Cu. These results demonstrate the importance of measuring extractable as well as total heavy metal concentrations in topsoils when assessing likely effects on plant yields and metal uptakes, and setting soil quality criteria.

  17. Quantification of new antiepileptic drugs by liquid chromatography/electrospray ionization tandem mass spectrometry and its application to cellular uptake experiment using human placental choriocarcinoma BeWo cells.

    PubMed

    Furugen, Ayako; Kobayashi, Masaki; Nishimura, Ayako; Takamura, Shigeo; Narumi, Katsuya; Yamada, Takehiro; Iseki, Ken

    2015-10-01

    A method for quantification of new antiepileptic drugs, including lamotrigine (LTG), levetiracetam (LEV), gabapentin (GBP), and topiramate (TPM), in cellular samples, using liquid chromatography/electrospray ionization tandem mass spectrometry was developed to better understand the membrane transport mechanisms of these drugs. Cell lysate was deproteinized by methanol containing LEV-d3 as an internal standard (IS). Chromatographic separation was performed on a C18 column using gradient elution with methanol-water-formic acid (10:90:0.1, v/v/v) and methanol-formic acid (100:0.1, v/v). Analytes were detected in positive ion electrospray mode with selected reaction monitoring (SRM). This method was applicable for a linear range of 5 to 500pmol for LTG; 5 to 1000pmol for LEV; 10 to 10,000pmol for GBP; and 5 to 5000pmol for TPM. The intra-day precision, inter-day precision, and accuracy data were assessed and found to be acceptable. This developed and validated method was then successfully applied to the investigation of uptake of the new antiepileptic drugs in placental choriocarcinoma BeWo cells. The intracellular concentration of these drugs in BeWo cells, accumulating over 30min at 37°C was in the order of GBP>LTG>LEV≈TPM. Furthermore, the uptake of GBP at 4°C was much lower than that at 37°C. The uptake of GBP was saturated at high concentrations. The kinetic parameters calculated for GBP uptake in BeWo cells were determined as Km of 105.4±6.4μM and Vmax at 8153±348pmol/mg protein/min. The novel method described here should enable investigators to elucidate the transport mechanisms of these antiepileptic drugs in BeWo cells.

  18. Characterization of Fluorescent Siderophore-Mediated Iron Uptake in Pseudomonas sp. Strain M114: Evidence for the Existence of an Additional Ferric Siderophore Receptor

    PubMed Central

    Morris, John; O'Sullivan, Daniel J.; Koster, Margot; Leong, John; Weisbeek, Peter J.; O'Gara, Fergal

    1992-01-01

    In Pseudomonas sp. strain M114, the outer membrane receptor for ferric pseudobactin M114 was shown to transport ferric pseudobactins B10 and A225, in addition to its own. The gene encoding this receptor, which was previously cloned on pCUP3, was localized by Tn5 mutagenesis to a region comprising >1.6 kb of M114 DNA. A mutant (strain M114R1) lacking this receptor was then created by a marker exchange technique. Characterization of this mutant by using purified pseudobactin M114 in radiolabeled ferric iron uptake studies confirmed that it was completely unable to utilize this siderophore for acquisition of iron. In addition, it lacked an outer membrane protein band of 89 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, growth of the mutant was severely restricted under low-iron conditions. However, this phenotype was reversed in the presence of another fluorescent siderophore (pseudobactin MT3A) from Pseudomonas sp. strain MT3A, suggesting the presence of a second receptor in strain M114. Furthermore, wild-type Pseudomonas sp. strain B24 was not able to utilize ferric pseudobactin MT3A, and this phenotype was not reversed upon expression of the M114 receptor encoded on pCUP3. However, a cosmid clone (pMS1047) that enabled strain B24 to utilize ferric pseudobactin MT3A was isolated from an M114 gene bank. Radiolabel transport assays with purified pseudobactin MT3A confirmed this event. Plasmid pMS1047 was shown to encode an outer membrane protein of 81 kDa in strain B24 under iron-limiting conditions; this protein corresponds to a similar protein in strain M114. Images PMID:16348650

  19. Single-cell irradiation from [211At] astatine-labeled C215 monoclonal antibody: improved estimates of radiosensitivity from measurements on cellular uptake and retention.

    PubMed

    Palm, Stig; Bäck, Tom; Claesson, Ingela; Delle, Ulla; Hultborn, Ragnar; Lindegren, Sture; Jacobsson, Lars

    2003-01-01

    New data on the biological effect of 211At-C215 monoclonal antibody in a slowly rotating, widely dispersed single-cell suspension of the human cancer cell line Colo-205 is presented. Cell growth curves of each experiment were used to calculate an apparent cell survival after irradiation. Uptake measurements provided the data needed to calculate the average number of 211At decays per cell in the cell suspension. The results from each experiment were then fit to a mono-exponential function. From the exponential fit, an average of 35 +/- 2 (SD) astatine-211 decays per cell are required for 37% apparent cell survival (D0). PMID:12820374

  20. Single-cell irradiation from [211At] astatine-labeled C215 monoclonal antibody: improved estimates of radiosensitivity from measurements on cellular uptake and retention.

    PubMed

    Palm, Stig; Bäck, Tom; Claesson, Ingela; Delle, Ulla; Hultborn, Ragnar; Lindegren, Sture; Jacobsson, Lars

    2003-01-01

    New data on the biological effect of 211At-C215 monoclonal antibody in a slowly rotating, widely dispersed single-cell suspension of the human cancer cell line Colo-205 is presented. Cell growth curves of each experiment were used to calculate an apparent cell survival after irradiation. Uptake measurements provided the data needed to calculate the average number of 211At decays per cell in the cell suspension. The results from each experiment were then fit to a mono-exponential function. From the exponential fit, an average of 35 +/- 2 (SD) astatine-211 decays per cell are required for 37% apparent cell survival (D0).

  1. Microstructural architecture developed in the fabrication of solid and open-cellular copper components by additive manufacturing using electron beam melting

    NASA Astrophysics Data System (ADS)

    Ramirez, Diana Alejandra

    The fabrication of Cu components were first built by additive manufacturing using electron beam melting (EBM) from low-purity, atomized Cu powder containing a high density of Cu2O precipitates leading to a novel example of precipitate-dislocation architecture. These microstructures exhibit cell-like arrays (1-3microm) in the horizontal reference plane perpendicular to the build direction with columnar-like arrays extending from ~12 to >60 microm in length and corresponding spatial dimensions of 1-3 microm. These observations were observed by the use of optical metallography, and scanning and transmission electron microscopy. The hardness measurements were taken both on the atomized powder and the Cu components. The hardness for these architectures ranged from ~HV 83 to 88, in contrast to the original Cu powder microindentation hardness of HV 72 and the commercial Cu base plate hardness of HV 57. These observations were utilized for the fabrication of open-cellular copper structures by additive manufacturing using EBM and illustrated the ability to fabricate some form of controlled microstructural architecture by EBM parameter alteration or optimizing. The fabrication of these structures ranged in densities from 0.73g/cm3 to 6.67g/cm3. These structures correspond to four different articulated mesh arrays. While these components contained some porosity as a consequence of some unmelted regions, the Cu2O precipitates also contributed to a reduced density. Using X-ray Diffraction showed the approximate volume fraction estimated to be ~2%. The addition of precipitates created in the EBM melt scan formed microstructural arrays which contributed to hardening contributing to the strength of mesh struts and foam ligaments. The measurements of relative stiffness versus relative density plots for Cu compared very closely with Ti-6Al-4V open cellular structures - both mesh and foams. The Cu reticulated mesh structures exhibit a slope of n = 2 in contrast to a slope of n = 2

  2. The role of the cell cycle in the cellular uptake of folate-modified poly(l-amino acid) micelles in a cell population

    NASA Astrophysics Data System (ADS)

    Tang, Jihui; Liu, Ziwei; Ji, Fenqi; Li, Yao; Liu, Junjie; Song, Jian; Li, Jun; Zhou, Jianping

    2015-12-01

    Nanoparticles are widely recognized as a vehicle for tumor-targeted therapies. There are many factors that can influence the uptake of nanoparticles, such as the size of the nanoparticles, and/or their shape, elasticity, surface charge and even the cell cycle phase. However, the influence of the cell cycle on the active targeting of a drug delivery system has been unknown until now. In this study, we initially investigated the folate receptor α (FR-α) expression in different phases of HeLa cells by flow cytometric and immunocytochemical methods. The results obtained showed that FR-α expression was cell cycle-dependent, i.e. the S cells' folate receptor expression was the highest as the cell progressed through its cycle. Then, we used folate modified poly(l-amino acid) micelles (FA-PM) as an example to investigate the influence of the cell cycle on the active targeting drug delivery system. The results obtained indicated that the uptake of FA-PM by cells was influenced by the cell cycle phase, and the S cells took up the greatest number of folate conjugated nanoparticles. Our findings suggest that future studies on ligand-mediated active targeting preparations should consider the cell cycle, especially when this system is used for a cell cycle-specific drug.

  3. Cellular uptake and cell-to-cell transfer of polyelectrolyte microcapsules within a triple co-culture system representing parts of the respiratory tract

    NASA Astrophysics Data System (ADS)

    Kuhn, Dagmar A.; Hartmann, Raimo; Fytianos, Kleanthis; Petri-Fink, Alke; Rothen-Rutishauser, Barbara; Parak, Wolfgang J.

    2015-06-01

    Polyelectrolyte multilayer microcapsules around 3.4 micrometers in diameter were added to epithelial cells, monocyte-derived macrophages, and dendritic cells in vitro and their uptake kinetics were quantified. All three cell types were combined in a triple co-culture model, mimicking the human epithelial alveolar barrier. Hereby, macrophages were separated in a three-dimensional model from dendritic cells by a monolayer of epithelial cells. While passing of small nanoparticles has been demonstrated from macrophages to dendritic cells across the epithelial barrier in previous studies, for the micrometer-sized capsules, this process could not be observed in a significant amount. Thus, this barrier is a limiting factor for cell-to-cell transfer of micrometer-sized particles.

  4. Alterations of nuclear DNA synthesis after irradiation of the cellular slime mold Dictyostelium discoideum: studies performed in a mutant strain displaying enhanced thymidine uptake

    SciTech Connect

    Hurley, D.L.

    1986-01-01

    The auxotrophic Dictyostelium discoideum strain HPS 401 was studied. Thymidine at 8 ..mu..g/ml or thymidylate at 50 ..mu..g/ml supported growth to maximal cell densities. Thin layer chromatography of cell extracts showed rapid intracellular accumulation of thymidine in HPS 401 vs slightly detectable accumulation in wild-type cells. Measurements showed that methionine and thymidylate were taken into all strains at a low rate, but HPS 401 had enhanced uptake of thymidine and uridine compared to wild-type. The HPS 401 phenotype is due to the efficient utilization of thymidine as a result of increased nucleoside uptake. Rapid nuclear purification removed mitochondrial DNA without decreasing the single-strand molecular weight of the nuclear DNA. The nuclear DNA peaks on alkaline sucrose gradients were identified using filter hybridization to cloned probes. As measured by pulse-chase labelling, production of full-sized main band DNA required 45-50 minutes. Pulse labelling of the cells immediately after ultraviolet irradiation caused the single-strand molecular weight of the DNA synthesized to decrease from 8 x 10/sup 6/ daltons at O J/m/sup 2/ to 3.9 x 10/sup 6/ daltons at 50 J/m/sup 2/ to 2.6 x 10/sup 6/ daltons at 200 J/m/sup 2/. The time required for maturation into full-sized DNA increased from 1 hour at O J/m/sup 2/ to 4 hours at 20 J/m/sup 2/ and to 21 hours at 200 J/m/sup 2/. Measured amounts of DNA synthesis at times after ultraviolet irradiation showed a period of reduced incorporation, followed by the resumption of control levels. The lag period ended at the same time as the production of full-sized DNA resumed.

  5. The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

    PubMed

    Isakoff, S J; Taha, C; Rose, E; Marcusohn, J; Klip, A; Skolnik, E Y

    1995-10-24

    Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

  6. Development of a novel nanoparticle by dual modification with the pluripotential cell-penetrating peptide PepFect6 for cellular uptake, endosomal escape, and decondensation of an siRNA core complex.

    PubMed

    Mitsueda, Asako; Shimatani, Yuri; Ito, Masahiro; Ohgita, Takashi; Yamada, Asako; Hama, Susumu; Gräslund, Astrid; Lindberg, Staffan; Langel, Ülo; Harashima, Hideyoshi; Nakase, Ikuhiko; Futaki, Shiroh; Kogure, Kentaro

    2013-11-01

    Development of novel devices for effective nucleotide release from nanoparticles is required to improve the functionality of nonviral delivery systems, because decondensation of nucleotide/polycation complexes is considered as a key step for cytoplasmic delivery of nucleotides. Previously, PepFect6 (PF6) comprised chloroquine analog moieties and a stearylated cell-penetrating peptide to facilitate endosomal escape and cellular uptake, respectively, was developed as a device for efficient siRNA delivery. As PF6 contains bulky chloroquine analog moieties, the polyplexes are expected to be loose structure, which facilitates decondensation. In the present study, siRNA was electrostatically condensed by PF6, and the PF6/siRNA complexes were coated with lipid membranes. The surface of the nanoparticles encapsulating the PF6/siRNA core (PF6-NP) was modified with PF6 for endosomal escape (PF6/PF6-NP). The RNAi effect of PF6/PF6-NP was compared with those of stearylated cell-penetrating peptide octaarginine (R8)-modified PF6-NP, R8-modified nanoparticles encapsulating the R8/siRNA core (R8-NP) and PF6-modified R8-NP. Nanoparticles encapsulating the PF6 polyplex, especially PF/PF-NP, showed a significant knockdown effect on luciferase activity of B16-F1 cells stably expressing luciferase. siRNA was widely distributed within the cytoplasm after transfection of the nanoparticles encapsulating the PF6 polyplex, while siRNA encapsulated in the R8-presenting nanoparticles was localized within the nuclei. Thus, the siRNA distribution was dependent on the manner of peptide-modification. In conclusion, we have successfully developed PF6/PF6-NP exhibiting a potent RNAi effect resulting from high cellular uptake, efficient endosomal escape and decondensation of the polyplexes based on the multifunctional cell penetrating peptide PF6. PF6 is therefore a useful pluripotential device for siRNA delivery. PMID:23893316

  7. Cytotoxicity of CdTe quantum dots in human umbilical vein endothelial cells: the involvement of cellular uptake and induction of pro-apoptotic endoplasmic reticulum stress

    PubMed Central

    Yan, Ming; Zhang, Yun; Qin, Haiyan; Liu, Kezhou; Guo, Miao; Ge, Yakun; Xu, Mingen; Sun, Yonghong; Zheng, Xiaoxiang

    2016-01-01

    Cadmium telluride quantum dots (CdTe QDs) have been proposed to induce oxidative stress, which plays a crucial role in CdTe QDs-mediated mitochondrial-dependent apoptosis in human umbilical vein endothelial cells (HUVECs). However, the direct interactions of CdTe QDs with HUVECs and their potential impairment of other organelles like endoplasmic reticulum (ER) in HUVECs are poorly understood. In this study, we reported that the negatively charged CdTe QDs (−21.63±0.91 mV), with good dispersity and fluorescence stability, were rapidly internalized via endocytosis by HUVECs, as the notable internalization could be inhibited up to 95.52% by energy depletion (NaN3/deoxyglucose or low temperature). The endocytosis inhibitors (methyl-β-cyclodextrin, genistein, sucrose, chlorpromazine, and colchicine) dramatically decreased the uptake of CdTe QDs by HUVECs, suggesting that both caveolae/raft- and clathrin-mediated endocytosis were involved in the endothelial uptake of CdTe QDs. Using immunocytochemistry, a striking overlap of the internalized CdTe QDs and ER marker was observed, which indicates that QDs may be transported to ER. The CdTe QDs also caused remarkable ER stress responses in HUVECs, confirmed by significant dilatation of ER cisternae, upregulation of ER stress markers GRP78/GRP94, and activation of protein kinase RNA-like ER kinase-eIF2α-activating transcription factor 4 pathway (including phosphorylation of both protein kinase RNA-like ER kinase and eIF2α and elevated level of activating transcription factor 4). CdTe QDs further promoted an increased C/EBP homologous protein expression, phosphorylation of c-JUN NH2-terminal kinase, and cleavage of ER-resident caspase-4, while the specific inhibitor (SP600125, Z-LEVD-fmk, or salubrinal) significantly attenuated QDs-triggered apoptosis, indicating that all three ER stress-mediated apoptosis pathways were activated and the direct participation of ER in the CdTe QDs-caused apoptotic cell death in HUVECs

  8. Cytotoxicity of CdTe quantum dots in human umbilical vein endothelial cells: the involvement of cellular uptake and induction of pro-apoptotic endoplasmic reticulum stress.

