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Sample records for protein delivery activity

  1. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

    SciTech Connect

    Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

    2008-08-01

    Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs.

  2. Transdermal delivery of proteins.

    PubMed

    Kalluri, Haripriya; Banga, Ajay K

    2011-03-01

    Transdermal delivery of peptides and proteins avoids the disadvantages associated with the invasive parenteral route of administration and other alternative routes such as the pulmonary and nasal routes. Since proteins have a large size and are hydrophilic in nature, they cannot permeate passively across the skin due to the stratum corneum which allows the transport of only small lipophilic drug molecules. Enhancement techniques such as chemical enhancers, iontophoresis, microneedles, electroporation, sonophoresis, thermal ablation, laser ablation, radiofrequency ablation and noninvasive jet injectors aid in the delivery of proteins by overcoming the skin barrier in different ways. In this review, these enhancement techniques that can enable the transdermal delivery of proteins are discussed, including a discussion of mechanisms, sterility requirements, and commercial development of products. Combination of enhancement techniques may result in a synergistic effect allowing increased protein delivery and these are also discussed.

  3. Near-Infrared Light Activation of Proteins Inside Living Cells Enabled by Carbon Nanotube-Mediated Intracellular Delivery.

    PubMed

    Li, He; Fan, Xinqi; Chen, Xing

    2016-02-01

    Light-responsive proteins have been delivered into the cells for controlling intracellular events with high spatial and temporal resolution. However, the choice of wavelength is limited to the UV and visible range; activation of proteins inside the cells using near-infrared (NIR) light, which has better tissue penetration and biocompatibility, remains elusive. Here, we report the development of a single-walled carbon nanotube (SWCNT)-based bifunctional system that enables protein intracellular delivery, followed by NIR activation of the delivered proteins inside the cells. Proteins of interest are conjugated onto SWCNTs via a streptavidin-desthiobiotin (SA-DTB) linkage, where the protein activity is blocked. SWCNTs serve as both a nanocarrier for carrying proteins into the cells and subsequently a NIR sensitizer to photothermally cleave the linkage and release the proteins. The released proteins become active and exert their functions inside the cells. We demonstrated this strategy by intracellular delivery and NIR-triggered nuclear translocation of enhanced green fluorescent protein, and by intracellular delivery and NIR-activation of a therapeutic protein, saporin, in living cells. Furthermore, we showed that proteins conjugated onto SWCNTs via the SA-DTB linkage could be delivered to the tumors, and optically released and activated by using NIR light in living mice.

  4. DELIVERY OF THERAPEUTIC PROTEINS

    PubMed Central

    Pisal, Dipak S.; Kosloski, Matthew P.; Balu-Iyer, Sathy V.

    2009-01-01

    The safety and efficacy of protein therapeutics are limited by three interrelated pharmaceutical issues, in vitro and in vivo instability, immunogenicity and shorter half-lives. Novel drug modifications for overcoming these issues are under investigation and include covalent attachment of poly(ethylene glycol) (PEG), polysialic acid, or glycolic acid, as well as developing new formulations containing nanoparticulate or colloidal systems (e.g. liposomes, polymeric microspheres, polymeric nanoparticles). Such strategies have the potential to develop as next generation protein therapeutics. This review includes a general discussion on these delivery approaches. PMID:20049941

  5. Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

    PubMed

    Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

    2017-01-01

    Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

  6. TARGETED DELIVERY OF INHALED PROTEINS

    EPA Science Inventory

    ETD-02-047 (Martonen) GPRA # 10108

    TARGETED DELIVERY OF INHALED PROTEINS
    T. B. Martonen1, J. Schroeter2, Z. Zhang3, D. Hwang4, and J. S. Fleming5
    1Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Research Triangle Park...

  7. Nanochemistry of Protein-Based Delivery Agents.

    PubMed

    Rajendran, Subin R C K; Udenigwe, Chibuike C; Yada, Rickey Y

    2016-01-01

    The past decade has seen an increased interest in the conversion of food proteins into functional biomaterials, including their use for loading and delivery of physiologically active compounds such as nutraceuticals and pharmaceuticals. Proteins possess a competitive advantage over other platforms for the development of nanodelivery systems since they are biocompatible, amphipathic, and widely available. Proteins also have unique molecular structures and diverse functional groups that can be selectively modified to alter encapsulation and release properties. A number of physical and chemical methods have been used for preparing protein nanoformulations, each based on different underlying protein chemistry. This review focuses on the chemistry of the reorganization and/or modification of proteins into functional nanostructures for delivery, from the perspective of their preparation, functionality, stability and physiological behavior.

  8. Nanochemistry of Protein-Based Delivery Agents

    PubMed Central

    Rajendran, Subin R. C. K.; Udenigwe, Chibuike C.; Yada, Rickey Y.

    2016-01-01

    The past decade has seen an increased interest in the conversion of food proteins into functional biomaterials, including their use for loading and delivery of physiologically active compounds such as nutraceuticals and pharmaceuticals. Proteins possess a competitive advantage over other platforms for the development of nanodelivery systems since they are biocompatible, amphipathic, and widely available. Proteins also have unique molecular structures and diverse functional groups that can be selectively modified to alter encapsulation and release properties. A number of physical and chemical methods have been used for preparing protein nanoformulations, each based on different underlying protein chemistry. This review focuses on the chemistry of the reorganization and/or modification of proteins into functional nanostructures for delivery, from the perspective of their preparation, functionality, stability and physiological behavior. PMID:27489854

  9. Nanochemistry of protein-based delivery agents

    NASA Astrophysics Data System (ADS)

    Rajendran, Subin; Udenigwe, Chibuike; Yada, Rickey

    2016-07-01

    The past decade has seen an increased interest in the conversion of food proteins into functional biomaterials, including their use for loading and delivery of physiologically active compounds such as nutraceuticals and pharmaceuticals. Proteins possess a competitive advantage over other platforms for the development of nanodelivery systems since they are biocompatible, amphipathic, and widely available. Proteins also have unique molecular structures and diverse functional groups that can be selectively modified to alter encapsulation and release properties. A number of physical and chemical methods have been used for preparing protein nanoformulations, each based on different underlying protein chemistry. This review focuses on the chemistry of the reorganization and/or modification of proteins into functional nanostructures for delivery, from the perspective of their preparation, functionality, stability and physiological behavior.

  10. Combined neurothrombectomy or thrombolysis with adjunctive delivery of 3K3A-activated protein C in acute ischemic stroke.

    PubMed

    Amar, Arun Paul; Griffin, John H; Zlokovic, Berislav V

    2015-01-01

    In the treatment of acute ischemic stroke (AIS), vessel recanalization correlates with improved functional status and reduced mortality. Mechanical neurothrombectomy achieves a higher likelihood of revascularization than intravenous thrombolysis (IVT), but there remains significant discrepancy between rates of recanalization and rates of favorable outcome. The poor neurological recovery among some stroke patients despite successful recanalization confirms the need for adjuvant therapy, such as pharmacological neuroprotection. Prior clinical trials of neuroprotectant drugs failed perhaps due to inability of the agent to reach the ischemic tissue beyond the occluded artery. A protocol that couples mechanical neurothrombectomy with concurrent delivery of a neuroprotectant overcomes this pitfall. Activated protein C (APC) exerts pleiotropic anti-inflammatory, anti-apoptotic, antithrombotic, cytoprotective, and neuroregenerative effects in stroke and appears a compelling candidate for this novel approach.

  11. Combined neurothrombectomy or thrombolysis with adjunctive delivery of 3K3A-activated protein C in acute ischemic stroke

    PubMed Central

    Amar, Arun Paul; Griffin, John H.; Zlokovic, Berislav V.

    2015-01-01

    In the treatment of acute ischemic stroke (AIS), vessel recanalization correlates with improved functional status and reduced mortality. Mechanical neurothrombectomy achieves a higher likelihood of revascularization than intravenous thrombolysis (IVT), but there remains significant discrepancy between rates of recanalization and rates of favorable outcome. The poor neurological recovery among some stroke patients despite successful recanalization confirms the need for adjuvant therapy, such as pharmacological neuroprotection. Prior clinical trials of neuroprotectant drugs failed perhaps due to inability of the agent to reach the ischemic tissue beyond the occluded artery. A protocol that couples mechanical neurothrombectomy with concurrent delivery of a neuroprotectant overcomes this pitfall. Activated protein C (APC) exerts pleiotropic anti-inflammatory, anti-apoptotic, antithrombotic, cytoprotective, and neuroregenerative effects in stroke and appears a compelling candidate for this novel approach. PMID:26388732

  12. Application of proteins in burst delivery systems

    NASA Astrophysics Data System (ADS)

    Freeman, E.; Weiland, L. M.; Meng, W. S.

    2010-09-01

    Biological proteins embedded in either a biological or an engineered membrane will actively maintain electrochemical balance across that membrane. In this study two applications will be examined. First a system of governing equations will be calibrated for a biological endosome. The endocytosis predictions presented then serve to validate the model. In addition, these predictions introduce new insights into endosome burst, which is of interest for advancing DNA vaccine delivery. The calibrated model is subsequently adapted to an analogous engineering scenario for targeted payload delivery. In the presence of a specific external stimulus, burst release of an arbitrary payload encased in a vesicle akin to an endosome is explored. Control of the process through manipulation of vesicle size, stimulus, and transporters is presented. A case is made for application of proteins as building blocks in the design of targeted response materials.

  13. Inhalation delivery of protein therapeutics.

    PubMed

    Kane, Colleen; O'Neil, Karyn; Conk, Michelle; Picha, Kristen

    2013-04-01

    Inhaled therapeutics are used routinely to treat a variety of pulmonary diseases including asthma, COPD and cystic fibrosis. In addition, biological therapies represent the fastest growing segment of approved pharmaceuticals. However, despite the increased availability of biological therapies, nearly all inhaled therapeutics are small molecule drugs with only a single inhaled protein therapeutic approved. There remains a significant unmet need for therapeutics in pulmonary diseases, and biological therapies with potential to alter disease progression represent a significant opportunity to treat these challenging diseases. This review provides a background into efforts to develop inhaled biological therapies and highlights some of the associated challenges. In addition, we speculate on the ideal properties of a biologic therapy for inhaled delivery.

  14. Agile delivery of protein therapeutics to CNS.

    PubMed

    Yi, Xiang; Manickam, Devika S; Brynskikh, Anna; Kabanov, Alexander V

    2014-09-28

    A variety of therapeutic proteins have shown potential to treat central nervous system (CNS) disorders. Challenge to deliver these protein molecules to the brain is well known. Proteins administered through parenteral routes are often excluded from the brain because of their poor bioavailability and the existence of the blood-brain barrier (BBB). Barriers also exist to proteins administered through non-parenteral routes that bypass the BBB. Several strategies have shown promise in delivering proteins to the brain. This review, first, describes the physiology and pathology of the BBB that underscore the rationale and needs of each strategy to be applied. Second, major classes of protein therapeutics along with some key factors that affect their delivery outcomes are presented. Third, different routes of protein administration (parenteral, central intracerebroventricular and intraparenchymal, intranasal and intrathecal) are discussed along with key barriers to CNS delivery associated with each route. Finally, current delivery strategies involving chemical modification of proteins and use of particle-based carriers are overviewed using examples from literature and our own work. Whereas most of these studies are in the early stage, some provide proof of mechanism of increased protein delivery to the brain in relevant models of CNS diseases, while in few cases proof of concept had been attained in clinical studies. This review will be useful to broad audience of students, academicians and industry professionals who consider critical issues of protein delivery to the brain and aim developing and studying effective brain delivery systems for protein therapeutics.

  15. Agile Delivery of Protein Therapeutics to CNS

    PubMed Central

    Yi, Xiang; Manickam, Devika S.; Brynskikh, Anna; Kabanov, Alexander V.

    2014-01-01

    A variety of therapeutic proteins have shown potential to treat central nervous system (CNS) disorders. Challenge to deliver these protein molecules to the brain is well known. Proteins administered through parenteral routes are often excluded from the brain because of their poor bioavailability and the existence of the blood-brain barrier (BBB). Barriers also exist to proteins administered through non-parenteral routes that bypass the BBB. Several strategies have shown promise in delivering proteins to the brain. This review, first, describes the physiology and pathology of the BBB that underscore the rationale and needs of each strategy to be applied. Second, major classes of protein therapeutics along with some key factors that affect their delivery outcomes are presented. Third, different routes of protein administration (parenteral, central intracerebroventricular and intraparenchymal, intranasal and intrathecal) are discussed along with key barriers to CNS delivery associated with each route. Finally, current delivery strategies involving chemical modification of proteins and use of particle-based carriers are overviewed using examples from literature and our own work. Whereas most of these studies are in the early stage, some provide proof of mechanism of increased protein delivery to the brain in relevant models of CNS diseases, while in few cases proof of concept had been attained in clinical studies. This review will be useful to broad audience of students, academicians and industry professionals who consider critical issues of protein delivery to the brain and aim developing and studying effective brain delivery systems for protein therapeutics. PMID:24956489

  16. Intracellular Protein Delivery for Treating Breast Cancer

    DTIC Science & Technology

    2014-08-01

    nanocapsules with specific cancer cell targeting ligands; Task 3. Preparing and testing of MMP activatable cell penetrating peptides (ACCPs)-coupled...AD_________________ Award Number: W81XWH-11-1-0371 TITLE: Intracellular Protein Delivery for Treating Breast Cancer PRINCIPAL INVESTIGATOR: Dr...SUBTITLE Intracellular Protein Delivery for Treating Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-11-1-0371 5c. PROGRAM ELEMENT NUMBER 6

  17. Long-Term Delivery of Protein Therapeutics

    PubMed Central

    Vaishya, Ravi; Khurana, Varun; Patel, Sulabh; Mitra, Ashim K

    2015-01-01

    Introduction Proteins are effective biotherapetics with applications in diverse ailments. Despite being specific and potent, their full clinical potential has not yet been realized. This can be attributed to short half-lives, complex structures, poor in vivo stability, low permeability frequent parenteral administrations and poor adherence to treatment in chronic diseases. A sustained release system, providing controlled release of proteins, may overcome many of these limitations. Areas covered This review focuses on recent development in approaches, especially polymer-based formulations, which can provide therapeutic levels of proteins over extended periods. Advances in particulate, gel based formulations and novel approaches for extended protein delivery are discussed. Emphasis is placed on dosage form, method of preparation, mechanism of release and stability of biotherapeutics. Expert opinion Substantial advancements have been made in the field of extended protein delivery via various polymer-based formulations over last decade despite the unique delivery-related challenges posed by protein biologics. A number of injectable sustained-release formulations have reached market. However, therapeutic application of proteins is still hampered by delivery related issues. A large number of protein molecules are under clinical trials and hence there is an urgent need to develop new methods to deliver these highly potent biologics. PMID:25251334

  18. Enzyme-Responsive Delivery of Multiple Proteins with Spatiotemporal Control

    PubMed Central

    Zhu, Suwei; Nih, Lina; Carmichael, S. Thomas; Lu, Yunfeng; Segura, Tatiana

    2015-01-01

    The growth of tissues and organs is regulated by orchestrated signals from biomolecules such as enzymes and growth factors. The ability to deliver signal molecules in response to particular biological events (e.g., enzyme expression and activation) holds great promise towards tissue healing and regeneration. The current delivery vehicles mainly rely on hydrolysable scaffolds and thin films of protein-containing polymers, which cannot be programmed to respond to biological signals. We report herein an injectable delivery platform based on enantiomeric protein nanocapsules, which can deliver multiple proteins with spatiotemporal control in response to the tissue proteases secreted during wound healing. Exemplified by stroke and diabetic wound healing in mice, sequential delivery of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) greatly enhances tissue revascularization and vessel maturation, providing effective delivery vehicles for tissue engineering and reparative medicine. PMID:25962336

  19. Liposomal dry powders as aerosols for pulmonary delivery of proteins.

    PubMed

    Lu, Dongmei; Hickey, Anthony J

    2005-12-21

    The purpose of this research was to develop liposomal dry powder aerosols for protein delivery. The delivery of stable protein formulations is essential for protein subunit vaccine delivery, which requires local delivery to macrophages in the lungs. Beta-glucuronidase (GUS) was used as a model protein to evaluate dry powder liposomes as inhaled delivery vehicles. Dimyristoyl phosphatylcholine:cholesterol (7:3) was selected as the liposome composition. The lyophilization of liposomes, micronization of the powders, aerosolization using a dry powder inhaler (DPI), and in vitro aerodynamic fine particle fraction upon collection in a twin-stage liquid impinger were evaluated. After lyophilization and jet-milling, the total amount of GUS and its activity, representing encapsulation efficiency and stability, were evaluated. The GUS amount and activity were measured and compared with freshly-prepared liposomes in the presence of mannitol, 43% of initial GUS amount, 29% of GUS activity after lyophilization and 36% of GUS amount, 22% of activity after micronization were obtained. Emitted doses from dry powder inhaler were 53%, 58%, 66%, and 73% for liposome powder:mannitol carrier ratios of 1:0, 1:4, 1:9, and 1:19. Fifteen percent of the liposome particles were less than 6.4 mum in aerodynamic diameter. The results demonstrate that milled liposome powders containing protein molecules can be aerosolized effectively at a fixed flow rate. Influences of different cryoprotectants on lyophilization of protein liposome formulations are reported. The feasibility of using liposomal dry powder aerosols for protein delivery has been demonstrated but further optimization is required in the context of specific therapeutic proteins.

  20. Rapid activity-dependent delivery of the neurotrophic protein CPG15 to the axon surface of neurons in intact Xenopus tadpoles.

    PubMed

    Cantallops, Isabel; Cline, Hollis T

    2008-05-01

    CPG15 (aka neuritin) is an activity-induced GPI-anchored axonal protein that promotes dendritic and axonal growth, and accelerates synaptic maturation in vivo. Here we show that CPG15 is distributed inside axons and on the axon surface. CPG15 is trafficked to and from the axonal surface by membrane depolarization. To assess CPG15 trafficking in vivo, we expressed an ecliptic pHluorin (EP)-CPG15 fusion protein in optic tectal explants and in retinal ganglion cells of intact Xenopus tadpoles. Depolarization by KCl increased EP-CPG15 fluorescence on axons. Intraocular kainic acid (KA) injection rapidly increased cell-surface EP-CPG15 in retinotectal axons, but coinjection of TTX and KA did not. Consistent with this, we find that intracellular CPG15 is localized to vesicles and endosomes in presynaptic terminals and colocalizes with synaptic vesicle proteins. The results indicate that the delivery of the neurotrophic protein CPG15 to the axon surface can be regulated on a rapid time scale by activity-dependent mechanisms in vivo.

  1. Dual delivery of active antibactericidal agents and bone morphogenetic protein at sustainable high concentrations using biodegradable sheath-core-structured drug-eluting nanofibers

    PubMed Central

    Hsu, Yung-Hen; Lin, Chang-Tun; Yu, Yi-Hsun; Chou, Ying-Chao; Liu, Shih-Jung; Chan, Err-Cheng

    2016-01-01

    In this study, we developed biodegradable sheath-core-structured drug-eluting nanofibers for sustainable delivery of antibiotics (vancomycin and ceftazidime) and recombinant human bone morphogenetic protein (rhBMP-2) via electrospinning. To prepare the biodegradable sheath-core nanofibers, we first prepared solutions of poly(d,l)-lactide-co-glycolide, vancomycin, and ceftazidime in 1,1,1,3,3,3-hexafluoro-2-propanol and rhBMP-2 in phosphate-buffered solution. The poly(d,l)-lactide-co-glycolide/antibiotics and rhBMP-2 solutions were then fed into two different capillary tubes controlled by two independent pumps for coaxial electrospinning. The electrospun nanofiber morphology was observed under a scanning electron microscope. We further characterized the in vitro antibiotic release from the nanofibers via high-performance liquid chromatography and that of rhBMP-2 via enzyme-linked immunosorbent assay and alkaline phosphatase activity. We showed that the biodegradable coaxially electrospun nanofibers could release high vancomycin/ceftazidime concentrations (well above the minimum inhibition concentration [MIC]90) and rhBMP-2 for >4 weeks. These experimental results demonstrate that novel biodegradable nanofibers can be constructed with various pharmaceuticals and proteins for long-term drug deliveries. PMID:27574423

  2. Dual delivery of active antibactericidal agents and bone morphogenetic protein at sustainable high concentrations using biodegradable sheath-core-structured drug-eluting nanofibers.

    PubMed

    Hsu, Yung-Hen; Lin, Chang-Tun; Yu, Yi-Hsun; Chou, Ying-Chao; Liu, Shih-Jung; Chan, Err-Cheng

    2016-01-01

    In this study, we developed biodegradable sheath-core-structured drug-eluting nanofibers for sustainable delivery of antibiotics (vancomycin and ceftazidime) and recombinant human bone morphogenetic protein (rhBMP-2) via electrospinning. To prepare the biodegradable sheath-core nanofibers, we first prepared solutions of poly(d,l)-lactide-co-glycolide, vancomycin, and ceftazidime in 1,1,1,3,3,3-hexafluoro-2-propanol and rhBMP-2 in phosphate-buffered solution. The poly(d,l)-lactide-co-glycolide/antibiotics and rhBMP-2 solutions were then fed into two different capillary tubes controlled by two independent pumps for coaxial electrospinning. The electrospun nanofiber morphology was observed under a scanning electron microscope. We further characterized the in vitro antibiotic release from the nanofibers via high-performance liquid chromatography and that of rhBMP-2 via enzyme-linked immunosorbent assay and alkaline phosphatase activity. We showed that the biodegradable coaxially electrospun nanofibers could release high vancomycin/ceftazidime concentrations (well above the minimum inhibition concentration [MIC]90) and rhBMP-2 for >4 weeks. These experimental results demonstrate that novel biodegradable nanofibers can be constructed with various pharmaceuticals and proteins for long-term drug deliveries.

  3. Intracellular Protein Delivery for Treating Breast Cancer

    DTIC Science & Technology

    2013-06-01

    delivery approach. This is a platform to deliver proteins in native forms into cells. The key design feature of our strategy is to first encapsulate ...with a Cyclodextrin ." J. Biol. Chem. 271: 21604-21613. Pelkmans, L. and A. Helenius (2002). "Endocytosis Via Caveolae." Traffic 3: 311-320

  4. Cry protein crystals: a novel platform for protein delivery.

    PubMed

    Nair, Manoj S; Lee, Marianne M; Bonnegarde-Bernard, Astrid; Wallace, Julie A; Dean, Donald H; Ostrowski, Michael C; Burry, Richard W; Boyaka, Prosper N; Chan, Michael K

    2015-01-01

    Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer's patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.

  5. Bioengineering novel floating nanoparticles for protein and drug delivery

    PubMed Central

    DasSarma, Priya; Karan, Ram; Kim, Jong-Myoung; Pecher, Wolf; DasSarma, Shiladitya

    2016-01-01

    Gas vesicle nanoparticles (GVNPs) are hollow protein nanoparticles produced by Halobacterium sp. NRC-1 which are being engineered for protein delivery. To advance the bioengineering potential of GVNPs, a strain of NRC-1 deleted for the gvpC gene (ΔgvpC) was constructed and a synthetic gene coding for Gaussia princeps luciferase was fused to an abbreviated gvpC gene on an expression plasmid. When introduced into theΔgvpC strain, an active GvpC-luciferase fusion protein bound to GVNPs resulted. These results represent both a technical improvement in the GVNP display system and its expansion for the display of active enzymes. PMID:27158595

  6. Antimalarial Activity of Granzyme B and Its Targeted Delivery by a Granzyme B–Single-Chain Fv Fusion Protein

    PubMed Central

    Kapelski, Stephanie; de Almeida, Melanie; Fischer, Rainer; Barth, Stefan

    2014-01-01

    We present here the first evidence that granzyme B acts against Plasmodium falciparum (50% inhibitory concentration [IC50], 1,590 nM; 95% confidence interval [95% CI], 1,197 to 2,112 nM). We created a novel antimalarial fusion protein consisting of granzyme B fused to a merozoite surface protein 4 (MSP4)-specific single-chain Fv protein (scFv), which targets the enzyme to infected erythrocytes, with up to an 8-fold reduction in the IC50 (176 nM; 95% CI, 154 to 202 nM). This study confirms the therapeutic efficacies of recombinant antibody-mediated antimalarial immunotherapeutics based on granzyme B. PMID:25313223

  7. Antimalarial activity of granzyme B and its targeted delivery by a granzyme B-single-chain Fv fusion protein.

    PubMed

    Kapelski, Stephanie; de Almeida, Melanie; Fischer, Rainer; Barth, Stefan; Fendel, Rolf

    2015-01-01

    We present here the first evidence that granzyme B acts against Plasmodium falciparum (50% inhibitory concentration [IC50], 1,590 nM; 95% confidence interval [95% CI], 1,197 to 2,112 nM). We created a novel antimalarial fusion protein consisting of granzyme B fused to a merozoite surface protein 4 (MSP4)-specific single-chain Fv protein (scFv), which targets the enzyme to infected erythrocytes, with up to an 8-fold reduction in the IC50 (176 nM; 95% CI, 154 to 202 nM). This study confirms the therapeutic efficacies of recombinant antibody-mediated antimalarial immunotherapeutics based on granzyme B.

  8. Intercellular trafficking and protein delivery by a herpesvirus structural protein.

    PubMed

    Elliott, G; O'Hare, P

    1997-01-24

    We show that the HSV-1 structural protein VP22 has the remarkable property of intercellular transport, which is so efficient that following expression in a subpopulation the protein spreads to every cell in a monolayer, where it concentrates in the nucleus and binds chromatin. VP22 movement was observed both after delivery of DNA by transfection or microinjection and during virus infection. Moreover, we demonstrate that VP22 trafficking occurs via a nonclassical Golgi-independent mechanism. Sensitivity to cytochalasin D treatment suggests that VP22 utilizes a novel trafficking pathway that involves the actin cytoskeleton. In addition, we demonstrate intercellular transport of a VP22 fusion protein after endogenous synthesis or exogenous application, indicating that VP22 may have potential in the field of protein delivery.

  9. Protein and Peptide Drug Delivery: Oral Approaches

    PubMed Central

    Shaji, Jessy; Patole, V.

    2008-01-01

    Till recent, injections remained the most common means for administering therapeutic proteins and peptides because of their poor oral bioavailability. However, oral route would be preferred to any other route because of its high levels of patient acceptance and long term compliance, which increases the therapeutic value of the drug. Designing and formulating a polypeptide drug delivery through the gastro intestinal tract has been a persistent challenge because of their unfavorable physicochemical properties, which includes enzymatic degradation, poor membrane permeability and large molecular size. The main challenge is to improve the oral bioavailability from less than 1% to at least 30-50%. Consequently, efforts have intensified over the past few decades, where every oral dosage form used for the conventional small molecule drugs has been used to explore oral protein and peptide delivery. Various strategies currently under investigation include chemical modification, formulation vehicles and use of enzyme inhibitors, absorption enhancers and mucoadhesive polymers. This review summarizes different pharmaceutical approaches which overcome various physiological barriers that help to improve oral bioavailability that ultimately achieve formulation goals for oral delivery. PMID:20046732

  10. Lack of Relationship between Cord Serum Angiopoietin-Like Protein 4 (ANGPTL4) and Lipolytic Activity in Human Neonates Born by Spontaneous Delivery

    PubMed Central

    Ortega-Senovilla, Henar; Schaefer-Graf, Ute; Meitzner, Katrin; Graf, Kristof; Abou-Dakn, Michael; Herrera, Emilio

    2013-01-01

    Background Ligands of peroxisome-proliferator activated receptors (PPARs), such as non-esterified fatty acids (NEFAs), induce expression of angiopoietin-like protein 4 (ANGPTL4). Recently ANGPTL4 has been reported to be a mediator of intracellular adipose lipolysis induced by glucocorticoids. Objective To determine the concentrations of ANGPTL4 in cord serum of neonates born by spontaneous vaginal delivery (SVD) and by pre-labor cesarean section (CS) from healthy women, and to relate them to parameters of neonatal lipolytic activity at birth. Measurements In 54 neonates born by SVD and in 56 neonates born by CS, arterial cord blood was drawn to determine insulin, cortisol, triacylglycerols (TAGs), glycerol, non-esterified fatty acids (NEFAs), individual fatty acids, ANGPTL4, adiponectin, retinol binding protein 4 (RBP4) and leptin. Results Birth weight and neonatal fat mass in SVD and CS showed no difference, but the concentrations of glycerol, adiponectin, RBP4, NEFAs and most individual fatty acids were higher in cord serum of neonates born by SVD compared to CS, indicating a higher adipose tissue breakdown in the SVD group. The concentrations of TAG and cortisol were also higher and that of insulin was lower in cord serum of SVD compared to the CS group. However, the concentration in cord serum of ANGPTL4 did not differ between the two groups and no positive correlation with either NEFA or glycerol concentrations were detected. Conclusion ANGPTL4 is known to stimulate lipolysis in adults, but does not appear to mediate the increased activity in SVD, indicating the presence of different regulatory inputs. PMID:24324678

  11. Continuous release of bone morphogenetic protein-2 through nano-graphene oxide-based delivery influences the activation of the NF-κB signal transduction pathway

    PubMed Central

    Zhong, Cheng; Feng, Jun; Lin, Xiangjin; Bao, Qi

    2017-01-01

    Graphene oxide (GO) has been used as a delivery vehicle for small molecule drugs and nucleotides. To further investigate GO as a smart biomaterial for the controlled release of cargo molecules, we hypothesized that GO may be an appropriate delivery vehicle because it releases bone morphogenetic protein 2 (BMP2). GO characterization indicated that the size distribution of the GO flakes ranged from 81.1 nm to 45,749.7 nm, with an approximate thickness of 2 nm. After BMP2 adsorption onto GO, Fourier-transformed infrared spectroscopy (FTIR) and thermal gravimetric analysis were performed. Compared to GO, BMP2-GO did not induce significant changes in the characteristics of the materials. GO continuously released BMP2 for at least 40 days. Bone marrow stem cells (BMSCs) and chondrocytes were treated with BMP2-GO in interleukin-1 media and assessed in terms of cell viability, flow cytometric characterization, and expression of particular mRNA. Compared to GO, BMP2-GO did not induce any significant changes in biocompatibility. We treated osteoarthritic rats with BMP2 and BMP2-GO, which showed significant differences in Osteoarthritis Research Society International (OARSI) scores (P<0.05). Quantitative assessment revealed significant differences compared to that using BMP2 and BMP2-GO (P<0.05). These findings indicate that GO may be potentially used to control the release of carrier materials. The combination of BMP2 and GO slowed the progression of NF-κB-activated degenerative changes in osteoarthritis. Therefore, we infer that our BMP2-GO strategy could alleviate the NF-κB pathway by inducing continuous BMP2 release. PMID:28243085

  12. Continuous release of bone morphogenetic protein-2 through nano-graphene oxide-based delivery influences the activation of the NF-κB signal transduction pathway.

    PubMed

    Zhong, Cheng; Feng, Jun; Lin, Xiangjin; Bao, Qi

    2017-01-01

    Graphene oxide (GO) has been used as a delivery vehicle for small molecule drugs and nucleotides. To further investigate GO as a smart biomaterial for the controlled release of cargo molecules, we hypothesized that GO may be an appropriate delivery vehicle because it releases bone morphogenetic protein 2 (BMP2). GO characterization indicated that the size distribution of the GO flakes ranged from 81.1 nm to 45,749.7 nm, with an approximate thickness of 2 nm. After BMP2 adsorption onto GO, Fourier-transformed infrared spectroscopy (FTIR) and thermal gravimetric analysis were performed. Compared to GO, BMP2-GO did not induce significant changes in the characteristics of the materials. GO continuously released BMP2 for at least 40 days. Bone marrow stem cells (BMSCs) and chondrocytes were treated with BMP2-GO in interleukin-1 media and assessed in terms of cell viability, flow cytometric characterization, and expression of particular mRNA. Compared to GO, BMP2-GO did not induce any significant changes in biocompatibility. We treated osteoarthritic rats with BMP2 and BMP2-GO, which showed significant differences in Osteoarthritis Research Society International (OARSI) scores (P<0.05). Quantitative assessment revealed significant differences compared to that using BMP2 and BMP2-GO (P<0.05). These findings indicate that GO may be potentially used to control the release of carrier materials. The combination of BMP2 and GO slowed the progression of NF-κB-activated degenerative changes in osteoarthritis. Therefore, we infer that our BMP2-GO strategy could alleviate the NF-κB pathway by inducing continuous BMP2 release.

  13. Polymeric-based particulate systems for delivery of therapeutic proteins.

    PubMed

    Akash, Muhammad Sajid Hamid; Rehman, Kanwal; Chen, Shuqing

    2016-01-01

    Polymeric-based particulate systems have been intensively developed to increase the short biological half-life and prevent enzymatic degradation of therapeutic proteins. These techniques demonstrate the useful characteristics for the delivery of therapeutic proteins and peptides to the targeted site of application and prevent the interaction of encapsulated drug with the normal cells. In this article, we have described the in depth of different pharmaceutical-based techniques that are currently being practiced for efficient delivery of therapeutic proteins and peptides. A comprehensive English literature was searched using different electronic search databases including PubMed, Science Direct, Web of Science, google scholar and library search. Different search terms and advanced search were made by combining all the search fields in abstract, keywords and/or titles. Findings of various studies that have been discussed in this article clearly indicate that polymeric-based techniques can significantly increase the therapeutic potentials of incorporated proteins with no known toxic effects. These techniques have shown to maintain the stability and retain biological activity of protein therapeutics. Hence it can be suggested that pharmaceutical-based techniques are promising drug carriers for efficient delivery of therapeutic proteins.

  14. Controlled delivery of peptides and proteins.

    PubMed

    Degim, I Tuncer; Celebi, Nevin

    2007-01-01

    The final aim/target of Pharmaceutical Sciences is to design successful dosage forms for effective therapy, considering individual patient needs and compliance. Development of new drug entities, particularly using peptides and proteins, is growing in importance and attracting increased interest, as they are specifically effective at a comparably low dose. These very potent and specific peptides and proteins can now be produced in large quantities due to increased knowledge and advancements in biotechnological and pharmaceutical applications. A number of peptide and protein products are now available on the market, and numerous studies investigating them have been published in the literature. Although many peptide/protein like products are generally designed for parenteral administration, some other noninvasive routes have also been used. For example, desmopressin is delivered nasally and deoxyribonuclease by inhalation. Although peptides and proteins are generally orally inactive, cyclosporine is an exception. In order to design and develop long-acting, more effective peptide/protein drugs, the controlled release mechanisms and effective parameters need to be understood and clarified. Therefore, we review herein various peptide/protein delivery systems, including biodegradable and nondegradable microspheres, microcapsules, nanocapsules, injectable implants, diffusion-controlled hydrogels and other hydrophilic systems, microemulsions and multiple emulsions, and the use of iontophoresis or electroporation, and discuss the results of recent researches.

  15. Protein-Based Nanomedicine Platforms for Drug Delivery

    SciTech Connect

    Ma Ham, Aihui; Tang, Zhiwen; Wu, Hong; Wang, Jun; Lin, Yuehe

    2009-08-03

    Drug delivery systems have been developed for many years, however some limitations still hurdle the pace of going to clinical phase, for example, poor biodistribution, drug molecule cytotoxicity, tissue damage, quick clearance from the circulation system, solubility and stability of drug molecules. To overcome the limitations of drug delivery, biomaterials have to be developed and applied to drug delivery to protect the drug molecules and to enhance the drug’s efficacy. Protein-based nanomedicine platforms for drug delivery are platforms comprised of naturally self-assembled protein subunits of the same protein or a combination of proteins making up a complete system. They are ideal for drug delivery platforms due to their biocompatibility and biodegradability coupled with low toxicity. A variety of proteins have been used and characterized for drug delivery systems including the ferritin/apoferritin protein cage, plant derived viral capsids, the small Heat shock protein (sHsp) cage, albumin, soy and whey protein, collagen, and gelatin. There are many different types and shapes that have been prepared to deliver drug molecules using protein-based platforms including the various protein cages, microspheres, nanoparticles, hydrogels, films, minirods and minipellets. There are over 30 therapeutic compounds that have been investigated with protein-based drug delivery platforms for the potential treatment of various cancers, infectious diseases, chronic diseases, autoimmune diseases. In protein-based drug delivery platforms, protein cage is the most newly developed biomaterials for drug delivery and therapeutic applications. Their uniform sizes, multifunctions, and biodegradability push them to the frontier for drug delivery. In this review, the recent strategic development of drug delivery has been discussed with a special emphasis upon the polymer based, especially protein-based nanomedicine platforms for drug delivery. The advantages and disadvantages are also

  16. Stimuli-Responsive Polymeric Systems for Controlled Protein and Peptide Delivery: Future Implications for Ocular Delivery.

    PubMed

    Mahlumba, Pakama; Choonara, Yahya E; Kumar, Pradeep; du Toit, Lisa C; Pillay, Viness

    2016-07-30

    Therapeutic proteins and peptides have become notable in the drug delivery arena for their compatibility with the human body as well as their high potency. However, their biocompatibility and high potency does not negate the existence of challenges resulting from physicochemical properties of proteins and peptides, including large size, short half-life, capability to provoke immune responses and susceptibility to degradation. Various delivery routes and delivery systems have been utilized to improve bioavailability, patient acceptability and reduce biodegradation. The ocular route remains of great interest, particularly for responsive delivery of macromolecules due to the anatomy and physiology of the eye that makes it a sensitive and complex environment. Research in this field is slowly gaining attention as this could be the breakthrough in ocular drug delivery of macromolecules. This work reviews stimuli-responsive polymeric delivery systems, their use in the delivery of therapeutic proteins and peptides as well as examples of proteins and peptides used in the treatment of ocular disorders. Stimuli reviewed include pH, temperature, enzymes, light, ultrasound and magnetic field. In addition, it discusses the current progress in responsive ocular drug delivery. Furthermore, it explores future prospects in the use of stimuli-responsive polymers for ocular delivery of proteins and peptides. Stimuli-responsive polymers offer great potential in improving the delivery of ocular therapeutics, therefore there is a need to consider them in order to guarantee a local, sustained and ideal delivery of ocular proteins and peptides, evading tissue invasion and systemic side-effects.

  17. Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

    PubMed Central

    Danyal, Karamatullah; Shaw, Sudipta; Page, Taylor R.; Duval, Simon; Horitani, Masaki; Marts, Amy R.; Lukoyanov, Dmitriy; Dean, Dennis R.; Raugei, Simone; Hoffman, Brian M.; Seefeldt, Lance C.; Antony, Edwin

    2016-01-01

    Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the α2β2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αβ active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves. PMID:27698129

  18. Nanostructured thin film polymer devices for constant-rate protein delivery.

    PubMed

    Bernards, Daniel A; Lance, Kevin D; Ciaccio, Natalie A; Desai, Tejal A

    2012-10-10

    Herein long-term delivery of proteins from biodegradable thin film devices is demonstrated, where a nanostructured polymer membrane controls release. Protein was sealed between two poly(caprolactone) films, which generated the thin film devices. Protein release for 210 days was shown in vitro, and stable activity was established through 70 days with a model protein. These thin film devices present a promising delivery platform for biologic therapeutics, particularly for application in constrained spaces.

  19. Targeted Intracellular Delivery of Proteins with Spatial and Temporal Control

    PubMed Central

    2015-01-01

    While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery. PMID:25490248

  20. Peptide-chaperone-directed transdermal protein delivery requires energy.

    PubMed

    Ruan, Renquan; Jin, Peipei; Zhang, Li; Wang, Changli; Chen, Chuanjun; Ding, Weiping; Wen, Longping

    2014-11-03

    The biologically inspired transdermal enhanced peptide TD1 has been discovered to specifically facilitate transdermal delivery of biological macromolecules. However, the biological behavior of TD1 has not been fully defined. In this study, we find that energy is required for the TD1-mediated transdermal protein delivery through rat and human skins. Our results show that the permeation activity of TD1-hEGF, a fusion protein composed of human epidermal growth factor (hEGF) and the TD1 sequence connected with a glycine-serine linker (GGGGS), can be inhibited by the energy inhibitor, rotenone or oligomycin. In addition, adenosine triphosphate (ATP), the essential energetic molecule in organic systems, can effectively facilitate the TD1 directed permeation of the protein-based drug into the skin in a dose-dependent fashion. Our results here demonstrate a novel energy-dependent permeation process during the TD1-mediated transdermal protein delivery that could be valuable for the future development of promising new transdermal drugs.