    PubMed

    Yan, Ming; Zhang, Yun; Qin, Haiyan; Liu, Kezhou; Guo, Miao; Ge, Yakun; Xu, Mingen; Sun, Yonghong; Zheng, Xiaoxiang

    2016-01-01

    Cadmium telluride quantum dots (CdTe QDs) have been proposed to induce oxidative stress, which plays a crucial role in CdTe QDs-mediated mitochondrial-dependent apoptosis in human umbilical vein endothelial cells (HUVECs). However, the direct interactions of CdTe QDs with HUVECs and their potential impairment of other organelles like endoplasmic reticulum (ER) in HUVECs are poorly understood. In this study, we reported that the negatively charged CdTe QDs (-21.63±0.91 mV), with good dispersity and fluorescence stability, were rapidly internalized via endocytosis by HUVECs, as the notable internalization could be inhibited up to 95.52% by energy depletion (NaN3/deoxyglucose or low temperature). The endocytosis inhibitors (methyl-β-cyclodextrin, genistein, sucrose, chlorpromazine, and colchicine) dramatically decreased the uptake of CdTe QDs by HUVECs, suggesting that both caveolae/raft- and clathrin-mediated endocytosis were involved in the endothelial uptake of CdTe QDs. Using immunocytochemistry, a striking overlap of the internalized CdTe QDs and ER marker was observed, which indicates that QDs may be transported to ER. The CdTe QDs also caused remarkable ER stress responses in HUVECs, confirmed by significant dilatation of ER cisternae, upregulation of ER stress markers GRP78/GRP94, and activation of protein kinase RNA-like ER kinase-eIF2α-activating transcription factor 4 pathway (including phosphorylation of both protein kinase RNA-like ER kinase and eIF2α and elevated level of activating transcription factor 4). CdTe QDs further promoted an increased C/EBP homologous protein expression, phosphorylation of c-JUN NH2-terminal kinase, and cleavage of ER-resident caspase-4, while the specific inhibitor (SP600125, Z-LEVD-fmk, or salubrinal) significantly attenuated QDs-triggered apoptosis, indicating that all three ER stress-mediated apoptosis pathways were activated and the direct participation of ER in the CdTe QDs-caused apoptotic cell death in HUVECs. Our

  9. Novel benzothiazinones (BTOs) as allosteric modulator or substrate competitive inhibitor of glycogen synthase kinase 3β (GSK-3β) with cellular activity of promoting glucose uptake.

    PubMed

    Zhang, Peng; Li, Shufen; Gao, Yang; Lu, Wenbo; Huang, Ke; Ye, Deyong; Li, Xi; Chu, Yong

    2014-12-15

    Glycogen synthase kinase 3β (GSK-3β) plays a key role in insulin metabolizing pathway and therefore inhibition of the enzyme might provide an important therapeutic approach for treatment of insulin resistance and type 2 diabetes. Recently, discovery of ATP noncompetitive inhibitors is gaining importance not only due to their generally increased selectivity but also for the potentially subtle modulation of the target. These kinds of compounds include allosteric modulators and substrate competitive inhibitors. Here we reported two benzothiazinone compounds (BTO), named BTO-5h (IC50=8 μM) and BTO-5s (IC50=10 μM) as novel allosteric modulator and substrate competitive inhibitor of GSK-3β, respectively. Their different action modes were proved by kinetic experiments. Furthermore, BTO-5s was selected to check the kinases profile and showed little or even no activity to a panel of ten protein kinases at 100 μM, indicating it has good selectivity. Docking studies were performed to give suggesting binding modes which can well explain their impacts on the enzyme. Moreover, cell experiments displayed both compounds reduced the phosphorylation level of glycogen synthase in an intact cell, and greatly enhanced the glucose uptake in both HpG2 and 3T3-L1 cells. All of these results suggested BTO-5s and BTO-5h maybe have potentially therapeutic value for anti-diabetes. The results also offer a new scaffold for designing and developing selective inhibitors with novel mechanisms of action.

  10. Human dermal fibroblasts HDFa can be used as an appropriate healthy control for PMMA nanoparticles-survivin molecular beacon cellular uptake studies.

    PubMed

    Adinolfi, Barbara; Pellegrino, Mario; Baldini, Francesco

    2015-02-01

    Survivin is a member of the inhibitor of apoptosis protein family (IAPs); besides its inhibitory action on apoptosis, it is also involved in the regulation of cell division. The protein expression is up-regulated in several cancers, being involved in the tumor progression and evasion of apoptosis. In line with its physiological roles, it is expressed also in several healthy tissues. The high expression of survivin in cancer cells correlates to poor prognosis and resistance to chemotherapeutic treatment, thus making this protein an attractive target in anticancer therapy. The dual role of survivin in cells, regulation of cell division and inhibition of apoptosis, combined with controversial data concerning the expression in normal tissues, emphasize the need to have an appropriate healthy control for in vitro studies. Aim of this study is to highlight this problem and to clarify the experimental conditions in which HDFa fibroblasts represent a negative control for survivin mRNA expression while ensuring the NPs-MB uptake. In this paper, by using confocal microscopy analysis supported by RT- and real-time-PCR experiments studies, we showed that HDFa fibroblasts represent a healthy negative control for survivin mRNA expression, only at very low cell density or at confluence. At the same time, we demonstrated that HDFa at any cell density are able to internalize NPs-MB after 6h of treatment.

  11. Physiological function and inflamed-brain migration of mouse monocyte-derived macrophages following cellular uptake of superparamagnetic iron oxide nanoparticles-Implication of macrophage-based drug delivery into the central nervous system.

    PubMed

    Tong, Hsin-I; Kang, Wen; Shi, Yingli; Zhou, Guangzhou; Lu, Yuanan

    2016-05-30

    This study was designed to use superparamagnetic iron oxide nanoparticles (SPIONs) as evaluating tools to study monocyte-derived macrophages (MDM)-mediated delivery of small molecular agents into the diseased brains. MDM were tested with different-configured SPIONs at selected concentrations for their impacts on carrier cells' physiological and migratory properties, which were found to depend largely on particle size, coating, and treatment concentrations. SHP30, a SPION of 30-nm core size with oleic acids plus amphiphilic polymer coating, was identified to have high cellular uptake efficiency and cause little cytotoxic effects on MDM. At lower incubation dose (25μg/mL), few alteration was observed in carrier cells' physiological and in vivo migratory functions, as tested in a lipopolysaccharide-induced acute neuroinflammation mouse model. Nevertheless, significant increase in monocyte-to-macrophage differentiation, and decrease in in vivo carrier MDM inflamed-brain homing ability were found in groups treated with a higher dose of SHP30at 100μg/mL. Overall, our results have identified MDM treatment at 25μg/mL SHP30 resulted in little functional changes, provided valuable parameters for using SPIONs as evaluating tools to study MDM-mediated therapeutics carriage and delivery, and supported the concepts of using monocytes-macrophages as cellular vehicles to transport small molecular agents to the brain. PMID:27001531

  12. Paclitaxel-Loaded TPGS-b-PCL Nanoparticles: In Vitro Cytotoxicity and Cellular Uptake in MCF-7 and MDA-MB-231 Cells versus mPEG-b-PCL Nanoparticles and Abraxane®.

    PubMed

    Bernabeu, Ezequiel; Gonzalez, Lorena; Legaspi, Maria J; Moretton, Marcela A; Chiappetta, Diego A

    2016-01-01

    Nanomedicines have become an attractive platform for the development of novel drug delivery systems in cancer chemotherapy. Polymeric nanoparticles (NPs) represent one of the best well-investigated nanosized carriers for delivery of antineoplastic compounds. The "Pegylation strategy" of drug delivery systems has been used in order to improve carrier biodistribution, however, some nanosized systems with PEG on their surface have exhibited poorly-cellular drug internalization. In this context, the purpose of the present study was to compare in vitro performance of two paclitaxel (PTX)-loaded NPs systems based on two biocompatible copolymers of alpha tocopheryl polyethylene glycol 1000 succinate-block-poly(ε-caprolactone) (TPGS-b-PCL) and methoxyPEG- block-poly(ε-caprolactone) (mPEG-b-PCL) in terms of citotoxicity and PTX cellular uptake. Fur- thermore, TPGS-b-PCL NPs were also copared with the commercially available PTX nano-sized formulation Abraxane®. Both TPGS-b-PCL and mPEG-b-PCL derivates were synthesized by ring opening polymerization of ε-caprolactone employing microwaved radiation. NPs were obtained by a solvent evaporation technique where the PTX content was determined by reverse-phase HPLC. The resulting NPs had an average size between 200 and 300 nm with a narrow size distribution. Also both NPs systems showed a spherical shape. The in vitro PTX release profile from the NPs was characterized employing the dialysis membrane method where all drug-loaded formulations showed a sustained and slow release of PTX. Finally, in vitro assays demonstrated that PTX-loaded TPGS- b-PCL exhibited a significant higher antitumor activity than PTX-loaded mPEG-b-PCL NPs and Abraxane® against an estrogen-dependent (MCF-7) and an estrogen independent (MDA-MB-231) breast cancer cells lines. Furthermore TPGS-b-PCL NPs showed a significant increase on PTX cellular uptake, for both breast cell lines, in comparison with mPEG-b-PCL NPs and Abraxane®. Overall findings confirmed

  13. Rg1 protects the MPP+-treated MES23.5 cells via attenuating DMT1 up-regulation and cellular iron uptake.

    PubMed

    Xu, Huamin; Jiang, Hong; Wang, Jun; Xie, Junxia

    2010-02-01

    Ginsenoside-Rg1 is one of the pharmacologically active component isolated from ginseng. Our previous study observed the protective effect of Rg1 on iron accumulation in the substantia nigra (SN) in 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-treated Parkinson's disease (PD) mice. However, the mechanisms of this neuroprotective effect of Rg1 are unknown. In this study, we elucidated possible mechanisms for this effect using 1-methyl-4-phenylpyridinium (MPP(+))-treated MES23.5 cells. Previous study showed MPP+ treatment induced up-regulation of divalent metal transporter 1 without iron responsive element (DMT1-IRE) in MES23.5 cells. In the present study, we observed that pretreatment with Rg1 could inhibit MPP+-induced up-regulation of DMT1-IRE in MES23.5 cells. Up-regulation of DMT1-IRE by MPP+ treatment was associated with ROS production and translocation of nuclear factor-kappaB (NF-kappaB) to nuclei, both of which were significantly inhibited by Rg1 pretreatment. The role of ROS and NF-kappaB in the up-regulation of DMT1-IRE was supported by application of an antioxidant NAC and BAY 11-7082, an inhibitor of IkappaBalpha phosphorylation. Furthermore, we also showed Rg1 could decrease DMT1-mediated ferrous iron uptake and iron-induced cell damage by inhibiting the up-regulation of DMT1-IRE. These results indicate that Rg1 protected the MPP+-treated MES23.5 cells via attenuating DMT1-IRE up-regulation likely through inhibition of ROS-NF-kappaB pathway; Attenuation of DMT1-IRE expression decreased the iron influx and iron-induced oxidative stress.

  14. Enhanced cellular uptake and phototoxicity of Verteporfin-conjugated gold nanoparticles as theranostic nanocarriers for targeted photodynamic therapy and imaging of cancers.

    PubMed

    Zhao, Linlin; Kim, Tae-Hyun; Kim, Hae-Won; Ahn, Jin-Chul; Kim, So Yeon

    2016-10-01

    Activatable theranostics with the capacity to respond to a given stimulus have recently been intensively explored to develop more specific, individualized therapies for various diseases, and to combine diagnostic and therapeutic capabilities into a single agent. In this work, we designed tumor-targeting ligand-conjugated block copolymer-gold nanoparticle (AuNP) conjugates as multifunctional nanocarriers of the hydrophobic photosensitizer (PS), verteporfin (Verte), for simultaneous photodynamic therapy and imaging of cancers. Folic acid (FA)-conjugated block copolymers composed of polyethylene glycol (PEG) and poly-β-benzyl-l-aspartate (PBLA) were attached to citrate-stabilized AuNPs through a bidentate dihydrolipoic acid (DHLA) linker. The resulting AuNP conjugates (FA-PEG-P(Asp-Hyd)-DHLA-AuNPs) were significantly more stable than unmodified AuNPs, and their optical properties were not affected by pH. The hydrophobic PS, Verte, was covalently incorporated onto the surfaces of the AuNP conjugates through a pH-sensitive linkage, which increased the water solubility of Verte from <1μg/ml to >2000μg/ml. The size of FA-PEG-P(Asp-Hyd)-DHLA-AuNPs-Verte as determined by light-scattering measurements was about 110.3nm, and FE-SEM and FE-TEM images showed that these nanoparticles were spherical and showed adequate dispersivity after modification. In particular, an in vitro cell study revealed high intracellular uptake of FA-PEG-P(Asp-Hyd)-DHLA-AuNPs-Verte (about 98.62%) and marked phototoxicity after laser irradiation compared with free Verte. These results suggest that FA-PEG-P(Asp-Hyd)-DHLA-AuNPs-Verte has great potential as an effective nanocarrier for dual imaging and photodynamic therapy. PMID:27287160

  15. Intermediate steps in cellular iron uptake from transferrin. II. A cytoplasmic pool of iron is released from cultured cells via temperature-dependent mechanical wounding.

    PubMed

    Richardson, D R; Dickson, L; Baker, E

    1996-09-01

    A previous study described a cytoplasmic, transferrin (Tf)-free, iron (Fe) pool that was detected only when cells were mechanically detached from the culture substratum at 4 degrees C, after initial incubation with 59Fe-125I-Tf at 37 degrees C (Richardson and Baker, 1992a). The release of this internalized 59Fe could be markedly reduced if the cells were treated with proteases or incubated at 37 degrees C prior to detachment. The present study was designed to characterize this Fe pool and understand the mechanism of its release. The results show that cellular 59Fe release increased linearly as a function of preincubation time with 59Fe-Tf subsequent to mechanical detachment at 4 degrees C using a spatula. These data suggest that the 59Fe release was largely composed of end product(s) and was not an "intermediate Fe pool." When the Fe(II) chelator, dipyridyl (DP), was incubated with 59Fe-Tf and the cells, it prevented the accumulation of 59Fe that was released following mechanical detachment at 4 degrees C. Other chelators had much less effect on the proportion of 59Fe released. Examination of the 59Fe released showed that after a 4-h preincubation with 59Fe-Tf, approximately 50% of the 59Fe was present in ferritin. These data indicate that mechanical detachment of cells at 4 degrees C resulted in membrane disruptions that allow the release of high M(r), molecules. Moreover, electron microscopy studies showed that detachment of cells from the substratum at 4 degrees C resulted in pronounced membrane damage. In contrast, when cells were detached at 37 degrees C, or at 4 degrees C after treatment with pronase, membrane damage was minimal or not apparent. These results may imply that temperature-dependent processes prevent the release of intracellular contents on membrane wounding, or alternatively, prevent wounding at 37 degrees C. The evidence also indicates that caution is required when interpreting data from experiments where cells have been mechanically detached

  16. Enhanced toxicity and cellular uptake of methotrexate-conjugated nanoparticles in folate receptor-positive cancer cells by decorating with folic acid-conjugated d-α-tocopheryl polyethylene glycol 1000 succinate.

    PubMed

    Junyaprasert, Varaporn Buraphacheep; Dhanahiranpruk, Sirithip; Suksiriworapong, Jiraphong; Sripha, Kittisak; Moongkarndi, Primchanien

    2015-12-01

    Folic acid-conjugated d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS-FOL) decorated methotrexate (MTX)-conjugated nanoparticles were developed for targeted delivery of MTX to folate receptor-expressed tumor cells. The synthesis of TPGS-FOL followed 3-step process. Firstly, the terminal hydroxyl group of TPGS was converted to sulfonyl chloride using mesyl chloride in comparison with nosyl and tosyl chlorides. The highest conversion efficiency and yield were obtained by mesyl chloride due to the formation of higher reactive intermediate in a presence of triethylamine. Secondly, the substitution of sulfonyl group by sodium azide produced considerably high yield with conversion efficiency of over 90%. Lastly, the coupling reaction of azido-substituted TPGS and propargyl folamide by click reaction resulted in 96% conjugation efficiency without polymer degradation. To fabricate the folate receptor-targeted nanoparticles, 10 and 20%mol MTX-conjugated PEGylated poly(ϵ-caprolactone) nanoparticles were decorated with TPGS-FOL. The size and size distribution of MTX-conjugated nanoparticles relatively increased with %MTX. The MTX release from the nanoparticles was accelerated in acidic medium with an increase of %MTX but retarded in physiological pH medium. The decoration of TPGS-FOL onto the nanoparticles slightly enlarged the size and size distribution of the nanoparticles; however, it did not affect the surface charge. The cytotoxicity and cellular uptake of MCF-7 cells demonstrated that 10% MTX-conjugated nanoparticles and FOL-decorated nanoparticles possessed higher toxicity and uptake efficiency than 20% MTX-conjugated nanoparticles and undecorated nanoparticles, respectively. The results indicated that FOL-10% MTX-conjugated nanoparticles exhibited potential targeted delivery of MTX to folate receptor-expressed cancer cells.

  17. Introduction of an N-glycan sequon into HEXA enhances human beta-hexosaminidase cellular uptake in a model of Sandhoff disease.

    PubMed

    Matsuoka, Kazuhiko; Tsuji, Daisuke; Aikawa, Sei-Ichi; Matsuzawa, Fumiko; Sakuraba, Hitoshi; Itoh, Kohji

    2010-08-01

    Human lysosomal beta-hexosaminidase A is a heterodimer composed of alpha- and beta-subunits encoded by HEXA and HEXB, respectively. We genetically introduced an additional N-glycosylation sequon into HEXA, which caused amino acid substitutions (S51 to N and A53 to T) at homologous positions to N84 and T86 in the beta-subunit. The mutant HexA (NgHexA) obtained from a Chinese hamster ovary (CHO) cell line co-expressing the mutated HEXA and wild-type HEXB complementary DNAs was demonstrated to contain an additional mannose-6-phosphate (M6P)-type-N-glycan. NgHexA was more efficiently taken up than the wild-type HexA and delivered to lysosomes, where it degraded accumulated substrates including GM2 ganglioside (GM2) when administered to cultured fibroblasts derived from a Sandhoff disease (SD) patient. On intracerebroventricular (i.c.v.) administration of NgHexA to SD model mice, NgHexA more efficiently restored the HexA activity and reduced the GM2 and GA2 (asialoGM2) accumulated in neural cells of the brain parenchyma than the wild-type HexA. These findings indicate that i.c.v. administration of the modified human HexA with an additional M6P-type N-glycan is applicable for enzyme replacement therapy (ERT) involving an M6P-receptor as a molecular target for HexA deficiencies including Tay-Sachs disease and SD. PMID:20571546

  18. Rapid Method To Determine Intracellular Drug Concentrations in Cellular Uptake Assays: Application to Metformin in Organic Cation Transporter 1-Transfected Human Embryonic Kidney 293 Cells.

    PubMed

    Chien, Huan-Chieh; Zur, Arik A; Maurer, Tristan S; Yee, Sook Wah; Tolsma, John; Jasper, Paul; Scott, Dennis O; Giacomini, Kathleen M

    2016-03-01

    Because of the importance of intracellular unbound drug concentrations in the prediction of in vivo concentrations that are determinants of drug efficacy and toxicity, a number of assays have been developed to assess in vitro unbound concentrations of drugs. Here we present a rapid method to determine the intracellular unbound drug concentrations in cultured cells, and we apply the method along with a mechanistic model to predict concentrations of metformin in subcellular compartments of stably transfected human embryonic kidney 293 (HEK293) cells. Intracellular space (ICS) was calculated by subtracting the [(3)H]-inulin distribution volume (extracellular space, ECS) from the [(14)C]-urea distribution volume (total water space, TWS). Values obtained for intracellular space (mean ± S.E.M.; μl/10(6) cells) of monolayers of HEK cells (HEK-empty vector [EV]) and cells overexpressing human organic cation transporter 1 (HEK-OCT1), 1.21± 0.07 and 1.25±0.06, respectively, were used to determine the intracellular metformin concentrations. After incubation of the cells with 5 µM metformin, the intracellular concentrations were 26.4 ± 7.8 μM and 268 ± 11.0 μM, respectively, in HEK-EV and HEK-OCT1. In addition, intracellular metformin concentrations were lower in high K(+) buffer (140 mM KCl) compared with normal K(+) buffer (5.4 mM KCl) in HEK-OCT1 cells (54.8 ± 3.8 μM and 198.1 ± 11.2 μM, respectively; P < 0.05). Our mechanistic model suggests that, depending on the credible range of assumed physiologic values, the positively charged metformin accumulates to particularly high levels in endoplasmic reticulum and/or mitochondria. This method together with the computational model can be used to determine intracellular unbound concentrations and to predict subcellular accumulation of drugs in other complex systems such as primary cells. PMID:26700958

  19. Divalent minerals decrease micellarization and uptake of carotenoids and digestion products into Caco-2 cells.

    PubMed

    Biehler, Eric; Hoffmann, Lucien; Krause, Elmar; Bohn, Torsten

    2011-10-01

    Carotenoids are lipophilic, dietary antioxidants with the potential to prevent chronic and age-related diseases. Prior to their availability for physiological functions, carotenoids require micellarization and intestinal uptake, both constituting marginally understood processes. Based on an in vitro digestion model coupled to Caco-2 cells, we assessed the effect of dietary abundant divalent ions on spinach-derived carotenoid micellarization and cellular uptake: Ca and Mg ranging from 7.5 to 25 mmol/L in the digesta and Zn and Fe ranging from 3.8 to 12.5 mmol/L. Both micellarization and uptake were significantly inhibited by minerals in a concentration-dependent manner, with stronger effects for Fe and Zn compared to Ca and Mg. Compared to controls (no mineral addition), fractional micellarization and uptake were decreased to the greatest extent (to 22.5 and 5.0%, respectively; P < 0.001) by 12.5 mmol/L Fe. Effects of Mg were of the least magnitude; at 25 mmol/L, only uptake was decreased significantly to 69.2% of the control value (P < 0.001). Total cellular carotenoid uptake from test meals decreased similarly compared to micellarization; however, decreased β-carotene micellarization was counterbalanced by improved fractional cellular uptakes from the micelles for all ions. Compared to controls, fractional β-carotene uptake from the micelles was greater in samples digested in the presence of Fe, Ca, and Zn, by up to 5-10 times at the highest concentrations of each ion (P < 0.001). Like for the above carotenoids, a high cellular uptake of the epoxycarotenoid conversion products neochrome (from neoxanthin) and luteoxanthin+auroxanthin (from violaxanthin) was also observed. The present results indicate that divalent ions may inhibit carotenoid micellarization and uptake.

  20. Cellular uptake and trafficking of polydiacetylene micelles

    NASA Astrophysics Data System (ADS)

    Gravel, Edmond; Thézé, Benoit; Jacques, Isabelle; Anilkumar, Parambath; Gombert, Karine; Ducongé, Frédéric; Doris, Eric

    2013-02-01

    Polydiacetylene (PDA) micelles coated with either carboxylate-, ammonium-, or methoxy-polyethyleneglycol (PEG) chains were assembled and loaded with a fluorescent dye (DiO). Their interaction with MCF-7 human breast tumor cells was investigated by epi-fluorescence microscopy and fluorescence-activated cell sorting (FACS) to determine their internalization pathway and intracellular fate. It was found that the ionic character of the micelles influenced their internalization kinetics through a caveolae-mediated pathway and that all micelle types behaved somewhat similarly inside cells.Polydiacetylene (PDA) micelles coated with either carboxylate-, ammonium-, or methoxy-polyethyleneglycol (PEG) chains were assembled and loaded with a fluorescent dye (DiO). Their interaction with MCF-7 human breast tumor cells was investigated by epi-fluorescence microscopy and fluorescence-activated cell sorting (FACS) to determine their internalization pathway and intracellular fate. It was found that the ionic character of the micelles influenced their internalization kinetics through a caveolae-mediated pathway and that all micelle types behaved somewhat similarly inside cells. Electronic supplementary information (ESI) available: Detailed synthetic procedures and supplementary figures. See DOI: 10.1039/c2nr34149b

  1. [Cellular delivery of modified peptide nucleic acids: a review].

    PubMed

    Liu, Chundong; Wang, Jianhua; Zeng, Fang

    2016-03-01

    Peptide nucleic acid (PNA) is a DNA surrogate in which the phosphate deoxyribose backbone of DNA is replaced by repeating N-(2-aminoethyl)glycine units. PNA can hybridize to the complementary DNA and RNA with higher affinity than their oligonucleotide counterparts. This character of PNA not only makes it a new tool for the studies of molecular biology but also the potential candidate for gene-targeting drugs. The non-ionic backbone of PNA leads to stable hybrids with the nucleic acids, but at the same time, the neutral backbone results in poor cellular uptake. To address this problem, studies on modified PNA progress rapidly in recent years. We reviewed literature reports combined with our study about the delivery methods, including backbone modified PNA and PNA-ligand conjugates, and the cellular uptake of modified PNA. In addition, we summarized the problems and future prospect of the cellular delivery of modified PNA.

  2. Integration of Peptides for Enhanced Uptake of PEGylayed Gold Nanoparticles.

    PubMed

    Cruje, C; Chithrani, B D

    2015-03-01

    Polyethylene glycol (PEG) has promoted the prospective applications of nanoparticles (NPs) in cancer therapy. PEG is used to evade the immune system allowing NPs accumulation within the tumor using its leaky vasculature. However, the cellular uptake of PEG-coated (PEGylated) NPs is lower in comparison to non-PEGylated NPs since PEG minimizes surface binding of ligands that mediate NP endocytosis. For improved outcome in therapeutic applications, it is necessary to enhance the uptake of PEGylated NPs. We added a peptide containing an integrin binding domain known as the RGD sequence to the NP surface in addition to PEG. We used gold NPs (GNPs) of sizes 14, 50, and 70 nm in this study. Our in vitro data for HeLa cells show enhanced uptake for NPs coated with both PEG and the peptide in comparison to PEGylated GNPs. NPs of size 50 nm had the highest uptake among the three sizes for all GNP surfaces. A similar size-dependent trend was observed for MDA-MB-231 cells for as-made GNPs with lower uptake in comparison to HeLa cells. However, only 14 nm peptide-modified PEGylated NPs had enhanced uptake. Hence, NP uptake was found dependent on cell type and NP surface properties. A properly designed NP system with both PEG and cell membrane targeting peptides can be used to protect it from the immune system and promote internalization by cells upon entry into tumor environment.

  3. Mitochondrial calcium uptake.

    PubMed

    Williams, George S B; Boyman, Liron; Chikando, Aristide C; Khairallah, Ramzi J; Lederer, W J

    2013-06-25

    Calcium (Ca(2+)) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca(2+) levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca(2+) ([Ca(2+)]i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca(2+) uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca(2+) uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca(2+) uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca(2+) influx into the mitochondria via the MCU is small relative to other cytosolic Ca(2+) extrusion pathways; (ii) single MCU conductance is ∼6-7 pS (105 mM [Ca(2+)]), and MCU flux appears to be modulated by [Ca(2+)]i, suggesting Ca(2+) regulation of MCU open probability (P(O)); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iv) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca(2+) critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca(2+) under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca(2+)]i, mitochondrial Ca(2+) uptake can increase 10- to 1,000-fold and begin to shape [Ca(2+)]i dynamics.