  1. Bioreducible Lipid-like Nanoparticles for Intracellular Protein Delivery

    NASA Astrophysics Data System (ADS)

    Arellano, Carlos Luis

    Protein-based therapy is one of the most direct ways to manipulate cell function and treat human disease. Although protein therapeutics has made its way to clinical practice, with five of the top fifteen global pharmaceuticals being peptide or protein-based drugs, one common limitation is that the effects of protein therapy are only achieved through the targeting of cell surface receptors and intracellular domains. Due to the impermeability of the cell membrane to most foreign materials, entire classes of potentially therapeutic proteins cannot thoroughly be studied without a safe and efficient method of transporting proteins into the cytosol. We report the use of a combinatorially-designed bioreducible lipid-like material (termed "lipidoid") - based protein delivery platform for the transfection of human cancer cell lines. Lipidoid nanoparticles are synthesized through a thin film dispersion method. The degradation of the bioreducible nanoparticles was observed when exposed to glutathione, a highly reductive compound present in the cytosol. We demonstrate that the nanoparticles are capable of transfecting a dose-dependent concentration of our model protein, beta-galactosidase into HeLa cells. Furthermore, formulations of the lipidoid containing the cytotoxic proteins saporin and RNase-A are both capable of inhibiting tumor cell proliferation as observed in in vitro treatment of different human cancer cell lines. There was no observed loss in protein activity after lyophilization and long--term storage, indicating the potential of pre-clinical applications. Overall, we demonstrate an effective approach to protein formulation and intracellular delivery. We believe that our formulations will lead to the study of a whole class of previously untapped therapeutics that may generate new solutions for previously untreatable diseases.

  2. Rational Design of a Polymer with Robust Efficacy for Intracellular Protein and Peptide Delivery.

    PubMed

    Chang, Hong; Lv, Jia; Gao, Xin; Wang, Xing; Wang, Hui; Chen, Hui; He, Xu; Li, Lei; Cheng, Yiyun

    2017-03-08

    The efficient delivery of biopharmaceutical drugs such as proteins and peptides into the cytosol of target cells poses substantial challenges owing to their large size and susceptibility to degradation. Current protein delivery vehicles have limitations such as the need for protein modification, insufficient delivery of large-size proteins or small peptides, and loss of protein function after the delivery. Here, we adopted a rational approach to design a polymer with robust efficacy for intracellular protein and peptide delivery. The polymer is composed of a dendrimer scaffold, a hydrophobic membrane-disruptive region, and a multivalent protein binding surface. It allows efficient protein/peptide binding, endocytosis, and endosomal disruption and is capable of efficiently delivering various biomacromolecules including bovine serum albumin, R-phycoerythrin, p53, saporin, β-galactosidase, and peptides into the cytosol of living cells. Transduction of apoptotic proteins and peptides successfully induces apoptosis in cancer cells, suggesting that the activities of proteins and peptides are maintained during the delivery. This technology represents an efficient and useful tool for intracellular protein and peptide delivery and has broad applicability for basic research and clinical applications.

  3. Peptide/protein vaccine delivery system based on PLGA particles

    PubMed Central

    Allahyari, Mojgan; Mohit, Elham

    2016-01-01

    abstract Due to the excellent safety profile of poly (D,L-lactide-co-glycolide) (PLGA) particles in human, and their biodegradability, many studies have focused on the application of PLGA particles as a controlled-release vaccine delivery system. Antigenic proteins/peptides can be encapsulated into or adsorbed to the surface of PLGA particles. The gradual release of loaded antigens from PLGA particles is necessary for the induction of efficient immunity. Various factors can influence protein release rates from PLGA particles, which can be defined intrinsic features of the polymer, particle characteristics as well as protein and environmental related factors. The use of PLGA particles encapsulating antigens of different diseases such as hepatitis B, tuberculosis, chlamydia, malaria, leishmania, toxoplasma and allergy antigens will be described herein. The co-delivery of antigens and immunostimulants (IS) with PLGA particles can prevent the systemic adverse effects of immunopotentiators and activate both dendritic cells (DCs) and natural killer (NKs) cells, consequently enhancing the therapeutic efficacy of antigen-loaded PLGA particles. We will review co-delivery of different TLR ligands with antigens in various models, highlighting the specific strengths and weaknesses of the system. Strategies to enhance the immunotherapeutic effect of DC-based vaccine using PLGA particles can be designed to target DCs by functionalized PLGA particle encapsulating siRNAs of suppressive gene, and disease specific antigens. Finally, specific examples of cellular targeting where decorating the surface of PLGA particles target orally administrated vaccine to M-cells will be highlighted. PMID:26513024

  4. Stimuli-Responsive Nanomaterials for Therapeutic Protein Delivery

    PubMed Central

    Lu, Yue; Sun, Wujin; Gu, Zhen

    2014-01-01

    Protein therapeutics have emerged as a significant role in treatment of a broad spectrum of diseases, including cancer, metabolic disorders and autoimmune diseases. The efficacy of protein therapeutics, however, is limited by their instability, immunogenicity and short half-life. In order to overcome these barriers, tremendous efforts have recently been made in developing controlled protein delivery systems. Stimuli-triggered release is an appealing and promising approach for protein delivery and has made protein delivery with both spatiotemporal- and dosage-controlled manners possible. This review surveys recent advances in controlled protein delivery of proteins or peptides using stimuli-responsive nanomaterials. Strategies utilizing both physiological and external stimuli are introduced and discussed. PMID:25151983

  5. Characterization of particulate drug delivery systems for oral delivery of Peptide and protein drugs.

    PubMed

    Christophersen, Philip Carsten; Fano, Mathias; Saaby, Lasse; Yang, Mingshi; Nielsen, Hanne Mørck; Mu, Huiling

    2015-01-01

    Oral drug delivery is a preferred route because of good patient compliance. However, most peptide/ protein drugs are delivered via parenteral routes because of the absorption barriers in the gastrointestinal (GI) tract such as enzymatic degradation by proteases and low permeability acrossthe biological membranes. To overcome these barriers, different formulation strategies for oral delivery of biomacromolecules have been proposed, including lipid based formulations and polymer-based particulate drug delivery systems (DDS). The aim of this review is to summarize the existing knowledge about oral delivery of peptide/protein drugs and to provide an overview of formulationand characterization strategies. For a better understanding of the challenges in oral delivery of peptide/protein drugs, the composition of GI fluids and the digestion processes of different kinds of excipients in the GI tract are summarized. Additionally, the paper provides an overview of recent studies on characterization of solid drug carriers for peptide/protein drugs, drug distribution in particles, drug release and stability in simulated GI fluids, as well as the absorption of peptide/protein drugs in cell-based models. The use of biorelevant media when applicable can increase the knowledge about the quality of DDS for oral protein delivery. Hopefully, the knowledge provided in this review will aid the establishment of improved biorelevant models capable of forecasting the performance of particulate DDS for oral peptide/protein delivery.

  6. Plasma-activated air mediates plasmid DNA delivery in vivo

    PubMed Central

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets. PMID:27110584

  7. Recent developments in protein and peptide parenteral delivery approaches

    PubMed Central

    Patel, Ashaben; Cholkar, Kishore; Mitra, Ashim K

    2014-01-01

    Discovery of insulin in the early 1900s initiated the research and development to improve the means of therapeutic protein delivery in patients. In the past decade, great emphasis has been placed on bringing protein and peptide therapeutics to market. Despite tremendous efforts, parenteral delivery still remains the major mode of administration for protein and peptide therapeutics. Other routes such as oral, nasal, pulmonary and buccal are considered more opportunistic rather than routine application. Improving biological half-life, stability and therapeutic efficacy is central to protein and peptide delivery. Several approaches have been tried in the past to improve protein and peptide in vitro/in vivo stability and performance. Approaches may be broadly categorized as chemical modification and colloidal delivery systems. In this review we have discussed various chemical approaches such as PEGylation, hyperglycosylation, mannosylation, and colloidal carriers including microparticles, nanoparticles, liposomes, carbon nanotubes and micelles for improving protein and peptide delivery. Recent developments on in situ thermosensitive gel-based protein and peptide delivery have also been described. This review summarizes recent developments on some currently existing approaches to improve stability, bioavailability and bioactivity of peptide and protein therapeutics following parenteral administration. PMID:24592957

  8. Liposome-Mediated Cellular Delivery of Active gp91phox

    PubMed Central

    Marques, Bruno; Liguori, Lavinia; Paclet, Marie-Hélène; Villegas-Mendéz, Ana; Rothe, Romy; Morel, Françoise; Lenormand, Jean-Luc

    2007-01-01

    Background Gp91phox is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms. Methodology Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins. Conclusions Using this system, we over-express truncated forms of the gp91phox protein under soluble form in the presence of detergents or lipids resulting in active proteins with a “native-like” conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67phox, p47phox, p40phox and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91phox protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91phox proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91phox protein. PMID:17848987

  9. Basics and recent advances in peptide and protein drug delivery

    PubMed Central

    Bruno, Benjamin J; Miller, Geoffrey D; Lim, Carol S

    2014-01-01

    While the peptide and protein therapeutic market has developed significantly in the past decades, delivery has limited their use. Although oral delivery is preferred, most are currently delivered intravenously or subcutaneously due to degradation and limited absorption in the gastrointestinal tract. Therefore, absorption enhancers, enzyme inhibitors, carrier systems and stability enhancers are being studied to facilitate oral peptide delivery. Additionally, transdermal peptide delivery avoids the issues of the gastrointestinal tract, but also faces absorption limitations. Due to proteases, opsonization and agglutination, free peptides are not systemically stable without modifications. This review discusses oral and transdermal peptide drug delivery, focusing on barriers and solutions to absorption and stability issues. Methods to increase systemic stability and site-specific delivery are also discussed. PMID:24228993

  10. Mechanisms allowing protein delivery in nasal mucosa using NPL nanoparticles.

    PubMed

    Bernocchi, B; Carpentier, R; Lantier, I; Ducournau, C; Dimier-Poisson, I; Betbeder, D

    2016-06-28

    The intranasal administration of proteins using nanoparticles is a promising approach for several applications, especially for mucosal vaccines. Delivery of protein within the epithelial barrier is a key point to elicit an immune response and nano-carrier has to show no toxicity. The aim of this work was to elucidate the interactions of cationic porous nanoparticles loaded with protein delivery for antigen delivery in the nose. We investigated the loading, the cellular delivery and the epithelial transcytosis of proteins associated to these nanoparticles containing an anionic lipid in their core (NPL). NPL were highly endocytosed by airway epithelial cells and significantly improved the protein delivery into the cell. In vitro transcytosis studies showed that NPL did not modify the in vitro epithelial permeability suggesting no toxicity of these carriers. Moreover protein and NPL did not translocate the epithelial barrier. In vivo studies demonstrated that NPL prolonged the nasal residence time of the protein and no NPL were found beyond the epithelial barrier in vivo, precluding a negative side effect. All together these results establish the NPL as a bio-eliminable and optimal vaccine carrier.

  11. Coacervate delivery systems for proteins and small molecule drugs

    PubMed Central

    Johnson, Noah R; Wang, Yadong

    2015-01-01

    Coacervates represent an exciting new class of drug delivery vehicles, developed in the past decade as carriers of small molecule drugs and proteins. This review summarizes several well-described coacervate systems, including Elastin-like peptides for delivery of anti-cancer therapeutics,Heparin-based coacervates with synthetic polycations for controlled growth factor delivery,Carboxymethyl chitosan aggregates for oral drug delivery,Mussel adhesive protein and hyaluronic acid coacervates. Coacervates present advantages in their simple assembly and easy incorporation into tissue engineering scaffolds or as adjuncts to cell therapies. They are also amenable to functionalization such as for targeting or for enhancing the bioactivity of their cargo. These new drug carriers are anticipated to have broad applications and noteworthy impact in the near future. PMID:25138695

  12. Protein Nanoparticles as Drug Delivery Carriers for Cancer Therapy

    PubMed Central

    Lohcharoenkal, Warangkana; Wang, Liying; Chen, Yi Charlie

    2014-01-01

    Nanoparticles have increasingly been used for a variety of applications, most notably for the delivery of therapeutic and diagnostic agents. A large number of nanoparticle drug delivery systems have been developed for cancer treatment and various materials have been explored as drug delivery agents to improve the therapeutic efficacy and safety of anticancer drugs. Natural biomolecules such as proteins are an attractive alternative to synthetic polymers which are commonly used in drug formulations because of their safety. In general, protein nanoparticles offer a number of advantages including biocompatibility and biodegradability. They can be prepared under mild conditions without the use of toxic chemicals or organic solvents. Moreover, due to their defined primary structure, protein-based nanoparticles offer various possibilities for surface modifications including covalent attachment of drugs and targeting ligands. In this paper, we review the most significant advancements in protein nanoparticle technology and their use in drug delivery arena. We then examine the various sources of protein materials that have been used successfully for the construction of protein nanoparticles as well as their methods of preparation. Finally, we discuss the applications of protein nanoparticles in cancer therapy. PMID:24772414

  13. Engineering protein self-assembling in protein-based nanomedicines for drug delivery and gene therapy.

    PubMed

    Ferrer-Miralles, Neus; Rodríguez-Carmona, Escarlata; Corchero, José Luis; García-Fruitós, Elena; Vázquez, Esther; Villaverde, Antonio

    2015-06-01

    Lack of targeting and improper biodistribution are major flaws in current drug-based therapies that prevent reaching high local concentrations of the therapeutic agent. Such weaknesses impose the administration of high drug doses, resulting in undesired side effects, limited efficacy and enhanced production costs. Currently, missing nanosized containers, functionalized for specific cell targeting will be then highly convenient for the controlled delivery of both conventional and innovative drugs. In an attempt to fill this gap, health-focused nanotechnologies have put under screening a growing spectrum of materials as potential components of nanocages, whose properties can be tuned during fabrication. However, most of these materials pose severe biocompatibility concerns. We review in this study how proteins, the most versatile functional macromolecules, can be conveniently exploited and adapted by conventional genetic engineering as efficient building blocks of fully compatible nanoparticles for drug delivery and how selected biological activities can be recruited to mimic viral behavior during infection. Although engineering of protein self-assembling is still excluded from fully rational approaches, the exploitation of protein nano-assemblies occurring in nature and the direct manipulation of protein-protein contacts in bioinspired constructs open intriguing possibilities for further development. These methodologies empower the construction of new and potent vehicles that offer promise as true artificial viruses for efficient and safe nanomedical applications.

  14. Intracellular Protein Delivery for Treating Breast Cancer

    DTIC Science & Technology

    2012-06-01

    um filters to fully clear cell lysate. The protein was then purified by an amylase column and MBP-APO was eluted from the column and buffer exchanged...lysate. Afterwards, the protein was then purified on an amylase column (New England BioLabs), which was passed over 5 times with lysate under gravity

  15. Intracellular Protein Delivery for Treating Breast Cancer

    DTIC Science & Technology

    2012-06-01

    chose to work with maltose -binding-protein fused apoptin (MBP-APO) which can be highly expressed in soluble form from Escherichia coli, whereas...MBP-APO was eluted from the column with 10 mM maltose buffer and buffer exchanged into PBS. The protein concentration was qualitatively assessed by

  16. Encapsulated Cellular Implants for Recombinant Protein Delivery and Therapeutic Modulation of the Immune System

    PubMed Central

    Lathuilière, Aurélien; Mach, Nicolas; Schneider, Bernard L.

    2015-01-01

    Ex vivo gene therapy using retrievable encapsulated cellular implants is an effective strategy for the local and/or chronic delivery of therapeutic proteins. In particular, it is considered an innovative approach to modulate the activity of the immune system. Two recently proposed therapeutic schemes using genetically engineered encapsulated cells are discussed here: the chronic administration of monoclonal antibodies for passive immunization against neurodegenerative diseases and the local delivery of a cytokine as an adjuvant for anti-cancer vaccines. PMID:26006227

  17. Maize arabinoxylan gels as protein delivery matrices.

    PubMed

    Berlanga-Reyes, Claudia M; Carvajal-Millán, Elizabeth; Lizardi-Mendoza, Jaime; Rascón-Chu, Agustin; Marquez-Escalante, Jorge A; Martínez-López, Ana Luisa

    2009-04-08

    The laccase induced gelation of maize bran arabinoxylans at 2.5% (w/v) in the presence of insulin or beta-lactoglobulin at 0.1% (w/v) was investigated. Insulin and beta-lacto-globulin did not modify either the gel elasticity (9 Pa) or the cross-links content (0.03 and 0.015 microg di- and triferulic acids/mg arabinoxylan, respectively). The protein release capability of the gel was also investigated. The rate of protein release from gels was dependent on the protein molecular weight. The apparent diffusion coefficient was 0.99 x 10(-7) and 0.79 x 10(-7) cm(2)/s for insulin (5 kDa) and beta-lactoglobulin (18 kDa), respectively. The results suggest that maize bran arabinoxylan gels can be potential candidates for the controlled release of proteins.

  18. Virus-Mimetic Fusogenic Exosomes for Direct Delivery of Integral Membrane Proteins to Target Cell Membranes.

    PubMed

    Yang, Yoosoo; Hong, Yeonsun; Nam, Gi-Hoon; Chung, Jin Hwa; Koh, Eunee; Kim, In-San

    2017-02-06

    An efficient system for direct delivery of integral membrane proteins is successfully developed using a new biocompatible exosome-based platform. Fusogenic exosomes harboring viral fusogen, vascular stomatitis virus (VSV)-G protein, can fuse with and modify plasma membranes in a process called "membrane editing." This can facilitate the transfer of biologically active membrane proteins into the target cell membranes both in vitro and in vivo.

  19. Enhancing the buccal mucosal delivery of peptide and protein therapeutics.

    PubMed

    Caon, Thiago; Jin, Liang; Simões, Cláudia M O; Norton, Raymond S; Nicolazzo, Joseph A

    2015-01-01

    With continuing advances in biotechnology and genetic engineering, there has been a dramatic increase in the availability of new biomacromolecules, such as peptides and proteins that have the potential to ameliorate the symptoms of many poorly-treated diseases. Although most of these macromolecular therapeutics exhibit high potency, their large molecular mass, susceptibility to enzymatic degradation, immunogenicity and tendency to undergo aggregation, adsorption, and denaturation have limited their ability to be administered via the traditional oral route. As a result, alternative noninvasive routes have been investigated for the systemic delivery of these macromolecules, one of which is the buccal mucosa. The buccal mucosa offers a number of advantages over the oral route, making it attractive for the delivery of peptides and proteins. However, the buccal mucosa still exhibits some permeability-limiting properties, and therefore various methods have been explored to enhance the delivery of macromolecules via this route, including the use of chemical penetration enhancers, physical methods, particulate systems and mucoadhesive formulations. The incorporation of anti-aggregating agents in buccal formulations also appears to show promise in other mucosal delivery systems, but has not yet been considered for buccal mucosal drug delivery. This review provides an update on recent approaches that have shown promise in enhancing the buccal mucosal transport of macromolecules, with a major focus on proteins and peptides.

  20. Sublingual delivery of insulin: effects of enhancers on the mucosal lipid fluidity and protein conformation, transport, and in vivo hypoglycemic activity.

    PubMed

    Cui, Chun-Ying; Lu, Wan-Liang; Xiao, Lan; Zhang, Shuang-Qing; Huang, Yan-Bei; Li, Sheng-Lin; Zhang, Rui-Juan; Wang, Gui-Ling; Zhang, Xuan; Zhang, Qiang

    2005-12-01

    The purposes of this study were to evaluate effects of enhancers for sublingual delivering insulin on the mucosal lipid fluidity and protein conformation, transport, and in vivo hypoglycemic activity in normal rats. The effects on sublingual mucosa, and aggregation states of insulin were estimated using fluorescence polarization, and circular dichroism method, respectively. The human immortalized oral epithelial cell monolayer was used for evaluating transport of insulin. Hydroxylpropyl-beta-cyclodextrin (HP-beta-CD), chitosan, polyethylene-polypropylene glycol, polyoxyethylene lauryl ether, polysorbate 80, egg lecithin, or oleic acid, was used as a penetration enhancer, respectively. The fluidity of sublingual mucosal lipid was markedly reduced by these enhancers excluding polysorbate 80, and the secondary structure of the mucosal proteins was also influenced by these enhancers. The hexamers of insulin were dissociated to monomers only by chitosan, polyoxyethylene lauryl ether, and egg lecithin. Nonetheless, plasma glucose levels in normal rats were significantly lowered after sublingual administration of insulin with an enhancer compared with those without an enhancer at the same time-point. The enhancing effects may be due to one or multiple factors: increasing the mucosal lipid fluidity, directly loosing the tight junction of epithelia, and dissociating the hexamers of insulin to monomers. Among these, the opened tight junction may correlate most with the enhancing effect in the mucosal permeability. Because the aggregates of insulin exist, the dissociation of the aggregates by an enhancer would benefit the permeability.

  1. Copper Delivery to Chloroplast Proteins and its Regulation.

    PubMed

    Aguirre, Guadalupe; Pilon, Marinus

    2015-01-01

    Copper is required for photosynthesis in chloroplasts of plants because it is a cofactor of plastocyanin, an essential electron carrier in the thylakoid lumen. Other chloroplast copper proteins are copper/zinc superoxide dismutase and polyphenol oxidase, but these proteins seem to be dispensable under conditions of low copper supply when transcripts for these proteins undergo microRNA-mediated down regulation. Two ATP-driven copper transporters function in tandem to deliver copper to chloroplast compartments. This review seeks to summarize the mechanisms of copper delivery to chloroplast proteins and its regulation. We also delineate some of the unanswered questions that still remain in this field.

  2. Copper Delivery to Chloroplast Proteins and its Regulation

    PubMed Central

    Aguirre, Guadalupe; Pilon, Marinus

    2016-01-01

    Copper is required for photosynthesis in chloroplasts of plants because it is a cofactor of plastocyanin, an essential electron carrier in the thylakoid lumen. Other chloroplast copper proteins are copper/zinc superoxide dismutase and polyphenol oxidase, but these proteins seem to be dispensable under conditions of low copper supply when transcripts for these proteins undergo microRNA-mediated down regulation. Two ATP-driven copper transporters function in tandem to deliver copper to chloroplast compartments. This review seeks to summarize the mechanisms of copper delivery to chloroplast proteins and its regulation. We also delineate some of the unanswered questions that still remain in this field. PMID:26793223

  3. Feasibility of Tissue Plasminogen Activator Formulated for Pulmonary Delivery

    PubMed Central

    Dunn, John S.; Nayar, Rajiv; Campos, Jackie; Hybertson, Brooks M.; Zhou, Yue; Manning, Mark Cornell; Repine, John E.; Stringer, Kathleen A.

    2007-01-01

    Purpose This study was conducted to assess the feasibility of a pulmonary formulation of tissue plasminogen activator (tPA) for nebulization into the airway by measuring protein stability, biologic activity, particle size, and estimating human lung distribution. Methods Formulations were derived by varying the surfactant and protein concentrations. Protein stability and recovery of each nebulized tPA formulation were assessed by ultraviolet spectroscopy. Formulations that met protein stability feasibility criteria were assessed for biologic and fibrinolytic activities. Biologic activity was determined by their ability to inhibit superoxide anion production by human neutrophils. Fibrinolytic activity was assessed by the cleavage of plasminogen to plasmin. Aerodynamic properties were assessed using a cascade impactor, and an estimation of human airway deposition was made via a human lung replica. Results Twenty-seven tPA formulations were initially assessed, 15 of which met protein stability criteria. Subsequently, three of these formulations maintained biologic and fibrinolytic activities. These formulations exhibited particle sizes of 2.4–3.1 μm, and had respirable doses ≥65%. A formulation of 1 mg mL−1 tPA and 0.1% Tween 80 exhibited a 45% deposition in the lower airways of a human lung replica. Conclusions A suitable pulmonary tPA formulation was identified that, following nebulization, maintained protein stability as well as biologic and fibrinolytic activities, and resulted in an optimal respirable dose and human airway deposition. This formulation may be applicable in the treatment of lung diseases, such as acute respiratory distress syndrome by permitting targeted pulmonary delivery of a therapeutic protein to the lungs. PMID:16180128

  4. Polymeric Nanoparticles for Pulmonary Protein and DNA Delivery

    PubMed Central

    Menon, Jyothi U.; Ravikumar, Priya; Pise, Amruta; Gyawali, Dipendra; Hsia, Connie C.W.; Nguyen, Kytai T.

    2014-01-01

    Polymeric nanoparticles (NPs) are promising carriers of biological agents to lung due to advantages including biocompatibility, ease of surface modification, localized action and reduced systemic toxicity. However, there have been no studies extensively characterizing and comparing the behavior of polymeric NPs for pulmonary protein/DNA delivery both in vitro and in vivo. We screened six polymeric NPs: gelatin, chitosan, alginate, poly lactic-co-glycolic acid (PLGA), PLGA-chitosan, and PLGA-polyethylene glycol (PEG), for inhalational protein/ DNA delivery. All NPs except PLGA-PEG and alginate were <300 nm in size with bi-phasic core compound release profile. Gelatin, PLGA NPs and PLGA-PEG NPs remained stable in deionized water, serum, saline and simulated lung fluid (Gamble’s solution) over 5 days. PLGA-based NPs and natural polymer NPs exhibited highest cytocompatibility and dose-dependent in vitro uptake respectively by human alveolar type-1 epithelial cells. Based on these profiles, gelatin and PLGA NPs were used to encapsulate a) plasmid DNA encoding yellow fluorescent protein (YFP) or b) rhodamine-conjugated erythropoietin (EPO) for inhalational delivery to rats. Following a single inhalation, widespread pulmonary EPO distribution persisted for up to 10 days while increasing YFP expression was observed for at least 7 days for both NPs. The overall results support both PLGA and gelatin NPs as promising carriers for pulmonary protein/DNA delivery. PMID:24512977

  5. Intracellular Protein Delivery for Treating Breast Cancer

    DTIC Science & Technology

    2014-08-01

    486. [12] J.G. Teodoro, D.W. Heilman, A.E. Parker , M.R. Green, Genes Dev. 18 (2004) 1952. [13] A.A.A.M. Danen-Van Oorschot, D.F. Fischer, J.M...53 poses signif eadily aggregate he three-dimen nd has been sho ence of DNA.24 oli, purified from ric protein (Figu onomers 1 and ize aggregation ar

  6. Intracellular Protein Delivery for treating Breast Cancer

    DTIC Science & Technology

    2013-06-01

    is included. Specific Aim 2: Equipping protein nanocapsules with specific cancer cell targeting ligands; Task 3. Preparing and testing of MMP...next tested cytotoxicity of the p53 delivered by using three different cancer cell lines, MDA-MB- 231, SK-BR-3, and BT-474. S-S p53 NC reduced viable...NCs, the selectivity is desirable. 10 Next, to test the selectivity of the LHRH tagged S121F p53 towards only the breast cancer cell lines that

  7. 77 FR 65900 - Agency Information Collection Activities: Delivery Ticket

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-31

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Delivery Ticket... and other Federal agencies to comment on an information collection requirement concerning the Delivery... concerning the following information collection: Title: Delivery Ticket. OMB Number: 1651-0081. Form...

  8. Issues in long-term protein delivery using biodegradable microparticles.

    PubMed

    Ye, Mingli; Kim, Sungwon; Park, Kinam

    2010-09-01

    Recently, a variety of bioactive protein drugs have been available in large quantities as a result of advances in biotechnology. Such availability has prompted development of long-term protein delivery systems. Biodegradable microparticulate systems have been used widely for controlled release of protein drugs for days and months. The most widely used biodegradable polymer has been poly(d,l-lactic-co-glycolic acid) (PLGA). Protein-containing microparticles are usually prepared by the water/oil/water (W/O/W) double emulsion method, and variations of this method, such as solid/oil/water (S/O/W) and water/oil/oil (W/O/O), have also been used. Other methods of preparation include spray drying, ultrasonic atomization, and electrospray methods. The important factors in developing biodegradable microparticles for protein drug delivery are protein release profile (including burst release, duration of release, and extent of release), microparticle size, protein loading, encapsulation efficiency, and bioactivity of the released protein. Many studies used albumin as a model protein, and thus, the bioactivity of the release protein has not been examined. Other studies which utilized enzymes, insulin, erythropoietin, and growth factors have suggested that the right formulation to preserve bioactivity of the loaded protein drug during the processing and storage steps is important. The protein release profiles from various microparticle formulations can be classified into four distinct categories (Types A, B, C, and D). The categories are based on the magnitude of burst release, the extent of protein release, and the protein release kinetics followed by the burst release. The protein loading (i.e., the total amount of protein loaded divided by the total weight of microparticles) in various microparticles is 6.7+/-4.6%, and it ranges from 0.5% to 20.0%. Development of clinically successful long-term protein delivery systems based on biodegradable microparticles requires

  9. Oral delivery of therapeutic proteins and peptides: a review on recent developments.

    PubMed

    Gupta, Sonal; Jain, Arushi; Chakraborty, Mainak; Sahni, Jasjeet K; Ali, Javed; Dang, Shweta

    2013-08-01

    Advent of recombinant technology in protein synthesis has given birth to a new range of biopharmaceuticals. These therapeutic peptides and proteins are now emerging as an imperative part of various treatment protocols especially in the cancer therapeutics. Despite extensive research efforts, oral delivery of therapeutic peptide or protein is still a challenge for pharmaceutical industries and researchers. Number of factors including high proteolytic activity and low pH conditions of gastrointestinal tract act as major barriers in the successful delivery of intact protein/peptide to the targeted site. Low permeability of protein/peptide across the intestinal barrier is also a factor adding to the low bioavailability. Therefore, because of the short circulatory half-life exhibited by peptides in vivo, they need to be administered frequently resulting in increased cost of treatment and low patient compliance. Nano-carrier-based delivery presents an appropriate choice of drug carriers owing to their property to protect proteins from degradation by the low pH conditions in stomach or by the proteolytic enzymes in the gastrointestinal tract. This review focuses on recent aspects and patents on oral delivery of therapeutic proteins and peptides with special emphasis on nano-carrier-based approach.

  10. Gene Delivery into Plant Cells for Recombinant Protein Production

    PubMed Central

    Chen, Qiang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  11. Systemic delivery of recombinant proteins by genetically modified myoblasts

    SciTech Connect

    Barr, E.; Leiden, J.M. )

    1991-12-06

    The ability to stably deliver recombinant proteins to the systemic circulation would facilitate the treatment of a variety of acquired and inherited diseases. To explore the feasibility of the use of genetically engineered myoblasts as a recombinant protein delivery system, stable transfectants of the murine C2C12 myoblast cell line were produced that synthesize and secrete high levels of human growth hormone (hGH) in vitro. Mice injected with hGH-transfected myoblasts had significant levels of hGH in both muscle and serum that were stable for at least 3 weeks after injection. Histological examination of muscles injected with {beta}-galactosidase-expressing C2C12 myoblasts demonstrated that many of the injected cells had fused to form multinucleated myotubes. Thus, genetically engineered myoblasts can be used for the stable delivery of recombinant proteins into the circulation.

  12. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

    PubMed

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Heo, Won Do; Choi, Chulhee

    2016-07-22

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

  13. Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

    NASA Astrophysics Data System (ADS)

    Shah, Dhiral Ashwin

    Intracellular delivery of specific proteins and peptides represents a novel method to influence stem cells for gain-of-function and loss-of-function. Signaling control is vital in stem cells, wherein intricate control of and interplay among critical pathways directs the fate of these cells into either self-renewal or differentiation. The most common route to manipulate cellular function involves the introduction of genetic material such as full-length genes and shRNA into the cell to generate (or prevent formation of) the target protein, and thereby ultimately alter cell function. However, viral-mediated gene delivery may result in relatively slow expression of proteins and prevalence of oncogene insertion into the cell, which can alter cell function in an unpredictable fashion, and non-viral delivery may lead to low efficiency of genetic delivery. For example, the latter case plagues the generation of induced pluripotent stem cells (iPSCs) and hinders their use for in vivo applications. Alternatively, introducing proteins into cells that specifically recognize and influence target proteins, can result in immediate deactivation or activation of key signaling pathways within the cell. In this work, we demonstrate the cellular delivery of functional proteins attached to hydrophobically modified silica (SiNP) nanoparticles to manipulate specifically targeted cell signaling proteins. In the Wnt signaling pathway, we have targeted the phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta) by designing a chimeric protein and delivering it in neural stem cells. Confocal imaging indicates that the SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, indicated by the elevation of beta-catenin levels, and increased transcription of Wnt target genes, such as c-MYC. The results presented here suggest that

  14. Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells

    PubMed Central

    Kwon, Kwang-Chul; Daniell, Henry

    2016-01-01

    Plants cells are now approved by the FDA for cost-effective production of protein drugs (PDs) in large-scale current Good Manufacturing Practice (cGMP) hydroponic growth facilities. In lyophilized plant cells, PDs are stable at ambient temperature for several years, maintaining their folding and efficacy. Upon oral delivery, PDs bioencapsulated in plant cells are protected in the stomach from acids and enzymes but are subsequently released into the gut lumen by microbes that digest the plant cell wall. The large mucosal area of the human intestine offers an ideal system for oral drug delivery. When tags (receptor-binding proteins or cell-penetrating peptides) are fused to PDs, they efficiently cross the intestinal epithelium and are delivered to the circulatory or immune system. Unique tags to deliver PDs to human immune or nonimmune cells have been developed recently. After crossing the epithelium, ubiquitous proteases cleave off tags at engineered sites. PDs are also delivered to the brain or retina by crossing the blood–brain or retinal barriers. This review highlights recent advances in PD delivery to treat Alzheimer's disease, diabetes, hypertension, Gaucher's or ocular diseases, as well as the development of affordable drugs by eliminating prohibitively expensive purification, cold chain and sterile delivery. PMID:27378236

  15. Multi-protein Delivery by Nanodiamonds Promotes Bone Formation

    PubMed Central

    Moore, L.; Gatica, M.; Kim, H.; Osawa, E.; Ho, D.

    2013-01-01

    Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE® for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation. PMID:24045646

  16. Multi-protein delivery by nanodiamonds promotes bone formation.

    PubMed

    Moore, L; Gatica, M; Kim, H; Osawa, E; Ho, D

    2013-11-01

    Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE(®) for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation.

  17. Effective polymer adjuvants for sustained delivery of protein subunit vaccines.

    PubMed

    Adams, Justin R; Haughney, Shannon L; Mallapragada, Surya K

    2015-03-01

    We have synthesized thermogelling cationic amphiphilic pentablock copolymers that have the potential to act as injectable vaccine carriers and adjuvants that can simultaneously provide sustained delivery and enhance the immunogenicity of released antigen. While these pentablock copolymers have shown efficacy in DNA delivery in past studies, the ability to deliver both DNA and protein for subunit vaccines using the same polymeric carrier can provide greater flexibility and efficacy. We demonstrate the ability of these pentablock copolymers, and the parent triblock Pluronic copolymers to slowly release structurally intact and antigenically stable protein antigens in vitro, create an antigen depot through long-term injection-site persistence and enhance the in vivo immune response to these antigens. We show release of the model protein antigen ovalbumin in vitro from the thermogelling block copolymers with the primary, secondary and tertiary structures of the released protein unchanged compared to the native protein, and its antigenicity preserved upon release. The block copolymers form a gel at physiological temperatures that serves as an antigenic depot and persists in vivo at the site of injection for over 50days. The pentablock copolymers show a significant fivefold enhancement in the immune response compared to soluble protein alone, even 6weeks after the administration, based on measurement of antibody titers. These results demonstrate the potential of these block copolymers hydrogels to persist for several weeks and sustain the release of antigen with minimal effects on protein stability and antigenicity; and their ability to be used simultaneously as a sustained delivery device as well as a subunit vaccine adjuvant platform.

  18. Bifunctional chimeric fusion proteins engineered for DNA delivery: Optimization of the protein to DNA ratio

    PubMed Central

    Gao, Shan; Simon, Melissa J.; Morrison, Barclay; Banta, Scott

    2009-01-01

    Background Cell penetrating peptides (CPPs) have been used to deliver nucleotide-based therapeutics to cells, but this approach has produced mixed results. Ionic interactions and covalent bonds between the CPPs and the cargos may inhibit the effectiveness of the CPPs or interfere with the bioactivity of the cargos. Methods We have created a bifunctional chimeric protein that binds DNA using the p50 domain of the NF-κB transcription factor and is functionalized for delivery with the TAT CPP. The green fluorescent protein (GFP) has been incorporated for tracking delivery. The new chimeric protein, p50-GFP-TAT, was compared to p50-GFP, GFP-TAT and GFP as controls for the ability to transduce PC12 cells with and without oligonucleotide cargos. Results The p50-GFP-TAT construct can deliver 30bp and 293bp oligonucleotides to PC12 cells with an optimal ratio of 1.89 protein molecules per base pair of DNA length. This correlation was validated through the delivery of a fluorescent protein transgene encoded in a plasmid to PC12 cells. Conclusion Self-assembling CPP-based bifunctional fusion proteins can be engineered for the non-viral delivery of nucleotide-based cargos to mammalian cells. General significance This work represents an important step forward in the rational design of protein-based systems for the delivery of macromolecular cargos. PMID:19402206

  19. Evaluation of interstitial protein delivery in multicellular layers model.

    PubMed

    Kim, Soo-Yeon; Kim, Tae Hyung; Choi, Jong Hoon; Lee, Kang Choon; Park, Ki Dong; Lee, Seung-Jin; Kuh, Hyo-Jeong

    2012-03-01

    The limited efficacy of anticancer protein drugs is related to their poor distribution in tumor tissue. We examined interstitial delivery of four model proteins of different molecular size and bioaffinity in multicellular layers (MCL) of human cancer cells. Model proteins were tumor necrosis factor-related apoptosis-including ligand (TRAIL), cetuximab, RNase A, and IgG. MCLs were cultured in Transwell inserts, exposed to drugs, then cryo-sectioned for image acquisition using fluorescence microscopy (fluorescent dye-labeled TRAIL, RNase A, IgG) or immunohistochemistry (cetuximab). TRAIL and cetuximab showed partial penetration into MCLs, whereas RNase A and IgG showed insignificant penetration. At 10-fold higher dose, a significant increase in penetration was observed for IgG only, while cetuximab showed an intense accumulation limited to the front layers. PEGylated TRAIL and RNase A formulated in a heparin-Pluronic (HP) nanogel showed significantly improved penetration attributable to increased stability and extracellular matrix binding, respectively. IgG penetration was significantly enhanced with paclitaxel pretreatment as a penetration enhancer. The present study suggests that MCL culture may be useful in evaluation of protein delivery in the tumor interstitium. Four model proteins showed limited interstitial penetration in MCL cultures. Bioaffinity, rather than molecular size, seems to have a positive effect on tissue penetration, although high binding affinity may lead to sequestration in the front cell layers. Polymer conjugation and nanoformulation, such as PEGylation and HP nanogel, or use of penetration enhancers are potential strategies to increase interstitial delivery of anticancer protein drugs.

  20. Remotely activated protein-producing nanoparticles.

    PubMed

    Schroeder, Avi; Goldberg, Michael S; Kastrup, Christian; Wang, Yingxia; Jiang, Shan; Joseph, Brian J; Levins, Christopher G; Kannan, Sneha T; Langer, Robert; Anderson, Daniel G

    2012-06-13

    The development of responsive nanomaterials, nanoscale systems that actively respond to stimuli, is one general goal of nanotechnology. Here we develop nanoparticles that can be controllably triggered to synthesize proteins. The nanoparticles consist of lipid vesicles filled with the cellular machinery responsible for transcription and translation, including amino acids, ribosomes, and DNA caged with a photolabile protecting group. These particles served as nanofactories capable of producing proteins including green fluorescent protein (GFP) and enzymatically active luciferase. In vitro and in vivo, protein synthesis was spatially and temporally controllable, and could be initiated by irradiating micrometer-scale regions on the time scale of milliseconds. The ability to control protein synthesis inside nanomaterials may enable new strategies to facilitate the study of orthogonal proteins in a confined environment and for remotely activated drug delivery.