  4. The uptake of HIV Tat peptide proceeds via two pathways which differ from macropinocytosis.

    PubMed

    Ben-Dov, Nadav; Korenstein, Rafi

    2015-03-01

    Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic molecules, yet the identity of their uptake routes remained unclear and is still under debate. In this study we provide new insights into CPP entry routes by quantitatively measuring the intracellular uptake of FAM-labeled Tat-peptide under rigorous kinetic and thermal conditions. The uptake of Tat-peptide between 4 and 15°C corresponds to Q10=1.1, proceeding through a prompt (<5 min), temperature-independent process, suggesting direct membrane translocation. At longer durations, Tat rate of uptake shows linear dependence on temperature with Q10=1.44, accompanied by activation energy Ea=4.45 Kcal/mole. These values are significantly lower than those we found for the macropinocytosis probe dextran (Q10=2.2 and Ea=7.2 Kcal/mole) which possesses an exponential dependence on temperature, characteristic of endocytosis processes. Tat-peptide and dextran do not interfere with each other's uptake rate and the ratio of Tat-peptide uptake to its extracellular concentration is ~15 times higher than that for dextran. In addition, Phloretin, a modulator of cell membrane dipole potential, is shown to increase dextran uptake but to reduce that of Tat. We conclude that the uptake of Tat differs from that of dextran in all parameters. Tat uptake proceeds by dual entry routes which differ by their energy dependence. PMID:25542781

  5. Janus magnetic cellular spheroids for vascular tissue engineering

    PubMed Central

    Mattix, Brandon M.; Olsen, Timothy R.; Casco, Megan; Reese, Laura; Poole, John T.; Zhang, Jing; Visconti, Richard P.; Simionescu, Agneta; Simionescu, Dan T.; Alexis, Frank

    2016-01-01

    Cell aggregates, or spheroids, have been used as building blocks to fabricate scaffold-free tissues that can closely mimic the native three-dimensional in vivo environment for broad applications including regenerative medicine and high throughput testing of drugs. The incorporation of magnetic nanoparticles (MNPs) into spheroids permits the manipulation of spheroids into desired shapes, patterns, and tissues using magnetic forces. Current strategies incorporating MNPs often involve cellular uptake, and should therefore be avoided because it induces adverse effects on cell activity, viability, and phenotype. Here, we report a Janus structure of magnetic cellular spheroids (JMCS) with spatial control of MNPs to form two distinct domains: cells and extracellular MNPs. This separation of cells and MNPs within magnetic cellular spheroids was successfully incorporated into cellular spheroids with various cellular and extracellular compositions and contents. The amount of cells that internalized MNPs was quantified and showed that JMCSs resulted in significantly lower internalization (35%) compared to uptake spheroids (83%, p < 0.05). Furthermore, the addition of MNPs to cellular spheroids using the Janus method has no adverse effects on cellular viability up to seven weeks, with spheroids maintaining at least 82% viability over 7 weeks when compared to control spheroids without MNPs. By safely incorporating MNPs into cellular spheroids, results demonstrated that JMCSs were capable of magnetic manipulation, and that magnetic forces used during magnetic force assembly mediate fusion into controlled patterns and complex tissues. Finally, JMCSs were assembled and fused into a vascular tissue construct 5 mm in diameter using magnetic force assembly. PMID:24183699

  6. Silicate reduces cadmium uptake into cells of wheat.

    PubMed

    Greger, Maria; Kabir, Ahmad H; Landberg, Tommy; Maity, Pooja J; Lindberg, Sylvia

    2016-04-01

    Cadmium (Cd) is a health threat all over the world and high Cd content in wheat causes high Cd intake. Silicon (Si) decreases cadmium content in wheat grains and shoot. This work investigates whether and how silicate (Si) influences cadmium (Cd) uptake at the cellular level in wheat. Wheat seedlings were grown in the presence or absence of Si with or without Cd. Cadmium, Si, and iron (Fe) accumulation in roots and shoots was analysed. Leaf protoplasts from plants grown without Cd were investigated for Cd uptake in the presence or absence of Si using the fluorescent dye, Leadmium Green AM. Roots and shoots of plants subjected to all four treatments were investigated regarding the expression of genes involved in the Cd uptake across the plasma membrane (i.e. LCT1) and efflux of Cd into apoplasm or vacuole from the cytosol (i.e. HMA2). In addition, phytochelatin (PC) content and PC gene (PCS1) expression were analysed. Expression of iron and metal transporter genes (IRT1 and NRAMP1) were also analysed. Results indicated that Si reduced Cd accumulation in plants, especially in shoot. Si reduced Cd transport into the cytoplasm when Si was added both directly during the uptake measurements and to the growth medium. Silicate downregulated LCT1 and HMA2 and upregulated PCS1. In addition, Si enhanced PC formation when Cd was present. The IRT1 gene, which was downregulated by Cd was upregulated by Si in root and shoot facilitating Fe transport in wheat. NRAMP1 was similarly expressed, though the effect was limited to roots. This work is the first to show how Si influences Cd uptake on the cellular level.

  7. A novel hNIS/tdTomato fusion reporter for visualizing the relationship between the cellular localization of sodium iodide symporter and its iodine uptake function under heat shock treatment.

    PubMed

    Yeom, Chan Joo; Chung, Taemoon; Youn, Hyewon; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key

    2015-01-01

    The function of membrane-localized sodium iodide symporter (NIS) determines the efficacy of radioiodine therapy in thyroid cancer. Here, we describe a dual mode reporter fused with human NIS (hNIS) and a red fluorescent protein named tandem dimeric Tomato (tdTomato) for the in vitro and in vivo imaging of hNIS protein expression, localization, and iodide uptake function. Human cervical epithelial adenocarcinoma cell line (HeLa)-hNIS/tdTomato cells were established by transducing a fusion gene expressing hNIS/tdTomato under the control of a cytomegalovirus promoter. Fluorescence imaging, confocal microscopy, and an 125I uptake assay were performed to validate the integrity of the fusion protein. Actinomycin D and cycloheximide were used to block newly synthesized hNIS proteins. In vivo images were acquired using a gamma camera and a Maestro fluorescence imaging device. The fluorescence intensity of membrane-localized hNIS and 125I uptake both were increased after heat shock. Scintigraphy and fluorescence imaging indicated specific accumulation of the hNIS/tdTomato fusion protein in xenografted tumors, supporting the utility of this system for in vivo monitoring of hNIS expression and activity. We developed a novel hNIS/tdTomato dual mode reporter that enables visualization of the expression, localization, and iodine uptake function of hNIS in vitro and in vivo. PMID:25773964

  8. A novel hNIS/tdTomato fusion reporter for visualizing the relationship between the cellular localization of sodium iodide symporter and its iodine uptake function under heat shock treatment.

    PubMed

    Yeom, Chan Joo; Chung, Taemoon; Youn, Hyewon; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key

    2015-01-01

    The function of membrane-localized sodium iodide symporter (NIS) determines the efficacy of radioiodine therapy in thyroid cancer. Here, we describe a dual mode reporter fused with human NIS (hNIS) and a red fluorescent protein named tandem dimeric Tomato (tdTomato) for the in vitro and in vivo imaging of hNIS protein expression, localization, and iodide uptake function. Human cervical epithelial adenocarcinoma cell line (HeLa)-hNIS/tdTomato cells were established by transducing a fusion gene expressing hNIS/tdTomato under the control of a cytomegalovirus promoter. Fluorescence imaging, confocal microscopy, and an 125I uptake assay were performed to validate the integrity of the fusion protein. Actinomycin D and cycloheximide were used to block newly synthesized hNIS proteins. In vivo images were acquired using a gamma camera and a Maestro fluorescence imaging device. The fluorescence intensity of membrane-localized hNIS and 125I uptake both were increased after heat shock. Scintigraphy and fluorescence imaging indicated specific accumulation of the hNIS/tdTomato fusion protein in xenografted tumors, supporting the utility of this system for in vivo monitoring of hNIS expression and activity. We developed a novel hNIS/tdTomato dual mode reporter that enables visualization of the expression, localization, and iodine uptake function of hNIS in vitro and in vivo.

  9. Cell uptake survey of pegylated nanographene oxide

    NASA Astrophysics Data System (ADS)

    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.

    2012-11-01

    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.

  10. Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids

    PubMed Central

    Luo, Shuanghui; Pal, Dhananjay; Shah, Sujay J.; Kwatra, Deep; Paturi, Kalyani D.; Mitra, Ashim. K.

    2010-01-01

    HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the cellular washing and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of PappB-A to PappA-B in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake, elevation of cyclosporine uptake in the presence of 5 μM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories. PMID:20163160

  11. Parameters and characteristics governing cellular internalization and trans-barrier trafficking of nanostructures

    PubMed Central

    Murugan, Karmani; Choonara, Yahya E; Kumar, Pradeep; Bijukumar, Divya; du Toit, Lisa C; Pillay, Viness

    2015-01-01

    Cellular internalization and trans-barrier transport of nanoparticles can be manipulated on the basis of the physicochemical and mechanical characteristics of nanoparticles. Research has shown that these factors significantly influence the uptake of nanoparticles. Dictating these characteristics allows for the control of the rate and extent of cellular uptake, as well as delivering the drug-loaded nanosystem intra-cellularly, which is imperative for drugs that require a specific cellular level to exert their effects. Additionally, physicochemical characteristics of the nanoparticles should be optimal for the nanosystem to bypass the natural restricting phenomena of the body and act therapeutically at the targeted site. The factors at the focal point of emerging smart nanomedicines include nanoparticle size, surface charge, shape, hydrophobicity, surface chemistry, and even protein and ligand conjugates. Hence, this review discusses the mechanism of internalization of nanoparticles and ideal nanoparticle characteristics that allow them to evade the biological barriers in order to achieve optimal cellular uptake in different organ systems. Identifying these parameters assists with the progression of nanomedicine as an outstanding vector of pharmaceuticals. PMID:25834433

  12. RGDS- and TAT-Conjugated Upconversion of NaYF4:Yb(3+)/Er(3+)&SiO2 Nanoparticles: In Vitro Human Epithelioid Cervix Carcinoma Cellular Uptake, Imaging, and Targeting.

    PubMed

    Kostiv, Uliana; Kotelnikov, Ilya; Proks, Vladimír; Šlouf, Miroslav; Kučka, Jan; Engstová, Hana; Ježek, Petr; Horák, Daniel

    2016-08-10

    Starting NaYF4:Yb(3+)/Er(3+) nanoparticles with size tuned from 24 to 33 nm were prepared by high-temperature coprecipitation of lanthanide chlorides in high-boiling organic solvents. To enhance colloidal stability in aqueous medium, an aminosilica shell was introduced on the surface by hydrolysis and condensation of tetramethyl orthosilicate and (3-aminopropyl)trimethoxysilane using a reverse microemulsion technique; to form alkyne groups, reaction with 4-pentynoic acid followed. Finally, the cell adhesive and cell penetrating azidopentanoyl-GGGRGDSGGGY-NH2 (RGDS) and azidopentanoyl-GGGRKKRRQRRR-NH2 (TAT) peptides were conjugated to the upconversion particles via Cu(I)-catalyzed alkyne-azide cycloaddition. The concentrations of the peptides bound to the nanoparticle surfaces and amount of adsorbed residual Cu(I) catalyst were determined using an (125)I-radiolabeled RGDS peptide and a (64)Cu(I)-doped catalyst, respectively. Targeting and uptake of the RGDS- and TAT-conjugated NaYF4:Yb(3+)/Er(3+)&SiO2 nanoparticles by human cervix carcinoma HeLa cells were monitored by confocal microscopy. RGDS-conjugated nanoparticle probes were mainly localized on the cell plasma membrane due to specific binding of the peptide to the corresponding integrins. In contrast, the TAT-conjugated nanoparticles were able to cross the cell membrane and accumulate in the cell cytoplasm. Thus, this new peptide bioconjugation approach supported both extra- and intracellular nanoparticle uptake, enabling targeting and imaging of the specific tumor phenotypes. PMID:27428386

  13. Examination of the mechanism of action of nitrogen monoxide on iron uptake from transferrin.

    PubMed

    Watts, R N; Richardson, D R

    2000-08-01

    Nitrogen monoxide (NO) exerts many of its functions by binding to iron (Fe) in the active sites of a number of key proteins. Previously we have shown that NO produced by NO-generating agents decreased cellular Fe uptake from transferrin (Tf). However, the mechanism of this effect was not elucidated. In this study we examined the possible mechanisms whereby NO could interfere with Fe uptake. Our experiments demonstrate that NO produced by the NO generator S-nitroso-N-acetylpenicillamine was slightly more effective than the Fe chelator deferoxamine at reducing iron 59 uptake from 59Fe-labeled Tf by LMTK- fibroblasts. Other NO generators including S-nitrosoglutathione (GSNO) and spermine-NONOate also decreased 59Fe uptake from 59Fe-labeled Tf. In contrast, precursors of these compounds that do not release NO had no effect. When the RAW264.7 macrophage cell line was activated to produce NO by incubation with lipopolysaccharide or lipopolysaccharide and interferon-gamma, a decrease in 59Fe uptake from 59Fe-labeled Tf was also observed. Experiments with electron paramagnetic resonance spectroscopy and ultraviolet-Vis spectrophotometry demonstrated that NO did not prevent Fe uptake by binding to the Fe-ligating sites of Tf, suggesting that it acted more distally. Because the uptake of Fe is an energy-dependent process, and since NO inhibits mitochondrial respiration, cellular adenosine triphosphate (ATP) was estimated after incubation with GSNO. In the presence of D-glucose (D-G), GSNO reduced ATP levels by 35% as compared with the control, while in the absence of D-G, GSNO reduced ATP by 72%. When the same experiments were performed with D-fructose (D-F), which cannot be efficiently metabolized by fibroblasts, no "rescue" effect was observed on ATP levels. The addition of D-G to GSNO prevented the decrease in 59Fe uptake from 59Fe-labeled Tf while D-F did not, in good correlation with their effects on ATP levels. These results suggest that D-G acts as a salvage

  14. Electrogenicity of hepatocellular fatty acid uptake.

    PubMed

    Elsing, C; Kassner, A; Gajdzik, L; Graf, J; Stremmel, W

    1998-08-18

    Sensitivity of cellular fatty acids uptake to the membrane potential difference is still a matter of controversy. For direct evaluation of potential sensitivity the effect of changing membrane potential on uptake of a fluorescent long chain fatty acid derivative, 12-NBD-stearate, in isolated rat hepatocytes, was examined. Changes in membrane potential were achieved by patch clamp procedures. Fatty acid influx was simultaneously determined by recording of cell fluorescence. Hyperpolarization from -30 to -70 mV accelerated fatty acid influx whereas depolarization to +50 mV reduced uptake. After obtaining equilibrium hyperpolarization increased cell fluorescence, whereas depolarization pushed NBD-stearate out of cells. Potential sensitivity of uptake was dependent on the fatty acid concentrations in the medium with most prominent effects at low unbound concentrations. These data show that, at low fatty acid concentrations, uptake is, in part, driven by an intracellular negative electric membrane potential.

  15. PEG functionalized luminescent lipid particles for cellular imaging

    NASA Astrophysics Data System (ADS)

    Rana, Suman; Barick, K. C.; Shetake, Neena G.; Verma, Gunjan; Aswal, V. K.; Panicker, Lata; Pandey, B. N.; Hassan, P. A.

    2016-08-01

    We report here the synthesis, characterization and cellular uptake of luminescent micelle-like particles with phospholipid core and non-ionic PEG based surfactant polysorbate 80 shell. The adsorption of polysorbate 80 at the interface of lipid containing microemulsion droplets and its solidification upon removal of solvent leads to anchoring of PEG chain to the lipid particles. Hydrophobic partitioning of luminescent molecules, sodium 3-hydroxynaphthalene-2-carboxylic acid to the phospholipid core offers additional functionality to these particles. Thus, the cooperative assembly of lipid, non-ionic amphiphile and organic luminescent probe leads to the formation of multifunctional biocompatible particles which are useful for simultaneous imaging and therapy.

  16. Architected Cellular Materials

    NASA Astrophysics Data System (ADS)

    Schaedler, Tobias A.; Carter, William B.

    2016-07-01

    Additive manufacturing enables fabrication of materials with intricate cellular architecture, whereby progress in 3D printing techniques is increasing the possible configurations of voids and solids ad infinitum. Examples are microlattices with graded porosity and truss structures optimized for specific loading conditions. The cellular architecture determines the mechanical properties and density of these materials and can influence a wide range of other properties, e.g., acoustic, thermal, and biological properties. By combining optimized cellular architectures with high-performance metals and ceramics, several lightweight materials that exhibit strength and stiffness previously unachievable at low densities were recently demonstrated. This review introduces the field of architected materials; summarizes the most common fabrication methods, with an emphasis on additive manufacturing; and discusses recent progress in the development of architected materials. The review also discusses important applications, including lightweight structures, energy absorption, metamaterials, thermal management, and bioscaffolds.

  17. Receptor-mediated uptake of ferritin-bound iron by human intestinal Caco-2 cells.

    PubMed

    Kalgaonkar, Swati; Lönnerdal, Bo

    2009-04-01

    Ferritin (Ft) is a large iron (Fe)-binding protein ( approximately 450 kDa) that is found in plant and animal cells and can sequester up to 4500 Fe atoms per Ft molecule. Our previous studies on intestinal Caco-2 cells have shown that dietary factors affect the uptake of Fe from Ft in a manner different from that of Fe from FeSO4, suggesting a different mechanism for cellular uptake. The objective of this study was to determine the mechanism for Ft-Fe uptake using Caco-2 cells. Binding of (59)Fe-labeled Ft at 4 degrees C showed saturable kinetics, and Scatchard analysis resulted in a K(d) of 1.6 muM, strongly indicating a receptor-mediated process. Competitive binding studies with excess unlabelled Ft significantly reduced binding, and uptake studies at 37 degrees C showed saturation after 4 h. Enhancing and blocking endocytosis using Mas-7 (a G-protein activator) and hypertonic medium (0.5 M sucrose), respectively, demonstrated that Ft-Fe uptake by Mas-7-treated cells was 140% of control cells, whereas sucrose treatment resulted in a statistically significant reduction in Ft-Fe uptake by 70% as compared to controls. Inhibition of macropinocytosis with 5-(N,N-dimethyl)-amiloride (Na+/H+ antiport blocker) resulted in a decrease (by approximately 20%) in Ft-Fe uptake at high concentrations of Ft, suggesting that enterocytes can use more than one Ft uptake mechanism in a concentration-dependent manner. These results suggest that Ft uptake by enterocytes is carried out via endocytosis when Ft levels are within a physiological range, whereas Ft at higher concentrations may be absorbed using the additional mechanism of macropinocytosis. PMID:18602806

  18. Multiplicity and specificity of siderophore uptake in the cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Rudolf, Mareike; Stevanovic, Mara; Kranzler, Chana; Pernil, Rafael; Keren, Nir; Schleiff, Enrico

    2016-09-01

    Many cyanobacteria secrete siderophores to sequester iron. Alternatively, mechanisms to utilize xenosiderophores have evolved. The overall uptake systems are comparable to that of other bacteria involving outer membrane transporters energized by TonB as well as plasma membrane-localized transporters. However, the function of the bioinformatically-inferred components is largely not established and recent studies showed a high diversity of the complexity of the uptake systems in different cyanobacteria. Thus, we approached the systems of the filamentous Anabaena sp. PCC 7120 as a model of a siderophore-secreting cyanobacterium. Anabaena sp. produces schizokinen and uptake of Fe-schizokinen involves the TonB-dependent transporter, schizokinen transporter (SchT), and the ABC-type transport system FhuBCD. We confirm that this system is also relevant for the uptake of structurally similar Fe-siderophore complexes like Fe-aerobactin. Moreover, we demonstrate a function of the TonB-dependent transporter IutA2 in Fe-schizokinen uptake in addition to SchT. The iutA2 mutant shows growth defects upon iron limitation, alterations in Fe-schizokinen uptake and in the transcription profile of the Fe-schizokinen uptake system. The physiological properties of the mutant confirm the importance of iron uptake for cellular function, e.g. for the Krebs cycle. Based on the relative relation of expression of schT and iutA2 as well as of the iron uptake rate to the degree of starvation, a model for the need of the co-existence of two different outer membrane transporters for the same substrate is discussed. PMID:27325117

  19. A Comparative Study of Cellular Uptake and Subcellular Localization of Doxorubicin Loaded in Self-Assemblies of Amphiphilic Copolymers with Pendant Dendron by MDA-MB-231 Human Breast Cancer Cells.

    PubMed

    Viswanathan, Geetha; Hsu, Yu-Hsuan; Voon, Siew Hui; Imae, Toyoko; Siriviriyanun, Ampornphan; Lee, Hong Boon; Kiew, Lik Voon; Chung, Lip Yong; Yusa, Shin-Ichi

    2016-06-01

    Previously synthesized amphiphilic diblock copolymers with pendant dendron moieties have been investigated for their potential use as drug carriers to improve the delivery of an anticancer drug to human breast cancer cells. Diblock copolymer (P71 D3 )-based micelles effectively encapsulate the doxorubicin (DOX) with a high drug-loading capacity (≈95%, 104 DOX molecules per micelle), which is approximately double the amount of drug loaded into the diblock copolymer (P296 D1 ) vesicles. DOX released from the resultant P71 D3 /DOX micelles is approximately 1.3-fold more abundant, at a tumoral acidic pH of 5.5 compared with a pH of 7.4. The P71 D3 /DOX micelles also enhance drug potency in breast cancer MDA-MB-231 cells due to their higher intracellular uptake, by approximately twofold, compared with the vesicular nanocarrier, and free DOX. Micellar nanocarriers are taken up by lysosomes via energy-dependent processes, followed by the release of DOX into the cytoplasm and subsequent translocation into the nucleus, where it exert its cytotoxic effect.

  20. Irradiation of Human Prostate Cancer Cells Increases Uptake of Antisense Oligodeoxynucleotide

    SciTech Connect

    Anai, Satoshi; Brown, Bob D.; Nakamura, Kogenta; Goodison, Steve; Hirao, Yoshihiko; Rosser, Charles J. . E-mail: charles.rosser@urology.ufl.edu

    2007-07-15

    Purpose: To investigate whether irradiation before antisense Bcl-2 oligodeoxynucleotide (ODN) administration enhances tissue uptake, and whether periodic dosing enhances cellular uptake of fluorescently labeled ODN relative to constant dosing. Methods and Materials: PC-3-Bcl-2 cells (prostate cancer cell line engineered to overexpress Bcl-2) were subjected to increasing doses of irradiation (0-10 Gy) with or without increasing concentrations of fluorescently labeled antisense Bcl-2 ODN (G4243). The fluorescent signal intensity was quantified as the total grain area with commercial software. In addition, PC-3-Bcl-2 subcutaneous xenograft tumors were treated with or without irradiation in combination with various dosing schemas of G4243. The uptake of fluorescent G4243 in tumors was quantitated. Results: The uptake of G4243 was increased in prostate cancer cells exposed to low doses of irradiation both in vitro and in vivo. Irradiation before G4243 treatment resulted in increased fluorescent signal intensity in xenograft tumors compared with those irradiated after G4243 treatment. A single weekly dose of G4243 produced higher G4243 uptake in xenograft tumors than daily dosing, even when the total dose administered per week was held constant. Conclusions: These findings suggest that ionizing radiation increases the uptake of therapeutic ODN in target tissues and, thus, has potential to increase the efficacy of ODN in clinical applications.

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

    SciTech Connect

    Levesque, Martine; Martineau, Corine; Jumarie, Catherine; Moreau, Robert

    2008-09-15

    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-dependent 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

  2. Post-uptake metabolism affects quantification of amino acid uptake.

    PubMed

    Warren, Charles R

    2012-01-01

    • The quantitative significance of amino acids to plant nutrition remains controversial. This experiment determined whether post-uptake metabolism and root to shoot export differ between glycine and glutamine, and examined implications for estimation of amino acid uptake. • Field soil containing a Eucalyptus pauciflora seedling was injected with uniformly (13)C- and (15)N-labelled glycine or glutamine. I quantified (15)N and (13)C excess in leaves and roots and intact labelled amino acids in leaves, roots and stem xylem sap. A tunable diode laser quantified fluxes of (12)CO(2) and (13)CO(2) from leaves and soil. • 60-360 min after addition of amino acid, intact molecules of U-(13)C,(15)N glutamine were < 5% of (15)N excess in roots, whereas U-(13)C,(15)N glycine was 30-100% of (15)N excess in roots. Intact molecules of glutamine, but not glycine, were exported from roots to shoots. • Post-uptake metabolism and transport complicate interpretation of isotope labelling such that root and shoot contents of intact amino acid, (13)C and (15)N may not reflect rates of uptake. Future experiments should focus on reconciling discrepancies between intact amino acid, (13)C and (15)N by determining the turnover of amino acids within roots. Alternatively, post-uptake metabolism and transport could be minimized by harvesting plants within minutes of isotope addition.

  3. Uptake of trimethoprim by renal cortex.

    PubMed

    Cacini, W; Myre, S A

    1985-10-01

    The purpose of this study was to examine the mechanisms involved in the uptake of the urinary antibacterial drug trimethoprim by incubated slices of rat renal cortex. Concentration-dependent studies of the uptake process demonstrated that a saturable component was involved. The results of inhibitor studies as well as the time-course pattern support the conclusion that at least two processes are involved in the uptake of trimethoprim. These include active transport via the organic cation system, accounting for about 40% of the total uptake, and a second component that continues to operate under conditions of inhibited cellular metabolism. Chromatographic examination of post-incubation bathing medium and slice extracts failed to demonstrate renal cortex metabolism of trimethoprim. PMID:4052093

  4. Rapid Responses to Reverse T3 Hormone in Immature Rat Sertoli Cells: Calcium Uptake and Exocytosis Mediated by Integrin

    PubMed Central

    Zanatta, Ana Paula; Zanatta, Leila; Gonçalves, Renata; Zamoner, Ariane; Silva, Fátima Regina Mena Barreto

    2013-01-01

    There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T3 (rT3) action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT3 are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT3 was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT3-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions). Therefore, our findings suggest that calcium uptake stimulated by rT3 may be mediated by integrin αvβ3. In addition, it was demonstrated that calcium uptake stimulated by rT3 is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT3 also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT3 modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology. PMID:24130850

  5. Modification of nitrate uptake pathway in plants affects the cadmium uptake by roots.

    PubMed

    Guan, Mei Yan; Fan, Shi Kai; Fang, Xian Zhi; Jin, Chong Wei

    2015-01-01

    NRT1.1 is a dual-affinity nitrate (NO3(-)) transporter involved in both high- and low-affinity NO3(-) uptake in Arabidopsis plants. In a recent study, we showed that, under cadmium (Cd) exposure, blocking the NRT1.1-mediated NO3(-) uptake reduces Cd entry into roots, thus lowing Cd levels in plants and improving plant growth. In addition, we also found that the Cd levels in edible parts of 11 Chinese cabbage (Brassica rapa L. ssp. pekinensis) cultivars correlated well with the NO3(-) uptake rates of their roots. These results suggested that the NO3(-) uptake of roots negatively regulate Cd uptake. Modification of NO3(-) uptake in crops by modulating NO3(-) uptake pathway might provide a biological engineering approach to reducing Cd accumulation in edible organs, thus improving food safety.

  6. Mechanism of poly-l-lysine-modified iron oxide nanoparticles uptake into cells.

    PubMed

    Li, Zheng; Shuai, Cijun; Li, Xiayu; Li, Xiaoling; Xiang, Juanjuan; Li, Guiyuan

    2013-10-01

    Poly-l-lysine-modified iron oxide nanoparticle (IONP-PLL), which is formed by modifying poly- l-lysine to the surface of iron oxide nanoparticles, can deliver exogenous genes to cells in vitro and in vivo. However, there is relatively little information available about how is IONP-PLL uptaken by cells. In this study, we are focusing on the transferrin receptor (TFR) mediated and TFR-independent cellular internalization of IONP-PLL. The cells were incubated with 1 µM of IONP-PLL with or without transferrin bound. Transferrin-TFR pathway blockers, such as NH4 Cl, CH3 NH2 , or trypsin, were added to the media and their effects were observed. Atomic absorption spectrophotometer was used to quantify the cellular concentration of iron. The cellular concentrations of iron were evaluated at 37°C or 4°C. (1) Transferrin-IONP-PLL uptake into cells was reliant on time and temperature. (2) The addition of blockers, either NH4 CL, CH3 NH2 , or trypsin, decreased the cellular transferrin-dependent IONP-PLL uptake, but not completely blocked the entry of IONP-PLL. (3) When the cells were culture at pH 6.5, under conditions which the binding of iron and transferrin were inhibited, IONP-PLL still had the capacity to enter into cells with time and temperature-dependent manner. These results suggest that the cellular internalization of IONP-PLL, much like iron ion, were mediated by TFR-dependent endocytosis and TFR-free uptake.

  7. The role of uncoupling protein 3 regulating calcium ion uptake into mitochondria during sarcopenia

    NASA Astrophysics Data System (ADS)

    Nikawa, Takeshi; Choi, Inho; Haruna, Marie; Hirasaka, Katsuya; Maita Ohno, Ayako; Kondo Teshima, Shigetada

    Overloaded mitochondrial calcium concentration contributes to progression of mitochondrial dysfunction in aged muscle, leading to sarcopenia. Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. Recently, it has been reported that UCP3 is associated with calcium uptake into mitochondria. However, the mechanisms by which UCP3 regulates mitochondrial calcium uptake are not well understood. Here we report that UCP3 interacts with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that is localized in mitochondria, which is involved in cellular responses to calcium ion. The hydrophilic sequences within the loop 2, matrix-localized hydrophilic domain of mouse UCP3 are necessary for binding to Hax-1 of the C-terminal domain in adjacent to mitochondrial innermembrane. Interestingly, these proteins interaction occur the calcium-dependent manner. Indeed, overexpression of UCP3 significantly enhanced calcium uptake into mitochondria on Hax-1 endogenously expressing C2C12 myoblasts. In addition, Hax-1 knock-down enhanced calcium uptake into mitochondria on both UCP3 and Hax-1 endogenously expressing C2C12 myotubes, but not myoblasts. Finally, the dissociation of UCP3 and Hax-1 enhances calcium uptake into mitochondria in aged muscle. These studies identify a novel UCP3-Hax-1 complex regulates the influx of calcium ion into mitochondria in muscle. Thus, the efficacy of UCP3-Hax-1 in mitochondrial calcium regulation may provide a novel therapeutic approach against mitochondrial dysfunction-related disease containing sarcopenia.

  8. Cellular dosimetry of diagnostic radionuclides for spherical and ellipsoidal geometry

    NASA Astrophysics Data System (ADS)

    Nettleton, Jo S.; Lawson, Richard S.

    1996-09-01

    Radionuclides which emit Auger electrons are widely used in diagnostic nuclear medicine. Studies have shown possible uptake of these in developing germ cells within the testes. In addition, mature sperm within the reproductive tract may be subject to uptake of radionuclides from the circulating blood pool. Though much work has been carried out concerning cellular dosimetry applied to spherical sources, such an approach may lead to significant errors when considering spermatids and spermatozoa, which are almost ellipsoidal in shape (with the long axis twice the short). A numerical method for determining geometrical reduction factors has been developed and used in conjunction with experimentally determined range - energy relationships for electrons, to determine dose gradients and S factors for homogeneous distributions of four commonly used diagnostic radionuclides (, , and ) throughout source regions of both spherical and ellipsoidal geometry at typical cellular dimensions. The results indicate that assumption of spherical geometry is acceptable when determining S factors for late-type germ cells, but introduces error into calculations of dose distribution towards the edge of the cell.

  9. Methylene blue protects astrocytes against glucose oxygen deprivation by improving cellular respiration.

    PubMed

    Roy Choudhury, Gourav; Winters, Ali; Rich, Ryan M; Ryou, Myoung-Gwi; Gryczynski, Zygmunt; Yuan, Fang; Yang, Shao-Hua; Liu, Ran

    2015-01-01

    Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration.