  1. Photo-synthesis of protein-based nanoparticles and the application in drug delivery

    SciTech Connect

    Xie, Jinbing; Wang, Hongyang; Cao, Yi; Qin, Meng Wang, Wei

    2015-07-15

    Recently, protein-based nanoparticles as drug delivery systems have attracted great interests due to the excellent behavior of high biocompatibility and biodegradability, and low toxicity. However, the synthesis techniques are generally costly, chemical reagents introduced, and especially present difficulties in producing homogeneous monodispersed nanoparticles. Here, we introduce a novel physical method to synthesize protein nanoparticles which can be accomplished under physiological condition only through ultraviolet (UV) illumination. By accurately adjusting the intensity and illumination time of UV light, disulfide bonds in proteins can be selectively reduced and the subsequent self-assembly process can be well controlled. Importantly, the co-assembly can also be dominated when the proteins mixed with either anti-cancer drugs, siRNA, or active targeting molecules. Both in vitro and in vivo experiments indicate that our synthesized protein–drug nanoparticles (drug-loading content and encapsulation efficiency being ca. 8.2% and 70%, respectively) not only possess the capability of traditional drug delivery systems (DDS), but also have a greater drug delivery efficiency to the tumor sites and a better inhibition of tumor growth (only 35% of volume comparing to the natural growing state), indicating it being a novel drug delivery system in tumor therapy.

  2. Secretion, delivery and function of oomycete effector proteins.

    PubMed

    Wawra, Stephan; Belmonte, Rodrigo; Löbach, Lars; Saraiva, Marcia; Willems, Ariane; van West, Pieter

    2012-12-01

    Oomycetes are responsible for multi-billion dollar damages in aquaculture, agriculture and forestry. One common strategy they share with most cellular disease agents is the secretion of effector proteins. Effectors are molecules that change host physiology by initiating and allowing an infection to develop. Oomycetes secrete both extracellular and intracellular effectors. Studying secretion, delivery and function of effectors will hopefully lead to alternative control measures, which is much needed as several chemicals to control plant and animal pathogenic oomycetes cannot be used anymore; due to resistance in the host, or because the control measures have been prohibited as a result of toxicity to the environment and/or consumers. Here the latest findings on oomycete effector secretion, delivery and function are discussed.

  3. Characterization of protein rheology and delivery forces for combination products.

    PubMed

    Rathore, Nitin; Pranay, Pratik; Bernacki, Joseph; Eu, Bruce; Ji, Wenchang; Walls, Ed

    2012-12-01

    Characterization of a protein-device combination product over a wide range of operating parameters defined by end-user requirements is critical for developing a product presentation that is convenient for patient use. In addition to the device components, several product attributes, such as product rheology and product-container interactions, govern the functionality of a delivery system. This article presents results from a characterization study conducted for a high-concentration antibody product in a prefilled syringe. Analytical models are used to study the rheological behavior and to estimate delivery forces over a broad design space comprising temperature, concentration, and shear stress. Data suggest that high-viscosity products may exhibit significant shear thinning under the shear rates encountered under desired injection times.

  4. Long acting porous microparticle for pulmonary protein delivery.

    PubMed

    Kwon, Min Jung; Bae, Jun Ho; Kim, Jung Ju; Na, Kun; Lee, Eun Seong

    2007-03-21

    This study investigated the porous-microparticle (PM) with low mass density and large size for pulmonary drug delivery. PM was prepared by the water-in-oil-in-water (W(1)/O/W(2)) multi-emulsion method with cyclodextrin derivative as a porogen and a stabilizer of peptide drugs. Herein, sucrose ethyl acetate (SAIB) was incorporated in PM for long acting protein release. In vitro release studies, the rapid release rate of proteins from PM was reduced due to the high viscosity of the added SAIB. As a result, BSA release from PM continued up to 7 days. This result suggests that PM having sustained release characteristics may be successfully applied for long-term pulmonary administration of protein or peptide drug. In addition, it is expected that these particles arrive at a deep lung epithelium due to low density (high porosity) and limit macrophage recognition because of big particle size (more than 5 microm).

  5. Review on the delivery of steroids by carrier proteins.

    PubMed

    Chanphai, P; Vesper, A R; Bariyanga, J; Bérubé, G; Tajmir-Riahi, H A

    2016-08-01

    Due to the poor solubility of steroids in aqueous solution, delivery of these biomaterials is of major biomedical importance. We have reviewed the conjugation of testosterone and it aliphatic dimer and aromatic dimer with several carrier proteins, human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (b-LG) in aqueous solution at physiological pH. The results of multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling were compared here. Steroid-protein bindings are via hydrophilic and H-bonding contacts. HSA forms more stable conjugate than BSA and b-LG. The stability of steroid-protein conjugates is testosterone>dimer-aromatic>dimer-aliphatic. Encapsulation of steroids by protein is shown by TEM images. Modeling showed the presence of H-bonding, which stabilized testosterone-protein complexes with the free binding energy of -12.95 for HSA and -11.55 for BSA and -8.92kcal/mol for b-LG conjugates. Steroid conjugation induced major perturbations of serum protein conformations. Serum proteins can transport steroids to the target molecules.

  6. Light activated liposomes: Functionality and prospects in ocular drug delivery.

    PubMed

    Lajunen, Tatu; Nurmi, Riikka; Kontturi, Leena; Viitala, Lauri; Yliperttula, Marjo; Murtomäki, Lasse; Urtti, Arto

    2016-12-28

    Ocular drug delivery, especially to the retina and choroid, is a major challenge in drug development. Liposome technology may be useful in ophthalmology in enabling new routes of delivery, prolongation of drug action and intracellular drug delivery, but drug release from the liposomes should be controlled. For that purpose, light activation may be an approach to release drug at specified time and site in the eye. Technical advances have been made in the field of light activated drug release, particularly indocyanine green loaded liposomes are a promising approach with safe materials and effective light triggered release of small and large molecules. This review discusses the liposomal drug delivery with light activated systems in the context of ophthalmic drug delivery challenges.

  7. Integrating Protein Engineering and Bioorthogonal Click Conjugation for Extracellular Vesicle Modulation and Intracellular Delivery

    PubMed Central

    Wang, Ming; Altinoglu, Sarah; Takeda, Yuji S.; Xu, Qiaobing

    2015-01-01

    Exosomes are small, cell-secreted vesicles that transfer proteins and genetic information between cells. This intercellular transmission regulates many physiological and pathological processes. Therefore, exosomes have emerged as novel biomarkers for disease diagnosis and as nanocarriers for drug delivery. Here, we report an easy-to-adapt and highly versatile methodology to modulate exosome composition and conjugate exosomes for intracellular delivery. Our strategy combines the metabolic labeling of newly synthesized proteins or glycan/glycoproteins of exosome-secreting cells with active azides and bioorthogonal click conjugation to modify and functionalize the exosomes. The azide-integrated can be conjugated to a variety of small molecules and proteins and can efficiently deliver conjugates into cells. The metabolic engineering of exosomes diversifies the chemistry of exosomes and expands the functions that can be introduced into exosomes, providing novel, powerful tools to study the roles of exosomes in biology and expand the biomedical potential of exosomes. PMID:26529317

  8. Carrier Mediated Systemic Delivery of Protein and Peptide Therapeutics.

    PubMed

    Zaman, Rahela; Othman, Iekhan; Chowdhury, Ezharul Hoque

    2016-01-01

    Over the last few decades proteins and peptide therapeutics have occupied an enormous fraction of pharmaceutical industry. Despite their high potential as therapeutics, the big challenge often encountered is the effective administration and bioavailability of protein therapeutics in vivo system. Peptide molecules are well known for their in vivo short half-lives. In addition, due to high molecular weight and susceptibility to enzymatic degradation, often it is not easy to administer peptides and proteins orally or through any other noninvasive routes. Conventional drug management system often demands for frequent and regular interval intravenous/subcutaneous administration, which decreases overall patient compliance and increases chances of side-effects related to dose-fluctuation in systemic circulation. A controlled mode of delivery system could address all these short-comings at a time. Therefore, long-acting sustained release formulations for both invasive and noninvasive routes are under rigorous study currently. Long-acting formulations through invasive routes can address patient compliance and dose-fluctuation issues by less frequent administration. Also, any new route of administration other than invasive routes will address cost-effectiveness of the therapeutic by lessening the need to deal with health professional and health care facility. Although a vast number of studies are dealing with novel drug delivery systems, till now only a handful of controlled release formulations for proteins and peptides have been approved by FDA. This study therefore focuses on current and perspective controlled release formulations of existing and novel protein/peptide therapeutics via conventional invasive routes as well as potential novel non-invasive routes of administration, e.g., oral, buccal, sublingual, nasal, ocular, rectal, vaginal and pulmonary.

  9. Sequence-defined shuttles for targeted nucleic acid and protein delivery.

    PubMed

    Röder, Ruth; Wagner, Ernst

    2014-01-01

    Molecular medicine opens into a space of novel specific therapeutic agents: intracellularly active drugs such as peptides, proteins or nucleic acids, which are not able to cross cell membranes and enter the intracellular space on their own. Through the development of cell-targeted shuttles for specific delivery, this restriction in delivery has the potential to be converted into an advantage. On the one hand, due to the multiple extra- and intracellular barriers, such carrier systems need to be multifunctional. On the other hand, they must be precise and reproducibly manufactured due to pharmaceutical reasons. Here we review the design of precise sequence-defined delivery carriers, including solid-phase synthesized peptides and nonpeptidic oligomers, or nucleotide-based carriers such as aptamers and origami nanoboxes.

  10. Cross-Linked Protein Crystals for Vaccine Delivery

    NASA Astrophysics Data System (ADS)

    St. Clair, Nancy; Shenoy, Bhami; Jacob, Lawrence D.; Margolin, Alexey L.

    1999-08-01

    The progress toward subunit vaccines has been limited by their poor immunogenicity and limited stability. To enhance the immune response, subunit vaccines universally require improved adjuvants and delivery vehicles. In the present paper, we propose the use of cross-linked protein crystals (CLPCs) as antigens. We compare the immunogenicity of CLPCs of human serum albumin with that of soluble protein and conclude that there are marked differences in the immune response to the different forms of human serum albumin. Relative to the soluble protein, crystalline forms induce and sustain over almost a 6-month study a 6- to 10-fold increase in antibody titer for highly cross-linked crystals and an approximately 30-fold increase for lightly cross-linked crystals. We hypothesize that the depot effect, the particulate structure of CLPCs, and highly repetitive nature of protein crystals may play roles in the enhanced production of circulating antibodies. Several features of CLPCs, such as their remarkable stability, purity, biodegradability, and ease of manufacturing, make them highly attractive for vaccine formulations. This work paves the way for a systematic study of protein crystallinity and cross-linking on enhancement of humoral and T cell responses.

  11. Cross-linked protein crystals for vaccine delivery

    PubMed Central

    St. Clair, Nancy; Shenoy, Bhami; Jacob, Lawrence D.; Margolin, Alexey L.

    1999-01-01

    The progress toward subunit vaccines has been limited by their poor immunogenicity and limited stability. To enhance the immune response, subunit vaccines universally require improved adjuvants and delivery vehicles. In the present paper, we propose the use of cross-linked protein crystals (CLPCs) as antigens. We compare the immunogenicity of CLPCs of human serum albumin with that of soluble protein and conclude that there are marked differences in the immune response to the different forms of human serum albumin. Relative to the soluble protein, crystalline forms induce and sustain over almost a 6-month study a 6- to 10-fold increase in antibody titer for highly cross-linked crystals and an approximately 30-fold increase for lightly cross-linked crystals. We hypothesize that the depot effect, the particulate structure of CLPCs, and highly repetitive nature of protein crystals may play roles in the enhanced production of circulating antibodies. Several features of CLPCs, such as their remarkable stability, purity, biodegradability, and ease of manufacturing, make them highly attractive for vaccine formulations. This work paves the way for a systematic study of protein crystallinity and cross-linking on enhancement of humoral and T cell responses. PMID:10449716

  12. On prilled Nanotubes-in-Microgel Oral Systems for protein delivery.

    PubMed

    de Kruif, Jan Kendall; Ledergerber, Gisela; Garofalo, Carla; Fasler-Kan, Elizaveta; Kuentz, Martin

    2016-04-01

    Newly discovered active macromolecules are highly promising for therapy, but poor bioavailability hinders their oral use. Microencapsulation approaches, such as protein prilling into microspheres, may enable protection from gastrointestinal (GI) enzymatic degradation. This would increase bioavailability mainly for local delivery to GI lumen or mucosa. This work's purpose was to design a novel architecture, namely a Nanotubes-in-Microgel Oral System, by prilling for protein delivery. Halloysite nanotubes (HNT) were selected as orally acceptable clay particles and their lumen was enlarged by alkaline etching. This chemical modification increased the luminal volume to a mean of 216.3 μL g(-1) (+40.8%). After loading albumin as model drug, the HNT were entrapped in microgels by prilling. The formation of Nanoparticles-in-Microsphere Oral System (NiMOS) yielded entrapment efficiencies up to 63.2%. NiMOS shape was spherical to toroidal, with a diameter smaller than 320 μm. Release profiles depended largely on the employed system and HNT type. Protein stability was determined throughout prilling and after in vitro enzymatic degradation. Prilling did not harm protein structure, and NiMOS demonstrated higher enzymatic protection than pure nanotubes or microgels, since up to 82% of BSA remained unscathed after in vitro digestion. Therefore, prilled NiMOS was shown to be a promising and flexible multi-compartment system for oral (local) macromolecular delivery.

  13. Smart polymer based delivery systems for peptides and proteins.

    PubMed

    Al-Tahami, Khaled; Singh, Jagdish

    2007-01-01

    Biodegradable polymeric systems represent promising means for delivering many bioactive agents, including peptide and protein drugs. The importance of these systems grew with the advancement in the understanding of peptide and protein pharmacology as well as the ability to mass-produce these compounds. Some polymers undergo sol-gel transition once administered. In situ gel formation happens in response to one or a combination of two or more stimuli. These stimuli include UV-irradiation, pH change, temperature change, and solvent exchange. These smart polymeric systems have several advantages over conventional methods, such as ease of manufacturing, ease of administration, biodegradability, and the ability to alter release profiles of the incorporated agents. In the past few years, an increasing number of in situ gel-forming systems have been investigated and many patents for their use in various biomedical applications, including drug delivery, have been reported. In this article, we introduce the different strategies that have been developed and patented for the use of smart polymers in delivering peptide and protein drugs. The advantage, disadvantages, possibilities, and limitations of each of the smart polymer systems have been discussed.

  14. Delivery of molecules into cells using carbon nanoparticles activated by femtosecond laser pulses.

    PubMed

    Chakravarty, Prerona; Qian, Wei; El-Sayed, Mostafa A; Prausnitz, Mark R

    2010-08-01

    A major barrier to drug and gene delivery is crossing the cell's plasma membrane. Physical forces applied to cells via electroporation, ultrasound and laser irradiation generate nanoscale holes in the plasma membrane for direct delivery of drugs into the cytoplasm. Inspired by previous work showing that laser excitation of carbon nanoparticles can drive the carbon-steam reaction to generate highly controlled shock waves, we show that carbon black nanoparticles activated by femtosecond laser pulses can facilitate the delivery of small molecules, proteins and DNA into two types of cells. Our initial results suggest that interaction between the laser energy and carbon black nanoparticles may generate photoacoustic forces by chemical reaction to create transient holes in the membrane for intracellular delivery.

  15. Self-assembling polymeric nanoparticles for enhanced intra-articular anti-inflammatory protein delivery

    NASA Astrophysics Data System (ADS)

    Whitmire, Rachel Elisabeth

    Osteoarthritis (OA) affects 26 million Americans, or approximately 14% of the adult population. The incidence of OA is predicted to dramatically increase in the next 20 years as the US grows older and the rate of obesity continues to increase. There are currently no clinical interventions that cure OA. Current biomaterial delivery systems exhibit several limitations. First, most drug-delivery particles are hydrophobic, which is not optimal for hydrophilic protein encapsulation. Second, hydrophobic particles, such as PLGA, could cause wear damage to the already-fragile OA cartilage structure. Additionally, these particles usually suffer from non-specific protein adsorption, which causes increased phagocytosis and can lead to increased inflammation. New therapies that increase the effectiveness of OA treatments or reverse OA disease progression will greatly decrease the economic costs and individual pain associated with this disease. The goal of this thesis was to develop a new drug-delivering material to deliver anti-inflammatory protein for treating OA. Our central hypothesis for this work is that a controlled release/presentation system will more effectively deliver anti-inflammatory protein therapies to the OA joint. The primary goal of this work was to synthesize a block copolymer that could self-assemble into injectable, sub-micron-scale particles and would allow an anti-inflammatory protein, IL-1ra, to be tethered to its surface for efficient protein delivery. The block copolymer incorporated an oligo-ethylene monomer for tissue compatibility and non-fouling behavior, a 4-nitrophenol group for efficient protein tethering, and cyclohexyl methacrylate, a hydrophobic monomer, for particle stability. We engineered the copolymer and tested it in both in vitro culture experiments and an in vivo model to evaluate protein retention in the knee joint. The rationale for this project was that the rational design and synthesis of a new drug- and protein

  16. Therapeutics and carriers: the dual role of proteins in nanoparticles for ocular delivery.

    PubMed

    Pescina, Silvia; Sonvico, Fabio; Santi, Patrizia; Nicoli, Sara

    2015-01-01

    Blindness and visual impairment affect millions of people worldwide and have a very important impact on patients quality of life. Proteins and peptides represent nowadays an important therapeutic tool for the treatment of ocular diseases but, despite their potential, have significant limitations, as the administration of protein-based pharmaceuticals represents a real challenge. Moreover, administration of ocular medications is difficult due to the peculiar structure of this organ and the presence of numerous barriers protecting the eye inner structure. Nanoencapsulation of peptides and proteins presents a number of advantages for their ocular delivery since it can protect the drug from metabolic activity, control and sustain the release and increase drug bioavailability after topical or intravitreal administration. In fact, nanoparticulate formulations are contributing to overcome ocular barriers, such as the corneal or the blood-retinal barrier, improve the residence time in the eye, increase local drug level, reduce the drug dosage and showing improved performance when compared to conventional formulations. Besides, proteins have also been proposed for the preparation of nanocarriers intended for ophthalmic administration, since they are highly biocompatible, biodegradable and easily modified to link surface ligands. The present review focuses the attention on the use of proteins in ocular drug delivery nanotechnology: their dual role as both therapeutics and carriers has been critically evaluated and discussed.

  17. Polysaccharides-based polyelectrolyte nanoparticles as protein drugs delivery system

    NASA Astrophysics Data System (ADS)

    Shu, Shujun; Sun, Lei; Zhang, Xinge; Wu, Zhongming; Wang, Zhen; Li, Chaoxing

    2011-09-01

    Polysaccharides-based nanoparticles were prepared by synthesized quaternized chitosan and dextran sulfate through simple ionic-gelation self-assembled method. Introduction of quaternized groups was intended to increase water solubility of chitosan and make the nanoparticles have broader pH sensitive range which can remain more stable in physiological pH and decrease the loss of protein drugs caused by the gastric cavity. The load of BSA was affected by molecular parameter, i.e., degree of substitution, and average molecular weight of quaternized chitosan, as well as concentration of BSA. Fast release occurred in phosphate buffer solution (pH 7.4) while the release was slow in hydrochloric acid (pH 1.4). The drug release mechanism is Fickian diffusion through release kinetics analysis. Cell uptake demonstrated nanoparicles can internalize into Caco-2 cells, which suggested that nanoparticles had good biocompatibility. No significant conformation change was noted for the released BSA in comparison with native BSA using circular dichroism spectroscopy. This kind of novel composite nanoparticles may be a promising delivery system for oral protein and peptide drugs.

  18. Recent Advances in Protein and Peptide Drug Delivery: A Special Emphasis on Polymeric Nanoparticles

    PubMed Central

    Patel, Ashaben; Patel, Mitesh; Yang, Xiaoyan; Mitra, Ashim K.

    2015-01-01

    Proteins and peptides are widely indicated in many diseased states. Parenteral route is the most commonly employed method of administration for therapeutic proteins and peptides. However, requirement of frequent injections due to short in vivo half-life results in poor patient compliance. Non-invasive drug delivery routes such as nasal, transdermal, pulmonary, and oral offer several advantages over parenteral administration. Intrinsic physicochemical properties and low permeability across biological membrane limit protein delivery via non-invasive routes. One of the strategies to improve protein and peptide absorption is by delivering through nanostructured delivery carriers. Among nanocarriers, polymeric nanoparticles (NPs) have demonstrated significant advantages over other delivery systems. This article summarizes the application of polymeric NPs for protein and peptide drug delivery following oral, nasal, pulmonary, parenteral, transdermal, and ocular administrations. PMID:25106908

  19. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

    PubMed

    Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

    2015-09-01

    Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.

  20. Smart Nanostructures for Cargo Delivery: Uncaging and Activating by Light.

    PubMed

    Karimi, Mahdi; Sahandi Zangabad, Parham; Baghaee-Ravari, Soodeh; Ghazadeh, Mehdi; Mirshekari, Hamid; Hamblin, Michael R

    2017-04-05

    Nanotechnology has begun to play a remarkable role in various fields of science and technology. In biomedical applications, nanoparticles have opened new horizons, especially for biosensing, targeted delivery of therapeutics, and so forth. Among drug delivery systems (DDSs), smart nanocarriers that respond to specific stimuli in their environment represent a growing field. Nanoplatforms that can be activated by an external application of light can be used for a wide variety of photoactivated therapies, especially light-triggered DDSs, relying on photoisomerization, photo-cross-linking/un-cross-linking, photoreduction, and so forth. In addition, light activation has potential in photodynamic therapy, photothermal therapy, radiotherapy, protected delivery of bioactive moieties, anticancer drug delivery systems, and theranostics (i.e., real-time monitoring and tracking combined with a therapeutic action to different diseases sites and organs). Combinations of these approaches can lead to enhanced and synergistic therapies, employing light as a trigger or for activation. Nonlinear light absorption mechanisms such as two-photon absorption and photon upconversion have been employed in the design of light-responsive DDSs. The integration of a light stimulus into dual/multiresponsive nanocarriers can provide spatiotemporal controlled delivery and release of therapeutic agents, targeted and controlled nanosystems, combined delivery of two or more agents, their on-demand release under specific conditions, and so forth. Overall, light-activated nanomedicines and DDSs are expected to provide more effective therapies against serious diseases such as cancers, inflammation, infections, and cardiovascular disease with reduced side effects and will open new doors toward the treatment of patients worldwide.

  1. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein–protein interaction module

    PubMed Central

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R.; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Do Heo, Won; Choi, Chulhee

    2016-01-01

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named ‘exosomes for protein loading via optically reversible protein–protein interactions' (EXPLORs). By integrating a reversible protein–protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. PMID:27447450

  2. Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

    SciTech Connect

    Danyal, Karamatullah; Shaw, Sudipta; Page, Taylor R.; Duval, Simon; Horitani, Masaki; Marts, Amy R.; Lukoyanov, Dmitriy; Dean, Dennis R.; Raugei, Simone; Hoffman, Brian M.; Seefeldt, Lance C.; Antony, Edwin

    2016-10-04

    Mo-dependent nitrogenase catalyzes the biological reduction of atmospheric dinitrogen (N2) to two ammonia (NH3) molecules, through the action of two component proteins, the MoFe protein and the Fe protein. The catalytic MoFe protein is a symmetric dimer of αβ units, each of which contains one active site FeMo-co (FeMo-co; [7Fe-9S-Mo-C-homocitrate]) and an electron-carrier P cluster. Each half of the nitrogenase ternary complex, in which one Fe protein with two bound ATP molecules has bound to each MoFe protein αβ unit, undergoes an electron transfer (ET) cycle with ET from a Fe protein [4Fe-4S] cluster into its αβ unit followed by the hydrolysis of the two ATP to two ADP and two Pi. The prevailing model holds that each αβ unit of the MoFe protein functions independently. We now report that the ET cycle exhibits negative cooperativity, with ET and ATP hydrolysis in one half of the ternary nitrogenase complex suppressing these processes in the other half. The observed ET, ATP hydrolysis, and Pi release behavior is captured in a global fit to a two-branch negative-cooperativity kinetic model. A possible mechanism for communication between the two halves of MoFe protein is suggested by normal mode analysis showing correlated and anti-correlated motions between the two nitrogenase αβ halves. EPR spectra furthermore show small differences between those of resting-state and singly-reduced MoFe protein that can be attributed to an intra-complex allosteric perturbation of the resting-state FeMo-co in one αβ unit by reduction of FeMo-co in the other. This work is supported as a part of the Biological and Electron Transfer and Catalysis (EFRC) program, an Energy Frontiers Research Center funded by the US Department of Energy (DOE), Office of Science (DE-SC0012518) to LCS, by National Institutes of Health (NIH) grants HL 63203 and GM 111097to BMH, and R15GM110671 to EA, and by the Division of Chemical Sciences, Geosciences, and Bio-Sciences, DOE to SR. The protein

  3. 78 FR 1219 - Agency Information Collection Activities: Delivery Ticket

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-08

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Delivery Ticket AGENCY: U.S. Customs and Border Protection, Department of Homeland Security. ACTION: 30-Day notice and request for comments; Extension of an existing information collection. SUMMARY: U.S. Customs and...

  4. In vitro and in vivo delivery of therapeutic proteins using cell penetrating peptides.

    PubMed

    Bolhassani, Azam; Jafarzade, Behnaz Sadat; Mardani, Golnaz

    2017-01-01

    The failure of proteins to penetrate mammalian cells or target tumor cells restricts their value as therapeutic tools in a variety of diseases such as cancers. Recently, protein transduction domains (PTDs) or cell penetrating peptides (CPPs) have been shown to promote the delivery of therapeutic proteins or peptides into live cells. The successful delivery of proteins mainly depends on their physicochemical properties. Although, linear cell penetrating peptides are one of the most effective delivery vehicles; but currently, cyclic CPPs has been developed to potently transport bioactive full-length proteins into cells. Up to now, several small protein transduction domains from viral proteins including Tat or VP22 could be fused to other peptides or proteins to entry them in various cell types at a dose-dependent approach. A major disadvantage of PTD-fusion proteins is primary uptake into endosomal vesicles leading to inefficient release of the fusion proteins into the cytosol. Recently, non-covalent complex formation (Chariot) between proteins and CPPs has attracted a special interest to overcome some delivery limitations (e.g., toxicity). Many preclinical and clinical trials of CPP-based delivery are currently under evaluation. Generally, development of more efficient protein transduction domains would significantly increase the potency of protein therapeutics. Moreover, the synergistic or combined effects of CPPs with other delivery systems for protein/peptide drug delivery would promote their therapeutic effects in cancer and other diseases. In this review, we will describe the functions and implications of CPPs for delivering the therapeutic proteins or peptides in preclinical and clinical studies.

  5. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

    NASA Astrophysics Data System (ADS)

    Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2015-05-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (A549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment.

  6. In vivo protein transduction: delivery of PEP-1-SOD1 fusion protein into myocardium efficiently protects against ischemic insult.

    PubMed

    Zhang, You-En; Wang, Jia-Ning; Tang, Jun-Ming; Guo, Ling-Yun; Yang, Jian-Ye; Huang, Yong-Zhang; Tan, Yan; Fu, Shou-Zhi; Kong, Xia; Zheng, Fei

    2009-02-28

    Myocardial ischemia-reperfusion injury is a medical problem occurring as damage to the myocardium following blood flow restoration after a critical period of coronary occlusion. Oxygen free radicals (OFR) are implicated in reperfusion injury after myocardial ischemia. The antioxidant enzyme, Cu, Zn-superoxide dismutase (Cu, Zn-SOD, also called SOD1) is one of the major means by which cells counteract the deleterious effects of OFR after ischemia. Recently, we reported that a PEP-1-SOD1 fusion protein was efficiently delivered into cultured cells and isolated rat hearts with ischemia-reperfusion injury. In the present study, we investigated the protective effects of the PEP-1-SOD1 fusion protein after ischemic insult. Immunofluorescecnce analysis revealed that the expressed and purified PEP-1-SOD1 fusion protein injected into rat tail veins was efficiently transduced into the myocardium with its native protein structure intact. When injected into Sprague-Dawley rat tail veins, the PEP-1- SOD1 fusion protein significantly attenuated myocardial ischemia-reperfusion damage; characterized by improving cardiac function of the left ventricle, decreasing infarct size, reducing the level of malondialdehyde (MDA), decreasing the release of creatine kinase (CK) and lactate dehydrogenase (LDH), and relieving cardiomyocyte apoptosis. These results suggest that the biologically active intact forms of PEP-1-SOD1 fusion protein will provide an efficient strategy for therapeutic delivery in various diseases related to SOD1 or to OFR.

  7. Calcium alginate/dextran methacrylate IPN beads as protecting carriers for protein delivery.

    PubMed

    D'Arrigo, Giorgia; Di Meo, Chiara; Pescosolido, Laura; Coviello, Tommasina; Alhaique, Franco; Matricardi, Pietro

    2012-07-01

    In the present study, mechanical and protein delivery properties of a system based on the interpenetration of calcium-alginate (Ca-Alg) and dextran-methacrylate (Dex-MA) networks are shown. Interpenetrated hydrogels beads were prepared by means of the alginate chains crosslinking with calcium ions, followed by the exposure to UV light that allows the Dex-MA network formation. Optical microscope analysis showed an average diameter of the IPN beads (Ca-Alg/Dex-MA) of 2 mm. This dimension was smaller than that of Ca-Alg beads because of the Dex-MA presence. Moreover, the strength of the IPN beads, and of their corresponding hydrogels, was influenced by the Dex-MA concentration and the crosslinking time. Model proteins (BSA and HRP) were successfully entrapped into the beads and released at a controlled rate, modulated by changing the Dex-MA concentration. The enzymatic activity of HRP released from the beads was maintained. These novel IPN beads have great potential as protein delivery system.

  8. Delivery of therapeutic peptides and proteins to the CNS.

    PubMed

    Salameh, Therese S; Banks, William A

    2014-01-01

    Peptides and proteins have potent effects on the brain after their peripheral administration, suggesting that they may be good substrates for the development of CNS therapeutics. Major hurdles to such development include their relation to the blood-brain barrier (BBB) and poor pharmacokinetics. Some peptides cross the BBB by transendothelial diffusion and others cross in the blood-to-brain direction by saturable transporters. Some regulatory proteins are also transported across the BBB and antibodies can enter the CNS via the extracellular pathways. Glycoproteins and some antibody fragments can be taken up and cross the BBB by mechanisms related to adsorptive endocytosis/transcytosis. Many peptides and proteins are transported out of the CNS by saturable efflux systems and enzymatic activity in the blood, CNS, or BBB are substantial barriers to others. Both influx and efflux transporters are altered by various substances and in disease states. Strategies that manipulate these interactions between the BBB and peptides and proteins provide many opportunities for the development of therapeutics. Such strategies include increasing transendothelial diffusion of small peptides, upregulation of saturable influx transporters with allosteric regulators and other posttranslational means, use of vectors and other Trojan horse strategies, inhibition of efflux transporters including with antisense molecules, and improvement in pharmacokinetic parameters to overcome short half-lives, tissue sequestration, and enzymatic degradation.

  9. Separating proteins with activated carbon.

    PubMed

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon.

  10. Catalytic Mesoporous Janus Nanomotors for Active Cargo Delivery

    PubMed Central

    2015-01-01

    We report on the synergy between catalytic propulsion and mesoporous silica nanoparticles (MSNPs) for the design of Janus nanomotors as active cargo delivery systems with sizes <100 nm (40, 65, and 90 nm). The Janus asymmetry of the nanomotors is given by electron beam (e-beam) deposition of a very thin platinum (2 nm) layer on MSNPs. The chemically powered Janus nanomotors present active diffusion at low H2O2 fuel concentration (i.e., <3 wt %). Their apparent diffusion coefficient is enhanced up to 100% compared to their Brownian motion. Due to their mesoporous architecture and small dimensions, they can load cargo molecules in large quantity and serve as active nanocarriers for directed cargo delivery on a chip. PMID:25844893

  11. Topical Delivery of Protein and Peptide Using Novel Cell Penetrating Peptide IMT-P8

    PubMed Central

    Gautam, Ankur; Nanda, Jagpreet Singh; Samuel, Jesse S.; Kumari, Manisha; Priyanka, Priyanka; Bedi, Gursimran; Nath, Samir K.; Mittal, Garima; Khatri, Neeraj; Raghava, Gajendra Pal Singh

    2016-01-01

    Skin, being the largest organ of the body, is an important site for drug administration. However, most of the drugs have poor permeability and thus drug delivery through the skin is very challenging. In this study, we examined the transdermal delivery capability of IMT-P8, a novel cell-penetrating peptide. We generated IMT-P8-GFP and IMT-P8-KLA fusion constructs and evaluated their internalization into mouse skin after topical application. Our results demonstrate that IMT-P8 is capable of transporting green fluorescent protein (GFP) and proapoptotic peptide, KLA into the skin and also in different cell lines. Interestingly, uptake of IMT-P8-GFP was considerably higher than TAT-GFP in HeLa cells. After internalization, IMT-P8-KLA got localized to the mitochondria and caused significant cell death in HeLa cells signifying an intact biological activity. Further in vivo skin penetration experiments revealed that after topical application, IMT-P8 penetrated the stratum corneum, entered into the viable epidermis and accumulated inside the hair follicles. In addition, both IMT-P8-KLA and IMT-P8-GFP internalized into the hair follicles and dermal tissue of the skin following topical application. These results suggested that IMT-P8 could be a potential candidate to be used as a topical delivery vehicle for various cosmetic and skin disease applications. PMID:27189051

  12. Cerebral Oxygen Delivery and Consumption During Evoked Neural Activity

    PubMed Central

    Vazquez, Alberto L.; Masamoto, Kazuto; Fukuda, Mitsuhiro; Kim, Seong-Gi

    2010-01-01

    Increases in neural activity evoke increases in the delivery and consumption of oxygen. Beyond observations of cerebral tissue and blood oxygen, the role and properties of cerebral oxygen delivery and consumption during changes in brain function are not well understood. This work overviews the current knowledge of functional oxygen delivery and consumption and introduces recent and preliminary findings to explore the mechanisms by which oxygen is delivered to tissue as well as the temporal dynamics of oxygen metabolism. Vascular oxygen tension measurements have shown that a relatively large amount of oxygen exits pial arterioles prior to capillaries. Additionally, increases in cerebral blood flow (CBF) induced by evoked neural activation are accompanied by arterial vasodilation and also by increases in arteriolar oxygenation. This increase contributes not only to the down-stream delivery of oxygen to tissue, but also to delivery of additional oxygen to extra-vascular spaces surrounding the arterioles. On the other hand, the changes in tissue oxygen tension due to functional increases in oxygen consumption have been investigated using a method to suppress the evoked CBF response. The functional decreases in tissue oxygen tension induced by increases in oxygen consumption are slow to evoked changes in CBF under control conditions. Preliminary findings obtained using flavoprotein autofluorescence imaging suggest cellular oxidative metabolism changes at a faster rate than the average changes in tissue oxygen. These issues are important in the determination of the dynamic changes in tissue oxygen metabolism from hemoglobin-based imaging techniques such as blood oxygenation-level dependent functional magnetic resonance imaging (fMRI). PMID:20616881

  13. Active Targeted Drug Delivery for Microbes Using Nano-Carriers

    PubMed Central

    Lin, Yung-Sheng; Lee, Ming-Yuan; Yang, Chih-Hui; Huang, Keng-Shiang

    2015-01-01

    Although vaccines and antibiotics could kill or inhibit microbes, many infectious diseases remain difficult to treat because of acquired resistance and adverse side effects. Nano-carriers-based technology has made significant progress for a long time and is introducing a new paradigm in drug delivery. However, it still has some challenges like lack of specificity toward targeting the infectious site. Nano-carriers utilized targeting ligands on their surface called ‘active target’ provide the promising way to solve the problems like accelerating drug delivery to infectious areas and preventing toxicity or side-effects. In this mini review, we demonstrate the recent studies using the active targeted strategy to kill or inhibit microbes. The four common nano-carriers (e.g. liposomes, nanoparticles, dendrimers and carbon nanotubes) delivering encapsulated drugs are introduced. PMID:25877093

  14. Protein Conjugation with Amphiphilic Block Copolymers for Enhanced Cellular Delivery

    PubMed Central

    Yi, Xiang; Batrakova, Elena; Banks, William A.; Vinogradov, Serguei; Kabanov, Alexander V.

    2008-01-01

    Modification of a model protein, horseradish peroxidase (HRP), with amphiphilic block copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic), was previously shown to enhance the transport of this protein across the blood—brain barrier in vivo and brain microvessel endothelial cells in vitro. This work develops procedures for synthesis and characterization of HRP with Pluronic copolymers, having different lengths of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks. Four monoamine Pluronic derivatives (L81, P85, L121, P123) were synthesized and successfully conjugated to a model protein, HRP, via biodegradable or nondegradable linkers (dithiobis(succinimidyl propionate) (DSP), dimethyl 3,3′-dithiobispropionimidate (DTBP), and disuccinimidyl propionate (DSS)). The conjugation was confirmed by HRP amino group titration, matrix-assisted laser desorption/ionization-time of flight spectroscopy, and cation-exchange chromatography. HRP conjugates containing an average of one to two Pluronic moieties and retaining in most cases over 70% of the activity were synthesized. Increased cellular uptake of these conjugates was demonstrated using the Mardin-Derby canine kidney cell line and primary bovine brain microvessel endothelial cells. The optimal modifications included Pluronic L81 and P85. These copolymers have shorter PPO chains compared to Pluronic P123 and L121, which were less efficient. There was little if any dependence of the uptake on the length of the hydrophilic PEO block for the optimal modifications. The proposed modifications may be used to increase cellular uptake of other proteins. PMID:18447367

  15. Delivery of a secreted soluble protein to the vacuole via a membrane anchor

    SciTech Connect

    Barrieu, F.; Chrispeels, M.J.

    1999-08-01

    To further understand how membrane proteins are sorted in the secretory system, the authors devised a strategy that involves the expression of a membrane-anchored yeast invertase in transgenic plants. The construct consisted of a signal peptide followed by the coding region of yeast invertase and the transmembrane domain and cytoplasmic tail of calnexin. The substitution of a lysine near the C terminus of calnexin with a glutamic acid residue ensured progression through the secretory system rather than retention in or return to the endoplasmic reticulum. In the transformed plants, invertase activity and a 70-kD cross-reacting protein were found in the vacuoles. This yeast invertase had plant-specific complex glycans, indicating that transport to the vacuole was mediated by the Golgi apparatus. The microsomal fraction contained a membrane-anchored 90-kD cross-reacting polypeptide, but was devoid of invertase activity. Their results indicate that this membrane-anchored protein proceeds in the secretory system beyond the point where soluble proteins are sorted for secretion, and is detached from its membrane anchor either just before or just after delivery to the vacuole.

  16. [Development of a novel transdermal delivery system of peptide and protein drugs using microneedle arrays].

    PubMed

    Katsumi, Hidemasa; Quan, Ying-Shu; Kamiyama, Fumio; Kusamori, Kosuke; Sakane, Toshiyasu; Yamamoto, Akira

    2014-01-01

    Transdermal delivery of peptide and protein drugs may be limited by the stratum corneum, which is a protective barrier against the entry of microorganisms and water. Many approaches have been utilized to promote peptide and protein drugs delivery across the stratum corneum, including chemical enhancer modification and physical disruption of barrier function. However, it has been difficult to achieve therapeutic levels of peptide and protein drugs via this route without any skin irritation. Recently, attention has been paid to the possibility of using microneedle arrays in delivering peptide and protein drugs into the skin. As a novel and minimally invasive approach, microneedle arrays are capable of creating superficial pathways across the skin for peptide and protein drugs to achieve enhanced transdermal drug delivery. This method combines the efficacy of conventional injection needles with the convenience of transdermal patches, while minimizing the disadvantages of these administration methods. Therefore, microneedle arrays are a very useful alternative method for delivering peptide and protein drugs from the skin into the systemic circulation without any serious damage to skin. In this review, recent challenges in the developments of microneedle arrays for the delivery of peptide and protein drugs are summarized. Then, future developments of microneedle arrays for the delivery of peptide and protein drugs are also discussed in order to improve their therapeutic efficacy and safety.

  17. Claudin 4-targeted protein incorporated into PLGA nanoparticles can mediate M cell targeted delivery

    PubMed Central

    Rajapaksa, Thejani E.; Stover-Hamer, Mary; Fernandez, Xiomara; Eckelhoefer, Holly A.; Lo, David D.