  10. Cellular Reprogramming

    PubMed Central

    Takahashi, Kazutoshi

    2014-01-01

    Nuclear reprogramming technology was first established more than 50 years ago. It can rejuvenate somatic cells by erasing the epigenetic memories and reconstructing a new pluripotent order. The recent discovery reviewed here that induced pluripotency can be achieved by a small set of transcription factors has opened up unprecedented opportunities in the pharmaceutical industry, the clinic, and laboratories. This technology allows us to access pathological studies by using patient-specific induced pluripotent stem (iPS) cells. In addition, iPS cells are also expected to be a rising star for regenerative medicine, as sources of transplantation therapy. PMID:24492711

  11. Relation between iron uptake, pH of growth medium, and penicillinase formation in Staphylococcus aureus.

    PubMed

    Cohen, S; Sweeney, H M; Leitner, F

    1967-04-01

    The uptake of iron and the formation of penicillinase was examined in cultures of wild-type Staphylococcus aureus. Uptake of iron was about twice as great at pH 4.7 as at pH 7.4 At pH 4.7, increase in iron uptake in the range of 1.0 to 4.0 mug per mg of bacterial protein was associated with a progressive increase in the rate of penicillinase formation, but a direct correlation between cellular iron content and rate of enzyme formation was not demonstrated. Addition of iron to deferrated medium enhanced penicillinase formation at pH 6.5 to 7.4 two- to fourfold in cultures induced with benzylpenicillin and in uninduced cultures. To demonstrate an effect on the uninduced cells, it was necessary to increase iron uptake by preliminary incubation of cells with iron in buffer. Calcium and certain other ions depressed iron uptake at acidic and at neutral pH, and, presumably as a result of this action, depressed the formation of penicillinase. Iron did not enhance penicillinase formation at pH 4.7 by two penicillinase constitutive mutants nor by wild-type cells undergoing induction at pH 6.5 by cephalosporin C or methicillin. After removal of cephalosporin C or methicillin during an early phase of induction, residual synthesis of enzyme was increased by prior uptake of iron. The results are considered compatible with the concept that uptake of iron, especially at acidic pH, interferes with the formation or function of penicillinase repressor. PMID:6032503

  12. Uptake epithelia behave in a cell-centric and not systems homeostatic manner in response to zinc depletion and supplementation† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c3mt00212h Click here for additional data file.

    PubMed Central

    Zheng, Dongling; Feeney, Graham P.; Handy, Richard D.; Kille, Peter

    2013-01-01

    Much remains to be understood about systemic regulation of zinc uptake in vertebrates, and adequate zinc status is far from always achieved in animals or human. In addition to absorbing zinc from the diet, fish are able to take up zinc directly from the water with the gills. This provides an elegant system to study zinc uptake, how it relates to zinc status, and the expression of genes for proteins involved in zinc acquisition. A 21-day experiment was conducted in which zebrafish were acclimated to deficient, control or excess zinc concentrations in the water and feed. Deficient provision of zinc reduced whole body zinc, potassium, sodium and calcium levels whilst zinc concentrations in the uptake epithelia (gills and gut) remained unchanged. Excess levels of zinc caused accumulation of zinc in the gills, intestine and carcass, but impaired whole body iron, sodium and calcium concentrations. Fish subjected to zinc deficiency had, surprisingly, a reduced zinc influx across the gill epithelium, even when tested at a high concentration of zinc in the water. Zinc influx in the excess group was indistinct from the control. Expression of genes for metallothionein-2 (Mt2) and zinc transporters-1, -2, and -8 (Znt1, Znt2, Znt8) in uptake epithelia showed in general a direct relationship with zinc supply, while mRNA for Zip4 was inversely related to zinc supply. Transcripts for the epithelial calcium channel (Ecac/Trpv6) showed time-dependent increased expression in the gills of the deficiency group, and a transient decrease of expression during zinc excess. Transcriptome profiling by microarrays showed that in both gills and intestine, the most markedly affected biological functions were those related to cell growth, proliferation and cancer, closely followed by processes of gene transcription and protein synthesis in general. Whilst changes in zinc supply had profound effects in the intestine on genes associated with uptake and metabolism of macronutrients, many of the

  13. Ligand modified nanoparticles increases cell uptake, alters endocytosis and elevates glioma distribution and internalization

    PubMed Central

    Gao, Huile; Yang, Zhi; Zhang, Shuang; Cao, Shijie; Shen, Shun; Pang, Zhiqing; Jiang, Xinguo

    2013-01-01

    Nanoparticles (NPs) were widely used in drugs/probes delivery for improved disease diagnosis and/or treatment. Targeted delivery to cancer cells is a highly attractive application of NPs. However, few studies have been performed on the targeting mechanisms of these ligand-modified delivery systems. Additional studies are needed to understand the transport of nanoparticles in the cancer site, the interactions between nanoparticles and cancer cells, the intracellular trafficking of nanoparticles within the cancer cells and the subcellular destiny and potential toxicity. Interleukin 13 (IL-13) peptide can specifically bind IL-13Rα2, a receptor that is highly expressed on glioma cells but is expressed at low levels on other normal cells. It was shown that the nanoparticels modification with the IL-13 peptide could improve glioma treatment by selectively increasing cellular uptake, facilitating cell internalization, altering the uptake pathway and increasing glioma localization. PMID:23982586

  14. 18F-FET and 18F-FCH uptake in human glioblastoma T98G cell lines

    PubMed Central

    Persico, Marco Giovanni; Buroni, Federica Eleonora; Pasi, Francesca; Aprile, Carlo; Nano, Rosanna; Hodolic, Marina

    2016-01-01

    . Conclusions 18F-FCH and 18F-FET are candidates for neuro-oncological PET imaging. 18F-FET could be the most useful oncological PET marker in the presence of reparative changes after therapy, where the higher affinity of 18F-FCH to inflammatory cells makes it more difficult to discriminate between tumour persistence and non-neoplastic changes. Additional studies on the influence of inflammatory tissue and radionecrotic cellular components on radiopharmaceutical uptake are necessary. PMID:27247547

  15. Characterization of a Dipartite Iron Uptake System from Uropathogenic Escherichia coli Strain F11*

    PubMed Central

    Koch, Doreen; Chan, Anson C. K.; Murphy, Michael E. P.; Lilie, Hauke; Grass, Gregor; Nies, Dietrich H.

    2011-01-01

    In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His44, Met90, His97, and His127, and CuB, a second degenerate octahedral geometry with the addition of Glu46. The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein. PMID:21596746

  16. Cellular: Toward personal communications

    NASA Astrophysics Data System (ADS)

    Heffernan, Stuart

    1991-09-01

    The cellular industry is one of the fastest growing segment of the telecommunications industry. With an estimated penetration rate of 20 percent in the near future, cellular is becoming an ubiquitous telecommunications service in the U.S. In this paper we will examine the major advancements in the cellular industry: customer equipment, cellular networks, engineering tools, customer support, and nationwide seamless service.

  17. Surfactant-Increased Glyphosate Uptake into Plasma Membrane Vesicles Isolated from Common Lambsquarters Leaves.

    PubMed Central

    Riechers, D. E.; Wax, L. M.; Liebl, R. A.; Bush, D. R.

    1994-01-01

    Plasma membrane vesicles were isolated from mature leaves of lambsquarters (Chenopodium album L.) to investigate whether this membrane is a barrier to glyphosate uptake and whether surfactants possess differential abilities to enhance glyphosate permeability. Amino acids representing several structural classes showed [delta]pH-dependent transport, indicating that the proteins necessary for active, proton-coupled amino acid transport were present and functional. Glyphosate uptake was very low compared to the acidic amino acid glutamate, indicating that glyphosate is not utilizing an endogenous amino acid carrier to enter the leaf cells and that the plasma membrane appears to be a significant barrier to cellular uptake. In addition, glyphosate flux was much lower than that measured for either bentazon or atrazine, both lipid-permeable herbicides that diffuse through the bilayer. Glyphosate uptake was stimulated by 0.01% (v:v) MON 0818, the cationic surfactant used in the commercial formulation of this herbicide for foliar application. This concentration of surfactant did not disrupt the integrity of the plasma membrane vesicles, as evidenced by the stability of imposed pH gradients and active amino acid transport. Nonionic surfactants that disrupt the cuticle but that do not promote glyphosate toxicity in the field also increased glyphosate transport into the membrane vesicles. Thus, no correlation was observed between whole plant toxicity and surfactant-aided uptake. Current data suggest that surfactant efficacy may be the result of charged surfactants' ability to diffuse away from the cuticle into the subtending apoplastic space, where they act directly on the plasma membrane to increase glyphosate uptake. PMID:12232297

  18. Cellular iron metabolism.

    PubMed

    Ponka, P

    1999-03-01

    Iron is essential for oxidation-reduction catalysis and bioenergetics, but unless appropriately shielded, iron plays a key role in the formation of toxic oxygen radicals that can attack all biological molecules. Hence, specialized molecules for the acquisition, transport (transferrin), and storage (ferritin) of iron in a soluble nontoxic form have evolved. Delivery of iron to most cells, probably including those of the kidney, occurs following the binding of transferrin to transferrin receptors on the cell membrane. The transferrin-receptor complexes are then internalized by endocytosis, and iron is released from transferrin by a process involving endosomal acidification. Cellular iron storage and uptake are coordinately regulated post-transcriptionally by cytoplasmic factors, iron-regulatory proteins 1 and 2 (IRP-1 and IRP-2). Under conditions of limited iron supply, IRP binding to iron-responsive elements (present in 5' untranslated region of ferritin mRNA and 3' untranslated region of transferrin receptor mRNA) blocks ferritin mRNA translation and stabilizes transferrin receptor mRNA. The opposite scenario develops when iron in the transit pool is plentiful. Moreover, IRP activities/levels can be affected by various forms of "oxidative stress" and nitric oxide. The kidney also requires iron for metabolic processes, and it is likely that iron deficiency or excess can cause disturbed function of kidney cells. Transferrin receptors are not evenly distributed throughout the kidney, and there is a cortical-to-medullary gradient in heme biosynthesis, with greatest activity in the cortex and least in the medulla. This suggests that there are unique iron/heme metabolism features in some kidney cells, but the specific aspects of iron and heme metabolism in the kidney are yet to be explained.

  19. Identification of novel vascular projections with cellular trafficking abilities on the microvasculature of pancreatic ductal adenocarcinoma

    PubMed Central

    Wu, Yanhua; Wei, Youheng; Fang, Yuan; Han, Xu; Li, Jianang; Zhou, Ping; Yi, Qing; Maitra, Anirban; Liu, Jun O; Tuveson, David A.; Lou, Wenhui; Yu, Long

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a nearly lethal neoplasm. It is a remarkably stroma-rich, vascular-poor, hypoperfused tumor, which prevents efficient drug delivery. Paradoxically, the neoplastic cells have robust glucose uptake, suggesting the microvasculature has adopted an alternative method for nutrient uptake and cellular trafficking. Using an adapted thick tumor section immunostaining and 3-dimensional (3D) construction imaging method in human tissue samples, we identified an undiscovered feature of the mature and angiostatic microvasculature in advanced PDAC tumors; long, “hairy”-like projections on the basal surface of microvessels that we refer to as “basal microvilli”. Functionally, these basal microvilli have an actin-rich cytoskeleton, endocytic and exocytic properties, and they contain glucose transporter-1 (GLUT-1) positive vesicles. Clinically, as was demonstrated by PET-CT, the tumor microvasculature with the longest and the most abundant basal microvilli positively correlated with the high glucose uptake of the PDAC tumor itself. In addition, these basal microvilli were found in regions of the tumor with low GLUT-1 expression, suggesting their presence could be dependent upon the glucose concentrations in the tumor milieu. Altogether, these basal microvilli mark a novel, pathological feature of the PDAC tumor microvasculature. Because these basal microvilli are pathological features with endo- and exocytic properties, they may provide a non-conventional method for cellular trafficking in PDAC tumors. PMID:25561062

  20. Charge, size, and cellular selectivity for multiwall carbon nanotubes by maize and soybean.

    PubMed

    Zhai, Guangshu; Gutowski, Sarah M; Walters, Katherine S; Yan, Bing; Schnoor, Jerald L

    2015-06-16

    Maize (Zea mays) and soybean (Glycine max) were used as model food-chain plants to explore vegetative uptake of differently charged multiwall carbon nanotubes (MWCNTs). Three types of MWCNTs, including neutral pristine MWCNT (p-MWCNT), positively charged MWCNT-NH2, and negatively charged MWCNT-COOH, were directly taken-up and translocated from hydroponic solution to roots, stems, and leaves of maize and soybean plants at the MWCNT concentrations ranging from 10.0 to 50.0 mg/L during 18-day exposures. MWCNTs accumulated in the xylem and phloem cells and within specific intracellular sites like the cytoplasm, cell wall, cell membrane, chloroplast, and mitochondria, which was observed by transmission electron microscopy. MWCNTs stimulated the growth of maize and inhibited the growth of soybean at the exposed doses. The cumulative transpiration of water in maize exposed to 50 mg/L of MWCNT-COOHs was almost twice as much as that in the maize control. Dry biomass of maize exposed to MWCNTs was greater than that of maize control. In addition, the uptake and translocation of these MWCNTs clearly exhibited cellular, charge, and size selectivity in maize and soybean, which could be important properties for nanotransporters. This is the first report of cellular, charge, and size selectivity on the uptake by whole food plants for three differently charged MWCNTs. PMID:26010305

  1. Microwave gallium-68 radiochemistry for kinetically stable bis(thiosemicarbazone) complexes: structural investigations and cellular uptake under hypoxia† †Electronic supplementary information (ESI) available. CCDC 1001632–1001634. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5dt02537k Click here for additional data file. Click here for additional data file.

    PubMed Central

    Alam, Israt S.; Arrowsmith, Rory L.; Cortezon-Tamarit, Fernando; Twyman, Frazer; Kociok-Köhn, Gabriele; Botchway, Stanley W.; Dilworth, Jonathan R.

    2016-01-01

    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. 68Ga 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. PMID:26583314

  2. 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

  3. Insight into the Modification of Polymeric Micellar and Liposomal Nanocarriers by Fluorescein-Labeled Lipids and Uptake-Mediating Lipopeptides.

    PubMed

    Draffehn, Sören; Eichhorst, Jenny; Wiesner, Burkhard; Kumke, Michael U

    2016-07-12

    Encapsulation of diagnostic and therapeutic compounds in transporters improves their delivery to the point of need. An even more efficient treatment of diseases can be achieved using carriers with targeting or protecting moieties. In the present work, we investigated micellar and liposomal nanocarriers modified with fluorescein, peptides, and polymers that are covalently bound to fatty acids or phospholipids to ensure a self-driven incorporation into the micelles or liposomes. First, we characterized the photophysics of the fluorescent probes in the absence and in the presence of nanocarriers. Changes in the fluorescence decay time, quantum yield, and intensity of a fluorescein-labeled fatty acid (fluorescein-labeled palmitic acid [fPA]) and a fluorescein-labeled lipopeptide (P2fA2) were found. By exploiting these changes, we investigated a lipopeptide (P2A2 as an uptake-mediating unit) in combination with different nanocarriers (micelles and liposomes) and determined the corresponding association constant Kass values, which were found to be very high. In addition, the mobility of fPA was exploited using fluorescence correlation spectroscopy (FCS) and fluorescence depolarization (FD) experiments to characterize the nanocarriers. Cellular uptake experiments with mouse brain endothelial cells provided information on the uptake behavior of liposomes modified by uptake-mediating P2A2 and revealed differences in the uptake behavior between pH-sensitive and pH-insensitive liposomes.

  4. Study on 99mTc-MAG3 and 99mTc-DMSA renal accumulation using in vitro cellular model.

    PubMed

    Nový, Zbynĕk; Mandíková, Jana; Trejtnar, Frantisek

    2011-02-01

    Mercaptoacetyltriglycine (MAG3) and dimercaptosuccinic acid (DMSA) labelled with technetium-99m belongs to standard renal radiodiagnostics. However, the renal transport mechanisms responsible for their high renal uptake have not been fully explained. In addition, no in vitro experimental study comparing the renal uptake of these radiopharmaceuticals at the cellular level has not been performed. The investigation compared the 99mTc-MAG3 and 99mTc-DMSA renal uptake using primary rat renal cells and evaluated contribution of active and passive transport processes to the renal accumulation. The renal cells were isolated from the rat kidneys by means of the two-phase collagenase perfusion method. The used experimental model showed to be useful tool for such type of investigation. The results documented significant quantitative and qualitative differences in the accumulation of 99mTc-DMSA and 99mTc-MAG3 in the rat isolated cells. The found experimental data indicated several times higher uptake of 99mTc-MAG3 than that found in 99mTc-DMSA. 99mTc-MAG3 cellular uptake was substantially decreased when active, energy-dependent processes were inhibited. However, 99mTc-DMSA accumulation in the renal cells demonstrated only a minor dependency on energy. These findings demonstrate a very different character of the membrane transport determining 99mTc-DMSA and 99mTc-MAG3 renal accumulation.

  5. Potassium Uptake Modulates Staphylococcus aureus Metabolism

    PubMed Central

    Gries, Casey M.; Sadykov, Marat R.; Bulock, Logan L.; Chaudhari, Sujata S.; Thomas, Vinai C.; Bose, Jeffrey L.

    2016-01-01

    ABSTRACT As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K+) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K+ uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K+ deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K+ uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization. PMID:27340697

  6. Potassium Uptake Modulates Staphylococcus aureus Metabolism.

    PubMed

    Gries, Casey M; Sadykov, Marat R; Bulock, Logan L; Chaudhari, Sujata S; Thomas, Vinai C; Bose, Jeffrey L; Bayles, Kenneth W

    2016-01-01

    As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K(+)) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K(+) uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K(+) deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K(+) uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K(+) uptake in S. aureus revealed that the Ktr-mediated K(+) transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K(+) uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K(+) uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K(+) uptake in establishing efficient carbon utilization. PMID:27340697

  7. Cellular Phone Towers

    MedlinePlus

    ... the call. How are people exposed to the energy from cellular phone towers? As people use cell ... where people can be exposed to them. The energy from a cellular phone tower antenna, like that ...

  8. Quercetin and epigallocatechin gallate inhibit glucose uptake and metabolism by breast cancer cells by an estrogen receptor-independent mechanism

    SciTech Connect

    Moreira, Liliana; Araújo, Isabel; Costa, Tito; Correia-Branco, Ana; Faria, Ana; Martel, Fátima; Keating, Elisa

    2013-07-15

    In this study we characterized {sup 3}H-2-deoxy-D-glucose ({sup 3}H -DG) uptake by the estrogen receptor (ER)-positive MCF7 and the ER-negative MDA-MB-231 human breast cancer cell lines and investigated the effect of quercetin (QUE) and epigallocatechin gallate (EGCG) upon {sup 3}H-DG uptake, glucose metabolism and cell viability and proliferation. In both MCF7 and MDA-MB-231 cells {sup 3}H-DG uptake was (a) time-dependent, (b) saturable with similar capacity (V{sub max}) and affinity (K{sub m}), (c) potently inhibited by cytochalasin B, an inhibitor of the facilitative glucose transporters (GLUT), (d) sodium-independent and (e) slightly insulin-stimulated. This suggests that {sup 3}H-DG uptake by both cell types is mediated by members of the GLUT family, including the insulin-responsive GLUT4 or GLUT12, while being independent of the sodium-dependent glucose transporter (SGLT1). QUE and EGCG markedly and concentration-dependently inhibited {sup 3}H-DG uptake by MCF7 and by MDA-MB-231 cells, and both compounds blocked lactate production by MCF7 cells. Additionally, a 4 h-treatment with QUE or EGCG decreased MCF7 cell viability and proliferation, an effect that was more potent when glucose was available in the extracellular medium. Our results implicate QUE and EGCG as metabolic antagonists in breast cancer cells, independently of estrogen signalling, and suggest that these flavonoids could serve as therapeutic agents/adjuvants even for ER-negative breast tumors. -- Highlights: • Glucose uptake by MCF7 and MDA-MB-231 cells is mainly mediated by GLUT1. • QUE and EGCG inhibit cellular glucose uptake thus abolishing the Warburg effect. • This process induces cytotoxicity and proliferation arrest in MCF7 cells. • The flavonoids’ effects are independent of estrogen receptor signalling.

  9. A Feedback Loop between Inflammation and Zn Uptake

    PubMed Central

    Bonaventura, Paola; Lamboux, Aline; Albarède, Francis; Miossec, Pierre

    2016-01-01

    Objective Zinc (Zn) has major effects on the immune system and inflammation is associated with systemic Zn deficiency. The aim of this work was to investigate how inflammation modifies Zn metabolism at the cellular level. Rheumatoid arthritis (RA) synoviocytes exposed to cytokines were used as a model of chronic inflammation. Osteoarthritis (OA) synoviocytes were used as control. Methods Zn levels were measured in medium and inside cells by Induced Coupled Plasma-Mass Spectrometry (ICP-MS), in the presence of minute quantities of stable spike 70Zn isotope and the addition or not of the pro-inflammatory cytokines interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-α). Gene expression of ZIP-8 importer, ZnT1 exporter and the homeostasis regulators metallothioneins (MTs) was evaluated after pre-exposure to cytokines, with or without exogenous Zn addition at increasing concentrations. IL-6 production was used as a marker of inflammation and measured by ELISA. Results Exposure to IL-17 and TNF-α enhanced expression of the Zn-importer ZIP-8, regardless of the concentration of Zn in the culture medium. In contrast, the expression of the Zn-exporter ZnT1 and of the MTs was primarily dependent on Zn levels. Addition of Zn also increased the production of IL-6, thus further stimulating the inflammatory response. Conclusion IL-17/TNF-mediated inflammation enhanced the intracellular Zn uptake by synoviocytes, further increasing inflammation. These observations document the existence of a feedback loop between inflammation and Zn uptake. Based on these results, a mathematical model was developed to represent the cytokine-mediated Zn homeostasis alterations. PMID:26845700

  10. Synthetic biology in cellular immunotherapy

    PubMed Central

    Chakravarti, Deboki; Wong, Wilson W.

    2015-01-01

    The adoptive transfer of genetically engineered T cells with cancer-targeting receptors has shown tremendous promise for eradicating tumors in clinical trials. This form of cellular immunotherapy presents a unique opportunity to incorporate advanced systems and synthetic biology approaches to create cancer therapeutics with novel functions. Here, we first review the development of synthetic receptors, switches, and circuits to control the location, duration, and strength of T cell activity against tumors. In addition, we discuss the cellular engineering and genome editing of host cells (or the chassis) to improve the efficacy of cell-based cancer therapeutics, and to reduce the time and cost of manufacturing. PMID:26088008

  11. Cellular senescence in aging primates.

    PubMed

    Herbig, Utz; Ferreira, Mark; Condel, Laura; Carey, Dee; Sedivy, John M

    2006-03-01

    The aging of organisms is characterized by a gradual functional decline of all organ systems. Mammalian somatic cells in culture display a limited proliferative life span, at the end of which they undergo an irreversible cell cycle arrest known as replicative senescence. Whether cellular senescence contributes to organismal aging has been controversial. We investigated telomere dysfunction, a recently discovered biomarker of cellular senescence, and found that the number of senescent fibroblasts increases exponentially in the skin of aging baboons, reaching >15% of all cells in very old individuals. In addition, the same cells contain activated ataxia-telangiectasia mutated kinase and heterochromatinized nuclei, confirming their senescent status. PMID:16456035

  12. Hierarchical cellular materials

    SciTech Connect

    Gibson, L.J.

    1991-12-31

    In this paper a method for estimating the contributions of both the composite and the cellular microstructures to the overall material properties and the mechanical efficiency of natural cellular solids will be described. The method will be demonstrated by focusing on the Young`s modulus; similar techniques can be used for other material properties. The results suggest efficient microstructures for engineered cellular materials.

  13. Hierarchical cellular materials

    SciTech Connect

    Gibson, L.J.

    1991-01-01

    In this paper a method for estimating the contributions of both the composite and the cellular microstructures to the overall material properties and the mechanical efficiency of natural cellular solids will be described. The method will be demonstrated by focusing on the Young's modulus; similar techniques can be used for other material properties. The results suggest efficient microstructures for engineered cellular materials.

  14. Myocardial Gene Transfer: Routes and Devices for Regulation of Transgene Expression by Modulation of Cellular Permeability

    PubMed Central

    Katz, Michael G.; Bridges, Charles R.

    2013-01-01

    Abstract Heart diseases are major causes of morbidity and mortality in Western society. Gene therapy approaches are becoming promising therapeutic modalities to improve underlying molecular processes affecting failing cardiomyocytes. Numerous cardiac clinical gene therapy trials have yet to demonstrate strong positive results and advantages over current pharmacotherapy. The success of gene therapy depends largely on the creation of a reliable and efficient delivery method. The establishment of such a system is determined by its ability to overcome the existing biological barriers, including cellular uptake and intracellular trafficking as well as modulation of cellular permeability. In this article, we describe a variety of physical and mechanical methods, based on the transient disruption of the cell membrane, which are applied in nonviral gene transfer. In addition, we focus on the use of different physiological techniques and devices and pharmacological agents to enhance endothelial permeability. Development of these methods will undoubtedly help solve major problems facing gene therapy. PMID:23427834

  15. General aspects of muscle glucose uptake.

    PubMed

    Alvim, Rafael O; Cheuhen, Marcel R; Machado, Silmara R; Sousa, André Gustavo P; Santos, Paulo C J L

    2015-03-01

    Glucose uptake in peripheral tissues is dependent on the translocation of GLUT4 glucose transporters to the plasma membrane. Studies have shown the existence of two major signaling pathways that lead to the translocation of GLUT4. The first, and widely investigated, is the insulin activated signaling pathway through insulin receptor substrate-1 and phosphatidylinositol 3-kinase. The second is the insulin-independent signaling pathway, which is activated by contractions. Individuals with type 2 diabetes mellitus have reduced insulin-stimulated glucose uptake in skeletal muscle due to the phenomenon of insulin resistance. However, those individuals have normal glucose uptake during exercise. In this context, physical exercise is one of the most important interventions that stimulates glucose uptake by insulin-independent pathways, and the main molecules involved are adenosine monophosphate-activated protein kinase, nitric oxide, bradykinin, AKT, reactive oxygen species and calcium. In this review, our main aims were to highlight the different glucose uptake pathways and to report the effects of physical exercise, diet and drugs on their functioning. Lastly, with the better understanding of these pathways, it would be possible to assess, exactly and molecularly, the importance of physical exercise and diet on glucose homeostasis. Furthermore, it would be possible to assess the action of drugs that might optimize glucose uptake and consequently be an important step in controlling the blood glucose levels in diabetic patients, in addition to being important to clarify some pathways that justify the development of drugs capable of mimicking the contraction pathway.

  16. Cellular-based preemption system

    NASA Technical Reports Server (NTRS)

    Bachelder, Aaron D. (Inventor)

    2011-01-01

    A cellular-based preemption system that uses existing cellular infrastructure to transmit preemption related data to allow safe passage of emergency vehicles through one or more intersections. A cellular unit in an emergency vehicle is used to generate position reports that are transmitted to the one or more intersections during an emergency response. Based on this position data, the one or more intersections calculate an estimated time of arrival (ETA) of the emergency vehicle, and transmit preemption commands to traffic signals at the intersections based on the calculated ETA. Additional techniques may be used for refining the position reports, ETA calculations, and the like. Such techniques include, without limitation, statistical preemption, map-matching, dead-reckoning, augmented navigation, and/or preemption optimization techniques, all of which are described in further detail in the above-referenced patent applications.

  17. 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...

  18. Interplay of drug metabolizing enzymes with cellular transporters.

    PubMed

    Böhmdorfer, Michaela; Maier-Salamon, Alexandra; Riha, Juliane; Brenner, Stefan; Höferl, Martina; Jäger, Walter

    2014-11-01

    Many endogenous and xenobiotic substances and their metabolites are substrates for drug metabolizing enzymes and cellular transporters. These proteins may not only contribute to bioavailability of molecules but also to uptake into organs and, consequently, to overall elimination. The coordinated action of uptake transporters, metabolizing enzymes, and efflux pumps, therefore, is a precondition for detoxification and elimination of drugs. As the understanding of the underlying mechanisms is important to predict alterations in drug disposal, adverse drug reactions and, finally, drug-drug interactions, this review illustrates the interplay between selected uptake/efflux transporters and phase I/II metabolizing enzymes.

  19. Cell Type-Dependent Changes in CdSe/ZnS Quantum Dot Uptake and Toxic Endpoints

    PubMed Central

    Soenen, Stefaan J.; Al-Ali, Abdullah; Brown, Andy; Hondow, Nicole; Wills, John; Jenkins, Gareth J. S.; Doak, Shareen H.

    2015-01-01

    Toxicity of nanoparticles (NPs) is often correlated with the physicochemical characteristics of the materials. However, some discrepancies are noted in in-vitro studies on quantum dots (QDs) with similar physicochemical properties. This is partly related to variations in cell type. In this study, we show that epithelial (BEAS-2B), fibroblast (HFF-1), and lymphoblastoid (TK6) cells show different biological responses following exposure to QDs. These cells represented the 3 main portals of NP exposure: bronchial, skin, and circulatory. The uptake and toxicity of negatively and positively charged CdSe:ZnS QDs of the same core size but with different surface chemistries (carboxyl or amine polymer coatings) were investigated in full and reduced serum containing media following 1 and 3 cell cycles. Following thorough physicochemical characterization, cellular uptake, cytotoxicity, and gross chromosomal damage were measured. Cellular damage mechanisms in the form of reactive oxygen species and the expression of inflammatory cytokines IL-8 and TNF-α were assessed. QDs uptake and toxicity significantly varied in the different cell lines. BEAS-2B cells demonstrated the highest level of QDs uptake yet displayed a strong resilience with minimal genotoxicity following exposure to these NPs. In contrast, HFF-1 and TK6 cells were more susceptible to toxicity and genotoxicity, respectively, as a result of exposure to QDs. Thus, this study demonstrates that in addition to nanomaterial physicochemical characterization, a clear understanding of cell type-dependent variation in uptake coupled to the inherently different capacities of the cell types to cope with exposure to these exogenous materials are all required to predict genotoxicity. PMID:25601991

  20. A Comparative Study of Iron Uptake Rates and Mechanisms amongst Marine and Fresh Water Cyanobacteria: Prevalence of Reductive Iron Uptake

    PubMed Central

    Lis, Hagar; Kranzler, Chana; Keren, Nir; Shaked, Yeala

    2015-01-01

    In this contribution, we address the question of iron bioavailability to cyanobacteria by measuring Fe uptake rates and probing for a reductive uptake pathway in diverse cyanobacterial species. We examined three Fe-substrates: dissolved inorganic iron (Fe') and the Fe-siderophores Ferrioxamine B (FOB) and FeAerobactin (FeAB). In order to compare across substrates and strains, we extracted uptake rate constants (kin = uptake rate/[Fe-substrate]). Fe' was the most bioavailable Fe form to cyanobacteria, with kin values higher than those of other substrates. When accounting for surface area (SA), all strains acquired Fe' at similar rates, as their kin/SA were similar. We also observed homogeneity in the uptake of FOB among strains, but with 10,000 times lower kin/SA values than Fe'. Uniformity in kin/SA suggests similarity in the mechanism of uptake and indeed, all strains were found to employ a reductive step in the uptake of Fe' and FOB. In contrast, different uptake pathways were found for FeAB along with variations in kin/SA. Our data supports the existence of a common reductive Fe uptake pathway amongst cyanobacteria, functioning alone or in addition to siderophore-mediated uptake. Cyanobacteria combining both uptake strategies benefit from increased flexibility in accessing different Fe-substrates. PMID:25768677

  1. Characterization of the Uptake of Quantum Dots by Algae

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Priyanka; Lin, Sijie; Sun, Xiaoqian; Brune, David; Ke, Pu-Chun

    2009-03-01

    The exposure of living systems to nanoparticles is inevitable due to a dramatic increase in their release into the environment, the most likely pathways being through inhalation, ingestion and skin uptake. The extremely small size of the nanoparticles may facilitate their tissue and cellular uptake by plants and animals, resulting in either positive (drug delivery, antioxidation) or negative (toxicity, cellular dysfunction) effects. Here we report the effects of quantum dots uptake by algae, the single-celled plant species and major food sources for aquatic organisms. In our studies, the presence of quantum dots in algal cells was detected using fluorescence microscopy and electron microscopy. Using spectrophotometry we found a supralinear increase of the uptake with the concentration of quantum dots, with a saturation of the uptake occurring beyond a concentration of 15 mg/mL. Using a bicarbonate indicator we further evaluated the effects of quantum dots uptake on algal photosynthesis and respiration. Such study facilitates our understanding of the environmental impact of nanomaterials.