    2009-01-01

    Polymer-based microparticles are in clinical use mainly for their ability to provide controlled release of peptides and compounds, but they are also being explored for their potential to deliver vaccines and drugs as suspensions directly into mucosal sites. It is generally assumed that uptake is mediated by epithelial M cells, but this is often not directly measured. To study the potential for optimizing M cell uptake of polymer microparticles in vivo, we produced sub-micron size PLGA particles incorporating a recombinant protein. This recombinant protein was produced with or without a c-terminal peptide previously shown to have high affinity binding to Claudin 4, a protein associated with M cell endocytosis. While the PLGA nanoparticles incorporate the protein throughout the matrix, much of the protein was also displayed on the surface, allowing us to take advantage of the binding activity of the targeting peptide. Accordingly, we found that instillation of these nanoparticles into the nasal passages or stomach of mice was found to significantly enhance their uptake by upper airway and intestinal M cells. Our results suggest that a reasonably simple nanoparticle manufacture method can provide insight into developing an effective needle-free delivery system. PMID:19896996

  18. Anti-interleukin-6 therapy through application of a monogenic protein inhibitor via gene delivery

    PubMed Central

    Görtz, Dieter; Braun, Gerald S.; Maruta, Yuichi; Djudjaj, Sonja; van Roeyen, Claudia R.; Martin, Ina V.; Küster, Andrea; Schmitz-Van de Leur, Hildegard; Scheller, Jürgen; Ostendorf, Tammo; Floege, Jürgen; Müller-Newen, Gerhard

    2015-01-01

    Anti-cytokine therapies have substantially improved the treatment of inflammatory and autoimmune diseases. Cytokine-targeting drugs are usually biologics such as antibodies or other engineered proteins. Production of biologics, however, is complex and intricate and therefore expensive which might limit therapeutic application. To overcome this limitation we developed a strategy that involves the design of an optimized, monogenic cytokine inhibitor and the protein producing capacity of the host. Here, we engineered and characterized a receptor fusion protein, mIL-6-RFP-Fc, for the inhibition of interleukin-6 (IL-6), a well-established target in anti-cytokine therapy. Upon application in mice mIL-6-RFP-Fc inhibited IL-6-induced activation of the transcription factor STAT3 and ERK1/2 kinases in liver and kidney. mIL-6-RFP-Fc is encoded by a single gene and therefore most relevant for gene transfer approaches. Gene transfer through hydrodynamic plasmid delivery in mice resulted in hepatic production and secretion of mIL-6-RFP-Fc into the blood in considerable amounts, blocked hepatic acute phase protein synthesis and improved kidney function in an ischemia and reperfusion injury model. Our study establishes receptor fusion proteins as promising agents in anti-cytokine therapies through gene therapeutic approaches for future targeted and cost-effective treatments. The strategy described here is applicable for many cytokines involved in inflammatory and other diseases. PMID:26423228

  19. Anticancer activity of stoppin based on a novel peptide delivery system.

    PubMed

    Gao, Yan-Fang; Wei, Xue-Ni; Ye, Xiao-Lei; Weng, Guo-Bin; Chen, Yi-Chen; Zhao, Ya-Rong; Ji, Hui

    2015-10-01

    Stoppin (L1) is a newly identified anticancer peptide, which is a potent p53‑MDM2/MDMX inhibitor. Due to its limitation in cell delivery efficiency, a new peptide delivery system was developed based on a nucleic acid‑polypeptide‑liposome complex and its stability and effectiveness in vitro was investigated. The nucleic acid‑stoppin‑liposome complex was prepared and characterization of the complex was conducted. The stability of the complex was evaluated by enzyme digestion. Following transfection of the A549 cells with the complex, detection of green fluorescent protein (GFP) and luciferase activity was conducted to evaluate transfection efficiency. In addition, the anticancer activity of the complex was determined by 3‑(4,5‑dimethyl‑thiazolyl‑2)‑2,5 diphenyltetrazolium bromide assay and apoptosis was detected by flow cytometry. The results indicated that the particle size of the complex was 102±10 nm and the encapsulation rate was ~100% when the ratio of liposome, L1 and plasmid was: 4 µl:1 µg:2 µg. The enzyme digestion experiment demonstrated that the complex was resistant to pancreatic and DNA enzyme degradation, indicating that the complex had biological stability. Cell transfection demonstrated that it had a mutual promotion effect on delivery, which could be confirmed by GFP fluorescence and luciferase assay. The cell‑killing efficiency of this novel delivery system was three times higher than with stoppin alone at a low concentration. In conclusion, this novel stoppin peptide delivery system was stable. The nucleic acid‑peptide‑liposome complex can protect the internal component from the degradation of enzymes, promote entry of the peptide into the cells and enhance the anti‑tumor activity of stoppin. Therefore, it is a promising approach for peptide delivery, which can be characterized and visualized using plasmids with GFP or luciferase.

  20. Bioengineered Vaults: Self-Assembling Protein Shell–Lipophilic Core Nanoparticles for Drug Delivery

    PubMed Central

    2015-01-01

    We report a novel approach to a new class of bioengineered, monodispersed, self-assembling vault nanoparticles consisting of a protein shell exterior with a lipophilic core interior designed for drug and probe delivery. Recombinant vaults were engineered to contain a small amphipathic α-helix derived from the nonstructural protein 5A of hepatitis C virus, thereby creating within the vault lumen a lipophilic microenvironment into which lipophilic compounds could be reversibly encapsulated. Multiple types of electron microscopy showed that attachment of this peptide resulted in larger than expected additional mass internalized within the vault lumen attributable to incorporation of host lipid membrane constituents spanning the vault waist (>35 nm). These bioengineered lipophilic vaults reversibly associate with a sample set of therapeutic compounds, including all-trans retinoic acid, amphotericin B, and bryostatin 1, incorporating hundreds to thousands of drug molecules per vault nanoparticle. Bryostatin 1 is of particular therapeutic interest because of its ability to potently induce expression of latent HIV, thus representing a preclinical lead in efforts to eradicate HIV/AIDS. Vaults loaded with bryostatin 1 released free drug, resulting in activation of HIV from provirus latency in vitro and induction of CD69 biomarker expression following intravenous injection into mice. The ability to preferentially and reversibly encapsulate lipophilic compounds into these novel bioengineered vault nanoparticles greatly advances their potential use as drug delivery systems. PMID:25061969

  1. Assembly and intracellular delivery of quantum dot-fluorescent protein bioconjugates

    NASA Astrophysics Data System (ADS)

    Medintz, Igor L.; Pons, Thomas; Delehanty, James B.; Susumu, Kimihiro; Dawson, Philip E.; Mattoussi, Hedi

    2008-02-01

    We have previously assembled semiconductor quantum dot (QD)-based fluorescence resonance energy transfer (FRET) sensors that can specifically detect nutrients, explosives or enzymatic activity. These sensors utilized the inherent benefits of QDs as FRET donors to optimize signal transduction. In this report we functionalize QDs with the multi-subunit multi-chromophore b-phycoerythrin (b-PE) light harvesting complex using biotin-Streptavidin binding. FRET and gel electrophoretic analyses were used to characterize and confirm the QD-b-PE self-assembly. We found that immobilizing additional cell-penetrating peptides on the nanocrystal surface along with the b-PE was the key factor allowing the mixed surface QD-cargos to undergo endocytosis and intracellular delivery. Our findings on the intracellular uptake promoted by CPP were compared to those collected using microinjection technique, where QD-assemblies were delivered directly into the cytoplasm; this strategy allows bypassing of the endocytic uptake pathway. Intracellular delivery of multifunctional QD-fluorescent protein assemblies has potential applications for use in protein tracking, sensing and diagnostics.

  2. Intranasal Delivery of Proteins and Peptides in the Treatment of Neurodegenerative Diseases.

    PubMed

    Meredith, M Elizabeth; Salameh, Therese S; Banks, William A

    2015-07-01

    The blood-brain barrier (BBB) is a major impediment to the therapeutic delivery of peptides and proteins to the brain. Intranasal delivery often provides a non-invasive means to bypass the BBB. Advantages of using intranasal delivery include minimizing exposure to peripheral organs and tissues, thus reducing systemic side effects. It also allows substances that typically have rapid degradation in the blood time to exert their effect. Intranasal delivery provides the ability to target proteins and peptides to specific regions of the brain when administered with substrates like cyclodextrins. In this review, we examined the use of intranasal delivery of various proteins and peptides that have implications in the treatment of neurodegenerative diseases, focusing especially on albumin, exendin/GLP-1, GALP, insulin, leptin, and PACAP. We have described their rationale for use, distribution in the brain after intranasal injection, how intranasal administration differed from other modes of delivery, and their use in clinical trials, if applicable. Intranasal delivery of drugs, peptides, and other proteins could be very useful in the future for the prevention or treatment of brain related diseases.

  3. Self-nanoemulsifying drug delivery systems (SNEDDS) for oral delivery of protein drugs: II. In vitro transport study.

    PubMed

    Rao, Sripriya Venkata Ramana; Agarwal, Payal; Shao, Jun

    2008-10-01

    To develop a self-nanoemulsifying drug delivery system (SNEDDS) for protein drugs, and particularly, to test the in vitro transport of beta-lactamase (BLM) by SNEDDS across the cell monolayer. Fluorescently labeled BLM (FITC-BLM), a model protein, formulated into 16 SNEDDS preparations through a solid dispersion technique were studied for transport across MDCK monolayer. All the SNEDDS nanoemulsions resulted in higher transport rate than the free solution. The transport rate by SNEDDS depends on the SNEDDS composition. SNEDDS NE-12-7 (oil: Lauroglycol FCC, surfactant: Cremophor EL and a cosurfactant: Transcutol HP) at the ratio of 5:4:3, rendered the highest transportation rate, 33% as compared to negligible transport by the free solution. FITC-BLM solution mixed with the surfactant and the cosurfactant of SNEDDS NE-12-7 or with blank SNEDDS NE-12-7 increased the transport only by 3.3 and 1.5 folds, respectively, compared to free solution alone. It was found that the monolayer integrity was not compromised in the presence of SNEDDS NE-12-7 or its surfactant/cosurfactant. The SNEDDS significantly increased the transport of FITC-BLM across MDCK monolayer in vitro. SNEDDS may be a potential effective delivery system for non-invasive protein drug delivery.

  4. A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins

    PubMed Central

    Tejeda-Dominguez, Farid; Huerta-Cantillo, Jazmin; Chavez-Dueñas, Lucia

    2017-01-01

    ABSTRACT The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia, require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens. PMID:28351918

  5. Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

    PubMed Central

    Wang, Ming; Zuris, John A.; Meng, Fantao; Rees, Holly; Sun, Shuo; Deng, Pu; Han, Yong; Gao, Xue; Pouli, Dimitra; Wu, Qi; Georgakoudi, Irene; Liu, David R.; Xu, Qiaobing

    2016-01-01

    A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing. PMID:26929348

  6. Novel Cationic Lipids with Enhanced Gene Delivery and Antimicrobial Activity

    PubMed Central

    Fein, David E.; Bucki, Robert; Byfield, Fitzroy; Leszczynska, Katarzyna; Janmey, Paul A.

    2010-01-01

    Cationic lipids facilitate plasmid delivery, and some cationic sterol-based compounds have antimicrobial activity because of their amphiphilic character. These dual functions are relevant in the context of local ongoing infection during intrapulmonary gene transfer for cystic fibrosis. The transfection activities of two cationic lipids, dexamethasone spermine (DS) and disubstituted spermine (D2S), were tested as individual components and mixtures in bovine aortic endothelial cells and A549 cells. The results showed a 3- to 7-fold improvement in transgene expression for mixtures of DS with 20 to 40 mol% D2S. D2S and coformulations with DS, dioleoyl phosphatidylethanolamine, and DNA exhibited potent bactericidal activity against Escherichia coli MG1655, Bacillus subtilis, and Pseudomonas aeruginosa PAO1, which was maintained in bronchoalveolar lavage fluid. Complete bacterial killing was demonstrated at ∼5 μM, including gene delivery formulations, with 2 orders of magnitude higher tolerance before eukaryotic membrane disruption (erythrocyte hemolysis). D2S also exhibited lipopolysaccharide (LPS) scavenging activity resulting in significant inhibition of LPS-mediated activation of human neutrophils with 85 and 65% lower interleukin-8 released at 12 and 24 h, respectively. Mixtures of DS and D2S can improve transfection activity over common lipofection reagents, and D2S has strong antimicrobial action suited for the suppression of bacterial-mediated inflammation. PMID:20573781

  7. Secretory delivery of recombinant proteins in attenuated Salmonella strains: potential and limitations of Type I protein transporters.

    PubMed

    Hahn, Heinz P; von Specht, Bernd Ulrich

    2003-07-15

    Live attenuated Salmonella strains have been extensively explored as oral delivery systems for recombinant vaccine antigens and effector proteins with immunoadjuvant and immunomodulatory potential. The feasibility of this approach was demonstrated in human vaccination trials for various antigens. However, immunization efficiencies with live vaccines are generally significantly lower compared to those monitored in parenteral immunizations with the same vaccine antigen. This is, at least partly, due to the lack of secretory expression systems, enabling large-scale extracellular delivery of vaccine and effector proteins by these strains. Because of their low complexity and the terminal location of the secretion signal in the secreted protein, Type I (ATP-binding cassette) secretion systems appear to be particularly suited for development of such recombinant extracellular expression systems. So far, the Escherichia coli hemolysin system is the only Type I secretion system, which has been adapted to recombinant protein secretion in Salmonella. However, this system has a number of disadvantages, including low secretion capacity, complex genetic regulation, and structural restriction to the secreted protein, which eventually hinder high-level in vivo delivery of recombinant vaccines and effector proteins. Thus, the development of more efficient recombinant protein secretion systems, based on Type I exporters can help to improve efficacies of live recombinant Salmonella vaccines. Type I secretion systems, mediating secretion of bacterial surface layer proteins, such as RsaA in Caulobacter crescentus, are discussed as promising candidates for improved secretory delivery systems.

  8. Insect GDNF:TTC fusion protein improves delivery of GDNF to mouse CNS

    SciTech Connect

    Li, Jianhong; Chian, Ru-Ju; Ay, Ilknur; Kashi, Brenda B.; Celia, Samuel A.; Tamrazian, Eric; Pepinsky, R. Blake; Fishman, Paul S.; Brown, Robert H.; Francis, Jonathan W.

    2009-12-18

    With a view toward improving delivery of exogenous glial cell line-derived neurotrophic factor (GDNF) to CNS motor neurons in vivo, we evaluated the bioavailability and pharmacological activity of a recombinant GDNF:tetanus toxin C-fragment fusion protein in mouse CNS. Following intramuscular injection, GDNF:TTC but not recombinant GDNF (rGDNF) produced strong GDNF immunostaining within ventral horn cells of the spinal cord. Intrathecal infusion of GDNF:TTC resulted in tissue concentrations of GDNF in lumbar spinal cord that were at least 150-fold higher than those in mice treated with rGDNF. While levels of immunoreactive choline acetyltransferase and GFR{alpha}-1 in lumbar cord were not altered significantly by intrathecal infusion of rGNDF, GDNF:TTC, or TTC, only rGDNF and GDNF:TTC caused significant weight loss following intracerebroventricular infusion. These studies indicate that insect cell-derived GDNF:TTC retains its bi-functional activity in mammalian CNS in vivo and improves delivery of GDNF to spinal cord following intramuscular- or intrathecal administration.

  9. A bacterial type III secretion-based protein delivery tool for broad applications in cell biology

    PubMed Central

    Ittig, Simon J.; Schmutz, Christoph; Kasper, Christoph A.; Amstutz, Marlise; Schmidt, Alexander; Sauteur, Loïc; Vigano, M. Alessandra; Low, Shyan Huey; Affolter, Markus; Cornelis, Guy R.; Nigg, Erich A.

    2015-01-01

    Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network. PMID:26598622

  10. Bicistronic gene transfer tools for delivery of miRNAs and protein coding sequences.

    PubMed

    Stoller, Michelle L; Chang, Henry C; Fekete, Donna M

    2013-09-05

    MicroRNAs (miRNAs) are a category of small RNAs that modulate levels of proteins via post-transcriptional inhibition. Currently, a standard strategy to overexpress miRNAs is as mature miRNA duplexes, although this method is cumbersome if multiple miRNAs need to be delivered. Many of these miRNAs are found within introns and processed through the RNA polymerase II pathway. We have designed a vector to exploit this naturally-occurring intronic pathway to deliver the three members of the sensory-specific miR-183 family from an artificial intron. In one version of the vector, the downstream exon encodes the reporter (GFP) while another version encodes a fusion protein created between the transcription factor Atoh1 and the hemaglutinin epitope, to distinguish it from endogenous Atoh1. In vitro analysis shows that the miRNAs contained within the artificial intron are processed and bind to their targets with specificity. The genes downstream are successfully translated into protein and identifiable through immunofluorescence. More importantly, Atoh1 is proven functional through in vitro assays. These results suggest that this cassette allows expression of miRNAs and proteins simultaneously, which provides the opportunity for joint delivery of specific translational repressors (miRNA) and possibly transcriptional activators (transcription factors). This ability is attractive for future gene therapy use.

  11. Liposome-protein corona in a physiological environment: challenges and opportunities for targeted delivery of nanomedicines.

    PubMed

    Caracciolo, Giulio

    2015-04-01

    Active targeting that exploits the (over)expression of surface receptors in target cells by ligand incorporation is a central concept in nanomedicine research. Despite unprecedented efforts, no targeted liposome-based therapeutics is commercially available for clinical practice. What is inhibiting the efficient translation of targeted liposome technology from bench to bedside? After introduction in the bloodstream, the lipid surface is immediately modified by the adsorption of a "protein corona" and preserving the surface functionality appears to be challenging. On the other hand, a long-standing corona with receptor-binding sites could associate with the target cell long enough to activate the cell's uptake machinery, triggering liposome endocytosis and intracellular cargo delivery. This opens the intriguing possibility to manipulate the corona composition by liposome design. This review will focus on the emerging field of liposome-protein corona research from basic, descriptive research to readily applicable knowledge and technologies for implementation in drug improvement and development. From the clinical editor: This review is addressing the liposome protein corona research concerning the potential gains in drug improvement and for drug development.

  12. Method for Targeted Therapeutic Delivery of Proteins into Cells | NCI Technology Transfer Center | TTC

    Cancer.gov

    The Protein Expression Laboratory at the National Cancer Institute in Frederick, MD is seeking statements of capability or interest from parties interested in collaborative research to further develop a platform technology for the targeted intra-cellular delivery of proteins using virus-like particles (VLPs).

  13. Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or an...

  14. Evaluation of biodegradable polyester-co-lactone microparticles for protein delivery.

    PubMed

    Tawfeek, Hesham M; Khidr, Sayed H; Samy, Eman M; Ahmed, Sayed M; Gaskell, Elsie E; Hutcheon, Gillian A

    2014-09-01

    Abstract Poly(glycerol adipate-co-ω-pentadecalactone) (PGA-co-PDL) was previously evaluated for the colloidal delivery of α-chymotrypsin. In this article, the effect of varying polymer molecular weight (MW) and chemistry on particle size and morphology; encapsulation efficiency; in vitro release; and the biological activity of α-chymotrypsin (α-CH) and lysozyme (LS) were investigated. Microparticles were prepared using emulsion solvent evaporation and evaluated by various methods. Altering the MW or monomer ratio of PGA-co-PDL did not significantly affect the encapsulation efficiency and overall poly(1,3-propanediol adipate-co-ω-pentadecalactone) (PPA-co-PDL) demonstrated the highest encapsulation efficiency. In vitro release varied between polymers, and the burst release for α-CH-loaded microparticles was lower when a higher MW PGA-co-PDL or more hydrophobic PPA-co-PDL was used. The results suggest that, although these co-polyesters could be useful for protein delivery, little difference was observed between the different PGA-co-PDL polymers and PPA-co-PDL generally provided a higher encapsulation and slower release of enzyme than the other polymers tested.

  15. A Solvent-Free Thermosponge Nanoparticle Platform for Efficient Delivery of Labile Proteins

    PubMed Central

    2015-01-01

    Protein therapeutics have gained attention recently for treatment of a myriad of human diseases due to their high potency and unique mechanisms of action. We present the development of a novel polymeric thermosponge nanoparticle for efficient delivery of labile proteins using a solvent-free polymer thermo-expansion mechanism with clinical potential, capable of effectively delivering a range of therapeutic proteins in a sustained manner with no loss of bioactivity, with improved biological half-lives and efficacy in vivo. PMID:25333768

  16. Outer eggshell membrane as delivery vehicle for polysaccharide/protein microcapsules incorporated with vitamin E.

    PubMed

    Chai, Zhi; Li, Yuanyuan; Liu, Fei; Du, Bingjian; Jiao, Tong; Zhang, Chunyue; Leng, Xiaojing

    2013-01-23

    This study investigates the features of a new type of delivery system prepared by combining a natural outer eggshell membrane (OESM) with emulsified microcapsules. The loading efficiency, controlled release properties, and forming mechanisms of the prepared system were studied. The polysaccharide/protein microcapsules incorporated with vitamin E can be attached to highly cross-linked protein fiber networks of OESM. This attachment could be reinforced more than 2-fold using glutaraldehyde as a cross-linking agent. The combined OESM/microcapsule delivery system significantly exhibited better controlled release properties than the microcapsules alone because of the steric blocking effect. Moreover, the OESM delivery system incorporated with microcapsules formed by pectin/protein as wall material showed more resistance against enzymatic attacks because of the formation of compact aggregates promoted by electrostatic effects.

  17. Tat-mediated protein delivery in living Caenorhabditis elegans

    SciTech Connect

    Delom, Frederic; Fessart, Delphine; Caruso, Marie-Elaine; Chevet, Eric . E-mail: eric.chevet@mcgill.ca

    2007-01-19

    The Tat protein from HIV-1 fused with heterologous proteins traverses biological membranes in a transcellular process called: protein transduction. This has already been successfully exploited in various biological models, but never in the nematode worm Caenorhabditis elegans. TAT-eGFP or GST-eGFP proteins were fed to C. elegans worms, which resulted in the specific localization of Tat-eGFP to epithelial intestinal cells. This system represents an efficient tool for transcellular transduction in C. elegans intestinal cells. Indeed, this approach avoids the use of tedious purification steps to purify the TAT fusion proteins and allows for rapid analyses of the transduced proteins. In addition, it may represent an efficient tool to functionally analyze the mechanisms of protein transduction as well as to complement RNAi/KO in the epithelial intestinal system. To sum up, the advantage of this technology is to combine the potential of bacterial expression system and the Tat-mediated transduction technique in living worm.

  18. Past, Present, and Future Technologies for Oral Delivery of Therapeutic Proteins

    PubMed Central

    SINGH, RAJESH; SINGH, SHAILESH; LILLARD, JAMES W.

    2015-01-01

    Biological drugs are usually complex proteins and cannot be orally delivered due to problems related to degradation in the acidic and protease-rich environment of the gastrointestinal (GI) tract. The high molecular weight of these drugs often results in poor absorption into the periphery when administered orally. The most common route of administration for these therapeutic proteins is injection. Most of these proteins have short serum half-lives and need to be administered frequently or in high doses to be effective. So, difficulties in the administration of protein-based drugs provides the motivation for developing drug delivery systems (DDSs) capable of maintaining therapeutic drug levels without side effects as well as traversing the deleterious mucosal environment. Employing a polymer as an entrapment matrix is a common feature among the different types of systems currently being pursued for protein delivery. Protein release from these matrices can occur through various mechanisms, such as diffusion through or erosion of the polymer matrix, and sometimes a combination of both. Encapsulation of proteins in liposomes has also been a widely investigated technology for protein delivery. All of these systems have merit and our worthy of pursuit. PMID:17918721

  19. Silk-elastin-like protein biomaterials for the controlled delivery of therapeutics

    PubMed Central

    Huang, Wenwen; Rollett, Alexandra; Kaplan, David L.

    2015-01-01

    Introduction Genetically engineered biomaterials are useful for controlled delivery owing to their rational design, tunable structure-function, biocompatibility, degradability and target specificity. Silk-elastin-like proteins (SELPs), a family of genetically engineered recombinant protein polymers, possess these properties. Additionally, given the benefits of combining semicrystalline silk-blocks and elastomeric elastin-blocks, SELPs possess multi-stimuli responsive properties and tenability, thereby, becoming promising candidates for targeted cancer therapeutics delivery and controlled gene release. Areas covered An overview of SELP biomaterials for drug delivery and gene release is provided. Biosynthetic strategies used for SELP production, fundamental physicochemical properties, and self-assembly mechanisms are discussed. The review focuses on sequence-structure-function relationships, stimuli responsive features, and current and potential drug delivery applications. Expert opinion The tunable material properties allow SELPs to be pursued as promising biomaterials for nano-carriers and injectable drug release systems. Current applications of SELPs have focused on thermally-triggered biomaterial formats for the delivery of therapeutics, based on local hyperthermia in tumors or infections. Other prominent controlled release applications of SELPs as injectable hydrogels for gene release have also been pursued. Further biomedical applications that utilize other stimuli to trigger the reversible material responses of SELPs for targeted delivery, including pH, ionic strength, redox, enzymatic stimuli and electric field, are in progress. Exploiting these additional stimuli responsive features will provide a broader range of functional biomaterials for controlled therapeutics release and tissue regeneration. PMID:25476201

  20. Systemic delivery of factor IX messenger RNA for protein replacement therapy.

    PubMed

    Ramaswamy, Suvasini; Tonnu, Nina; Tachikawa, Kiyoshi; Limphong, Pattraranee; Vega, Jerel B; Karmali, Priya P; Chivukula, Pad; Verma, Inder M

    2017-03-07

    Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4-6 h) that remains stable for up to 4-6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA-LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable.

  1. Systemic delivery of factor IX messenger RNA for protein replacement therapy

    PubMed Central

    Ramaswamy, Suvasini; Tonnu, Nina; Tachikawa, Kiyoshi; Limphong, Pattraranee; Vega, Jerel B.; Karmali, Priya P.; Chivukula, Pad; Verma, Inder M.

    2017-01-01

    Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4–6 h) that remains stable for up to 4–6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA–LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable. PMID:28202722

  2. Cell-penetrating DNA-binding protein as a safe and efficient naked DNA delivery carrier in vitro and in vivo

    SciTech Connect

    Kim, Eun-Sung; Yang, Seung-Woo; Hong, Dong-Ki; Kim, Woo-Taek; Kim, Ho-Guen; Lee, Sang-Kyou

    2010-01-29

    Non-viral gene delivery is a safe and suitable alternative to viral vector-mediated delivery to overcome the immunogenicity and tumorigenesis associated with viral vectors. Using the novel, human-origin Hph-1 protein transduction domain that can facilitate the transduction of protein into cells, we developed a new strategy to deliver naked DNA in vitro and in vivo. The new DNA delivery system contains Hph-1-GAL4 DNA-binding domain (DBD) fusion protein and enhanced green fluorescent protein (EGFP) reporter plasmid that includes the five repeats of GAL4 upstream activating sequence (UAS). Hph-1-GAL4-DBD protein formed complex with plasmid DNA through the specific interaction between GAL4-DBD and UAS, and delivered into the cells via the Hph-1-PTD. The pEGFP DNA was successfully delivered by the Hph-1-GAL4 system, and the EGFP was effectively expressed in mammalian cells such as HeLa and Jurkat, as well as in Bright Yellow-2 (BY-2) plant cells. When 10 {mu}g of pEGFP DNA was intranasally administered to mice using Hph-1-GAL4 protein, a high level of EGFP expression was detected throughout the lung tissue for 7 days. These results suggest that an Hph-1-PTD-mediated DNA delivery strategy may be an useful non-viral DNA delivery system for gene therapy and DNA vaccines.

  3. Clinical translation of controlled protein delivery systems for tissue engineering

    PubMed Central

    Spiller, Kara L.; Vunjak-Novakovic, Gordana

    2013-01-01

    Strategies that utilize controlled release of drugs and proteins for tissue engineering have enormous potential to regenerate damaged organs and tissues. The multiple advantages of controlled release strategies merit overcoming the significant challenges to translation, including high costs and long, difficult regulatory pathways. This review highlights the potential of controlled release of proteins for tissue engineering and regenerative medicine. We specifically discuss treatment modalities that have reached preclinical and clinical trials, with emphasis on controlled release systems for bone tissue engineering, the most advanced application with several products already in clinic. Possible strategies to address translational and regulatory concerns are also discussed. PMID:25787736

  4. Non-destructively shattered mesoporous silica for protein drug delivery

    SciTech Connect

    Lei, Chenghong; Chen, Baowei; Li, Xiaolin; Qi, Wen N.; Liu, Jun

    2013-07-15

    Mesoporous silicas have been extensively used for entrapping small chemical molecules and biomacromolecules. We hypothesize that the loading density of biomacromlecules such as proteins in mesoporous silicas could be limited due to mesopore disorderness and depth because of some pore volume inaccessible. We innovatively shattered mesoporous silicas resulting in reduced particle sizes and improved intramesoporous structures in aqueous solution by a powerful sonication, where the mesoporous structures were still well maintained. The sonication-shattered mesoporous silicas can allow protein loading densities to be increased by more than 170%, demonstrating that significantly more mesoporous room of the silicas could become accessible for biomacromolecule loading after the sonication-shattering.

  5. Light-activated, in situ forming gel for sustained suprachoroidal delivery of bevacizumab.

    PubMed

    Tyagi, Puneet; Barros, Matthew; Stansbury, Jeffrey W; Kompella, Uday B

    2013-08-05

    after release from the gel. As the cross-linking duration was increased to 10 min, the gel/antibody was better confined at the injection site in excised rabbit eye suprachoroidal space. Delivery of Alexa Fluor 488 dye conjugated bevacizumab was sustained for at least 60 days in the suprachoroidal space of SD rats. PCM and HEMA gel sustained bevacizumab release for 4 months and maintained the stability and VEGF-binding activity of bevacizumab. Therefore, light-activated PCM and HEMA gel is suitable for in situ gel formation and sustained protein delivery in the suprachoroidal space.

  6. The uptake machinery of clostridial actin ADP-ribosylating toxins--a cell delivery system for fusion proteins and polypeptide drugs.

    PubMed

    Barth, Holger; Blöcker, Dagmar; Aktories, Klaus

    2002-12-01

    Several bacterial protein toxins, including Clostridium botulinum C2 toxin, Clostridum perfringens iota toxin, Clostridium difficile ADP-ribosyltransferase, and the Bacillus-produced vegetative insecticidal proteins, target the cytoskeleton by ADP-ribosylation of actin. All these toxins are binary in structure and consist of an enzyme component, possessing ADP-ribosyltransferase activity and a separated binding and translocation component, which is involved in the delivery of the enzyme component into the cell. The toxins are not only important virulence factors but also cell biological tools to study the function of the actin cytoskeleton. Moreover, the binary toxins turned out to be effective transporter systems for the delivery of specific fusion toxins (e.g., Rho-ADP-ribosylating C3 exoenzyme) into cells. The present review describes the biological functions of the toxins, focuses on recent studies on the uptake and delivery mechanism and discusses the usage as a drug delivery system.

  7. Delivery

    PubMed Central

    Miller, Thomas A

    2013-01-01

    Enthusiasm greeted the development of synthetic organic insecticides in the mid-twentieth century, only to see this give way to dismay and eventually scepticism and outright opposition by some. Regardless of how anyone feels about this issue, insecticides and other pesticides have become indispensable, which creates something of a dilemma. Possibly as a result of the shift in public attitude towards insecticides, genetic engineering of microbes was first met with scepticism and caution among scientists. Later, the development of genetically modified crop plants was met with an attitude that hardened into both acceptance and hard-core resistance. Transgenic insects, which came along at the dawn of the twenty-first century, encountered an entrenched opposition. Those of us responsible for studying the protection of crops have been affected more or less by these protagonist and antagonistic positions, and the experiences have often left one thoughtfully mystified as decisions are made by non-participants. Most of the issues boil down to concerns over delivery mechanisms. © 2013 Society of Chemical Industry PMID:23852646

  8. The augmentation of intracellular delivery of peptide therapeutics by artificial protein transduction domains.

    PubMed

    Yoshikawa, Tomoaki; Sugita, Toshiki; Mukai, Yohei; Abe, Yasuhiro; Nakagawa, Shinsaku; Kamada, Haruhiko; Tsunoda, Shin-ichi; Tsutsumi, Yasuo

    2009-07-01

    Protein transduction domains (PTDs), such as HIV-derived Tat, have been successfully used as functional biomaterials for intracellular delivery of anti-cancer macromolecular drugs (protein, peptides, and oligonucleotides). Although there were therefore great expectations regarding the therapeutic potential of PTDs for the development of anti-cancer therapeutics, their clinical application so far has been extremely limited because of the relatively high concentrations required to mediate any effects on cancer cells in vitro or in vivo. In this context, improving the transduction efficiency of PTDs using phage display-based molecular evolution techniques may be useful for creating artificial PTDs with high efficiency and safety. Here, we report an evaluation of transduction efficiency and toxicity of such artificial PTDs (designated mT02 and mT03) compared with Tat. The internalization of mT02 was the most rapid and efficient by a mechanism different from the usual macropinocytosis. Furthermore, we found that artificial PTDs fused with survivin antagonistic peptide potentiate tumor cell-cytostatic activity. Thus, the results of this work provide new insights for designing new-generation peptide therapeutics for a wide variety of cancers as well as those expressing survivin.

  9. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

  10. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  11. Topical delivery of active principles: the field of dermatological research.

    PubMed

    Nino, Massimiliano; Calabrò, Gabriella; Santoianni, Pietro

    2010-01-15

    To be effective an active drug or principle must cross the stratum corneum barrier; this process can be influenced to obtain better functional and therapeutical effects. In spite of the wide variety of the methods studied in order to improve the transdermal transfer to obtain systemic effects, the applicability is limited in this field. Attention to the epidermal barrier and penetration of active principles has been reported mostly in studies concerning dermocosmetics. Studies regarding methods of penetration are gaining experimental and clinical interest. Cutaneous bioavailability of most commercially available dermatological formulations is low. Increase of intradermal delivery can relate to chemical, biochemical, or physical manipulations. Chemical enhancers have been adopted to: (a) increase the diffusibility of the substance across the barrier; (b) increase product solubility in the vehicle; (c) improve the partition coefficient. Moreover methods of interference with the biosynthesis of some lipids allow the modification of the structure of the barrier to increase the penetration. The main physical techniques that increase cutaneous penetration of substances are: iontophoresis (that increases the penetration of ionized substances), electroporation (that electrically induces penetration through the barrier), and sonophoresis, based on 20 to 25 KHz ultrasound that induces alterations of the horny barrier, allowing penetration of active principles. Recent development of these methods are here reported and underline the importance and role of vehicles and other factors that determine effects of partition and diffusion, crucial to absorption.

  12. Spatiotemporally synchronized cancer combination therapy using photo-activated nanoparticle drug delivery systems (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hasan, Tayyaba

    2016-03-01

    This talk will introduce a new nanotechnology platform for cancer combination therapy that utilizes near infrared light activation not only for photodynamic damage but also as an extrinsic mechanism to initiate release of complimentary drugs to suppress dynamic bursts in molecular signaling networks that promote tumor cell survival and treatment escape. The goal is to achieve co-delivery with concomitant activity of photodynamic, molecular inhibitor and chemotherapeutic agents, selectively within the tumor. This approach overcomes challenges in achieving synergistic interactions using sequential drug delivery. Conventional drug delivery is compromised by the differential pharmacokinetics of individual agents and potentially antagonistic effects—such as vascular shutdown by one agent that limits delivery of the second. Here, photodynamic damage—which efficiently kills drug-resistant cells via damage of common proteins involved in drug-resistance (such as anti-apoptosis factors and drug-efflux transporters)—is synchronized spatially and temporally with the photo-initiated release of complimentary agents—to enable full interaction amongst the individual therapies. This spatiotemporal synchronization offers new prospects for exploiting time-sensitive synergistic interactions. Specific implementations of these concepts will be presented in preclinical models of cancer. Strategies to enable molecular-targeting of cancer cells via site-specific attachment of targeting moieties to the outer lipid shell of these nanovehicles will also be discussed. If successful in humans, this new paradigm for synchronized, tumor-focused combination therapy will ultimately supersede the present use of chronic drug injection by increasing efficacy per cycle whilst reducing systemic exposure to toxic drugs.

  13. Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins.

    PubMed

    Chen, Qiang; Lai, Huafang; Hurtado, Jonathan; Stahnke, Jake; Leuzinger, Kahlin; Dent, Matthew

    2013-06-01

    Current human biologics are most commonly produced by mammalian cell culture-based fermentation technologies. However, its limited scalability and high cost prevent this platform from meeting the ever increasing global demand. Plants offer a novel alternative system for the production of pharmaceutical proteins that is more scalable, cost-effective, and safer than current expression paradigms. The recent development of deconstructed virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins, and in turn, provided a preferred plant based production platform. One of the remaining challenges for the commercial application of this platform was the lack of a scalable technology to deliver the transgene into plant cells. Therefore, this review focuses on the development of an effective and scalable technology for gene delivery in plants. Direct and indirect gene delivery strategies for plant cells are first presented, and the two major gene delivery technologies based on agroinfiltration are subsequently discussed. Furthermore, the advantages of syringe and vacuum infiltration as gene delivery methodologies are extensively discussed, in context of their applications and scalability for commercial production of human pharmaceutical proteins in plants. The important steps and critical parameters for the successful implementation of these strategies are also detailed in the review. Overall, agroinfiltration based on syringe and vacuum infiltration provides an efficient, robust and scalable gene-delivery technology for the transient expression of recombinant proteins in plants. The development of this technology will greatly facilitate the realization of plant transient expression systems as a premier platform for commercial production of pharmaceutical proteins.

  14. Quantitative ToF-SIMS Studies of Protein Drug Release from Biodegradable Polymer Drug Delivery Membranes

    PubMed Central

    Burns, Sarah A.; Gardella, Joseph A.

    2008-01-01

    Biodegradable polymers are of interest in developing strategies to control protein drug delivery. The protein that was used in this study is Keratinocyte Growth Factor (KGF) which is a protein involved in the re-epithelialization process. The protein is stabilized in the biodegradable polymer matrix during formulation and over the course of polymer degradation with the use of an ionic surfactant Aerosol-OT (AOT) which will encapsulate the protein in an aqueous environment. The release kinetics of the protein from the surface of these materials requires precise timing which is a crucial factor in the efficacy of this drug delivery system. Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) was used in the same capacity to identify the molecular ion peak of the surfactant and polymer and use this to determine surface concentration. In the polymer matrix, the surfactant molecular ion peak was observed in the positive and negative mode at m/z 467 and 421, respectively. These peaks were determined to be [AOT + Na+] and [AOT−Na+]-. These methods are used to identify the surfactant and protein from the polymer matrix and are used to measure the rate of surface accumulation. The second step was to compare this accumulation rate with the release rate of the protein into an aqueous solution during the degradation of the biodegradable film. This rate is compared to that from fluorescence spectroscopy measurements using the protein autofluorescence from that released into aqueous solution. PMID:20016666

  15. Food-grade protein-based nanoparticles and microparticles for bioactive delivery: fabrication, characterization, and utilization.

    PubMed

    Davidov-Pardo, Gabriel; Joye, Iris J; McClements, David Julian

    2015-01-01

    Proteins can be used to fabricate nanoparticles and microparticles suitable for use as delivery systems for bioactive compounds in pharmaceutical, food, cosmetic, and other products. Food proteins originate from various animal or vegetal sources and exhibit a wide diversity of molecular and physicochemical characteristics, e.g., molecular weight, conformation, flexibility, polarity, charge, isoelectric point, solubility, and interactions. As a result, protein particles can be assembled using numerous different preparation methods, from one or more types of protein or from a combination of a protein and another type of biopolymer (usually a polysaccharide). The final characteristics of the particles produced are determined by the proteins and/or polysaccharides used, as well as the fabrication techniques employed. This chapter provides an overview of the functional properties of food proteins that can be used to assemble nanoparticles and microparticles, the fabrication techniques available to create those particles, the factors that influence their stability, and their potential applications within the food industry.

  16. Superporous polyacrylate/chitosan IPN hydrogels for protein delivery.

    PubMed

    Gümüşderelioğlu, Menemşe; Erce, Deniz; Demirtaş, T Tolga

    2011-11-01

    In this study, poly(acrylamide), poly(AAm), and poly(acrylamide-co-acrylic acid), poly(AAm-co-AA) superporous hydrogels (SPHs) were synthesized by radical polymerization in the presence of gas blowing agent, sodium bicarbonate. In addition, ionically crosslinked chitosan (CH) superporous hydrogels were synthesized to form interpenetrating superporous hydrogels, i.e. poly(AAm)-CH and poly(AAm-co-AA)-CH SPH-IPNs. The hydrogels have a structure of interconnected pores with pore sizes of approximately 100-150 μm. Although the extent of swelling increased when AA were incorporated to the poly(AAm) structure, the time to reach the equilibrium swelling (~30 s) was not affected so much. In the presence of chitosan network mechanical properties significantly improved when compared with SPHs, however, equilibrium swelling time (~30 min) was prolonged significantly as due to the lower porosities and pore sizes of SPH-IPNs than that of SPHs. Model protein bovine serum albumin (BSA) was loaded into SPHs and SPH-IPNs by solvent sorption in very short time (<1 h) and very high capacities (~30-300 mg BSA/g dry gel) when compared to conventional hydrogels. BSA release profiles from SPHs and SPH-IPNs were characterized by an initial burst of protein during the first 20 min followed by a completed release within 1 h. However, total releasable amount of BSA from SPH-IPNs was lower than that of SPHs as due to the electrostatic interactions between chitosan and BSA.

  17. Controlled, sustained release of proteins via an injectable, mineral-coated microsphere delivery vehicle

    NASA Astrophysics Data System (ADS)

    Franklin-Ford, Travelle

    Hydroxyapatite interfaces have demonstrated strong protein binding and protein selection from a passing solution and can serve as a biocompatible carrier for controlled protein delivery. Hydroxyapatite is a major component of long bones and tooth enamel and is the most stable of all calcium phosphate isoforms in aqueous solutions at physiologic pH, providing a sensitive chromatographic mechanism for separating proteins. Here we describe an approach to create a synthetic hydroxyapatite coating through a biomimetic, heterogeneous nucleation from a modified simulated body fluid--supersaturated with calcium and phosphate ions on the surface of injectable polymer microspheres. We are able to bind and release bioactive growth factors into a variety of in vitro and in vivo conditions, demonstrating the functionality and advantage of the biomaterial. Creating a hydroxyapatite layer on the Poly(D,L-lactide-co-glycolide) (PLG) microsphere surface, avails the microsphere interior for another application that will not compete with protein binding and release. Encapsulating an imaging agent within the aqueous phase of the emulsion provides a visual reference for the injectable therapy upon microsphere fabrication. Another advantage of this system is that the mineral coating and subsequent protein binding is not compromised by the encapsulated imaging agent. This dual function delivery vehicle is not only advantageous for spatial tracking therapeutic applications, but also determining the longevity of the delivery vehicle once injected. In the broader sense, providing a mechanism to image and track our temporally controlled, sustained delivery system gives more evidence to support the effects of released protein on in vivo responses (bioactivity) and locate microspheres within different biological systems.

  18. Design and Characterization of Novel Recombinant Listeriolysin O–Protamine Fusion Proteins for Enhanced Gene Delivery

    PubMed Central

    2015-01-01

    To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO–protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems. PMID:25521817

  19. Heterologous Expression and Delivery of Biologically Active Exendin-4 by Lactobacillus paracasei L14.

    PubMed

    Zeng, Zhu; Yu, Rui; Zuo, Fanglei; Zhang, Bo; Peng, Deju; Ma, Huiqin; Chen, Shangwu

    2016-01-01

    Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, is an excellent therapeutic peptide drug for type 2 diabetes due to longer lasting biological activity compared to GLP-1. This study explored the feasibility of using probiotic Lactobacillus paracasei as an oral vector for recombinant exendin-4 peptide delivery, an alternative to costly chemical synthesis and inconvenient administration by injection. L. paracasei transformed with a plasmid encoding the exendin-4 gene (L. paracasei L14/pMG76e-exendin-4) with a constitutive promotor was successfully constructed and showed efficient secretion of exendin-4. The secreted exendin-4 significantly enhanced insulin secretion of INS-1 β-cells, along with an increment in their proliferation and inhibition of their apoptosis, corresponding to the effect of GLP-1 on these cells. The transcription level of the pancreatic duodenal homeobox-1 gene (PDX-1), a key transcription factor for cellular insulin synthesis and secretion, was upregulated by the treatment with secreted exendin-4, paralleling the upregulation of insulin gene expression. Caco-2 cell monolayer permeability assay showed a 34-fold increase in the transport of exendin-4 delivered by L. paracasei vs. that of free exendin-4 (control), suggesting effective facilitation of exendin-4 transport across the intestinal barrier by this delivery system. This study demonstrates that the probiotic Lactobacillus can be engineered to secrete bioactive exendin-4 and facilitate its transport through the intestinal barrier, providing a novel strategy for oral exendin-4 delivery using this lactic acid bacterium.

  20. Heterologous Expression and Delivery of Biologically Active Exendin-4 by Lactobacillus paracasei L14

    PubMed Central

    Zeng, Zhu; Yu, Rui; Zuo, Fanglei; Zhang, Bo; Peng, Deju; Ma, Huiqin; Chen, Shangwu

    2016-01-01

    Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, is an excellent therapeutic peptide drug for type 2 diabetes due to longer lasting biological activity compared to GLP-1. This study explored the feasibility of using probiotic Lactobacillus paracasei as an oral vector for recombinant exendin-4 peptide delivery, an alternative to costly chemical synthesis and inconvenient administration by injection. L. paracasei transformed with a plasmid encoding the exendin-4 gene (L. paracasei L14/pMG76e-exendin-4) with a constitutive promotor was successfully constructed and showed efficient secretion of exendin-4. The secreted exendin-4 significantly enhanced insulin secretion of INS-1 β-cells, along with an increment in their proliferation and inhibition of their apoptosis, corresponding to the effect of GLP-1 on these cells. The transcription level of the pancreatic duodenal homeobox-1 gene (PDX-1), a key transcription factor for cellular insulin synthesis and secretion, was upregulated by the treatment with secreted exendin-4, paralleling the upregulation of insulin gene expression. Caco-2 cell monolayer permeability assay showed a 34-fold increase in the transport of exendin-4 delivered by L. paracasei vs. that of free exendin-4 (control), suggesting effective facilitation of exendin-4 transport across the intestinal barrier by this delivery system. This study demonstrates that the probiotic Lactobacillus can be engineered to secrete bioactive exendin-4 and facilitate its transport through the intestinal barrier, providing a novel strategy for oral exendin-4 delivery using this lactic acid bacterium. PMID:27764251

  1. Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity

    NASA Astrophysics Data System (ADS)

    Whitehead, Kathryn A.; Dorkin, J. Robert; Vegas, Arturo J.; Chang, Philip H.; Veiseh, Omid; Matthews, Jonathan; Fenton, Owen S.; Zhang, Yunlong; Olejnik, Karsten T.; Yesilyurt, Volkan; Chen, Delai; Barros, Scott; Klebanov, Boris; Novobrantseva, Tatiana; Langer, Robert; Anderson, Daniel G.

    2014-06-01

    One of the most significant challenges in the development of clinically viable delivery systems for RNA interference therapeutics is to understand how molecular structures influence delivery efficacy. Here, we have synthesized 1,400 degradable lipidoids and evaluate their transfection ability and structure-function activity. We show that lipidoid nanoparticles mediate potent gene knockdown in hepatocytes and immune cell populations on IV administration to mice (siRNA EC50 values as low as 0.01 mg kg-1). We identify four necessary and sufficient structural and pKa criteria that robustly predict the ability of nanoparticles to mediate greater than 95% protein silencing in vivo. Because these efficacy criteria can be dictated through chemical design, this discovery could eliminate our dependence on time-consuming and expensive cell culture assays and animal testing. Herein, we identify promising degradable lipidoids and describe new design criteria that reliably predict in vivo siRNA delivery efficacy without any prior biological testing.

  2. A genetic and structural analysis of the yeast Vps15 protein kinase: evidence for a direct role of Vps15p in vacuolar protein delivery.

    PubMed Central

    Herman, P K; Stack, J H; Emr, S D

    1991-01-01

    The yeast VPS15 gene encodes a novel protein kinase homolog that is required for the sorting of soluble hydrolases to the yeast vacuole. In this study, we extend our previous mutational analysis of the VPS15 gene and show that alterations of specific Gps15p residues, that are highly conserved among all protein kinase molecules, result in the biological inactivation of Vps15p. Furthermore, we demonstrate here that short C-terminal deletions of Vps15p result in a temperature-conditional defect in vacuolar protein sorting. Immediately following the temperature shift, soluble vacuolar hydrolases, such as carboxypeptidase Y and proteinase A, accumulate as Golgi-modified precursors within a saturable intracellular compartment distinct from the vacuole. This vacuolar protein sorting block is efficiently reversed when mutant cells are shifted back to the permissive temperature; the accumulated precursors are rapidly processed to their mature forms indicating that they have been delivered to the vacuole. This rapid and efficient reversal suggests that the accumulated vacuolar protein precursors were present within a normal transport intermediate in the vacuolar protein sorting pathway. In addition, this protein delivery block shows specificity for soluble vacuolar enzymes as the membrane protein, alkaline phosphatase, is efficiently delivered to the vacuole at the non-permissive temperature. Interestingly, the C-terminal Vps15p truncations are not phosphorylated in vivo suggesting that the phosphorylation of Vps15p may be critical for its biological activity at elevated temperatures. The rapid onset and high degree of specificity of the vacuolar protein delivery block in these mutants suggests that the primary role of Vps15p is to regulate the sorting of soluble hydrolases to the yeast vacuolar compartment. Images PMID:1756716

  3. Halloysite clay nanotubes for controlled delivery of chemically active agents

    NASA Astrophysics Data System (ADS)

    Abdullayev, Elshard

    In this work we explored the capabilities of halloysite nanotubes as capsules for encapsulation and controlled delivery of the chemically and biologically active substances. Halloysite is a two-layered aluminosilicate which has a predominantly hollow tubular structure in the submicron range and is chemically similar to kaolinite [1, 2]. In the first section of this work, we analyzed the structure of the halloysite nanotubes as well as its capability to encapsulate and deliver biologically and chemically active agents, similarities and differences between release characteristics of different agents and how these differences relate with their chemical structure. Models were used to describe the release characteristics of the active agents. Study of the interaction between loaded agents and halloysite nanotubes provides better understanding of the release characteristics of the loaded agents and how halloysite can be implemented for technological and medical applications. The second part of the work deals with self-healing coatings produced on the basis of halloysite nanotubes loaded with corrosion inhibitors. Self-healing coatings are one of the effective methods to protect metals from corrosion and deterioration. The difference between self-healing coatings and the usual coatings is the ability of the first to recover after the formation of the damages due to external or internal stresses. High efficiency of the self- healing coatings produced by halloysite nanotubes were demonstrated on 110 Copper alloys and 2024 aluminum alloys. Controlled delivery of the corrosion inhibitors with additional encapsulation of the halloysite nanotubes by synthesizing stoppers at tube endings was also demonstrated. Additional encapsulation of the halloysite nanotubes may be necessary when slow release of the loaded agents is required or rapid convection of the liquid in the surrounding environment takes place (since this may cause rapid release of the loaded agents without additional

  4. Oral Delivery of Bioencapsulated Proteins Across Blood–Brain and Blood–Retinal Barriers

    PubMed Central

    Kohli, Neha; Westerveld, Donevan R; Ayache, Alexandra C; Verma, Amrisha; Shil, Pollob; Prasad, Tuhina; Zhu, Ping; Chan, Sic L; Li, Qiuhong; Daniell, Henry

    2014-01-01

    Delivering neurotherapeutics to target brain-associated diseases is a major challenge. Therefore, we investigated oral delivery of green fluorescence protein (GFP) or myelin basic protein (MBP) fused with the transmucosal carrier cholera toxin B subunit (CTB), expressed in chloroplasts (bioencapsulated within plant cells) to the brain and retinae of triple transgenic Alzheimer's disease (3×TgAD) mice, across the blood–brain barriers (BBB) and blood–retinal barriers (BRB). Human neuroblastoma cells internalized GFP when incubated with CTB-GFP but not with GFP alone. Oral delivery of CTB-MBP in healthy and 3×TgAD mice shows increased MBP levels in different regions of the brain, crossing intact BBB. Thioflavin S–stained amyloid plaque intensity was reduced up to 60% by CTB-MBP incubation with human AD and 3×TgAD mice brain sections ex vivo. Amyloid loads were reduced in vivo by 70% in hippocampus and cortex brain regions of 3×TgAD mice fed with bioencapsulated CTB-MBP, along with reduction in the ratio of insoluble amyloid β 42 (Aβ42) to soluble fractions. CTB-MBP oral delivery reduced Aβ42 accumulation in retinae and prevented loss of retinal ganglion cells in 3×TgAD mice. Lyophilization of leaves increased CTB-MBP concentration by 17-fold and stabilized it during long-term storage in capsules, facilitating low-cost oral delivery of therapeutic proteins across the BBB and BRB. PMID:24281246

  5. Controlled protein delivery from electrospun non-wovens: novel combination of protein crystals and a biodegradable release matrix.

    PubMed

    Puhl, Sebastian; Li, Linhao; Meinel, Lorenz; Germershaus, Oliver

    2014-07-07

    Poly-ε-caprolactone (PCL) is an excellent polymer for electrospinning and matrix-controlled drug delivery combining optimal processability and good biocompatibility. Electrospinning of proteins has been shown to be challenging via the use of organic solvents, frequently resulting in protein unfolding or aggregation. Encapsulation of protein crystals represents an attractive but largely unexplored alternative to established protein encapsulation techniques because of increased thermodynamic stability and improved solvent resistance of the crystalline state. We herein explore the electrospinning of protein crystal suspensions and establish basic design principles for this novel type of protein delivery system. PCL was deployed as a matrix, and lysozyme was used as a crystallizing model protein. By rational combination of lysozyme crystals 0.7 or 2.1 μm in diameter and a PCL fiber diameter between 1.6 and 10 μm, release within the first 24 h could be varied between approximately 10 and 100%. Lysozyme loading of PCL microfibers between 0.5 and 5% was achieved without affecting processability. While relative release was unaffected by loading percentage, the amount of lysozyme released could be tailored. PCL was blended with poly(ethylene glycol) and poly(lactic-co-glycolic acid) to further modify the release rate. Under optimized conditions, an almost constant lysozyme release over 11 weeks was achieved.

  6. Intrathecal delivery of protein therapeutics to the brain: a critical reassessment.

    PubMed

    Calias, Pericles; Banks, William A; Begley, David; Scarpa, Maurizio; Dickson, Patricia

    2014-11-01

    Disorders of the central nervous system (CNS), including stroke, neurodegenerative diseases, and brain tumors, are the world's leading causes of disability. Delivery of drugs to the CNS is complicated by the blood-brain barriers that protect the brain from the unregulated leakage and entry of substances, including proteins, from the blood. Yet proteins represent one of the most promising classes of therapeutics for the treatment of CNS diseases. Many strategies for overcoming these obstacles are in development, but the relatively straightforward approach of bypassing these barriers through direct intrathecal administration has been largely overlooked. Originally discounted because of its lack of usefulness for delivering small, lipid-soluble drugs to the brain, the intrathecal route has emerged as a useful, in some cases perhaps the ideal, route of administration for certain therapeutic protein and targeted disease combinations. Here, we review blood-brain barrier functions and cerebrospinal fluid dynamics and their relevance to drug delivery via the intrathecal route, discuss animal and human studies that have investigated intrathecal delivery of protein therapeutics, and outline several characteristics of protein therapeutics that can allow them to be successfully delivered intrathecally.

  7. Generation and remote delivery of plasma activated species

    NASA Astrophysics Data System (ADS)

    Maguire, Paul; Mahony, Charles; Kelsey, Colin; Rutherford, David; Mariotti, Davide; Macias-Montero, Manuel; Perez-Martin, Fatima; Diver, Declan

    2016-09-01

    Plasma interactions with microdroplets offer new opportunities to deliver active chemical agents and nanoparticles to remote substrates downstream with many potential applications from cancer theranostics and wound healing in biomedicine, gentle food decontamination and seed germination in plasma agriculture to catalyst production and photonic structures fabrication, among others. We demonstrate plasma-liquid based pristine nanomaterials synthesis in flight and subsequent delivery up to 120mm from the atmospheric pressure plasma source. Monosized and non-aggregating metal nanoparticles are formed in the rf plasma in less than 100us, representing an increase in precursor reduction rate that is many (>4) orders of magnitude faster than that observed with standard colloidal chemistry or via high energy radiolytic techniques. Also the collection and purification limitations of the latter are avoided. Plasma activated liquid including OH radicals and H2O2 are transported over 120mm and have demonstrated high efficacy bacterial decontamination. These results will be compared with charge species and radical transport from the rf plasma without microdroplets. Reaction models based on high solvated surface electron concentrations will be presented. Funding from EPSRC acknowledged (Grants EP/K006088/1 and EP/K006142/1).

  8. Near-infrared light activated delivery platform for cancer therapy.

    PubMed

    Lin, Min; Gao, Yan; Hornicek, Francis; Xu, Feng; Lu, Tian Jian; Amiji, Mansoor; Duan, Zhenfeng

    2015-12-01

    Cancer treatment using conventional drug delivery platforms may lead to fatal damage to normal cells. Among various intelligent delivery platforms, photoresponsive delivery platforms are becoming popular, as light can be easily focused and tuned in terms of power intensity, wavelength, and irradiation time, allowing remote and precise control over therapeutic payload release both spatially and temporally. This unprecedented controlled delivery manner is important to improve therapeutic efficacy while minimizing side effects. However, most of the existing photoactive delivery platforms require UV/visible excitation to initiate their function, which suffers from phototoxicity and low level of tissue penetration limiting their practical applications in biomedicine. With the advanced optical property of converting near infrared (NIR) excitation to localized UV/visible emission, upconversion nanoparticles (UCNPs) have emerged as a promising photoactive delivery platform that provides practical applications for remote spatially and temporally controlled release of therapeutic payload molecules using low phototoxic and high tissue penetration NIR light as the excitation source. This article reviews the state-of-the-art design, synthesis and therapeutic molecular payload encapsulation strategies of UCNP-based photoactive delivery platforms for cancer therapy. Challenges and promises for engineering of advanced delivery platforms are also highlighted.

  9. PLGA/alginate composite microspheres for hydrophilic protein delivery.

    PubMed

    Zhai, Peng; Chen, X B; Schreyer, David J

    2015-11-01

    Poly(lactic-co-glycolic acid) (PLGA) microspheres and PLGA/alginate composite microspheres were prepared by a novel double emulsion and solvent evaporation technique and loaded with bovine serum albumin (BSA) or rabbit anti-laminin antibody protein. The addition of alginate and the use of a surfactant during microsphere preparation increased the encapsulation efficiency and reduced the initial burst release of hydrophilic BSA. Confocal laser scanning microcopy (CLSM) of BSA-loaded PLGA/alginate composite microspheres showed that PLGA, alginate, and BSA were distributed throughout the depths of microspheres; no core/shell structure was observed. Scanning electron microscopy revealed that PLGA microspheres erode and degrade more quickly than PLGA/alginate composite microspheres. When loaded with anti-laminin antibody, the function of released antibody was well preserved in both PLGA and PLGA/alginate composite microspheres. The biocompatibility of PLGA and PLGA/alginate microspheres were examined using four types of cultured cell lines, representing different tissue types. Cell survival was variably affected by the inclusion of alginate in composite microspheres, possibly due to the sensitivity of different cell types to excess calcium that may be released from the calcium cross-linked alginate.

  10. Delivery Mode, Duration of Labor, and Cord Blood Adiponectin, Leptin, and C-Reactive Protein: Results of the Population-Based Ulm Birth Cohort Studies

    PubMed Central

    Logan, Chad A.; Thiel, Larissa; Bornemann, Rebecca; Koenig, Wolfgang; Reister, Frank; Brenner, Hermann; Rothenbacher, Dietrich; Genuneit, Jon

    2016-01-01

    Background Numerous studies have reported associations between delivery mode and health outcomes in infancy and later life. Previous smaller studies indicated a relationship between delivery mode and newborn inflammation potentially constituting a mediating factor. We aimed to determine the influence of delivery mode and duration of labor on cord blood concentrations of adiponectin, leptin, and high-sensitivity C-reactive protein (hs-CRP). Methods In the Ulm SPATZ Health Study, 934 singleton newborns and their mothers were recruited during their hospital stay in the University Medical Center Ulm, Southern Germany, from 04/2012-05/2013. Inflammatory biomarkers were measured by ELISAs (n = 836). Delivery mode was analyzed categorically (elective cesarean (reference), active labor delivery: emergency cesarean, assisted vaginal, and spontaneous vaginal); duration of labor continuously. Following log-transformation, linear regression was used to estimate geometric means ratios (GMR) adjusted for potential confounders for the effects of delivery mode and duration of labor on each biomarker separately. Independent replication was sought in the similarly conducted Ulm Birth Cohort Study recruited from 11/2000-11/2001. Results Individually, active labor delivery modes as well as increasing duration of labor were associated with higher leptin and hs-CRP concentrations. After mutual adjustment, the associations with delivery modes were attenuated but those for duration of labor remained statistically significant (GMR (95%CI) 1.10 (1.00; 1.21) and 1.15 (1.04; 1.27) for leptin and hs-CRP per hour of labor, respectively). No significant adjusted associations were observed between delivery modes and adiponectin concentrations. These findings were replicated in an independent birth cohort study. Conclusions Cord blood leptin and hs-CRP concentrations were associated with duration of labor rather than delivery mode. Further research is warranted to investigate these associations

  11. Hybrid nanosystems based on natural polymers as protein carriers for respiratory delivery: Stability and toxicological evaluation.

    PubMed

    Rodrigues, Susana; Cordeiro, Clara; Seijo, Begoña; Remuñán-López, Carmen; Grenha, Ana

    2015-06-05

    Chitosan/carrageenan/tripolyphosphate nanoparticles were previously presented as holding potential for an application in transmucosal delivery of macromolecules, with tripolyphosphate demonstrating to contribute for both size reduction and stabilisation of the nanoparticles. This work was aimed at evaluating the capacity of the nanoparticles as protein carriers for pulmonary and nasal transmucosal delivery, further assessing their biocompatibility pattern regarding that application. Nanoparticles demonstrated stability in presence of lysozyme, while freeze-drying was shown to preserve their characteristics when glucose or sucrose were used as cryoprotectants. Bovine serum albumin was associated to the nanoparticles, which were successfully microencapsulated by spray-drying to meet the aerodynamic requirements inherent to pulmonary delivery. Finally, a satisfactory biocompatibility profile was demonstrated upon exposure of two respiratory cell lines (Calu-3 and A549 cells) to the carriers. A negligible effect on cell viability along with no alterations on transepithelial electrical resistance and no induction of inflammatory response were observed.

  12. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  13. Differential analysis of "protein corona" profile adsorbed onto different nonviral gene delivery systems.

    PubMed

    Capriotti, Anna Laura; Caracciolo, Giulio; Caruso, Giuseppe; Foglia, Patrizia; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2011-12-15

    A shotgun proteomics approach was used to characterize and compare the proteins that lead to the formation of a rich "protein corona" adsorbed onto the surfaces of cationic liposomes (CLs), lipoplexes, and lipid/polycation/DNA (LPD) complexes, when they come into contact with plasma. After separation of the nanoparticle-protein complex from plasma, the protein mixture was digested, and peptides were analyzed by nanoliquid chromatography-Orbitrap LTQ-XL mass spectrometry. The number of proteins bound to lipoplexes was double that of those identified in the corona of CLs (208 vs 105), while 77 proteins were common to both coronas. The number of proteins bound to the surface of the LPD complexes (158, 133 of which are common to lipoplexes) is intermediate between those found in the protein corona of both CLs and lipoplexes. About half of them were found in the protein corona of CLs. By overlapping the three formulations, it can be seen that only 12 proteins are peculiar to LPD complexes. These results may help in designing gene delivery systems capable of binding the minimum possible quantity of proteins that influence transfection negatively, binding selectively proteins capable of helping in steering in vivo the vector toward the target, and obtaining more efficient and effective gene therapy.

  14. Globular protein-coated Paclitaxel nanosuspensions: interaction mechanism, direct cytosolic delivery, and significant improvement in pharmacokinetics.

    PubMed

    Li, Yongji; Wu, Zhannan; He, Wei; Qin, Chao; Yao, Jing; Zhou, Jianping; Yin, Lifang

    2015-05-04

    , respectively, and prolonged T1/2 by 314-fold. As expected, PTX-NS had better in vitro and in vivo antitumor activity compared to PTX alone. Additionally, β-LG is cyto- and bio-compatible, and PTX-NS is not toxic to healthy tissues. In conclusion, the present study has suggested the high potency of globular proteins, such as β-LG, as novel biomaterials for nanosuspension platform to improve the drug delivery for disease treatment.

  15. Emulsomes Meet S-layer Proteins: An Emerging Targeted Drug Delivery System

    PubMed Central

    Ucisik, Mehmet H.; Sleytr, Uwe B.; Schuster, Bernhard

    2015-01-01

    Here, the use of emulsomes as a drug delivery system is reviewed and compared with other similar lipidic nanoformulations. In particular, we look at surface modification of emulsomes using S-layer proteins, which are self-assembling proteins that cover the surface of many prokaryotic organisms. It has been shown that covering emulsomes with a crystalline S-layer lattice can protect cells from oxidative stress and membrane damage. In the future, the capability to recrystallize S-layer fusion proteins on lipidic nanoformulations may allow the presentation of binding functions or homing protein domains to achieve highly specific targeted delivery of drug-loaded emulsomes. Besides the discussion on several designs and advantages of composite emulsomes, the success of emulsomes for the delivery of drugs to fight against viral and fungal infections, dermal therapy, cancer, and autoimmunity is summarized. Further research might lead to smart, biocompatible emulsomes, which are able to protect and reduce the side effects caused by the drug, but at the same time are equipped with specific targeting molecules to find the desired site of action. PMID:25697368

  16. SPE-39 Family Proteins Interact with the HOPS Complex and Function in Lysosomal Delivery

    PubMed Central

    Zhu, Guang-dan; Salazar, Gloria; Zlatic, Stephanie A.; Fiza, Babar; Doucette, Michele M.; Heilman, Craig J.; Levey, Allan I.

    2009-01-01

    Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here, we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, RAB7-, and RAB11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. Although Caenorhabditis elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B, whereas RNA interference of VPS33B causes spe-39–like spermatogenesis defects. The human SPE-39 orthologue C14orf133 also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, syntaxin 8-, and syntaxin 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis. PMID:19109425

  17. Physical and chemical gels of lipid nanoparticles for controlled delivery of lipophilic drugs and proteins

    NASA Astrophysics Data System (ADS)

    Couffin, Anne-Claude; Delmas, Thomas; Thomann, Jean-Sébastien; Cheibani, Ismail; Bayma, Eric; Heinrich, Emilie; Escudé, Marie; Courant, Thomas; Hoang, Antoine; Auzély, Rachel; Texier, Isabelle

    2013-05-01

    The controlled delivery of drugs and biologicals (proteins, antibodies, DNA and derivatives) is a growing need to take the full benefit of new therapeutic strategies. However these new molecules or biomolecules display solubility issues, or high degradation rates once injected. Therefore, both suitable delivery materials for their encapsulation and protection from the surrounding environment, and smart delivery devices (such as micro-needles or implanted pumps) are necessary to achieve controlled delivery of these precious therapeutic agents. We have developed bio-inspired gel materials, based on lipid nanoparticles which act as reservoirs for lipophilic drugs. The lipid nanoparticles, termed lipidots™, are biocompatible, colloidally stable, non-immunogenic, and obtained from a cheap and simple solvent-free process. The particles can be assembled to form physical or chemical gels, with tunable rheological properties. Physico-chemical studies have been carried out to determine the limits of the stability domains for colloidal and gel formulations (choice of surfactants for nanoparticle surface, and composition ratios of lipids, surfactants and co-surfactants). In particular, it is demonstrated that lipid nanoparticles keep their integrity in the gels. Gels of lipidots™ could therefore constitute biocompatible materials for the efficient encapsulation and tuned delivery of lipophilic drugs and biomolecules.

  18. Enzymatic biodegradation of hydrogels for protein delivery targeted to the small intestine.

    PubMed

    Knipe, Jennifer M; Chen, Frances; Peppas, Nicholas A

    2015-03-09

    Multiresponsive poly(methacrylic acid-co-N-vinylpyrrolidone) hydrogels were synthesized with biodegradable oligopeptide crosslinks. The oligopeptide crosslinks were incorporated using EDC-NHS zero-length links between the carboxylic acid groups of the polymer and free primary amines on the peptide. The reaction of the peptide was confirmed by primary amine assay and IR spectroscopy. The microgels exhibited pH-responsive swelling as well as enzyme-catalyzed degradation targeted by trypsin present in the small intestine, as demonstrated upon incubation with gastrointestinal fluids from rats. Relative turbidity was used to evaluate enzyme-catalyzed degradation as a function of time, and initial trypsin concentration controlled both the degradation mechanism as well as the extent of degradation. Trypsin activity was effectively extinguished by incubation at 70 °C, and both the microgels and degradation products posed no cytotoxic effect toward two different cell lines. The microgels demonstrated pH-dependent loading of the protein insulin for oral delivery to the small intestine.

  19. Oral films as breakthrough tools for oral delivery of proteins/peptides.

    PubMed

    Castro, Pedro M; Fonte, Pedro; Sousa, Flávia; Madureira, Ana Raquel; Sarmento, Bruno; Pintado, Manuela E

    2015-08-10

    Therapeutic proteins and peptides demonstrate unique, peerless, pharmacological characteristics such as high specificity to receptors and superior biological mimicking of physiological mechanisms, resulting in a better therapeutic index compared to conventional chemical-derived drugs. However, proteins also present inherent bioavailability limitations. Thus, this paper proposes several effective tools to improve protein/peptide drugs stability, permeability and pharmacokinetics with special emphasis on oral polymeric films as oral delivery platforms. Indeed, oral films present inherent characteristics that can greatly enhance biological performance of proteins and peptides and patient compliance along with other advantages that are critically discussed in this review. A rational choice of excipients addressed in and manufacture processes are also focused. In addition, possible toxicity issues to be overtaken and critical analysis regarding current market tendencies respecting oral films and protein/peptides along with future prospects are disclosed.

  20. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease.

    PubMed

    Dinca, Ana; Chien, Wei-Ming; Chin, Michael T

    2016-02-22

    Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.

  1. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease

    PubMed Central

    Dinca, Ana; Chien, Wei-Ming; Chin, Michael T.

    2016-01-01

    Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects. PMID:26907261

  2. pH-responsive and enzymatically-responsive hydrogel microparticles for the oral delivery of therapeutic proteins: Effects of protein size, crosslinking density, and hydrogel degradation on protein delivery.

    PubMed

    Koetting, Michael Clinton; Guido, Joseph Frank; Gupta, Malvika; Zhang, Annie; Peppas, Nicholas A

    2016-01-10

    Two potential platform technologies for the oral delivery of protein therapeutics were synthesized and tested. pH-responsive poly(itaconic acid-co-N-vinyl-2-pyrrolidone) (P(IA-co-NVP)) hydrogel microparticles were tested in vitro with model proteins salmon calcitonin, urokinase, and rituximab to determine the effects of particle size, protein size, and crosslinking density on oral delivery capability. Particle size showed no significant effect on overall delivery potential but did improve percent release of encapsulated protein over the micro-scale particle size range studied. Protein size was shown to have a significant impact on the delivery capability of the P(IA-co-NVP) hydrogel. We show that when using P(IA-co-NVP) hydrogel microparticles with 3 mol% tetra(ethylene glycol) dimethacrylate crosslinker, a small polypeptide (salmon calcitonin) loads and releases up to 45 μg/mg hydrogel while the mid-sized protein urokinase and large monoclonal antibody rituximab load and release only 19 and 24 μg/mg hydrogel, respectively. We further demonstrate that crosslinking density offers a simple method for tuning hydrogel properties to variously sized proteins. Using 5 mol% TEGDMA crosslinker offers optimal performance for the small peptide, salmon calcitonin, whereas lower crosslinking density of 1 mol% offers optimal performance for the much larger protein rituximab. Finally, an enzymatically-degradable hydrogels of P(MAA-co-NVP) crosslinked with the peptide sequence MMRRRKK were synthesized and tested in simulated gastric and intestinal conditions. These hydrogels offer ideal loading and release behavior, showing no degradative release of encapsulated salmon calcitonin in gastric conditions while yielding rapid and complete release of encapsulated protein within 1h in intestinal conditions.

  3. Ultrasonic-Activated Micellar Drug Delivery for Cancer Treatment

    PubMed Central

    Husseini, Ghaleb A.; Pitt, William G.

    2008-01-01

    The use of nanoparticles and ultrasound in medicine continues to evolve. Great strides have been made in the areas of producing micelles, nanoemulsions and solid nanoparticles that can be used in drug delivery. An effective nanocarrier allows for the delivery of a high concentration of potent medications to targeted tissue while minimizing the side effect of the agent to the rest of the body. Polymeric micelles have been shown to encapsulate therapeutic agents and maintain their structural integrity at lower concentrations. Ultrasound is currently being used in drug delivery as well as diagnostics, and has many advantages that elevate its importance in drug delivery. The technique is non-invasive, thus no surgery is needed; the ultrasonic waves can be easily controlled by advanced electronic technology so that they can be focused on the desired target volume. Additionally, the physics of ultrasound are widely used and well understood; thus ultrasonic application can be tailored towards a particular drug delivery system. In this article, we review the recent progress made in research that utilizes both polymeric micelles and ultrasonic power in drug delivery. PMID:18506804

  4. Quantitative ToF-SIMS studies of protein drug release from biodegradable polymer drug delivery membranes

    NASA Astrophysics Data System (ADS)

    Burns, Sarah A.; Gardella, Joseph A.

    2008-12-01

    Biodegradable polymers are of interest in developing strategies to control protein drug delivery. The protein that was used in this study is Keratinocyte Growth Factor (KGF) which is a protein involved in the re-epithelialization process. The protein is stabilized in the biodegradable polymer matrix during formulation and over the course of polymer degradation with the use of an ionic surfactant Aerosol-OT (AOT) which will encapsulate the protein in an aqueous environment. The release kinetics of the protein from the surface of these materials requires precise timing which is a crucial factor in the efficacy of this drug delivery system. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used in the same capacity to identify the molecular ion peak of the surfactant and polymer and use this to determine surface concentration. In the polymer matrix, the surfactant molecular ion peak was observed in the positive and negative mode at m/ z 467 and 421, respectively. These peaks were determined to be [AOT + Na +] and [AOT - Na +]. These methods are used to identify the surfactant and protein from the polymer matrix and are used to measure the rate of surface accumulation. The second step was to compare this accumulation rate with the release rate of the protein into an aqueous solution during the degradation of the biodegradable film. This rate is compared to that from fluorescence spectroscopy measurements using the protein autofluorescence from that released into aqueous solution [C.M. Mahoney, J. Yu, A. Fahey, J.A.J. Gardella, SIMS depth profiling of polymer blends with protein based drugs, Appl. Surf. Sci. 252 (2006), 6609-6614.].

  5. Formation of multimeric antibodies for self-delivery of active monomers.

    PubMed

    Dekel, Yaron; Machluf, Yossy; Gefen, Tal; Eidelshtein, Gennady; Kotlyar, Alexander; Bram, Yaron; Shahar, Ehud; Reslane, Farah; Aizenshtein, Elina; Pitcovski, Jacob

    2017-11-01

    Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.

  6. Delivery Systems for Bone Morphogenetic Protein (BMP) for Repair of Battle Incurred Bone Injuries.

    DTIC Science & Technology

    1987-11-01

    infections, congenital malformations that fail to heal are eligible for BMP treatment. I (my child/my ward) will be one of 50 patients to be treated with...Fusions in Dogs 6. Craniotomy Defects in Sheep t0 7. Craniotomy Defects in Monkeys 10 8. BMP Delivery System of Bone Matrix Non Collagenous 11 Proteins...effects. The most important and indispensptle substitutes for experiments in human beings are adult mongrel dogs, monkeys, and sheep . Experimental .S

  7. Peptides and proteins used to enhance gold nanoparticle delivery to the brain: preclinical approaches.

    PubMed

    Velasco-Aguirre, Carolina; Morales, Francisco; Gallardo-Toledo, Eduardo; Guerrero, Simon; Giralt, Ernest; Araya, Eyleen; Kogan, Marcelo J

    2015-01-01

    An exciting and emerging field in nanomedicine involves the use of gold nanoparticles (AuNPs) in the preclinical development of new strategies for the treatment and diagnosis of brain-related diseases such as neurodegeneration and cerebral tumors. The treatment of many brain-related disorders with AuNPs, which possess useful physical properties, is limited by the blood-brain barrier (BBB). The BBB highly regulates the substances that can permeate into the brain. Peptides and proteins may represent promising tools to improve the delivery of AuNPs to the central nervous system (CNS). In this review, we summarize the potential applications of AuNPs to CNS disorders, discuss different strategies based on the use of peptides or proteins to improve the delivery of AuNPs to the brain, and examine the intranasal administration route, which bypasses the BBB. We also analyze the potential neurotoxicity of AuNPs and the perspectives and new challenges concerning the use of peptides and proteins to enhance the delivery of AuNPs to the brain. The majority of the work described in this review is in a preclinical stage of experimentation, or in select cases, in clinical trials in humans. We note that the use of AuNPs still requires substantial study before being translated into human applications. However, for further clinical research, the issues related to the potential use of AuNPs must be analyzed.

  8. Targeting of gastrointestinal tract for amended delivery of protein/peptide therapeutics: strategies and industrial perspectives.

    PubMed

    Pawar, Vivek K; Meher, Jaya Gopal; Singh, Yuvraj; Chaurasia, Mohini; Surendar Reddy, B; Chourasia, Manish K

    2014-12-28

    Delivery of proteins/peptides to the gastrointestinal (GI) tract via peroral/oral route involves tremendous challenges due to unfavorable environmental conditions like harsh pH, presence of proteolytic enzymes and absorption barriers. Detailed research is being conducted at the academic and industrial levels to diminish these troubles and various products are under clinical trials. Several approaches have been established to optimize oral delivery of proteins and peptides and can be broadly categorized into chemical and physical strategies. Chemical strategies include site specific mutagenesis, proteinylation, glycosylation, PEGylation and prodrug approaches, whereas physical strategies comprise formulation based approaches including application of absorption enhancers and metabolism modifiers along with delivering them via colloidal carrier systems such as nanoparticles, liposomes, microparticles, and micro- and nano-emulsions. This review stands to accomplish the diverse aspects of oral delivery of proteins/peptides and summarizes the key concepts involved in targeting the biodrugs to specific sites of the GI tract such as the intestine and colon. Furthermore some light has also been shed on the current industrial practices followed in developing oral formulations of such bioactives.