  2. Glycosylation of Sodium/Iodide Symporter (NIS) Regulates Its Membrane Translocation and Radioiodine Uptake

    PubMed Central

    Chung, Taemoon; Youn, Hyewon; Yeom, Chan Joo; Kang, Keon Wook; Chung, June-Key

    2015-01-01

    Purpose Human sodium/iodide symporter (hNIS) protein is a membrane glycoprotein that transports iodide ions into thyroid cells. The function of this membrane protein is closely regulated by post-translational glycosylation. In this study, we measured glycosylation-mediated changes in subcellular location of hNIS and its function of iodine uptake. Methods HeLa cells were stably transfected with hNIS/tdTomato fusion gene in order to monitor the expression of hNIS. Cellular localization of hNIS was visualized by confocal microscopy of the red fluorescence of tdTomato. The expression of hNIS was evaluated by RT-PCR and immunoblot analysis. Functional activity of hNIS was estimated by radioiodine uptake. Cyclic AMP (cAMP) and tunicamycin were used to stimulate and inhibit glycosylation, respectively. In vivo images were obtained using a Maestro fluorescence imaging system. Results cAMP-mediated Glycosylation of NIS resulted in increased expression of hNIS, stimulating membrane translocation, and enhanced radioiodine uptake. In contrast, inhibition of glycosylation by treatment with tunicamycin dramatically reduced membrane translocation of intracellular hNIS, resulting in reduced radioiodine uptake. In addition, our hNIS/tdTomato fusion reporter successfully visualized cAMP-induced hNIS expression in xenografted tumors from mouse model. Conclusions These findings clearly reveal that the membrane localization of hNIS and its function of iodine uptake are glycosylation-dependent, as our results highlight enhancement of NIS expression and glycosylation with subsequent membrane localization after cAMP treatment. Therefore, enhancing functional NIS by the increasing level of glycosylation may be suggested as a promising therapeutic strategy for cancer patients who show refractory response to conventional radioiodine treatment. PMID:26599396

  3. 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

    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

  4. Cellular interactions of lauric acid and dextran-coated magnetite nanoparticles

    NASA Astrophysics Data System (ADS)

    Pradhan, Pallab; Giri, Jyotsnendu; Banerjee, Rinti; Bellare, Jayesh; Bahadur, Dhirendra

    2007-04-01

    In vitro cytocompatibility and cellular interactions of lauric acid and dextran-coated magnetite nanoparticles were evaluated with two different cell lines (mouse fibroblast and human cervical carcinoma). Lauric acid-coated magnetite nanoparticles were less cytocompatible than dextran-coated magnetite nanoparticles and cellular uptake of lauric acid-coated magnetic nanoparticles was more than that of dextran-coated magnetite nanoparticles. Lesser cytocompatibility and higher uptake of lauric acid-coated magnetite nanoparticles as compared to dextran-coated magnetic nanoparticles may be due to different cellular interactions by coating material. Thus, coating plays an important role in modulation of biocompatibility and cellular interaction of magnetic nanoparticles.

  5. Food additives

    PubMed Central

    Spencer, Michael

    1974-01-01

    Food additives are discussed from the food technology point of view. The reasons for their use are summarized: (1) to protect food from chemical and microbiological attack; (2) to even out seasonal supplies; (3) to improve their eating quality; (4) to improve their nutritional value. The various types of food additives are considered, e.g. colours, flavours, emulsifiers, bread and flour additives, preservatives, and nutritional additives. The paper concludes with consideration of those circumstances in which the use of additives is (a) justified and (b) unjustified. PMID:4467857

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

    SciTech Connect

    Hossain, Ekhtear; Ota, Akinobu; Karnan, Sivasundaram; Damdindorj, Lkhagvasuren; Takahashi, Miyuki; Konishi, Yuko; Konishi, Hiroyuki; Hosokawa, Yoshitaka

    2013-12-15

    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, an 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.

  7. Uptake coefficients for biosolids-amended dryland winter wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biosolids regulations developed in the United States employed risk assessment impacts of trace element additions on plant uptake. The US Environmental Protection Agency adapted the uptake coefficient (ratio of plant concentration to quantity of element added) when developing limitations on selected...

  8. Overview of cellular CDMA

    NASA Astrophysics Data System (ADS)

    Lee, William C. Y.

    1991-05-01

    A general description of code division multiple access (CDMA) is presented. This overview of CDMA highlights the potential of increasing capacity in future cellular communications. The author describes the mobile radio environment and its impact on narrowband and wideband propagation. The advantage of having CDMA in cellular systems is discussed, and the concept of radio capacity in cellular is introduced. The power control schemes in CDMA are analyzed in detail.

  9. Nitrogen Uptake in Spinach

    NASA Astrophysics Data System (ADS)

    Ramirez, J.; VanBenthem, P.

    2013-12-01

    A plant's absorption of nitrogen can be encouraged by a variety of environmental factors, especially the application of fertilizers. As a common limiting factor in plant growth, not up taking enough nitrogen can be a result of an unhealthy plant. Moreover, as farmers seek out methods to increase growth of plants, fertilizers are used as a solution to the issue of nitrogen deficiency to incorporate additional nitrogen from chemical or organic sources, by not using the right fertilizer can greatly affect the plats. The point of this research project is to determine the effect of various fertilizers on the plant growth, and to correlate the measured nitrogen, water and chlorophyll content in spinach leaves. Spinach leaves were used because they are known to quickly uptake chemicals in the environment. The spinach plants were exposed to four different growing parameters, which are referred to as control, ammonium nitrate, MiracleGro , and organic. The spinach was originally placed in nitrogen deficient soil with only 2.2x10 4 weight percent (wt. %) nitrogen. The leaves in the control group were grown in this nitrogen deficient soil without any fertilizer added. Ammomium nitrate and MiracleGro were added to the spinach in the A and MG groups, respectively, and organic chicken stool was used for the O group. By using a spectral imaging system and flame combustion techniques, the chlorophyll content can be related to the nitrogen content in the spinach leaves. In these spinach leaves, nitrogen and chlorophyll content were measured, chlorophyll is a green pigment that plays a crucial role in producing nutrients for green plants. The lack of chlorophyll will allow the plant to become susceptible to diseases, so it is extremely important that the plants have a high content of chlorophyll. The role of nitrogen in chlorophyll is very important and helps in the creation of chlorophyll; therefore it is necessary that an appropriate amount of nitrogen is added for optimal growth

  10. Cadmium uptake and resistance among selected bacteria

    SciTech Connect

    Burke, B.E.

    1987-01-01

    The purpose of this research was to determine the relationship between Cd resistance and Cd uptake by lake sediment bacteria. For the Gram positive and gram negative sediment bacteria that were tested, the relationship between resistance and Cd uptake varied and was dependent on the isolate under consideration. Results of this study indicated that bacterial communities in lake sediments may influence the concentration and availability of Cd in sediments and the water column. In addition, results of this study did not support the theory that the genes encoding for Cd resistance are usually carried on antibiotic resistance plasmids.

  11. Ocean uptake of carbon dioxide

    SciTech Connect

    Peng, Tsung-Hung; Takahashi, Taro

    1993-06-01

    Factors controlling the capacity of the ocean for taking up anthropogenic C0{sup 2} include carbon chemistry, distribution of alkalinity, pCO{sup 2} and total concentration of dissolved C0{sup 2}, sea-air pCO{sup 2} difference, gas exchange rate across the sea-air interface, biological carbon pump, ocean water circulation and mixing, and dissolution of carbonate in deep sea sediments. A general review of these processes is given and models of ocean-atmosphere system based on our understanding of these regulating processes axe used to estimate the magnitude of C0{sup 2} uptake by the ocean. We conclude that the ocean can absorb up to 35% of the fossil fuel emission. Direct measurements show that 55% Of C0{sup 2} from fossil fuel burning remains in the atmosphere. The remaining 10% is not accounted for by atmospheric increases and ocean uptake. In addition, it is estimated that an amount equivalent to 30% of recent annual fossil fuel emissions is released into the atmosphere as a result of deforestation and farming. To balance global carbon budget, a sizable carbon sink besides the ocean is needed. Storage of carbon in terrestrial biosphere as a result of C0{sup 2} fertilization is a potential candidate for such missing carbon sinks.

  12. Ocean uptake of carbon dioxide

    SciTech Connect

    Peng, Tsung-Hung ); Takahashi, Taro . Lamont-Doherty Earth Observatory)

    1993-01-01

    Factors controlling the capacity of the ocean for taking up anthropogenic C0[sup 2] include carbon chemistry, distribution of alkalinity, pCO[sup 2] and total concentration of dissolved C0[sup 2], sea-air pCO[sup 2] difference, gas exchange rate across the sea-air interface, biological carbon pump, ocean water circulation and mixing, and dissolution of carbonate in deep sea sediments. A general review of these processes is given and models of ocean-atmosphere system based on our understanding of these regulating processes axe used to estimate the magnitude of C0[sup 2] uptake by the ocean. We conclude that the ocean can absorb up to 35% of the fossil fuel emission. Direct measurements show that 55% Of C0[sup 2] from fossil fuel burning remains in the atmosphere. The remaining 10% is not accounted for by atmospheric increases and ocean uptake. In addition, it is estimated that an amount equivalent to 30% of recent annual fossil fuel emissions is released into the atmosphere as a result of deforestation and farming. To balance global carbon budget, a sizable carbon sink besides the ocean is needed. Storage of carbon in terrestrial biosphere as a result of C0[sup 2] fertilization is a potential candidate for such missing carbon sinks.

  13. Deuterated carbohydrate probes as ‘label-free’ substrates for probing nutrient uptake in mycobacteria by nuclear reaction analysis† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4cc09588j Click here for additional data file.

    PubMed Central

    Lowery, R.; Gibson, M. I.; Thompson, R. L.

    2015-01-01

    Understanding and probing small molecule uptake in cells is challenging, requiring sterically large chemical labels, or radioactive isotopes. Here, the uptake of deuterated sugars by Mycobacterium smegmatis, a non-pathogenic model of Mycobacterium tuberculosis, has been investigated using ion-beam (nuclear reaction) analysis demonstrating a new technique for label-free nutrient acquisition measurement. PMID:25695462

  14. 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.

  15. 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. PMID:10865941

  16. Mitochondrial Ca(2+) uptake in skeletal muscle health and disease.

    PubMed

    Zhou, Jingsong; Dhakal, Kamal; Yi, Jianxun

    2016-08-01

    Muscle uses Ca(2+) as a messenger to control contraction and relies on ATP to maintain the intracellular Ca(2+) homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca(2+) from their surroundings, a process called mitochondrial Ca(2+) uptake. Under physiological conditions, Ca(2+) uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca(2+) overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca(2+) uptake could shape spatio-temporal patterns of intracellular Ca(2+) signaling. Malfunction of mitochondrial Ca(2+) uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca(2+) levels. Besides the sudden elevation of Ca(2+) level induced by action potentials, Ca(2+) transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as minutes during tetanic contraction, which raises the question whether mitochondrial Ca(2+) uptake is fast and big enough to shape intracellular Ca(2+) signaling during excitation-contraction coupling and creates technical challenges for quantification of the dynamic changes of Ca(2+) inside mitochondria. This review focuses on characterization of mitochondrial Ca(2+) uptake in skeletal muscle and its role in muscle physiology and diseases. PMID:27430885

  17. Similar specificities of symbiont uptake by adults and larvae in an anemone model system for coral biology.

    PubMed

    Hambleton, Elizabeth A; Guse, Annika; Pringle, John R

    2014-05-01

    Reef-building corals depend for much of their energy on photosynthesis by symbiotic dinoflagellate algae (genus Symbiodinium) that live within their gastrodermal cells. However, the cellular mechanisms underpinning this ecologically critical symbiosis, including those governing the specificity of symbiont uptake by the host, remain poorly understood, in part because of the difficulties of working with corals in the laboratory. Here, we used the small symbiotic sea anemone Aiptasia as an experimentally tractable model system to analyze the specificity and timing of symbiosis onset in larval and adult animals under controlled laboratory conditions. Using four clonal, axenic Symbiodinium strains, we found no difference in uptake specificity between larvae (even when very young) and adults. Although both compatible and incompatible algal strains were found within the larval guts, only the former appeared to be internalized by gastrodermal cells, and they (but not incompatible algae) proliferated rapidly within the larvae in the absence of detectable exchange with other larvae. Older larvae showed reduced ingestion of both compatible and incompatible algae, and the addition of food failed to promote the uptake of an incompatible algal strain. Thus, Aiptasia adults and larvae appear to have similar mechanisms for discriminating between compatible and incompatible dinoflagellate types prior to phagocytosis by host gastrodermal cells. Whether a particular algal strain is compatible or incompatible appears to be stable during years of axenic culture in the absence of a host. These studies provide a foundation for future analyses of the mechanisms of symbiont-uptake specificity in this emerging model system. PMID:24526722

  18. Plant Water Uptake in Drying Soils1

    PubMed Central

    Lobet, Guillaume; Couvreur, Valentin; Meunier, Félicien; Javaux, Mathieu; Draye, Xavier

    2014-01-01

    Over the last decade, investigations on root water uptake have evolved toward a deeper integration of the soil and roots compartment properties, with the goal of improving our understanding of water acquisition from drying soils. This evolution parallels the increasing attention of agronomists to suboptimal crop production environments. Recent results have led to the description of root system architectures that might contribute to deep-water extraction or to water-saving strategies. In addition, the manipulation of root hydraulic properties would provide further opportun