  9. Peptides and proteins used to enhance gold nanoparticle delivery to the brain: preclinical approaches

    PubMed Central

    Velasco-Aguirre, Carolina; Morales, Francisco; Gallardo-Toledo, Eduardo; Guerrero, Simon; Giralt, Ernest; Araya, Eyleen; Kogan, Marcelo J

    2015-01-01

    An exciting and emerging field in nanomedicine involves the use of gold nanoparticles (AuNPs) in the preclinical development of new strategies for the treatment and diagnosis of brain-related diseases such as neurodegeneration and cerebral tumors. The treatment of many brain-related disorders with AuNPs, which possess useful physical properties, is limited by the blood–brain barrier (BBB). The BBB highly regulates the substances that can permeate into the brain. Peptides and proteins may represent promising tools to improve the delivery of AuNPs to the central nervous system (CNS). In this review, we summarize the potential applications of AuNPs to CNS disorders, discuss different strategies based on the use of peptides or proteins to improve the delivery of AuNPs to the brain, and examine the intranasal administration route, which bypasses the BBB. We also analyze the potential neurotoxicity of AuNPs and the perspectives and new challenges concerning the use of peptides and proteins to enhance the delivery of AuNPs to the brain. The majority of the work described in this review is in a preclinical stage of experimentation, or in select cases, in clinical trials in humans. We note that the use of AuNPs still requires substantial study before being translated into human applications. However, for further clinical research, the issues related to the potential use of AuNPs must be analyzed. PMID:26300639

  10. A Universal Protein Tag for Delivery of SiRNA-Aptamer Chimeras

    PubMed Central

    Liu, Hong Yan; Gao, Xiaohu

    2013-01-01

    siRNA-aptamer chimeras have emerged as one of the most promising approaches for targeted delivery of siRNA due to the modularity of their diblock RNA structure, relatively lower cost over other targeted delivery approaches, and, most importantly, the outstanding potential for clinical translation. However, additional challenges must be addressed for efficient RNA interference (RNAi), in particular, endosomal escape. Currently, vast majority of siRNA delivery vehicles are based on cationic materials, which form complexes with negatively charged siRNA. Unfortunately, these approaches complicate the formulations again by forming large complexes with heterogeneous sizes, unfavorable surface charges, colloidal instability, and poor targeting ligand orientation. Here, we report the development of a small and simple protein tag that complements the therapeutic and targeting functionalities of chimera with two functional domains: a dsRNA binding domain (dsRBD) for siRNA docking and a pH-dependent polyhistidine to disrupt endosomal membrane. The protein selectively tags along the siRNA block of individual chimera, rendering the overall size of the complex small, desirable for deep tissue penetration, and the aptamer block accessible for target recognition. More interestingly, we found that extending the c-terminal polyhistidine segment in the protein tag to 18 amino acids completely abolishes the RNA binding function of dsRBD. PMID:24196104

  11. Comparative analysis of the methods of drug and protein delivery for the treatment of cancer, genetic diseases and diagnostics.

    PubMed

    Todorova, Roumiana

    2011-11-01

    The methods of protein and drug delivery for the treatment of cancer, genetic diseases and diagnostics were summarized. The potential of protein transduction is discussed and the recent developments in the field are reviewed. An overview is provided of the non-viral delivery methods such as liposomes, polymer-based delivery, cell-penetrating peptides, bacterial secretion, cells, virosomes, physical methods including electroporation, microinjection, osmotic lysis, nanoparticles, sonoporation to locally inject therapeutic molecules. The characteristic properties of non-viral vectors and their use for the delivery of therapeutic molecules for the diagnosis and treatment of disorders and to target tumors are also discussed. The potential of the transduced peptides and proteins was used as new therapeutic compounds against infectious diseases, to complement deficiencies in specific genes, to specifically kill tumour cells, for gene therapy. The protein delivery vectors can enhance the transfection at low concentrations and help to develop future gene delivery systems with reduced toxicity. Vitamin B12, folic acid, biotin, and riboflavin are essential in the treatment of cancer. Ultrasound has a potential in the delivery of therapeutic agents. The new developing technologies of drug delivery and targeting offer the possibility to improve the therapeutic possibilities of the existing drugs and to develop novel therapeutics.

  12. Rice seed ER-derived protein body as an efficient delivery vehicle for oral tolerogenic peptides.

    PubMed

    Takagi, Hidenori; Hiroi, Takachika; Hirose, Sakiko; Yang, Lijun; Takaiwa, Fumio

    2010-08-01

    Mucosal delivery of peptide/protein therapeutics via the oral route is a desirable strategy in human immunotherapy. A key step for enhancing the bioavailability of orally administered therapeutics is to protect them from enzymatic digestion in the gastrointestinal tract. Here, we generated transgenic rice seeds accumulating allergen-derived T cell epitopes, a model tolerogen for the control of pollen allergy, in either ER-derived protein body-I (PB-I) or protein storage vacuole protein body-II (PB-II). Compared with PB-II-localized or chemically synthesized forms, PB-I-localized T cell epitopes showed higher resistance to enzymatic digestion in simulated gastric fluid. Moreover, the dose of T cell epitope required for suppression of allergen-specific IgE in mice was about 20-fold lower when fed in PB-I localized form than when unprotected. These findings demonstrate the potential of bioencapsulation in PB-I for broad applications as a viable strategy to achieve efficient mucosal delivery of oral peptide/protein therapeutics.

  13. Multifunctional Delivery Systems for Advanced oral Uptake of Peptide/Protein Drugs.

    PubMed

    Park, Jin Woo; Kim, Sun Jin; Kwag, Dong Sup; Kim, Sol; Park, Jeyoung; Youn, Yu Seok; Bae, You Han; Lee, Eun Seong

    2015-01-01

    In recent years, advances in biotechnology and protein engineering have enabled the production of large quantities of proteins and peptides as important therapeutic agents. Various researchers have used biocompatible functional polymers to prepare oral dosage forms of proteins and peptides for chronic use and for easier administration to enhance patient compliance. However, there is a need to enhance their safety and effectiveness further. Most macromolecules undergo severe denaturation at low pH and enzymatic degradation in the gastrointestinal tract. The macromolecules' large molecular size and low lipophilicity cause low permeation through the intestinal membrane. The major strategies that have been used to overcome these challenges (in oral drug carrier systems) can be classified as follows: enteric coating or encapsulation with pH-sensitive polymers or mucoadhesive polymers, co-administration of protease inhibitors, incorporation of absorption enhancers, modification of the physicochemical properties of the macromolecules, and site-specific delivery to the colon. This review attempts to summarize the various advanced oral delivery carriers, including nanoparticles, lipid carriers, such as liposomes, nano-aggregates using amphiphilic polymers, complex coacervation of oppositely charged polyelectrolytes, and inorganic porous particles. The particles were formulated and/or surface modified with functional polysaccharides or synthetic polymers to improve oral bioavailability of proteins and peptides. We also discuss formulation strategies to overcome barriers, therapeutic efficacies in vivo, and potential benefits and issues for successful oral dosage forms of the proteins and peptides.

  14. PEG-Immobilized Keratin for Protein Drug Sequestration and pH-Mediated Delivery

    PubMed Central

    de Guzman, Roche C.; Rabbany, Sina Y.

    2016-01-01

    Protein drugs like growth factors are promising therapeutics for damaged-tissue repair. Their local delivery often requires biomaterial carriers for achieving the therapeutic dose range while extending efficacy. In this study, polyethylene glycol (PEG) and keratin were crosslinked and used as sponge-like scaffolds (KTN-PEG) to absorb test proteins with different isoelectric points (pI): albumin (~5), hemoglobin (~7), and lysozyme (~11). The protein release kinetics was influenced by charge at physiological pH 7.4. The keratin network, with pI 5.3, electrostatically attracted lysozyme and repulsed albumin generating the release rate profile: albumin > hemoglobin > lysozyme. However, under acidic conditions (pH 4), all proteins including keratins were positively charged and consequently intermolecular repulsion altered the release hierarchy, now determined by size (MW) diffusion: lysozyme (14 kDa) > hemoglobin (64 kDa) > albumin (66 kDa). Vascular endothelial growth factor C (VEGF-C), with properties comparable to lysozyme, was absorbed into the KTN-PEG scaffold. Endothelial cells cultured on this substrate had significantly larger numbers than on scaffolds without VEGF-C suggesting that the ionically bound and retained growth factor at neutral pH indirectly increased acute cell attachment and viability. PEG and keratin based sequestrations of proteins with basic pIs are therefore a feasible strategy with potential applications for selective biologics delivery. PMID:26904294

  15. Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines

    PubMed Central

    2011-01-01

    Food-grade Lactic Acid Bacteria (LAB) have been safely consumed for centuries by humans in fermented foods. Thus, they are good candidates to develop novel oral vectors, constituting attractive alternatives to attenuated pathogens, for mucosal delivery strategies. Herein, this review summarizes our research, up until now, on the use of LAB as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Most of our work has been based on the model LAB Lactococcus lactis, for which we have developed efficient genetic tools, including expression signals and host strains, for the heterologous expression of therapeutic proteins such as antigens, cytokines and enzymes. Resulting recombinant lactococci strains have been tested successfully for their prophylactic and therapeutic effects in different animal models: i) against human papillomavirus type 16 (HPV-16)-induced tumors in mice, ii) to partially prevent a bovine β-lactoglobulin (BLG)-allergic reaction in mice and iii) to regulate body weight and food consumption in obese mice. Strikingly, all of these tools have been successfully transposed to the Lactobacillus genus, in recent years, within our laboratory. Notably, anti-oxidative Lactobacillus casei strains were constructed and tested in two chemically-induced colitis models. In parallel, we also developed a strategy based on the use of L. lactis to deliver DNA at the mucosal level, and were able to show that L. lactis is able to modulate the host response through DNA delivery. Today, we consider that all of our consistent data, together with those obtained by other groups, demonstrate and reinforce the interest of using LAB, particularly lactococci and lactobacilli strains, to develop novel therapeutic protein mucosal delivery vectors which should be tested now in human clinical trials. PMID:21995317

  16. [Protein nutrition and physical activity].

    PubMed

    Navarro, M P

    1992-09-01

    The relationship between physical exercise and diet in order to optimize performance is getting growing interest. This review examines protein needs and protein intakes as well as the role of protein in the body and the metabolic changes occurring at the synthesis and catabolic levels during exercise. Protein synthesis in muscle or liver, amino acids oxidation, glucose production via gluconeogenesis from amino acids, etc., are modified, and consequently plasma and urinary nitrogen metabolites are affected. A brief comment on the advantages, disadvantages and forms of different protein supplements for sportsmen is given.

  17. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  18. Progress in nanoparticulate systems for peptide, proteins and nucleic acid drug delivery.

    PubMed

    Slomkowski, Stanislaw; Gosecki, Mateusz

    2011-11-01

    Progress in many therapies, in particular in the therapies based on peptides, proteins and nucleic acids used as bioactive compounds, strongly depends on development of appropriate carriers which would be suitable for controlled delivery of the intact abovementioned compounds to required tissues, cells and intracellular compartments. This review presents last ten years' achievements and problems in development and application of synthetic polymer nanoparticulate carriers for oral, pulmonary and nasal delivery routes of oligopeptides and proteins. Whereas some traditional synthetic polymer carriers are only briefly recalled the main attention is concentrated on nanoparticles produced from functional copolymers mostly with hydroxyl, carboxyl and amino groups, suitable for immobilization of targeting moieties and for assuring prolonged circulation of nanoparticles in blood. Formulations of various nanoparticulate systems are described, including solid particles, polymer micelles, nanovesicles and nanogels, especially systems allowing drug release induced by external stimuli. Discussed are properties of these species, in particular stability in buffers and models of body fluids, loading with drugs and with drug models, drug release processes and results of biological studies. There are also discussed systems for gene delivery with special attention devoted to polymers suitable for compacting nucleic acids into nanoparticles as well as the relations between chemical structure of polymer carriers and ability of the latter for crossing cell membranes and for endosomal escape.

  19. Self-Assembling Peptide Amphiphiles for Therapeutic Delivery of Proteins, Drugs, and Stem Cells

    NASA Astrophysics Data System (ADS)

    Lee, Sungsoo Seth

    Biomaterials are used to help regenerate or replace the structure and function of damaged tissues. In order to elicit desired therapeutic responses in vivo, biomaterials are often functionalized with bioactive agents, such as growth factors, small molecule drugs, or even stem cells. Therefore, the strategies used to incorporate these bioactive agents in the microstructures and nanostructures of biomaterials can strongly influence the their therapeutic efficacy. Using self-assembling peptide amphiphiles (PAs), this work has investigated supramolecular nanostructures with improved interaction with three types of therapeutic agents: bone morphogenetic protein 2 (BMP-2) which promotes osteogenic differentiation and bone growth, anti-inflammatory drug naproxen which is used to treat osteo- and rheumatoid arthritis, and neural stem cells that could differentiate into neurons to treat neurodegenerative diseases. For BMP-2 delivery, two specific systems were investigated with affinity for BMP-2: 1) heparin-binding nanofibers that display the natural ligand of the osteogenic protein, and 2) nanofibers that display a synthetic peptide ligand discovered in our laboratory through phage display to directly bind BMP-2. Both systems promoted enhanced osteoblast differentiation of pluripotent C2C12 cells and augmented bone regeneration in two in vivo models, a rat critical-size femur defect model and spinal arthrodesis model. The thesis also describes the use of PA nanofibers to improve the delivery of the anti-inflammatory drug naproxen. To promote a controlled release, naproxen was chemically conjugated to the nanofiber surface via an ester bond that would only be cleaved by esterases, which are enzymes found naturally in the body. In the absence of esterases, the naproxen remained conjugated to the nanofibers and was non-bioactive. On the other hand, in the presence of esterases, naproxen was slowly released and inhibited cyclooxygenase-2 (COX-2) activity, an enzyme responsible

  20. A review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules.

    PubMed

    Choonara, Bibi F; Choonara, Yahya E; Kumar, Pradeep; Bijukumar, Divya; du Toit, Lisa C; Pillay, Viness

    2014-11-15

    The oral delivery of proteins and peptides is a dynamic research field despite the numerous challenges limiting their effective delivery. Successful oral delivery of proteins and peptides requires the accomplishment of three key tasks: protection of the macromolecules from degradation in the gastrointestinal tract (GIT), permeation through the intestinal barrier and absorption of molecules into the systemic circulation. Currently, no clinically useful oral formulations have been developed but several attempts have been made to overcome the challenges of low oral bioavailability resulting from poor absorption, poor permeation and enzymatic degradation of the proteins and peptides in the GIT. Present strategies attempt to provide structural protection of the proteins and peptides and improved absorption through the use of enzyme inhibitors, absorption enhancers, novel polymeric delivery systems and chemical modification. However, each of these technologies has their limitations despite showing positive results. This review attempts to discuss the physical and chemical barriers of the GIT with particular emphasis on the current approaches employed to overcome these barriers, including the evaluation of other non-parenteral routes of protein and peptide delivery. In addition, this review assimilates oral formulation strategies under development and within the clinical trial stage in relation to their benefits and drawbacks with regard to facilitating optimal protection and absorption of proteins and peptides, as well as pertinent future challenges and opportunities governing oral drug delivery.

  1. Injectable hydrogels embedded with alginate microspheres for controlled delivery of bone morphogenetic protein-2.

    PubMed

    Zhu, Youjia; Wang, Jiulong; Wu, Jingjing; Zhang, Jun; Wan, Ying; Wu, Hua

    2016-03-23

    Some delivery carriers with injectable characteristics were built using the thermosensitive chitosan/dextran-polylactide/glycerophosphate hydrogel and selected alginate microspheres for the controllable release of bone morphogenetic protein-2 (BMP-2). BMP-2 was first loaded into the microspheres with an average size of around 20 μm and the resulting microspheres were then embedded into the gel in order to achieve well-controlled BMP-2 release. The microsphere-embedded gels show their incipient gelation temperature at around 32 °C and pH at about 7.1. Some gels had their elastic modulus close to 1400 Pa and the ratio of elastic modulus to viscous modulus at around 34, revealing that they behaved like mechanically strong gels. Optimized microsphere-embedded gels were found to be able to administer the BMP-2 release without significant initial burst release in an approximately linear manner over a period of time longer than four weeks. The release rate and the released amount of BMP-2 from these gels could be regulated individually or cooperatively by the initial BMP-2 load and the dextran-polylactide content in the gels. Measurements of the BMP-2 induced alkaline phosphatase activity in C2C12 cells confirmed that C2C12 cells responded to BMP-2 in a dose-dependent way and the released BMP-2 from the microsphere-embedded gels well retained their bioactivity. In vivo assessment of some gels revealed that the released BMP-2 maintained its osteogenesis functions.

  2. Biologically active proteins from natural product extracts.

    PubMed

    O'Keefe, B R

    2001-10-01

    The term "biologically active proteins" is almost redundant. All proteins produced by living creatures are, by their very nature, biologically active to some extent in their homologous species. In this review, a subset of these proteins will be discussed that are biologically active in heterologous systems. The isolation and characterization of novel proteins from natural product extracts including those derived from microorganisms, plants, insects, terrestrial vertebrates, and marine organisms will be reviewed and grouped into several distinct classes based on their biological activity and their structure.

  3. Cellular Delivery and Photochemical Activation of Antisense Agents through a Nucleobase Caging Strategy

    PubMed Central

    Govan, Jeane M.; Uprety, Rajendra; Thomas, Meryl; Lusic, Hrvoje; Lively, Mark O.; Deiters, Alexander

    2013-01-01

    Antisense oligonucleotides are powerful tools to regulate gene expression in cells and model organisms. However, a transfection or microinjection is needed for efficient delivery of the antisense agent. We report the conjugation of multiple HIV TAT peptides to a hairpin-protected antisense agent through a light-cleavable nucleobase caging group. This conjugation allows for the facile delivery of the antisense agent without a transfection reagent and photochemical activation offers precise control over gene expression. The developed approach is highly modular, as demonstrated by the conjugation of folic acid to the caged antisense agent. This enabled targeted cell delivery through cell-surface folate receptors followed by photochemical triggering of antisense activity. Importantly, the presented strategy delivers native oligonucleotides after light-activation, devoid of any delivery functionalities or modifications that could otherwise impair their antisense activity. PMID:23915424

  4. Comparison of different cationized proteins as biomaterials for nanoparticle-based ocular gene delivery.

    PubMed

    Zorzi, Giovanni K; Párraga, Jenny E; Seijo, Begoña; Sanchez, Alejandro

    2015-11-01

    Cationized polymers have been proposed as transfection agents for gene therapy. The present work aims to improve the understanding of the potential use of different cationized proteins (atelocollagen, albumin and gelatin) as nanoparticle components and to investigate the possibility of modulating the physicochemical properties of the resulting nanoparticle carriers by selecting specific protein characteristics in an attempt to improve current ocular gene-delivery approaches. The toxicity profiles, as well as internalization and transfection efficiency, of the developed nanoparticles can be modulated by modifying the molecular weight of the selected protein and the amine used for cationization. The most promising systems are nanoparticles based on intermediate molecular weight gelatin cationized with the endogenous amine spermine, which exhibit an adequate toxicological profile, as well as effective association and protection of pDNA or siRNA molecules, thereby resulting in higher transfection efficiency and gene silencing than the other studied formulations.

  5. Soy protein/soy polysaccharide complex nanogels: folic acid loading, protection, and controlled delivery.

    PubMed

    Ding, Xuzhe; Yao, Ping

    2013-07-09

    In this study, we developed a facile approach to produce nanogels via self-assembly of folic acid, soy protein, and soy polysaccharide. High-pressure homogenization was introduced to break down the original aggregates of soy protein, which benefits the binding of soy protein with soy polysaccharide and folic acid at pH 4.0. After a heat treatment that causes the soy protein denaturation and gelation, folic acid-loaded soy protein/soy polysaccharide complex nanogels were fabricated. The nanogels have a polysaccharide surface that makes the nanogels dispersible in acidic conditions where folic acid is insoluble and soy protein forms precipitates after heating. More importantly, the protein and polysaccharide can inhibit the reactions between dissolved oxygen and folic acid during UV irradiation. After the preparation and storage of the nanogels in the presence of heat, oxygen, and light in acidic conditions, most of the folic acid molecules in the nanogels remain in their natural structure and can be released rapidly at neutral pH, that is, in the intestine. Because most food and beverages are acidic, the nanogels are a suitable delivery system of folic acid in food and beverages.

  6. A translocation signal for delivery of oomycete effector proteins into host plant cells.

    PubMed

    Whisson, Stephen C; Boevink, Petra C; Moleleki, Lucy; Avrova, Anna O; Morales, Juan G; Gilroy, Eleanor M; Armstrong, Miles R; Grouffaud, Severine; van West, Pieter; Chapman, Sean; Hein, Ingo; Toth, Ian K; Pritchard, Leighton; Birch, Paul R J

    2007-11-01

    Bacterial, oomycete and fungal plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK--representing a change that conserves physicochemical properties of the protein--P. infestans fails to deliver Avr3a or an Avr3a-GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.

  7. Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates.

    PubMed

    Seras-Franzoso, Joaquin; Peebo, Karl; García-Fruitós, Elena; Vázquez, Esther; Rinas, Ursula; Villaverde, Antonio

    2014-03-01

    Bacterial inclusion bodies (IBs) have recently been used to generate biocompatible cell culture interfaces, with diverse effects on cultured cells such as cell adhesion enhancement, stimulation of cell growth or induction of mesenchymal stem cell differentiation. Additionally, novel applications of IBs as sustained protein delivery systems with potential applications in regenerative medicine have been successfully explored. In this scenario, with IBs gaining significance in the biomedical field, the fine tuning of this functional biomaterial is crucial. In this work, the effect of temperature on fibroblast growth factor-2 (FGF-2) IB production and performance has been evaluated. FGF-2 was overexpressed in Escherichia coli at 25 and 37 °C, producing IBs with differences in size, particle structure and biological activity. Cell culture topographies made with FGF-2 IBs biofabricated at 25 °C showed higher levels of biological activity as well as a looser supramolecular structure, enabling a higher protein release from the particles. In addition, the controlled use of FGF-2 protein particles enabled the generation of functional topographies with multiple biological activities being effective on diverse cell types.

  8. Low-activity waste feed delivery -- Minimum duration between successive batches

    SciTech Connect

    Peters, B.B.

    1998-08-25

    The purpose of this study is to develop a defensible basis for establishing what ``minimum duration`` will provide acceptable risk mitigation for low-activity waste feed delivery to the privatization vendors. The study establishes a probabilistic-based duration for staging of low-activity waste feed batches. A comparison is made of the durations with current feed delivery plans and potential privatization vendor facility throughput rates.

  9. Rational Design of Protein C Activators

    PubMed Central

    Barranco-Medina, Sergio; Murphy, Mary; Pelc, Leslie; Chen, Zhiwei; Di Cera, Enrico; Pozzi, Nicola

    2017-01-01

    In addition to its procoagulant and proinflammatory functions mediated by cleavage of fibrinogen and PAR1, the trypsin-like protease thrombin activates the anticoagulant protein C in a reaction that requires the cofactor thrombomodulin and the endothelial protein C receptor. Once in the circulation, activated protein C functions as an anticoagulant, anti-inflammatory and regenerative factor. Hence, availability of a protein C activator would afford a therapeutic for patients suffering from thrombotic disorders and a diagnostic tool for monitoring the level of protein C in plasma. Here, we present a fusion protein where thrombin and the EGF456 domain of thrombomodulin are connected through a peptide linker. The fusion protein recapitulates the functional and structural properties of the thrombin-thrombomodulin complex, prolongs the clotting time by generating pharmacological quantities of activated protein C and effectively diagnoses protein C deficiency in human plasma. Notably, these functions do not require exogenous thrombomodulin, unlike other anticoagulant thrombin derivatives engineered to date. These features make the fusion protein an innovative step toward the development of protein C activators of clinical and diagnostic relevance. PMID:28294177

  10. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system

    PubMed Central

    Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

    2012-01-01

    Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins. PMID:16603603

  11. Enhanced protein delivery by multi-ion containing eggshell derived apatitic-alginate composite nanocarriers.

    PubMed

    Sampath Kumar, T S; Madhumathi, K; Rajkamal, B; Zaheatha, S; Rajathi Malar, A; Alamelu Bai, S

    2014-11-01

    Eggshell is an attractive natural source of calcium for the synthesis of hydroxyapatite (HA) as it contains minor amounts of biologically relevant elements such as Mg, Sr, and Si. The mineral phase of the human bone is essentially a calcium deficient hydroxyapatite (CDHA) which shows more bioactivities and absorbance than stoichiometric HA does. Hence, we have attempted to develop a protein delivery system based on eggshell derived CDHA (ECDHA) nanoparticles for bone tissue engineering. Nanoparticles with Ca/P molar ratio of 1.67, 1.61 and 1.51 to form CDHAs with compositions covering the properties of stable HA phase (Ca/P=1.67) to degradable tricalcium phosphate (TCP) phase (Ca/P=1.5) were synthesized by microwave-accelerated wet chemical synthesis using eggshell as well as synthetic calcium hydroxide as calcium precursors. The delivery profiles of bovine serum albumin (BSA), a model protein by the nanocarriers, were studied. Both eggshells derived and synthetic CDHA samples showed maximum amount of loading of 57% and 37%, respectively at a Ca/P ratio of 1.51, comparing to stoichiometric HA. ECDHA also showed a much more BSA release (25%) than synthetically derived CDHA (6.5%) did. To further improve the release profile, alginate coating was carried out on CDHA nanoparticles and the BSA release profiles were evaluated. A maximum release of 65% was observed for alginate coated ECDHA at a Ca/P ratio of 1.51 for a period of 2 days. The ECDHA nanoparticle with a Ca/P ratio similar to degradable TCP and with alginate coating seems to be an ideal protein delivery agent.

  12. Protein-lipid nanohybrids as emerging platforms for drug and gene delivery: Challenges and outcomes.

    PubMed

    Gaber, Mohamed; Medhat, Waseem; Hany, Mark; Saher, Nourhan; Fang, Jia-You; Elzoghby, Ahmed

    2017-03-30

    Nanoparticulate drug delivery systems have been long used to deliver a vast range of drugs and bioactives owing to their ability to demonstrate novel physical, chemical, and/or biological properties. An exponential growth has spurred in research and development of these nanocarriers which led to the evolution of a great number of diverse nanosystems including liposomes, nanoemulsions, solid lipid nanoparticles (SLNs), micelles, dendrimers, polymeric nanoparticles (NPs), metallic NPs, and carbon nanotubes. Among them, lipid-based nanocarriers have made the largest progress whether commercially or under development. Despite this progress, these lipid-based nanocarriers suffer from several limitations that led to the development of many protein-coated lipid nanocarriers. To less extent, protein-based nanocarriers suffer from limitations that led to the fabrication of some lipid bilayer enveloping protein nanocarriers. This review discusses in-depth some limitations associated with the lipid-based or protein-based nanocarriers and the fruitful outcomes brought by protein-lipid hybridization. Also discussed are the various hybridization techniques utilized to formulate these protein-lipid nanohybrids and the mechanisms involved in the drug loading process.

  13. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    PubMed Central

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  14. Silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus

    PubMed Central

    Price, Daniel L.; Li, Pingdong; Chen, Chun-Hao; Wong, Danni; Yu, Zhenkun; Chen, Nanhai G.; Yu, Yong A.; Szalay, Aladar A.; Cappello, Joseph; Fong, Yuman; Wong, Richard J.

    2016-01-01

    Background Oncolytic viral efficacy may be limited by the penetration of the virus into tumors. This may be enhanced by intraoperative application of virus immediately after surgical resection. Methods Oncolytic vaccinia virus GLV-1h68 was delivered in silk-elastin-like protein polymer (SELP) in vitro and in vivo in anaplastic thyroid carcinoma cell line 8505c in nude mice. Results GLV-1h68 in SELP infected and lysed anaplastic thyroid cancer cells in vitro equally as effectively as in phosphate-buffered saline (PBS), and at 1 week retains a thousand fold greater infectious plaque-forming units. In surgical resection models of residual tumor, GLV-1h68 in SELP improves tumor control and shows increased viral β-galactosidase expression as compared to PBS. Conclusion The use of SELP matrix for intraoperative oncolytic viral delivery protects infectious viral particles from degradation, facilitates sustained viral delivery and transgene expression, and improves tumor control. Such optimization of methods of oncolytic viral delivery may enhance therapeutic outcomes. PMID:25244076

  15. Delivery of Membrane Impermeable Cargo into CHO Cells by Peptide Nanoparticles Targeted by a Protein Corona

    PubMed Central

    Dittrich, Christian; Burckhardt, Christoph J.; Danuser, Gaudenz

    2012-01-01

    Nanocarriers can fulfill essential functions in the stabilization and delivery of drugs: they prevent solubility issues and degradation, reduce side effects and modify the pharmacokinetic profile. However, particle based pharmaceuticals are very complex and thus challenging to scale up. As formulation routines account for a large fraction of production costs, reducing complexity in the process of assembly, loading and functionalization of nanoparticles is very desirable. Unlike existing approaches with similar goals, our protocol is designed to minimize usage of material and time. Prerequisite to this elegant one-step procedure is the controlled phase-separation of a hydrophobic peptide to nanoparticles, inducing concurrent cargo-entrapment and association of a protein corona. We demonstrate the process by assembling Flutax-2 containing peptide nanoparticles functionalized with transferrin. Cellular uptake of the particles and cargo release depend on specific particle-cell interactions via transferrin receptor. These data indicate corona-mediated delivery of membrane impermeable cargo in vitro by a particulate delivery system entirely composed of amino acids. PMID:22226586

  16. Synthesis of protein-loaded hydrogel particles in an aqueous two-phase system for coincident antigen and CpG oligonucleotide delivery to antigen-presenting cells.

    PubMed

    Jain, Siddhartha; Yap, Woon Teck; Irvine, Darrell J

    2005-01-01

    Materials that effectively deliver protein antigens together with activating ligands to antigen-presenting cells are sought for improved nonviral vaccines. To this end, we synthesized protein-loaded poly(ethylene glycol) (PEG)-based hydrogel particles by cross-linking PEG within the polymer-rich phase of an emulsion formed by a poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) triblock copolymer in saturated aqueous salt solution. These particles (500-nm diameter) contained high levels of encapsulated protein (approximately 75% of dry mass), which was selectively released by proteolytic enzymes normally present in the phagosomal/endosomal compartments of dendritic cells (DCs). For co-delivery of cellular activation signals, gel particles were surface-modified by sequential adsorption of poly(l-arginine) and CpG oligonucleotides. DCs pulsed with protein-loaded particles activated naïve T cells in vitro approximately 10-fold more efficiently than DCs incubated with soluble protein. This organic solvent-free strategy for protein encapsulation within submicron-sized hydrophilic particles is attractive for macromolecule delivery to a variety of phagocytic and nonphagocytic cells.

  17. Cbl proteins in platelet activation.

    PubMed

    Buitrago, Lorena; Tsygankov, Alexander; Sanjay, Archana; Kunapuli, Satya P

    2013-01-01

    Platelets play a fundamental role in hemostasis. Their functional responses have to be tightly controlled as any disturbance may lead to bleeding disorders or thrombosis. It is thus important to clearly identify and understand the signaling mechanisms involved in platelet function. An important role of c-Cbl and Cbl-b ubiquitin ligases in platelet functional responses and in hematological malignancies has been recently described. Cbl proteins perform negative and positive regulation of several signaling pathways in platelets. In this review, we explore the role of Cbl proteins in platelet functional responses.

  18. Identification of intracellular receptor proteins for activated protein kinase C.

    PubMed Central

    Mochly-Rosen, D; Khaner, H; Lopez, J

    1991-01-01

    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC. Images PMID:1850844

  19. Activity-Based Protein Profiling of Microbes

    SciTech Connect

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  20. Biodegradable Magnetic Silica@Iron Oxide Nanovectors with Ultra-Large Mesopores for High Protein Loading, Magnetothermal Release, and Delivery.

    PubMed

    Omar, Haneen; Croissant, Jonas G; Alamoudi, Kholod; Alsaiari, Shahad; Alradwan, Ibrahim; Majrashi, Majed A; Anjum, Dalaver H; Martins, Patricia; Moosa, Basem; Almalik, Abdulaziz; Khashab, Niveen M

    2016-11-29

    The delivery of large cargos of diameter above 15nm for biomedical applications has proved challenging since it requires biocompatible, stably-loaded, and biodegradable nanomaterials. In this study, we describe the design of biodegradable silica-iron oxide hybrid nanovectors with large mesopores for large protein delivery in cancer cells. The mesopores of the nanomaterials spanned from 20 to 60nm in diameter and post-functionalization allowed the electrostatic immobilization of large proteins (e.g. mTFP-Ferritin, ~534kDa). Half of the content of the nanovectors was based with iron oxide nanophases which allowed the rapid biodegradation of the carrier in fetal bovine serum and a magnetic responsiveness. The nanovectors released large protein cargos in aqueous solution under acidic pH or magnetic stimuli. The delivery of large proteins was then autonomously achieved in cancer cells via the silica-iron oxide nanovectors, which is thus a promising for biomedical applications.

  1. Impact of Absorption and Transport on Intelligent Therapeutics and Nano-scale Delivery of Protein Therapeutic Agents

    PubMed Central

    Peppas, Nicholas A.; Carr, Daniel A

    2009-01-01

    The combination of materials design and advances in nanotechnology has led to the development of new therapeutic protein delivery systems. The pulmonary, nasal, buccal and other routes have been investigated as delivery options for protein therapy, but none result in improved patient compliances and patient quality of life as the oral route. For the oral administration of these new systems, an understanding of protein transport is essential because of the dynamic nature of the gastrointestinal tract and the barriers to transport that exist. Models have been developed to describe the transport between the gastrointestinal lumen and the bloodstream, and laboratory techniques like cell culture provide a means to investigate the absorption and transport of many therapeutic agents. Biomaterials, including stimuli-sensitive complexation hydrogels, have been investigated as promising carriers for oral delivery. However, the need to develop models that accurately predict protein blood concentration as a function of the material structure and properties still exists. PMID:20161384

  2. Nanoporous membrane based on block copolymer thin film for protein drug delivery

    NASA Astrophysics Data System (ADS)

    Yang, Seung Yun; Yang, Jeong-A.; Kim, Eung-Sam; Jeon, Gumhye; Oh, Eun Ju; Choi, Kwan Yong; Hahn, Sei Kwang; Kim, Jin Kon

    2010-03-01

    We studied long term and controlled release of protein drugs by using nanoporous membranes with various pore sizes. Nanoporous membrane consists of the separation layer prepared by polystyrene-block-poly(methylmethacrylate) copolymer thin film and conventional microfiltration membrane as a support. We demonstrate a long-term constant in vitro release of bovine serum albumin (BSA)and human growth hormone ) (hGH) without their denaturation up to 2 months. A nearly constant serum concentration of hGH was maintained up to 3 weeks in SD rats. The long-term constant delivery based on this membrane for protein drugs within the therapeutic range can be highly appreciated for the patients with hormone- deficiency.

  3. Design and characterization of calcium alginate microparticles coated with polycations as protein delivery system.

    PubMed

    Zarate, J; Virdis, L; Orive, G; Igartua, M; Hernández, R M; Pedraz, J L

    2011-01-01

    Bovine serum albumin (BSA) loaded calcium alginate microparticles (MPs) produced in this study by a w/o emulsification and external gelation method exhibited spherical and fairly smooth and porous morphology with 1.052 ± 0.057 µm modal particle size. The high permeability of the calcium alginate hydrogel lead to a potent burst effect and too fast protein release. To overcome these problems, MPs were coated with polycations, such as chitosan, poly-L-lysine and DEAE-dextran. Our results demonstrated that coated MPs showed slower release and were able to significantly reduce the release of BSA in the first hour. Therefore, this method can be applied to prepare coated alginate MPs which could be an optimal system for the controlled release of biotherapeutic molecules. Nevertheless, further studies are needed to optimize delivery properties which could provide a sustained release of proteins.

  4. The use of colloidal microgels for the controlled delivery of proteins and peptides

    NASA Astrophysics Data System (ADS)

    Cornelius, Victoria J.; Snowden, Martin J.; Mitchell, John C.

    2007-01-01

    Colloidal microgels may be used for the absorption and controlled release of confirmationally sensitive molecules such as proteins and peptides. These monodisperse microgels are easily prepared in a single pot reaction from e.g. Nisopropylacrylamide, butyl acrylate and methacrylic acid in the presence of a cross-linking agent and a suitable free radical initiator. The resultant materials display dramatic conformational changes in aqueous dispersion in response to changes in e.g. environmental pH. Colloidal microgels are capable of absorbing a range of different proteins and peptides at one pH, affording them protection by changing the conformation of the microgel following a pH change. A further change in environmental pH will allow the microgel to adopt a more extended confirmation and therefore allow the release of the encapsulated material. In the case of e.g. insulin this would offer the possibility of an oral delivery route. At the pH of stomach the microgel adopts a compact conformation, "protecting" the protein from denaturation. As the pH increases passing into the GI tract, the microgel changes its conformation to a more expanded form and thereby allows the protein to be released. Colloidal microgels offer an opportunity for the controlled release of conformationally sensitive protein and peptide molecules via an oral route.

  5. Transcript-activated collagen matrix as sustained mRNA delivery system for bone regeneration.

    PubMed

    Badieyan, Zohreh Sadat; Berezhanskyy, Taras; Utzinger, Maximilian; Aneja, Manish Kumar; Emrich, Daniela; Erben, Reinhold; Schüler, Christiane; Altpeter, Philipp; Ferizi, Mehrije; Hasenpusch, Günther; Rudolph, Carsten; Plank, Christian

    2016-10-10

    Transcript therapies using chemically modified messenger RNAs (cmRNAs) are emerging as safe and promising alternatives for gene and recombinant protein therapies. However, their applications have been limited due to transient translation and relatively low stability of cmRNAs compared to DNA. Here we show that vacuum-dried cmRNA-loaded collagen sponges, termed transcript activated matrices (TAMs), can serve as depots for sustained delivery of cmRNA. TAMs provide steady state protein production for up to six days, and substantial residual expression until 11days post transfection. Another advantage of this technology was nearly 100% transfection efficiency as well as low toxicity in vitro. TAMs were stable for at least 6months at room temperature. Human BMP-2-encoding TAMs induced osteogenic differentiation of MC3T3-E1 cells in vitro and bone regeneration in a non-critical rat femoral bone defect model in vivo. In summary, TAMs are a promising tool for bone regeneration and potentially also for other applications in regenerative medicine and tissue engineering.

  6. Blood-brain barrier transport of cationized immunoglobulin G: Enhanced delivery compared to native protein

    SciTech Connect

    Triguero, D.; Buciak, J.B.; Yang, J.; Pardridge, W.M.

    1989-06-01

    IgG molecules are potential neuropharmaceuticals that may be used for therapeutic or diagnostic purposes. However, IgG molecules are excluded from entering brain, owing to a lack of transport of these plasma proteins through the brain capillary wall, or blood-brain barrier (BBB). The possibility of enhanced IgG delivery through the BBB by cationization of the proteins was explored in the present studies. Native bovine IgG molecules were cationized by covalent coupling of hexamethylenediamine and the isoelectric point was raised to greater than 10.7 based on isoelectric focusing studies. Native and cationized IgG molecules were radiolabeled with /sup 125/I and chloramine T. Cationized IgG, but not native IgG, was rapidly taken up by isolated bovine brain microvessels, which were used as an in vitro model system of the BBB. Cationized IgG binding was time and temperature dependent and was saturated by increasing concentrations of unlabeled cationized IgG (dissociation constant of the high-affinity binding site, 0.90 +/- 0.37 microM; Bmax, 1.4 +/- 0.4 nmol per mg of protein). In vivo studies documented enhanced brain uptake of 125I-labeled cationized IgG relative to (3H)albumin, and complete transcytosis of the 125I-labeled cationized IgG molecule through the BBB and into brain parenchyma was demonstrated by thaw-mount autoradiography of frozen sections of rat brain obtained after carotid arterial infusions of 125I-labeled cationized IgG. These studies demonstrate that cationization of IgG molecules greatly facilitates the transport of these plasma proteins through the BBB in vivo, and this process may provide a new strategy for IgG delivery through the BBB.

  7. Blood-Brain Barrier Transport of Cationized Immunoglobulin G: Enhanced Delivery Compared to Native Protein

    NASA Astrophysics Data System (ADS)

    Triguero, Domingo; Buciak, Jody B.; Yang, Jing; Pardridge, William M.

    1989-06-01

    IgG molecules are potential neuropharmaceuticals that may be used for therapeutic or diagnostic purposes. However, IgG molecules are excluded from entering brain, owing to a lack of transport of these plasma proteins through the brain capillary wall, or blood-brain barrier (BBB). The possibility of enhanced IgG delivery through the BBB by cationization of the proteins was explored in the present studies. Native bovine IgG molecules were cationized by covalent coupling of hexamethylenediamine and the isoelectric point was raised to >10.7 based on isoelectric focusing studies. Native and cationized IgG molecules were radiolabeled with 125I and chloramine T. Cationized IgG, but not native IgG, was rapidly taken up by isolated bovine brain microvessels, which were used as an in vitro model system of the BBB. Cationized IgG binding was time and temperature dependent and was saturated by increasing concentrations of unlabeled cationized IgG (dissociation constant of the high-affinity binding site, 0.90 ± 0.37 μ M; Bmax, 1.4 ± 0.4 nmol per mg of protein). In vivo studies documented enhanced brain uptake of 125I-labeled cationized IgG relative to [3H]albumin, and complete transcytosis of the 125I-labeled cationized IgG molecule through the BBB and into brain parenchyma was demonstrated by thaw-mount autoradiography of frozen sections of rat brain obtained after carotid arterial infusions of 125I-labeled cationized IgG. These studies demonstrate that cationization of IgG molecules greatly facilitates the transport of these plasma proteins through the BBB in vivo, and this process may provide a new strategy for IgG delivery through the BBB.

  8. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    PubMed

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.

  9. Processive degradation of unstructured protein by Escherichia coli Lon occurs via the slow, sequential delivery of multiple scissile sites followed by rapid and synchronized peptide bond cleavage events.

    PubMed

    Mikita, Natalie; Cheng, Iteen; Fishovitz, Jennifer; Huang, Jonathan; Lee, Irene

    2013-08-20

    Processive protein degradation is a common feature found in ATP-dependent proteases. This study utilized a physiological substrate of Escherichia coli Lon protease known as the lambda N protein (λN) to initiate the first kinetic analysis of the proteolytic mechanism of this enzyme. To this end, experiments were designed to determine the timing of three selected scissile sites in λN approaching the proteolytic site of ELon and their subsequent cleavages to gain insight into the mechanism by which ATP-dependent proteases attain processivity in protein degradation. The kinetic profile of peptide bond cleavage at different regions of λN was first detected by the iTRAQ/mass spectrometry technique. Fluorogenic λN constructs were then generated as reporter substrates for transient kinetic characterization of the ATP- versus AMPPNP-dependent peptide bond cleavage and the delivery of the scissile sites near the amino- versus carboxyl-terminal of the λN protein to the proteolytic site of ELon. Collectively, our results support a mechanism by which the cleavage of multiple peptide bonds awaits the "almost complete" delivery of all the scissile sites in λN to the proteolytic site in an ATP-dependent manner. Comparing the time courses of delivery to the active site of the selected scissile sites further implicates the existence of a preferred directionality in the final stage of substrate delivery, which begins at the carboxyl-terminal. The subsequent cleavage of the scissile sites in λN, however, appears to lack a specific directionality and occurs at a much faster rate than the substrate delivery step.

  10. A novel protocol allowing oral delivery of a protein complement inhibitor that subsequently targets to inflamed colon mucosa and ameliorates murine colitis

    PubMed Central

    Elvington, M; Blichmann, P; Qiao, F; Scheiber, M; Wadsworth, C; Luzinov, I; Lucero, J; Vertegel, A; Tomlinson, S

    2014-01-01

    While there is evidence of a pathogenic role for complement in inflammatory bowel disease, there is also evidence for a protective role that relates to host defence and protection from endotoxaemia. There is thus concern regarding the use of systemic complement inhibition as a therapeutic strategy. Local delivery of a complement inhibitor to the colon by oral administration would ameliorate such concerns, but while formulations exist for oral delivery of low molecular weight drugs to the colon, they have not been used successfully for oral delivery of proteins. We describe a novel pellet formulation consisting of cross-linked dextran coated with an acrylic co-polymer that protects the complement inhibitor CR2-Crry from destruction in the gastrointestinal tract. CR2-Crry containing pellets administered by gavage, were characterized using a therapeutic protocol in a mouse model of dextran sulphate sodium (DSS)-induced colitis. Oral treatment of established colitis over a 5-day period significantly reduced mucosal inflammation and injury, with similar therapeutic benefit whether or not the proton pump inhibitor, omeprazole, was co-administered. Reduction in injury was associated with the targeting of CR2-Crry to the mucosal surface and reduced local complement activation. Treatment had no effect on systemic complement activity. This novel method for oral delivery of a targeted protein complement inhibitor will reduce systemic effects, thereby decreasing the risk of opportunistic infection, as well as lowering the required dose and treatment cost and improving patient compliance. Furthermore, the novel delivery system described here may provide similar benefits for administration of other protein-based drugs, such as anti-tumour necrosis factor-α antibodies. PMID:24730624

  11. Dietary protein considerations to support active aging.

    PubMed

    Wall, Benjamin T; Cermak, Naomi M; van Loon, Luc J C

    2014-11-01

    Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the application of dietary protein supplementation to support exercise-induced gains in muscle mass in younger individuals. In contrast, the role of dietary protein in supporting the maintenance (or gain) of skeletal muscle mass in active older persons has received less attention. Older individuals display a blunted muscle protein synthetic response to dietary protein ingestion. However, this reduced anabolic response can largely be overcome when physical activity is performed in close temporal proximity to protein consumption. Moreover, recent evidence has helped elucidate the optimal type and amount of dietary protein that should be ingested by the older adult throughout the day in order to maximize the skeletal muscle adaptive response to physical activity. Evidence demonstrates that when these principles are adhered to, muscle maintenance or hypertrophy over prolonged periods can be further augmented in active older persons. The present review outlines the current understanding of the role that dietary protein occupies in the lifestyle of active older adults as a means to increase skeletal muscle mass, strength and function, and thus support healthier aging.

  12. Collagen peptide-based biomaterials for protein delivery and peptide-promoted self-assembly of gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Ernenwein, Dawn M.

    2011-12-01

    Bottom-up self-assembly of peptides has driven the research progress for the following two projects: protein delivery vehicles of collagen microflorettes and the assembly of gold nanoparticles with coiled-coil peptides. Collagen is the most abundant protein in the mammals yet due to immunogenic responses, batch-to-batch variability and lack of sequence modifications, synthetic collagen has been designed to self-assemble into native collagen-like structures. In particular with this research, metal binding ligands were incorporated on the termini of collagen-like peptides to generate micron-sized particles, microflorettes. The over-arching goal of the first research project is to engineer MRI-active microflorettes, loaded with His-tagged growth factors with differential release rates while bound to stem cells that can be implemented toward regenerative cell-based therapies. His-tagged proteins, such as green fluorescent protein, have successfully been incorporated on the surface and throughout the microflorettes. Protein release was monitored under physiological conditions and was related to particle degradation. In human plasma full release was obtained within six days. Stability of the microflorettes under physiological conditions was also examined for the development of a therapeutically relevant delivery agent. Additionally, MRI active microflorettes have been generated through the incorporation of a gadolinium binding ligand, DOTA within the collagen-based peptide sequence. To probe peptide-promoted self-assemblies of gold nanoparticles (GNPs) by non-covalent, charge complementary interactions, a highly anionic coiled-coil peptide was designed and synthesized. Upon formation of peptide-GNP interactions, the hydrophobic domain of the coiled-coil were shown to promote the self-assembly of peptide-GNPs clustering. Hydrophobic forces were found to play an important role in the assembly process, as a peptide with an equally overall negative charge, but lacking an

  13. Breadboard activities for advanced protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Banish, Michael

    1993-01-01

    The proposed work entails the design, assembly, testing, and delivery of a turn-key system for the semi-automated determination of protein solubilities as a function of temperature. The system will utilize optical scintillation as a means of detecting and monitoring nucleation and crystallite growth during temperature lowering (or raising, with retrograde solubility systems). The deliverables of this contract are: (1) turn-key scintillation system for the semi-automatic determination of protein solubilities as a function of temperature, (2) instructions and software package for the operation of the scintillation system, and (3) one semi-annual and one final report including the test results obtained for ovostatin with the above scintillation system.

  14. Consuming viscous prey: a novel protein-secreting delivery system in neotropical snail-eating snakes

    PubMed Central

    2014-01-01

    Background Efficient venom delivery systems are known to occur only in varanoid lizards and advanced colubroidean snakes among squamate reptiles. Although components of these venomous systems might have been present in a common ancestor, the two lineages independently evolved strikingly different venom gland systems. In snakes, venom is produced exclusively by serous glands in the upper jaw. Within the colubroidean radiation, lower jaw seromucous infralabial glands are known only in two distinct lineages–the basal pareatids and the more advanced Neotropical dipsadines known as “goo-eating snakes”. Goo-eaters are a highly diversified, ecologically specialized clade that feeds exclusively on invertebrates (e.g., gastropod molluscs and annelids). Their evolutionary success has been attributed to their peculiar feeding strategies, which remain surprisingly poorly understood. More specifically, it has long been thought that the more derived Dipsadini genera Dipsas and Sibynomorphus use glandular toxins secreted by their infralabial glands to extract snails from their shells. Results Here, we report the presence in the tribe Dipsadini of a novel lower jaw protein-secreting delivery system effected by a gland that is not functionally related to adjacent teeth, but rather opens loosely on the oral epithelium near the tip of the mandible, suggesting that its secretion is not injected into the prey as a form of envenomation but rather helps control the mucus and assists in the ingestion of their highly viscous preys. A similar protein-secreting system is also present in the goo-eating genus Geophis and may share the same adaptive purpose as that hypothesized for Dipsadini. Our phylogenetic hypothesis suggests that the acquisition of a seromucous infralabial gland represents a uniquely derived trait of the goo-eating clade that evolved independently twice within the group as a functionally complex protein-secreting delivery system. Conclusions The acquisition by snail

  15. Drug Delivery to Extravascular Tissue by Ultrasound-activated Microbubbles

    NASA Astrophysics Data System (ADS)

    Kooiman, Klazina; Harteveld, Miranda; de Jong, Nico; van Wamel, Annemieke

    2007-05-01

    Drugs will be delivered to tissue more efficiently if the vascular endothelial permeability is increased. Although recent studies have established that the permeability of single-cell membranes is increased by ultrasound in combination with contrast agents, it is not known whether this combination can also increase the permeability of an endothelial layer. To investigate endothelial layer permeability, we treated monolayers of human umbilical vein endothelial cells with ultrasound and the contrast agent BR14. Endothelial layer permeability was assessed by measuring the transendothelial electrical resistance (TEER) and the transendothelial transport of fluorescein. Ultrasound in combination with BR14 significantly decreased TEER to 68.0 ± 3.1% of initial values and temporally increased endothelial permeability for fluorescein by 38.1 ± 16.4 %. After treatment, no cell loss or damage was observed. In conclusion, ultrasound in combination with BR14 increased the endothelial layer permeability. This feature may be used for future ultrasound-guided drug delivery systems.

  16. Efficient induction of antimicrobial activity with vancomycin nanoparticle-loaded poly(trimethylene carbonate) localized drug delivery system

    PubMed Central

    Zhang, Yang; Liang, Ruo-jia; Xu, Jiao-jiao; Shen, Li-feng; Gao, Jian-qing; Wang, Xu-ping; Wang, Na-ni; Shou, Dan; Hu, Ying

    2017-01-01

    Surgery and the local placement of an antibiotic are the predominant therapies to treat chronic osteomyelitis. Vancomycin-loaded N-trimethyl chitosan nanoparticles (VCM/TMC NPs) as a potential drug delivery system have high intracellular penetration and effective intracellular antibacterial activity. This study investigated the effects of a biocompatible material, poly(trimethylene carbonate) (PTMC), to increase the sustained effectiveness of an intracellular antibiotic and its potential application in antibiotic delivery. VCM/TMC NP-PTMC was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy to determine the morphology, stability and chemical interaction of the drug with the polymer. Further, the biodegradation, antibacterial activity, protein adsorption, cell proliferation and drug release characteristics were evaluated. In addition, a Staphylococcus aureus-induced osteomyelitis rabbit model was used to investigate the antibiotic activity and bone repair capability of VCM/TMC NP-PTMC. The results showed that the composite beads of VCM/TMC NPs followed a sustained and slow release pattern and had excellent antibacterial activity and a higher protein adsorption and cell proliferation rate than the VCM-PTMC in vitro. Furthermore, VCM/TMC NP-PTMC inhibits bacteria and promotes bone repair in vivo. Thus, VCM/TMC NP-PTMC might be beneficial in periodontal management to reduce the bacterial load at the infection site and promote bone repair. PMID:28243084

  17. Protein delivery of a Ni catalyst to photosystem I for light-driven hydrogen production.

    PubMed

    Silver, Sunshine C; Niklas, Jens; Du, Pingwu; Poluektov, Oleg G; Tiede, David M; Utschig, Lisa M

    2013-09-11

    The direct conversion of sunlight into fuel is a promising means for the production of storable renewable energy. Herein, we use Nature's specialized photosynthetic machinery found in the Photosystem I (PSI) protein to drive solar fuel production from a nickel diphosphine molecular catalyst. Upon exposure to visible light, a self-assembled PSI-[Ni(P2(Ph)N2(Ph))2](BF4)2 hybrid generates H2 at a rate 2 orders of magnitude greater than rates reported for photosensitizer/[Ni(P2(Ph)N2(Ph))2](BF4)2 systems. The protein environment enables photocatalysis at pH 6.3 in completely aqueous conditions. In addition, we have developed a strategy for incorporating the Ni molecular catalyst with the native acceptor protein of PSI, flavodoxin. Photocatalysis experiments with this modified flavodoxin demonstrate a new mechanism for biohybrid creation that involves protein-directed delivery of a molecular catalyst to the reducing side of Photosystem I for light-driven catalysis. This work further establishes strategies for constructing functional, inexpensive, earth-abundant solar fuel-producing PSI hybrids that use light to rapidly produce hydrogen directly from water.

  18. Effective delivery of recombinant proteins to rod photoreceptors via lipid nanovesicles

    SciTech Connect

    Asteriti, Sabrina; Dal Cortivo, Giuditta; Pontelli, Valeria; Cangiano, Lorenzo; Buffelli, Mario; Dell’Orco, Daniele

    2015-06-12

    The potential of liposomes to deliver functional proteins in retinal photoreceptors and modulate their physiological response was investigated by two experimental approaches. First, we treated isolated mouse retinas with liposomes encapsulating either recoverin, an important endogenous protein operating in visual phototransduction, or antibodies against recoverin. We then intravitrally injected in vivo liposomes encapsulating either rhodamin B or recoverin and we investigated the distribution in retina sections by confocal microscopy. The content of liposomes was found to be released in higher amount in the photoreceptor layer than in the other regions of the retina and the functional effects of the release were in line with the current model of phototransduction. Our study sets the basis for quantitative investigations aimed at assessing the potential of intraocular protein delivery via biocompatible nanovesicles, with promising implications for the treatment of retinal diseases affecting the photoreceptor layer. - Highlights: • Recombinant proteins encapsulated in nano-sized liposomes injected intravitreally reach retinal photoreceptors. • The phototransduction cascade in rods is modulated by the liposome content. • Mathematical modeling predicts the alteration of the photoresponses following liposome fusion.

  19. Genetic delivery of an immunoRNase by an oncolytic adenovirus enhances anticancer activity.

    PubMed

    Fernández-Ulibarri, Inés; Hammer, Katharina; Arndt, Michaela A E; Kaufmann, Johanna K; Dorer, Dominik; Engelhardt, Sarah; Kontermann, Roland E; Hess, Jochen; Allgayer, Heike; Krauss, Jürgen; Nettelbeck, Dirk M

    2015-05-01

    Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase-encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody-fusion protein for targeted bystander killing of tumor cells (viro-antibody therapy). Specifically, we explore genetic delivery of a small immunoRNase consisting of an EGFR-binding scFv antibody fragment fused to the RNase Onconase (ONC(EGFR)) that induces tumor cell death by RNA degradation after cellular internalization. Onconase is a frog RNase that combines lack of immunogenicity and excellent safety in patients with high tumor killing potency due to its resistance to the human cytosolic RNase inhibitor. We show that ONC(EGFR) expression by oncolytic adenoviruses is feasible with an optimized, replication-dependent gene expression strategy. Virus-encoded ONC(EGFR) induces potent and EGFR-dependent bystander killing of tumor cells. Importantly, the ONC(EGFR)-encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR-positive tumor cells in vitro and significantly enhanced therapeutic activity in a mouse xenograft tumor model. The latter demonstrates that ONC(EGFR) is expressed at levels sufficient to trigger tumor cell killing in vivo. The established ONC(EGFR)-encoding oncolytic adenovirus represents a novel agent for treatment of EGFR-positive tumors. This viro-antibody therapy platform can be further developed for targeted/personalized cancer therapy by exploiting antibody diversity to target further established or emerging tumor markers or combinations thereof.

  20. PLGA-Mesoporous Silicon Microspheres for the in Vivo Controlled Temporospatial Delivery of Proteins.

    PubMed

    Minardi, Silvia; Pandolfi, Laura; Taraballi, Francesca; De Rosa, Enrica; Yazdi, Iman K; Liu, Xeuwu; Ferrari, Mauro; Tasciotti, Ennio

    2015-08-05

    In regenerative medicine, the temporospatially controlled delivery of growth factors (GFs) is crucial to trigger the desired healing mechanisms in the target tissues. The uncontrolled release of GFs has been demonstrated to cause severe side effects in the surrounding tissues. The aim of this study was to optimize a translational approach for the fine temporal and spatial control over the release of proteins, in vivo. Hence, we proposed a newly developed multiscale composite microsphere based on a core consisting of the nanostructured silicon multistage vector (MSV) and a poly(dl-lactide-co-glycolide) acid (PLGA) outer shell. Both of the two components of the resulting composite microspheres (PLGA-MSV) can be independently tailored to achieve multiple release kinetics contributing to the control of the release profile of a reporter protein in vitro. The influence of MSV shape (hemispherical or discoidal) and size (1, 3, or 7 μm) on PLGA-MSV's morphology and size distribution was investigated. Second, the copolymer ratio of the PLGA used to fabricate the outer shell of PLGA-MSV was varied. The composites were fully characterized by optical microscopy, scanning electron microscopy, ζ potential, Fourier transform infrared spectroscopy, and thermogravimetric analysis-differential scanning calorimetry, and their release kinetics over 30 days. PLGA-MSV's biocompatibility was assessed in vitro with J774 macrophages. Finally, the formulation of PLGA-MSV was selected, which concurrently provided the most consistent microsphere size and allowed for a zero-order release kinetic. The selected PLGA-MSVs were injected in a subcutaneous model in mice, and the in vivo release of the reporter protein was followed over 2 weeks by intravital microscopy, to assess if the zero-order release was preserved. PLGA-MSV was able to retain the payload over 2 weeks, avoiding the initial burst release typical of most drug delivery systems. Finally, histological evaluation assessed the

  1. Particle designs for the stabilization and controlled-delivery of protein drugs by biopolymers: a case study on insulin.

    PubMed

    Lim, Hui-Peng; Tey, Beng-Ti; Chan, Eng-Seng

    2014-07-28

    Natural biopolymers have attracted considerable interest for the development of delivery systems for protein drugs owing to their biocompatibility, non-toxicity, renewability and mild processing conditions. This paper offers an overview of the current status and future perspectives of particle designs using biopolymers for the stabilization and controlled-delivery of a model protein drug--insulin. We first describe the design criteria for polymeric encapsulation and subsequently classify the basic principles of particle fabrication as well as the existing particle designs for oral insulin encapsulation. The performances of these existing particle designs in terms of insulin stability and in vitro release behavior in acidic and alkaline media, as well as their in vivo performance are compared and reviewed. This review forms the basis for future works on the optimization of particle design and material formulation for the development of an improved oral delivery system for protein drugs.

  2. Active site proton delivery and the lyase activity of human CYP17A1

    SciTech Connect

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G.

    2014-01-03

    Highlights: •The disruption of PREG/PROG hydroxylation activity by T306A showed the participation of Cpd I. •T306A supports the involvement of a nucleophilic peroxo-anion during lyase activity. •The presence of cytochrome b{sub 5} augments C–C lyase activity. •Δ5-Steroids are preferred substrates for CYP17 catalysis. -- Abstract: Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing

  3. Challenging delivery of VLHL NS plasminogen activator inhibitor-1 by osmotic pumps in diabetic mouse: A case report.

    PubMed

    Jankun, Jerzy

    2012-10-01

    ALZET(®) osmotic pumps are implantable devices used in animals for the continuous infusion of drugs or proteins at controlled rates from 1 day to 4 weeks. Pumps have been used successfully in a number of studies on the effects of controlled delivery of a wide range of experimental agents, independent of their properties. In the present study, use of these pumps was made in mice with diabetic nephropathy. Plasminogen activator inhibitor-1 (PAI-1) mediates diabetic nephropathy, which is characterized by the excessive accumulation of extracellular matrix (ECM) in the kidney. Disproportionate PAI-1 inactivates tissue plasminogen activator, which is one of the proteolytic enzymes in a cascade responsible for ECM remodeling in the kidney. The decrease of PAI-1 in the kidney has been shown to arrest the progression of nephropathy in experimental animals. This was achieved using inactive PAI-1R which increased the clearance of wild-type PAI-1 in order to protect net proteolytic activity and ECM clearance. However, this protein has a brief half-life in vivo, therefore, high and frequent doses are required. Thus, VLHL NS PAI-1 protein with a long half-life of over 700 h (Gln197Cys, Gly355Cys) inactivated by single point mutation (Arg369Ala) was used. Following the sacrifice of animals the tips of the flow moderators of the osmotic pumps in the treated animals were found to be clogged. In addition, from each pump from the treatment group, but not controls, we collected 50-150 μl of clear liquid containing VLHL NS PAI-1, cellular and serum proteins suggesting early pump sealing by cellular material. In conclusion, despite encouraging results obtained for the PAI-1R protein, the method of VLHL PAI-1 delivery should be ameliorated.

  4. Promotion of the transdermal delivery of protein drugs by N-trimethyl chitosan nanoparticles combined with polypropylene electret

    PubMed Central

    Tu, Ye; Wang, Xinxia; Lu, Ying; Zhang, He; Yu, Yuan; Chen, Yan; Liu, Junjie; Sun, Zhiguo; Cui, Lili; Gao, Jing; Zhong, Yanqiang

    2016-01-01

    We recently reported that electret, which was prepared by a corona charging system with polypropylene film, could enhance the transdermal delivery of several drugs of low molecular weight. The aim of this study was to investigate whether electret could enhance the transdermal delivery of protein drugs by N-trimethyl chitosan nanoparticles (TMC NPs) prepared by an ionic gelation method. A series of experiments were performed, including in vitro skin permeation assays and anti-inflammatory effects, to evaluate the transdermal delivery of protein drugs by TMC NPs in the presence of electret. The results showed that in the presence of electret, the transdermal delivery of protein drugs in TMC NPs was significantly enhanced, as demonstrated by in vitro permeation studies and confocal laser scanning microscopy. Notably, superoxide dismutase-loaded TMC NPs combined with electret exhibited the best inhibitory effect on the edema of the mouse ear. TMC NPs combined with electret represent a novel platform for the transdermal delivery of protein drugs. PMID:27822034

  5. Development of Cy5.5-Labeled Hydrophobically Modified Glycol Chitosan Nanoparticles for Protein Delivery

    NASA Astrophysics Data System (ADS)

    Chin, Amanda

    Therapeutic proteins are often highly susceptible to enzymatic degradation, thus restricting their in vivo stability. To overcome this limitation, delivery systems designed to promote uptake and reduce degradation kinetics have undergone a rapid shift from macro-scale systems to nanomaterial based carriers. Many of these nanomaterials, however, elicit immune responses and may have cytotoxic effects both in vitro and in vivo. The naturally derived polysaccharide chitosan has emerged as a promising biodegradable material and has been utilized for many biomedical applications; nevertheless, its function is often constrained by poor solubility. Glycol chitosan, a derivative of chitosan, can be hydrophobically modified to impart amphiphilic properties that enable the self-assembly into nanoparticles in aqueous media at neutral pH. This nanoparticle system has shown initial success as a therapeutic agent in several model cell culture systems, but little is known about its stability against enzymatic degradation. Therefore, the goal of this research was to investigate the resistance of hydrophobically modified glycol chitosan against enzyme-catalyzed degradation using an in vivo simulated system containing lysozyme. To synthesize the nanoparticles, hydrophobic cholanic acid was first covalently conjugated to glycol chitosan using of N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Conjugates were purified by dialysis, lyophilized, and ultra-sonicated to form nanoparticles. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of 5beta-cholanic acid to the glycol chitosan. Particle size and stability over time were determined with dynamic light scattering (DLS), and particle morphology was evaluated by transmission electron microscopy (TEM). The average diameter of the nanoparticles was approximately 200 nm, which remained stable at 4°C for up to 10 days. Additionally, a near infrared fluorescent (NIRF) dye

  6. Microencapsulation of protein drugs for drug delivery: strategy, preparation, and applications.

    PubMed

    Ma, Guanghui

    2014-11-10

    Bio-degradable poly(lactide) (PLA)/poly(lactide-glycolide) (PLGA) and chitosan microspheres (or microcapsules) have important applications in Drug Delivery Systems (DDS) of protein/peptide drugs. By encapsulating protein/peptide drugs in the microspheres, the serum drug concentration can be maintained at a higher constant value for a prolonged time, or injection formulation can be changed to orally or mucosally administered formulation. PLA/PLGA and chitosan are most often used in injection formulation and oral formulation. However, in the preparation and applications of PLA/PLGA and chitosan microspheres containing protein/peptide drugs, the problems of broad size distribution and poor reproducibility of microspheres, and deactivation of protein during the preparation, storage and release, are still big challenges. In this article, the techniques for control of the diameter of microspheres and microcapsules will be introduced at first, then the strategies about how to maintain the bioactivity of protein drugs during preparation and drug release will be reviewed and developed in our research group. The membrane emulsification techniques including direct membrane emulsification and rapid membrane emulsification processes were developed to prepare uniform-sized microspheres, the diameter of microspheres can be controlled from submicron to 100μm by these two processes, and the reproducibility of products can be guaranteed. Furthermore, compared with conventional stirring method, the big advantages of membrane emulsification process were that the uniform microspheres with much higher encapsulation efficiency can be obtained, and the release behavior can be adjusted by selecting microsphere size. Mild membrane emulsification condition also can prevent the deactivation of proteins, which frequently occurred under high shear force in mechanical stirring, sonification, and homogenization methods. The strategies for maintaining the bioactivity of protein drug were

  7. Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines

    NASA Astrophysics Data System (ADS)

    Nochi, Tomonori; Yuki, Yoshikazu; Takahashi, Haruko; Sawada, Shin-Ichi; Mejima, Mio; Kohda, Tomoko; Harada, Norihiro; Kong, Il Gyu; Sato, Ayuko; Kataoka, Nobuhiro; Tokuhara, Daisuke; Kurokawa, Shiho; Takahashi, Yuko; Tsukada, Hideo; Kozaki, Shunji; Akiyoshi, Kazunari; Kiyono, Hiroshi

    2010-07-01

    Nanotechnology is an innovative method of freely controlling nanometre-sized materials. Recent outbreaks of mucosal infectious diseases have increased the demands for development of mucosal vaccines because they induce both systemic and mucosal antigen-specific immune responses. Here we developed an intranasal vaccine-delivery system with a nanometre-sized hydrogel (`nanogel') consisting of a cationic type of cholesteryl-group-bearing pullulan (cCHP). A non-toxic subunit fragment of Clostridium botulinum type-A neurotoxin BoHc/A administered intranasally with cCHP nanogel (cCHP-BoHc/A) continuously adhered to the nasal epithelium and was effectively taken up by mucosal dendritic cells after its release from the cCHP nanogel. Vigorous botulinum-neurotoxin-A-neutralizing serum IgG and secretory IgA antibody responses were induced without co-administration of mucosal adjuvant. Importantly, intranasally administered cCHP-BoHc/A did not accumulate in the olfactory bulbs or brain. Moreover, intranasally immunized tetanus toxoid with cCHP nanogel induced strong tetanus-toxoid-specific systemic and mucosal immune responses. These results indicate that cCHP nanogel can be used as a universal protein-based antigen-delivery vehicle for adjuvant-free intranasal vaccination.

  8. Protein transduction domain-containing microemulsions as cutaneous delivery systems for an anticancer agent.

    PubMed

    Pepe, Dominique; McCall, Melissa; Zheng, Haian; Lopes, Luciana B

    2013-05-01

    In this study, we developed cationic microemulsions containing a protein transduction domain (penetratin) for optimizing paclitaxel localization within the skin. Microemulsions were prepared by mixing a surfactant blend (BRIJ:ethanol:propylene glycol 2:1:1, w/w/w) with monocaprylin (oil phase) at 1.3:1 ratio, and adding water at 30% (ME-30), 43% (ME-43), and 50% (ME-50). Electrical conductivity and viscosity measurements indicated that ME-30 is most likely a bicontinuous system, whereas ME-43 and ME-50 are water continuous. Their irritation potential, studied in bioengineered skin equivalents, decreased as aqueous content increased. Because ME-50 was not stable in the presence of paclitaxel (0.5%), ME-43 was selected for penetratin incorporation (0.4%). The microemulsion containing penetratin (ME-P) displayed zeta potential of +5.2 mV, and promoted a 1.8-fold increase in paclitaxel cutaneous (but not transdermal) delivery compared with the plain ME-43, whereas the enhancement promoted by another cationic microemulsion containing phytosphingosine was 1.3-fold. Compared with myvacet oil, ME-P promoted a larger increase on transepidermal water loss (twofold) than the plain or the phytosphingosine-containing microemulsions (1.5-fold), suggesting that penetratin addition increases the barrier-disrupting and penetration-enhancing effects of microemulsions. The ratio Δcutaneous/Δtransdermal delivery promoted by ME-P was the highest among the formulations, suggesting its potential for drug localization within cutaneous tumor lesions.

  9. Polysaccharide nanoparticles for protein and Peptide delivery: exploring less-known materials.

    PubMed

    dos Santos, Marlise A; Grenha, Ana

    2015-01-01

    Finding adequate carriers for protein and peptide delivery has become an urgent need, owing to the growing number of macromolecules identified as having therapeutic potential. Nanoparticles have emerged in the field as very promising vehicles and much work has been directed to testing the capacity of different materials to compose the matrix of these carriers. Natural materials and, specifically, polysaccharides have been taking the forefront of the challenge, because of several favoring properties that include the higher propensity to exhibit biodegradability and biocompatibility, and also the high structural flexibility. The majority of works found in the literature regarding polysaccharide nanoparticles uses very popular materials like chitosan or hyaluronic acid. This review is aimed at describing and exploring the potential of polysaccharides that are not so well known or that are less explored. For those, the main properties will be described, together with an overview of the reported applications as nanoparticle matrix materials.

  10. Preparation and Characterization of P(MAA-g-EG) Nanospheres for Protein Delivery Applications

    NASA Astrophysics Data System (ADS)

    Torres-Lugo, Madeline; Peppas, Nicholas A.

    2002-04-01

    Novel complexation hydrogel nanospheres of poly(methacrylic acid-grafted-poly(ethylene glycol)) (P(MAA-g-EG)) were prepared by dispersion polymerization to be used for protein delivery applications. Polymerization was conducted in solvents such as deionized water, ethanol/water, sodium hydroxide, hydrochloric acid, and acetic acid solutions. When polymerizing in deionized water we produced nanospheres without agglomeration. Photon correlation spectroscopy studies revealed that the nanospheres possessed a narrow particle size distribution and the size was inversely proportional to the concentration of poly(ethylene glycol) incorporated in the monomer mixture. These nanospheres exhibited pH-sensitivity comparable to that encountered in hydrogel films with the same composition. The composition of the nanospheres was investigated by transmission Fourier transform infrared spectroscopy. The comparison between hydrogel films and nanospheres with the same monomer composition revealed that nanospheres possessed similar spectral characteristics than hydrogel films prepared by the same techniques. These nanospheres could be used for calcitonin release under physiological conditions.

  11. Development of a disposable infusion system for the delivery of protein therapeutics.

    PubMed

    Eddington, David T; Beebe, David J

    2005-09-01

    This paper describes the development and optimization of a low flow open-loop infusion device for continuous delivery of protein therapeutics. Specifically, a non-electronic polymer device is actuated with responsive hydrogels to infuse at 2 microL/hr for 12 hours. Hydrogel actuators transduce a chemical signal (change in pH of the local environment) into a mechanical response (swelling) generating the pressure to drive the infusion. The hydrogel actuators are separated from the drug reservoir by an elastomeric impermeable membrane. As the hydrogel actuators expand, the expansion deflects the flexible membrane down and reduces the volume of the drug reservoir causing the infusion of drug through the needle that is the only outlet for the reservoir.

  12. The impact of nanoparticle protein corona on cytotoxicity, immunotoxicity and target drug delivery

    PubMed Central

    Corbo, Claudia; Molinaro, Roberto; Parodi, Alessandro; Toledano Furman, Naama E; Salvatore, Francesco; Tasciotti, Ennio

    2016-01-01

    In a perfect sequence of events, nanoparticles (NPs) are injected into the bloodstream where they circulate until they reach the target tissue. The ligand on the NP surface recognizes its specific receptor expressed on the target tissue and the drug is released in a controlled manner. However, once injected in a physiological environment, NPs interact with biological components and are surrounded by a protein corona (PC). This can trigger an immune response and affect NP toxicity and targeting capabilities. In this review, we provide a survey of recent findings on the NP–PC interactions and discuss how the PC can be used to modulate both cytotoxicity and the immune response as well as to improve the efficacy of targeted delivery of nanocarriers. PMID:26653875

  13. The impact of nanoparticle protein corona on cytotoxicity, immunotoxicity and target drug delivery.

    PubMed

    Corbo, Claudia; Molinaro, Roberto; Parodi, Alessandro; Toledano Furman, Naama E; Salvatore, Francesco; Tasciotti, Ennio

    2016-01-01

    In a perfect sequence of events, nanoparticles (NPs) are injected into the bloodstream where they circulate until they reach the target tissue. The ligand on the NP surface recognizes its specific receptor expressed on the target tissue and the drug is released in a controlled manner. However, once injected in a physiological environment, NPs interact with biological components and are surrounded by a protein corona (PC). This can trigger an immune response and affect NP toxicity and targeting capabilities. In this review, we provide a survey of recent findings on the NP-PC interactions and discuss how the PC can be used to modulate both cytotoxicity and the immune response as well as to improve the efficacy of targeted delivery of nanocarriers.

  14. Dopamine-conjugated poly(lactic-co-glycolic acid) nanoparticles for protein delivery to macrophages.

    PubMed

    Lee, Song Yi; Cho, Hyun-Jong

    2017-03-15

    Poly(lactic-co-glycolic acid)-dopamine (PLGA-D)-based nanoparticles (NPs) were developed for the delivery of protein to macrophages. PLGA-D was synthesized via amide bond formation between the amine group of D and the carboxylic acid group of PLGA. Bovine serum albumin (BSA, model protein) was encapsulated in PLGA NPs and PLGA-D NPs, which had an approximately 200nm mean diameter, <0.2 polydispersity index, and negative zeta potential value. There was no increment in the mean diameters of BSA-loaded NPs after 24h of incubation in biological fluid-simulated media (i.e., aqueous buffer and serum media). The primary, secondary, and tertiary structures of BSA released from the NPs were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism, and fluorescence spectrophotometry; the structural stability of BSA was preserved during its encapsulation in the NPs and release from the NPs. PLGA/BSA NPs and PLGA-D/BSA NPs did not induce serious cytotoxicity in RAW 264.7 cells (mouse macrophage cell line) in an established concentration range. In RAW 264.7 cells, the intracellular accumulation of PLGA-D NPs was 2-fold higher than that of PLGA NPs. All of these findings indicated that PLGA-D NPs are a promising system for delivering proteins to macrophages.

  15. A novel cell penetrating peptide carrier for the delivery of nematocidal proteins drug

    NASA Astrophysics Data System (ADS)

    Kim, Jea Hyun

    Nematodes have recently become a primary source of harmful diseases to the environment that inflict harsh damages to pine trees and marine species. However, nematodes cannot be killed by normal pesticides or chemicals due to their thick outer protective layer mainly composed of collagen and cuticles. Thus, a novel approach to trigger intracellular delivery of chemicals through the layers of nematodes is required. In this study, the selection of the novel CPP was carefully progressed through protein database and serial digested fragmentation, internalization of each amino sequence was analyzed through flow cytometry and confocal microscope. As one of the most effective CPP material, JH 1.6 was compared with other major CPPs and its cellular toxicity was investigated. Furthermore, JH 1.6 was attached to various RNA, DNA, and proteins and internalization efficiency was evaluated for mammalian cells. To examine its effects on nematodes in vivo, JH 1.6 was conjugated with nematocidal protein - botulinum neurotoxin (BnT) and treated in C.elegans as a model animal. The results showed that JH 1.6 had high relative internalization rate and low cellular toxicity compared to other major CPP such as TAT and GV1001 peptides.

  16. MiniTn7-transposon delivery vectors for inducible or constitutive fluorescent protein expression in Enterobacteriaceae

    PubMed Central

    Remus-Emsermann, Mitja N. P.; Gisler, Pascal; Drissner, David

    2016-01-01

    Here we present the generation and function of two sets of bacterial plasmids that harbor fluorescent genes encoding either blue, cyan, yellow or red fluorescent proteins. In the first set, protein expression is controlled by the strong and constitutive nptII promoter whereas in the second set, the strong tac promoter was chosen that underlies LacIq regulation. Furthermore, the plasmids are mobilizable, contain Tn7 transposons and a temperature-sensitive origin of replication. Using Escherichia coli S17-1 as donor strain, the plasmids allow fast and convenient Tn7-transposon delivery into many enterobacterial hosts, such as the here-used E. coli O157:H7. This procedure omits the need of preparing competent recipient cells and antibiotic resistances are only transiently conferred to the recipients. As the fluorescence proteins show little to no overlap in fluorescence emission, the constructs are well suited for the study of multicolored synthetic bacterial communities during biofilm production or in host colonization studies, e.g. of plant surfaces. Furthermore, tac promoter-reporter constructs allow the generation of so-called reproductive success reporters, which allow to estimate past doublings of bacterial individuals after introduction into environments, emphasizing the role of individual cells during colonization. PMID:27445318

  17. Whey protein mucoadhesive properties for oral drug delivery: Mucin-whey protein interaction and mucoadhesive bond strength.

    PubMed

    Hsein, Hassana; Garrait, Ghislain; Beyssac, Eric; Hoffart, Valérie

    2015-12-01

    Whey protein is a natural polymer recently used as an excipient in buccoadhesive tablets but its mucoadhesive properties were barely studied. In this work, we characterize mucoadhesion of whey protein in order to determine the mechanisms and optimal conditions for use as excipient in oral drug delivery. Thus, native and denatured whey protein (NWP and DWP) were investigated and the effect of concentration and pH were also studied. Many methods of characterization were selected to allow the study of chemical and physical interactions with mucin and then the results were bound with an ex vivo experiments. Turbidity of WP-mucin mixture increased at acidic pH 1.2 till 4.5 indicating interaction with mucin but not at pH 6.8. No interaction with mucin was also found by ITC method at pH 6.8 for native and denatured whey protein used at 1% (w/w). Forces of bioadhesion evaluated by viscosity measurements were the best for high concentrated (10.8%) DWP solutions at pH 6.8 and were low at pH 1.2 for NWP and DWP solutions. Addition of chemical blockers indicated that hydrogen bondings and disulfide bridges were the main mechanisms of interactions with mucin. Reticulation of DWP with calcium ions to obtain microparticles (MP) did not influence the ability of interaction with mucin as shown by FTIR analysis. These results correlated with ex vivo study on rat tissue demonstrating important adhesion (75%) of WP MP on the intestine and null on the stomach after 2h of deposit.

  18. Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

    PubMed Central

    Ishchenko, Andrii; Cherezov, Vadim; Liu, Wei

    2016-01-01

    Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include

  19. Novel xylan-controlled delivery of therapeutic proteins to inflamed colon by the human anaerobic commensal bacterium

    PubMed Central

    2013-01-01

    Introduction Growth factors such as keratinocyte growth factor-2 (KGF-2) and transforming growth factor-beta (TGF-β) are important immunoregulatory and epithelial growth factors. They are also potential therapeutic proteins for inflammatory bowel disease. However, owing to protein instability in the upper gastrointestinal tract, it is difficult to achieve therapeutic levels of these proteins in the injured colon when given orally. Furthermore, the short half-life necessitates repeated dosage with large amounts of the growth factor, which may have dangerous side effects, hence the importance of temporal and spatial control of growth factor delivery. Methods The human commensal gut bacterium, Bacteroides ovatus, was genetically engineered to produce human KGF-2 or TGF-β 1 (BO-KGF or BO-TGF) in a regulated manner in response to the dietary polysaccharide, xylan. The successful application of BO-KGF or BO-TGF in the prevention of dextran sodium sulphate induced murine colitis is presented here. Results This novel drug delivery system had a significant prophylactic effect, limiting the development of intestinal inflammation both clinically and histopathologically. The ability to regulate heterologous protein production by B ovatus using xylan is both unique and an important safety feature of this drug delivery system. Conclusions The use of genetically engineered B ovatus for the controlled and localised delivery of epithelial growth promoting and immunomodulatory proteins has potential clinical applications for the treatment of various diseases targeting the colon. PMID:23676805

  20. Transdermal delivery of a approximately 13 kDa protein--an in vivo comparison of physical enhancement methods.

    PubMed

    Katikaneni, Sahitya; Li, Guohua; Badkar, Advait; Banga, Ajay K

    2010-02-01

    The availability of several enhancement techniques has made it possible to study delivery of macromolecules through skin. This study was conducted to evaluate the transdermal delivery of a ~13 kDa protein using iontophoresis, sonophoresis, and microneedles alone or in combination. In vivo delivery experiments were carried out using hairless rats with daniplestim (DP) as the model protein (molecular weight: 12.760 kDa; isoelectric point, 6.2). Delivery enhancement abilities of the above techniques were evaluated at two different drug concentrations in the patch: 2 mg/mL and 5 mg/mL. At a drug loading concentration of 2 mg/mL maximum delivery was seen with the combination of microneedles and iontophoresis. At 5 mg/mL, sonophoresis alone gave a C(max) of 8.22 +/- 5.9 ng/mL and a combination of sonophoresis and iontophoresis gave a C(max) of 4.9 +/- 1.8 ng/mL. The results of this study suggest that combination of microneedles and iontophoresis was the most effective approach in delivering a 13 kDa protein through the skin.

  1. Self-Nanoemulsified Drug Delivery System of Hydrochlorothiazide for Increasing Dissolution Rate and Diuretic Activity.

    PubMed

    Mendes, Cassiana; Buttchevitz, Aline; Kruger, Jéssica Henriques; Caon, Thiago; de Oliveira Benedet, Patricia; Lemos-Senna, Elenara; Silva, Marcos Antônio Segatto

    2017-02-17

    Hydrochlorothiazide (HCTZ) is a class IV drug according to the Biopharmaceutical Classification System. This study aimed the development of self-nanoemulsifying drug delivery system (SNEDDS) for HCTZ as an approach to overcome the biopharmaceutical limitations. Pre-formulation screening and ternary phase diagrams were carried out to select the oil phase, the surfactant, and the co-surfactant as the amount of each constituent. The optimized formulations, with reduced amount of surfactant, and composed of medium chain triglycerides, Cremophor EL and Transcutol P did not affect the pH or show drug incompatibilities. The SNEDDS were stabilized by the nanoscale globules and high negative zeta potential. All the physicochemical characterization assays were performed in biorelevant media to better predict the in vivo performance. The enhanced dissolution rate of the SNEDDS reflected in the in vivo diuretic activity, presenting a natriuresis, kaliuresis, and chloriuresis at early stages and an increased volume of total urine compared with HCTZ alone. The designed SNEDDS produced an improvement in the pharmacodynamics due to high dissolution and probable inhibition of intestinal efflux protein by Cremophor EL. The use of SNEDDS demonstrated to be an efficient approach to modulate the absorption of HCTZ and drug therapeutics.

  2. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  3. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  4. Whey protein/polysaccharide-stabilized emulsions: Effect of polymer type and pH on release and topical delivery of salicylic acid.

    PubMed

    Combrinck, Johann; Otto, Anja; du Plessis, Jeanetta

    2014-06-01

    Emulsions are widely used as topical formulations in the pharmaceutical and cosmetic industries. They are thermodynamically unstable and require emulsifiers for stabilization. Studies have indicated that emulsifiers could affect topical delivery of actives, and this study was therefore designed to investigate the effects of different polymers, applied as emulsifiers, as well as the effects of pH on the release and topical delivery of the active. O/w emulsions were prepared by the layer-by-layer technique, with whey protein forming the first layer around the oil droplets, while either chitosan or carrageenan was subsequently adsorbed to the protein at the interface. Additionally, the emulsions were prepared at three different pH values to introduce different charges to the polymers. The active ingredient, salicylic acid, was incorporated into the oil phase of the emulsions. Physical characterization of the resulting formulations, i.e., droplet size, zeta potential, stability, and turbidity in the water phase, was performed. Release studies were conducted, after which skin absorption studies were performed on the five most stable emulsions, by using Franz type diffusion cells and utilizing human, abdominal skin membranes. It was found that an increase in emulsion droplet charge could negatively affect the release of salicylic acid from these formulations. Contrary, positively charged emulsion droplets were found to enhance dermal and transdermal delivery of salicylic acid from emulsions. It was hypothesized that electrostatic complex formation between the emulsifier and salicylic acid could affect its release, whereas electrostatic interaction between the emulsion droplets and skin could influence dermal/transdermal delivery of the active.

  5. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  6. Nitroxide delivery system for Nrf2 activation and skin protection.

    PubMed

    Ben Yehuda Greenwald, Maya; Frušić-Zlotkin, Marina; Soroka, Yoram; Sasson, Shmuel Ben; Bianco-Peled, Havazelet; Bitton, Ronit; Kohen, Ron

    2015-08-01

    Cyclic nitroxides are a large group of compounds composed of diverse stable radicals also known as synthetic antioxidants. Although nitroxides are valuable for use in several skin conditions, in in vivo conditions they have several drawbacks, such as nonspecific dispersion in normal tissue, preferential renal clearance and rapid reduction of the nitroxide to the corresponding hydroxylamine. However, these drawbacks can be easily addressed by encapsulating the nitroxides within microemulsions. This approach would allow nitroxide activity and therefore their valuable effects (e.g. activation of the Keap1-Nrf2-EpRE pathway) to continue. In this work, nitroxides were encapsulated in a microemulsion composed of biocompatible ingredients. The nanometric size and shape of the vehicle microemulsion and nitroxide microemulsion displayed high similarity, indicating that the stability of the microemulsions was preserved. Our studies demonstrated that nitroxide microemulsions were more potent inducers of the Keap1-Nrf2-EpRE pathway than the free nitroxides, causing the activation of phase II enzymes. Moreover, microemulsions containing nitroxides significantly reduced UVB-induced cytotoxicity in the skin. Understanding the mechanism of this improved activity may expand the usage of many other Nrf2 modulating molecules in encapsulated form, as a skin protection strategy against oxidative stress-related conditions.

  7. Keratin sponge/hydrogel II, active agent delivery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Keratin sponge/hydrogels from oxidation and reduction hydrolysis of fine and coarse wool fibers were formed to behave as cationic hydrogels to swell and release active agents in the specific region of the gastro-intestinal (GI) tract. Their porous, interpenetrating networks (IPN) were effective for...

  8. CW/pulsed NIR irradiation of gold nanorods: effect on transdermal protein delivery mediated by photothermal ablation.

    PubMed

    Tang, Hengmin; Kobayashi, Hiroaki; Niidome, Yasuro; Mori, Takeshi; Katayama, Yoshiki; Niidome, Takuro

    2013-10-28

    Transdermal delivery is a useful and attractive method for drug delivery, even though the stratum corneum is a major barrier of protein translocation into the skin. To achieve protein delivery through the stratum corneum, we first cast gold nanorods, acting as a heating device in response to near-infrared light irradiation, onto the skin surface. After applying an aqueous solution of ovalbumin to the skin, the skin was irradiated by near-infrared laser light. Irradiation of the skin using a continuous-wave laser increased the skin temperature resulting in an efficient translocation of ovalbumin into the skin. Furthermore, migration of inflammation cells and induction heat shock protein 70 (HSP70) were observed. Irradiation of the skin using a pulsed laser caused an enhanced permeability of the stratum corneum without an increase in skin temperature, migration of inflammation cells, or HSP70 induction. This effect is due to the pulsed-laser irradiation increasing the temperature of a limited part of the skin surface. Thus, the physiological response of skin is dependent on the type of laser light used. It is anticipated that this phenomenon will find wide application in such applications as, for example, general transdermal protein delivery and transdermal vaccination.

  9. Application of elastin-mimetic recombinant proteins in chemotherapeutics delivery, cellular engineering, and regenerative medicine.

    PubMed

    Jeon, Won Bae

    2013-01-01

    With the remarkable increase in the fields of biomedical engineering and regenerative medicine, biomaterial design has become an indispensable approach for developing the biocompatible carriers for drug or gene cargo and extracellular matrix (ECM) for cell survival, proliferation and differentiation. Native ECM materials derived from animal tissues were believed to be the best choices for tissue engineering. However, possible pathogen contamination by cellular remnants from foreign animal tissues is an unavoidable issue that has limited the use of native ECM for human benefit. Some synthetic polymers have been used as alternative materials for manufacturing native ECM because of the biodegradability and ease of large-scale production of the polymers. However, the inherent polydispersity of the polymers causes batch-to-batch variation in polymer composition and possible cytotoxic interactions between chemical matrices and neighboring cells or tissues have not yet been fully resolved. Elastin-like proteins (ELPs) are genetically engineered biopolymers modeled after the naturally occurring tropoelastin and have emerged as promising materials for biomedical applications because they are biocompatible, non-immunogenic and biodegradable, and their composition, mechanical stiffness and even fate within the cell can be controlled at the gene level. This commentary highlights the recent progresses in the development of the ELP-based recombinant proteins that are being increasingly used for the delivery of chemotherapeutics and to provide a cell-friendly ECM environment.

  10. Protective effect of post-ischaemic viral delivery of heat shock proteins in vivo

    PubMed Central

    Badin, Romina A; Modo, Michael; Cheetham, Mike; Thomas, David L; Gadian, David G; Latchman, David S; Lythgoe, Mark F

    2009-01-01

    Heat shock proteins (HSPs) function as molecular chaperones involved in protein folding, transport and degradation and, in addition, they can promote cell survival both in vitro and in vivo after a range of stresses. Although some in vivo studies have suggested that HSP27 and HSP70 can be neuroprotective, current evidence is limited, particularly when HSPs have been delivered after an insult. The effect of overexpressing HSPs after transient occlusion of the middle cerebral artery in rats was investigated by delivering an attenuated herpes simplex viral vector (HSV-1) engineered to express HSP27 or HSP70 30 mins after tissue reperfusion. Magnetic resonance imaging scans were used to determine lesion size and cerebral blood flow at six different time points up to 1 month after stroke. Animals underwent two sensorimotor tests at the same time points to assess the relationship between lesion size and function. Results indicate that post-ischaemic viral delivery of HSP27, but not of HSP70, caused a statistically significant reduction in lesion size and induced a significant behavioural improvement compared with controls. This is the first evidence of effective post-ischaemic gene therapy with a viral vector expressing HSP27 in an experimental model of stroke. PMID:18781161

  11. Protein-engineered block-copolymers as stem cell delivery vehicles

    NASA Astrophysics Data System (ADS)

    Heilshorn, Sarah

    2015-03-01

    Stem cell transplantation is a promising therapy for a myriad of debilitating diseases and injuries; however, current delivery protocols are inadequate. Transplantation by direct injection, which is clinically preferred for its minimal invasiveness, commonly results in less than 5% cell viability, greatly inhibiting clinical outcomes. We demonstrate that mechanical membrane disruption results in significant acute loss of viability at clinically relevant injection rates. As a strategy to protect cells from these damaging forces, we show that cell encapsulation within hydrogels of specific mechanical properties will significantly improve viability. Building on these fundamental studies, we have designed a reproducible, bio-resorbable, customizable hydrogel using protein-engineering technology. In our Mixing-Induced Two-Component Hydrogel (MITCH), network assembly is driven by specific and stoichiometric peptide-peptide binding interactions. By integrating protein science methodologies with simple polymer physics models, we manipulate the polypeptide chain interactions and demonstrate the direct ability to tune the network crosslinking density, sol-gel phase behavior, and gel mechanics. This is in contrast to many other physical hydrogels, where predictable tuning of bulk mechanics from the molecular level remains elusive due to the reliance on non-specific and non-stoichiometric chain interactions for network formation. Furthermore, the hydrogel network can be easily modified to deliver a variety of bioactive payloads including growth factors, peptide drugs, and hydroxyapatite nanoparticles. Through a series of in vitro and in vivo studies, we demonstrate that these materials may significantly improve transplanted stem cell retention and function.

  12. Development of a Gastric Absorptive, Immediate Responsive, Oral Protein-Loaded Versatile Polymeric Delivery System.

    PubMed

    Kondiah, Pierre P D; Choonara, Yahya E; Tomar, Lomas K; Tyagi, Charu; Kumar, Pradeep; du Toit, Lisa C; Marimuthu, Thashree; Modi, Girish; Pillay, Viness

    2017-02-15

    A multifunctional platform to deliver three diverse proteins of insulin, interferon beta (INF-β) and erythropoietin (EPO), using a novel copolymeric microparticulate system of TMC-PEGDMA-MAA, was synthesised as an intelligent pH-responsive 2-fold gastric and intestinal absorptive system. Physiochemical and physicomechanical studies proved the degree of crystallinity that supported the controlled protein delivery of the microparticulate system. The copolymer was tableted before undertaking in vitro and in vivo analysis. After 2.5 h in simulated gastric fluid (SGF), insulin showed a fractional release of 3.2% in comparison to simulated intestinal fluid (SIF), in which a maximum of 83% of insulin was released. Similarly, INF-β and EPO released 3 and 9.7% in SGF and a maximum of 74 and 81.3% in SIF, respectively. In vivo studies demonstrated a significant decrease in blood glucose by 54.19% within 4 h post-dosing, and the comparator formulation provided 74.6% decrease in blood glucose within the same time period. INF-β peak bioavailable dose in serum was calculated to be 1.3% in comparison to an SC formulation having a peak concentration of 0.9%, demonstrating steady-state release for 24 h. EPO-loaded copolymeric microparticles had a 1.6% peak bioavailable concentration, in comparison to the 6.34% peak concentration after 8 h from the SC comparator formulation.

  13. Image-guided convection-enhanced delivery of GDNF protein into monkey putamen.

    PubMed

    Gimenez, Francisco; Krauze, Michal T; Valles, Francisco; Hadaczek, Piotr; Bringas, John; Sharma, Nitasha; Forsayeth, John; Bankiewicz, Krystof S

    2011-01-01

    Recently, we developed an MRI-based method that enables tracking of parenchymal infusions of therapeutic agents by inclusion of a contrast reagent in the infusate. We show that both liposomal Gadoteridol (GDL) and free Gadoteridol (Gd) can be used for MRI-monitored infusions into the non-human primate (NHP) putamen to predict the distribution of GDNF protein after convection-enhanced delivery (CED). GDNF and both MRI tracers showed good co-distribution within the putamen and other brain regions. Although the CED infusion technique can distribute GDNF protein over large brain regions, continuous administration of GDNF could cause undesired effects that could counteract the benefits of CED as demonstrated in this study when large volumes of GDNF were delivered that lead to GDNF leakage into CSF. These limitations can be addressed by employing an intermittent CED schedule that permits consistent target coverage without GDNF leakage into CSF or white matter. We present an approach intracranial GDNF infusions that can be optimized by means of real-time monitoring via MRI. Adoption of this new standard, along with advanced, reflux-resistant cannulae, may permit reconsideration of direct GDNF infusion into parenchyma as a clinical strategy, since previous clinical studies involving chronic infusion of recombinant glial cell line-derived neurotrophic factor (GDNF) to the putamen for the treatment of Parkinson's disease have yielded mixed results, a state of affairs that may in part be attributed to suboptimal infusion parameters.

  14. Tragacanth as an oral peptide and protein delivery carrier: Characterization and mucoadhesion.

    PubMed

    Nur, M; Ramchandran, L; Vasiljevic, T

    2016-06-05

    Biopolymers such as tragacanth, an anionic polysaccharide gum, can be alternative polymeric carrier for physiologically important peptides and proteins. Characterization of tragacanth is thus essential for providing a foundation for possible applications. Rheological studies colloidal solution of tragacanth at pH 3, 5 or 7 were carried out by means of steady shear and small amplitude oscillatory measurements. Tragacanth mucoadhesivity was also analyzed using an applicable rheological method and compared to chitosan, alginate and PVP. The particle size and zeta potential were measured by a zetasizer. Thermal properties of solutions were obtained using a differential scanning calorimetry. The solution exhibited shear-thinning characteristics. The value of the storage modulus (G') and the loss modulus (G″) increased with an increase in angular frequency (Ω). In all cases, loss modulus values were higher than storage values (G″>G') and viscous character was, therefore, dominant. Tragacanth and alginate showed a good mucoadhesion. Tragacanth upon dispersion created particles of a submicron size with a negative zeta potential (-7.98 to -11.92 mV). These properties were pH dependant resulting in acid gel formation at pH 3.5. Tragacanth has thus a potential to be used as an excipient for peptide/protein delivery.

  15. Protein encapsulated magnetic carriers for micro/nanoscale drug delivery systems.

    SciTech Connect

    Xie, Y.; Kaminski, M. D.; Mertz, C. J.; Finck, M. R.; Guy, S. G.; Chen, H.; Rosengart, A. J.; Chemical Engineering; Univ. of Chicago, Pritzker School of Medicine

    2005-01-01

    Novel methods for drug delivery may be based on nanotechnology using non-invasive magnetic guidance of drug loaded magnetic carriers to the targeted site and thereafter released by external ultrasound energy. The key building block of this system is to successfully synthesize biodegradable, magnetic drug carriers. Magnetic carriers using poly(D,L-lactide-co-glycolide) (PLGA) or poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) as matrix materials were loaded with bovine serum albumin (BSA) by a double-emulsion technique. BSA-loaded magnetic microspheres were characterized for size, morphology, surface charge, and magnetization. The BSA encapsulation efficiency was determined by recovering albumin from the microspheres using dimethyl sulfoxide and 0.05N NaOH/0.5% SDS then quantifying with the Micro-BCA protein assay. BSA release profiles were also determined by the Micro-BCA protein assay. The microspheres had drug encapsulation efficiencies up to 90% depending on synthesis parameters. Particles were spherical with a smooth or porous surface having a size range less than 5 {mu}m. The surface charge (expressed as zeta potential) was near neutral, optimal for prolonged intravascular survival. The magnetization of these BSA loaded magnetic carriers was 2 to 6 emu/g, depending on the specific magnetic materials used during synthesis.

  16. Nanoparticle formation by using shellac and chitosan for a protein delivery system.

    PubMed

    Kraisit, Pakorn; Limmatvapirat, Sontaya; Nunthanid, Jurairat; Sriamornsak, Pornsak; Luangtana-anan, Manee

    2013-01-01

    The potential of using two natural polymers (chitosan and shellac) for the formation of nanoparticles by the process of ionic cross-linking to encapsulate bovine serum albumin, a model protein was investigated. Depending on the concentrations of chitosan, shellac and bovine serum albumin, three physical states - nanoparticle, aggregation, and solution could be observed as a result of the electrostatic force. The formation of nanoparticles was due to the balance between the repulsion force and attractive force while the imbalance between both forces resulted in the formation of aggregation and solution. The Fourier transform infrared spectroscopy and differential scanning calorimetry were applied to prove the nanoparticle formation. The particle size was characterized by the light scattering technique and was found in the range between 100 and 300 nm. The morphology of the particles, detected by transmission electron microscopy was spherical shape. The result showed that the zeta potential of the nanoparticles possessed positive charges. The concentrations of chitosan, shellac and bovine serum albumin had an influence on the physicochemical properties of the nanoparticles such as the particle size, the zeta potential, the encapsulation, the loading efficiencies and the cumulative release. Therefore, chitosan and shellac could be used to form nanoparticles for protein delivery by the ionic cross-linking method.

  17. Online Delivery as a Course Adjunct Promotes Active Learning and Student Success

    ERIC Educational Resources Information Center

    Lewis, J. Scott; Harrison, Marissa A.

    2012-01-01

    Chickering and Gamson's notable summary of the best practices of undergraduate teaching include promoting active learning, cooperation, and student-faculty contact. The present study hypothesized that online delivery of lecture prior to course meetings allows more in-class time to achieve these goals. Students in a control group received a…

  18. Active scaffolds for on-demand drug and cell delivery

    PubMed Central

    Zhao, Xuanhe; Kim, Jaeyun; Cezar, Christine A.; Huebsch, Nathaniel; Lee, Kangwon; Bouhadir, Kamal; Mooney, David J.

    2011-01-01

    Porous biomaterials have been widely used as scaffolds in tissue engineering and cell-based therapies. The release of biological agents from conventional porous scaffolds is typically governed by molecular diffusion, material degradation, and cell migration, which do not allow for dynamic external regulation. We present a new active porous scaffold that can be remotely controlled by a magnetic field to deliver various biological agents on demand. The active porous scaffold, in the form of a macroporous ferrogel, gives a large deformation and volume change of over 70% under a moderate magnetic field. The deformation and volume variation allows a new mechanism to trigger and enhance the release of various drugs including mitoxantrone, plasmid DNA, and a chemokine from the scaffold. The porous scaffold can also act as a depot of various cells, whose release can be controlled by external magnetic fields. PMID:21149682

  19. Physical Activity Program Delivery by Professionals versus Volunteers: the TEAM Randomized Trial

    PubMed Central

    Castro, Cynthia M.; Pruitt, Leslie A.; Buman, Matthew P.; King, Abby C.

    2010-01-01

    Background Older adults have low rates of physical activity participation but respond positively to telephone-mediated support programs. Programs are often limited by reliance on professional staff. This study tested telephone-based physical activity advice delivered by professional staff versus trained volunteer peer mentors. Design A 12-month, randomized, controlled clinical trial was executed from 2003–2008. Setting/participants: Twelve volunteer peer mentors and 181 initially inactive adults ages 50 years and older were recruited from the San Francisco Bay Area. Intervention Participants were randomized to: (1) telephone-based physical activity advice delivered by professional staff, (2) telephone-based physical activity advice delivered by trained volunteer peers, or (3) an attention-control arm of staff-delivered telephone support for nutrition. Main Outcome Measures: Moderate-intensity or more vigorous physical activity (MVPA) was assessed at baseline, 6, and 12 months with the CHAMPS Questionnaire, with accelerometry validation (Actigraph) in a randomly selected subsample. Treatment fidelity was examined through analysis of quantity and quality of intervention delivery. Results At 6 and 12 months, both physical activity arms significantly increased MVPA relative to the control arm. Both physical activity arms were comparable in quantity of intervention delivery, but peers demonstrated more versatility and comprehensiveness in quality of intervention content. Conclusions This study demonstrates that trained peer volunteers can effectively promote physical activity increases through telephone-based advice. The results support a program delivery model with good dissemination potential for a variety of community settings. PMID:21553972

  20. Hydrogel Delivery of Mesenchymal Stem Cell–Expressing Bone Morphogenetic Protein-2 Enhances Bone Defect Repair

    PubMed Central

    Hsiao, Hui-Yi; Yang, Shu-Rui; Brey, Eric M.; Chu, I-Ming

    2016-01-01

    Background: The application of bone tissue engineering for repairing bone defects has gradually shown some satisfactory progress. One of the concerns raising scientific attention is the poor supply of growth factors. A number of growth factor delivery approaches have been developed for promoting bone formation. However, there is no systematic comparison of those approaches on efficiency of neobone formation. In this study, the approaches using periosteum, direct supply of growth factors, or gene transfection of growth factors were evaluated to determine the osteogenic capacity on the repair of bone defect. Methods: In total, 42 male 21-week-old Sprague-Dawley rats weighing 250 to 400 g were used as the bone defect model to evaluate the bone repair efficiency. Various tissue engineered constructs of poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLLA) copolymer hydrogel with periosteum, with external supply of bone morphogenetic protein-2 (BMP2), or with BMP2-transfected bone marrow–derived mesenchymal stem cells (BMMSCs) were filled in a 7-mm bone defect region. Animals were euthanized at 3 months, and the hydrogel constructs were harvested. The evaluation with histological staining and radiography analysis were performed for the volume of new bone formation. Results: The PEG-PLLA scaffold with BMMSCs promotes bone regeneration with the addition of periosteum. The group with BMP2-transfected BMMSCs demonstrated the largest volume of new bone among all the testing groups. Conclusions: Altogether, the results of this study provide the evidence that the combination of PEG-PLLA hydrogels with BMMSCs and sustained delivery of BMP2 resulted in the maximal bone regeneration. PMID:27622106

  1. Delivery of a protein transduction domain-mediated Prdx6 protein ameliorates oxidative stress-induced injury in human and mouse neuronal cells

    PubMed Central

    Singh, Shatrunjai P.; Chhunchha, Bhavana; Fatma, Nigar; Kubo, Eri; Singh, Sanjay P.

    2015-01-01

    Oxidative stress or reduced expression of naturally occurring antioxidants during aging has been identified as a major culprit in neuronal cell/tissue degeneration. Peroxiredoxin (Prdx) 6, a protective protein with GSH peroxidase and acidic calcium-independent phospholipase A2 activities, acts as a rheostat in regulating cellular physiology by clearing reactive oxygen species (ROS) and thereby optimizing gene regulation. We found that under stress, the neuronal cells displayed reduced expression of Prdx6 protein and mRNA with increased levels of ROS, and the cells subsequently underwent apoptosis. Using Prdx6 fused to TAT transduction domain, we showed evidence that Prdx6 was internalized in human brain cortical neuronal cells, HCN-2, and mouse hippocampal cells, HT22. The cells transduced with Prdx6 conferred resistance against the oxidative stress inducers paraquat, H2O2, and glutamate. Furthermore, Prdx6 delivery ameliorated damage to neuronal cells by optimizing ROS levels and overstimulation of NF-κB. Intriguingly, transduction of Prdx6 increased the expression of endogenous Prdx6, suggesting that protection against oxidative stress was mediated by both extrinsic and intrinsic Prdx6. The results demonstrate that Prdx6 expression is critical to protecting oxidative stress-evoked neuronal cell death. We propose that local or systemic application of Prdx6 can be an effective means of delaying/postponing neuronal degeneration. PMID:26447207

  2. Activating PKC-β1 at the blood-brain barrier reverses induction of P-glycoprotein activity by dioxin and restores drug delivery to the CNS.

    PubMed

    Wang, Xueqian; Hawkins, Brian T; Miller, David S

    2011-06-01

    Upregulation of blood-brain barrier (BBB) P-glycoprotein expression causes central nervous system (CNS) pharmacoresistance. However, activation of BBB protein kinase C-β1 (PKC-β1) rapidly reduces basal P-glycoprotein transport activity. We tested whether PKC-β1 activation would reverse CNS drug resistance caused by dioxin acting through aryl hydrocarbon receptor. A selective PKC-β1 agonist abolished the increase in P-glycoprotein activity induced by dioxin in isolated rat brain capillaries and reversed the effect of dioxin on brain uptake of verapamil in dioxin-dosed rats. Thus, targeting BBB PKC-β1 may be an effective strategy to improve drug delivery to the brain, even in drug-resistant individuals.

  3. Development and evaluation of mucoadhesive nanoparticles based on thiolated Eudragit for oral delivery of protein drugs

    NASA Astrophysics Data System (ADS)

    Zhang, Yan; Yang, Zhijie; Hu, Xi; Zhang, Ling; Li, Feng; Li, Meimei; Tang, Xing; Xiao, Wei

    2015-02-01

    The objective of this study was to develop pH-sensitive Eudragit L100-cysteine/reduced glutathione (Eul-cys/GSH) nanoparticles (NPs), which provided the mucoadhesion and protection for protein drugs against enzymatic degradation. Insulin was chosen as a model biomolecule for testing this system. The Eul-cys conjugate, which was obtained by grafting cysteine onto the carboxy group of Eudragit L100, was analyzed by HNMR and SEM, and the swelling degree (SD), cation binding, and enzymatic inhibition were also determined. The results obtained showed that the Eul-cys conjugate represent a pH-sensitive delivery system which effectively protected the insulin from being degraded by the proteases, and this is related to the mechanism of Ca2+ binding. Insulin-loaded Eul-cys/GSH NPs were prepared by a diffusion method involving an electrostatic interaction between the network structure of the polymer and the embedded proteins, including insulin and GSH. TEM images indicated that Eul-cys/GSH existed as smooth and spherical NPs in aqueous solution with particle sizes of 260 ± 20 nm. The X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) findings showed the presence of amorphous insulin in thiolated NPs and higher free thiol oxidation than the result obtained by Ellman's reagent method. In addition, thiolated NPs showed excellent binding efficiency to the mucin in rat intestine, indicating that Eul-cys/GSH NPs have great potential to be applied as safe carriers for the oral administration of protein drugs.

  4. Protein Delivery of an Artificial Transcription Factor Restores Widespread Ube3a Expression in an Angelman Syndrome Mouse Brain.

    PubMed

    Bailus, Barbara J; Pyles, Benjamin; McAlister, Michelle M; O'Geen, Henriette; Lockwood, Sarah H; Adams, Alexa N; Nguyen, Jennifer Trang T; Yu, Abigail; Berman, Robert F; Segal, David J

    2016-03-01

    Angelman syndrome (AS) is a neurological genetic disorder caused by loss of expression of the maternal copy of UBE3A in the brain. Due to brain-specific genetic imprinting at this locus, the paternal UBE3A is silenced by a long antisense transcript. Inhibition of the antisense transcript could lead to unsilencing of paternal UBE3A, thus providing a therapeutic approach for AS. However, widespread delivery of gene regulators to the brain remains challenging. Here, we report an engineered zinc finger-based artificial transcription factor (ATF) that, when injected i.p. or s.c., crossed the blood-brain barrier and increased Ube3a expression in the brain of an adult mouse model of AS. The factor displayed widespread distribution throughout the brain. Immunohistochemistry of both the hippocampus and cerebellum revealed an increase in Ube3a upon treatment. An ATF containing an alternative DNA-binding domain did not activate Ube3a. We believe this to be the first report of an injectable engineered zinc finger protein that can cause widespread activation of an endogenous gene in the brain. These observations have important implications for the study and treatment of AS and other neurological disorders.

  5. A new targeted drug delivery method using ultrasound and acoustically active lipospheres

    NASA Astrophysics Data System (ADS)

    Shortencarier, Michaelann; Bloch, Susannah; Dayton, Paul; Ferrara, Kathy; Matsunaga, Terry; Labell, Rachel; Schumann, Patricia

    2005-04-01

    The goal is to create a strategy for localized drug delivery using engineered delivery vehicles and ultrasound energy. These drug delivery vehicles, referred to as acoustically active lipospheres (AALs), consist of small gas bubbles surrounded by thick oil shells (where drugs can be carried) and are enclosed by an outermost lipid layer. Ultrasound radiation force can be used to displace these vehicles near the blood vessel wall, after which a higher intensity pulse can fragment the vehicle and transfer its contents to the endothelium. Blood velocity ranges from 1 to 10 mm/s in the microvasculature. Therefore, agents in capillaries insonified by a transducer with a 1 mm focal beam width for 0.1 to 1 seconds can be displaced of 1 to 2 mm. Delivery vehicles containing various fluorescent dyes within the oil layer are exposed to ultrasound in cell chambers or flowing vessels. Fluorescence intensity increases more than 10-fold with application of radiation force and fragmentation, both over no exposure and over fragmentation pulses alone (both P<0.001). This implies radiation force is necessary to bring AALs into proximity of the cell monolayer before their destruction in order for drug delivery to occur.

  6. Plasmid DNA delivery into MDA-MB-453 cells mediated by recombinant Her-NLS fusion protein.

    PubMed

    Jeyarajan, Sivakumar; Xavier, Jennifer; Rao, N Madhusudhana; Gopal, Vijaya

    2010-10-05

    A major rate-limiting step in nonviral gene delivery is the entry of nucleic acids across various membrane barriers and eventually into the nucleus where it must be transcribed. Cell-penetrating peptides and proteins are employed to generate formulations that overcome these challenges to facilitate DNA delivery into cells efficiently. However, these are limited by their inability to deliver nucleic acids selectively due to lack of specificity because they deliver to both cancer and normal cells. In this study, through modular design, we generated a recombinant fusion protein designated as Her-nuclear localization sequence (Her-NLS), where heregulin-α (Her), a targeting moiety, was cloned in frame with cationic NLS peptide to obtain a cell-specific targeting biomolecule for nucleic acid delivery. The heregulin-α(1) isoform possesses the epidermal growth factor-like domain and binds to HER2/3 heterodimers which are overexpressed in certain breast cancers. Purified recombinant Her-NLS fusion protein binds plasmid DNA and specifically transfects MDA-MB-453 cells overexpressing the epidermal growth factor receptors HER2/3 in vitro. The approach described would also permit replacement of heregulin ligand with other targeting moieties that would be suited to cell-specific nucleic acid delivery mediated via receptor-ligand interactions.

  7. Coated whey protein/alginate microparticles as oral controlled delivery systems for probiotic yeast.

    PubMed

    Hébrard, Géraldine; Hoffart, Valérie; Beyssac, Eric; Cardot, Jean-Michel; Alric, Monique; Subirade, Muriel

    2010-01-01

    Viable Saccharomyces boulardii, used as a biotherapeutic agent, was encapsulated in food-grade whey protein isolate (WP) and alginate (ALG) microparticles, in order to protect and vehicle them in gastrointestinal environment. Yeast-loaded microparticles with a WP/ALG ratio of 62/38 were produced with high encapsulation efficiency (95%) using an extrusion/cold gelation method and coated with ALG or WP by a simple immersion method. Swelling, yeast survival, WP loss and yeast release in simulated gastric and intestinal fluids (SGF and SIF, pH 1.2 and 7.5) with and without their respective digestive enzymes (pepsin and pancreatin) were investigated. In SGF, ALG network shrinkage limited enzyme diffusion into the WP/ALG matrix. Coated and uncoated WP/ALG microparticles were resistant in SGF even with pepsin. Survival of yeast cells in microparticles was 40% compared to 10% for free yeast cells and was improved to 60% by coating. In SIF, yeast cell release followed coated microparticle swelling with a desirable delay. Coated WP/ALG microparticles appear to have potential as oral delivery systems for Saccharomyces boulardii or as encapsulation means for probiotic cells in pharmaceutical or food processing applications.

  8. Stimulation of bone healing by sustained bone morphogenetic protein 2 (BMP-2) delivery.

    PubMed

    Faßbender, Mirja; Minkwitz, Susann; Strobel, Catrin; Schmidmaier, Gerhard; Wildemann, Britt

    2014-05-14

    The aim of the study was to investigate the effect of a sustained release of bone morphogenetic protein2 (BMP-2) incorporated in a polymeric implant coating on bone healing. In vitro analysis revealed a sustained, but incomplete BMP-2 release until Day 42. For the in vivo study, the rat tibia osteotomy was stabilized either with control or BMP-2 coated wires, and the healing progress was followed by micro computed tomography (µCT), biomechanical testing and histology at Days 10, 28, 42 and 84. MicroCT showed an accelerated formation of mineralized callus, as well as remodeling and an increase of mineralized/total callus volume (p=0.021) at Day 42 in the BMP-2 group compared to the control. Histology revealed an increased callus mineralization at Days 42 and 84 (p=0.006) with reduced cartilage at Day 84 (p=0.004) in the BMP-2 group. Biomechanical stiffness was significantly higher in the BMP-2 group (p=0.045) at Day 42. In summary, bone healing was enhanced after sustained BMP-2 application compared to the control. Using the same drug delivery system, but a burst release of BMP-2, a previous published study showed a similar positive effect on bone healing. Distinct differences in the healing outcome might be explained due to the different BMP release kinetics and dosages. However, further studies are necessary to adapt the optimal release profiles to physiological mechanisms.

  9. The Potential Role of Cell Penetrating Peptides in the Intracellular Delivery of Proteins for Therapy of Erythroid Related Disorders

    PubMed Central

    Papadopoulou, Lefkothea C.; Tsiftsoglou, Asterios S.

    2013-01-01

    The erythroid related disorders (ERDs) represent a large group of hematological diseases, which in most cases are attributed either to the deficiency or malfunction of biosynthetic enzymes or oxygen transport proteins. Current treatments for these disorders include histo-compatible erythrocyte transfusions or allogeneic hematopoietic stem cell (HSC) transplantation. Gene therapy delivered via suitable viral vectors or genetically modified HSCs have been under way. Protein Transduction Domain (PTD) technology has allowed the production and intracellular delivery of recombinant therapeutic proteins, bearing Cell Penetrating Peptides (CPPs), into a variety of mammalian cells. Remarkable progress in the field of protein transduction leads to the development of novel protein therapeutics (CPP-mediated PTs) for the treatment of monogenetic and/or metabolic disorders. The “concept” developed in this paper is the intracellular protein delivery made possible via the PTD technology as a novel therapeutic intervention for treatment of ERDs. This can be achieved via four stages including: (i) the production of genetically engineered human CPP-mediated PT of interest, since the corresponding native protein either is missing or is mutated in the erythroid progenitor cell (ErPCs) or mature erythrocytes of patients; (ii) isolation of target cells from the peripheral blood of the selected patients; (iii) ex vivo transduction of cells with the CPP-mediated PT of interest; and (iv) re-administration of the successfully transduced cells back into the same patients. PMID:24275786

  10. Zwitterionic Phosphorylcholine-TPE Conjugate for pH-Responsive Drug Delivery and AIE Active Imaging.

    PubMed

    Chen, Yangjun; Han, Haijie; Tong, Hongxin; Chen, Tingting; Wang, Haibo; Ji, Jian; Jin, Qiao

    2016-08-24

    Polymeric micelles have emerged as a promising nanoplatform for cancer theranostics. Herein, we developed doxorubicin (DOX) encapsulated pH-responsive polymeric micelles for combined aggregation induced emission (AIE) imaging and chemotherapy. The novel zwitterionic copolymer poly(2-methacryloyloxyethylphosphorylcholine-co-2-(4-formylphenoxy)ethyl methacrylate) (poly(MPC-co-FPEMA)) was synthesized via RAFT polymerization and further converted to PMPC-hyd-TPE after conjugation of tetraphenylethene (TPE, a typical AIE chromophore) via acid-cleavable hydrazone bonds. The AIE activatable copolymer PMPC-hyd-TPE could self-assemble into spherical PC-hyd-TPE micelles, and DOX could be loaded through hydrophobic interactions. The zwitterionic micelles exhibited excellent physiological stability and low protein adsorption due to the stealthy phosphorylcholine (PC) shell. In addition, the cleavage of hydrophobic TPE molecules under acidic conditions could induce swelling of micelles, which was verified by size changes with time at pH 5.0. The in vitro DOX release profile also exhibited accelerated release rate with pH value decreasing from 7.4 to 5.0. Fluorescent microscopy and flow cytometry studies further demonstrated fast internalization and accumulation of drug loaded PC-hyd-TPE-DOX micelles in HepG2 cells, resulting in considerable time/dose-dependent cytotoxicity. Meanwhile, high-quality AIE imaging of PC-hyd-TPE micelles was confirmed in HepG2 cells. Notably, ex vivo imaging study exhibited efficient accumulation and drug release of PC-hyd-TPE-DOX micelles in the tumor tissue. Consequently, the multifunctional micelles with combined nonfouling surface, AIE active imaging, and pH-responsive drug delivery showed great potential as novel nanoplatforms for a new generation of cancer theranostics.

  11. Intra-tumoral delivery of functional ID4 protein via PCL/maltodextrin nano-particle inhibits prostate cancer growth

    PubMed Central

    Morton, Derrick; Sharma, Pankaj; Gorantla, Yamini; Joshi, Jugal; Nagappan, Perri; Pallaniappan, Ravi; Chaudhary, Jaideep

    2016-01-01

    ID4, a helix loop helix transcriptional regulator has emerged as a tumor suppressor in prostate cancer. Epigenetic silencing of ID4 promotes prostate cancer whereas ectopic expression in prostate cancer cell lines blocks cancer phenotype. To directly investigate the anti-tumor property, full length human recombinant ID4 encapsulated in biodegradable Polycaprolactone/Maltodextrin (PCL-MD) nano-carrier was delivered to LNCaP cells in which the native ID4 was stably silenced (LNCaP(-)ID4). The cellular uptake of ID4 resulted in increased apoptosis, decreased proliferation and colony formation. Intratumoral delivery of PCL-MD ID4 into growing LNCaP(-)ID4 tumors in SCID mice significantly reduced the tumor volume compared to the tumors treated with chemotherapeutic Docetaxel. The study supports the feasibility of using nano-carrier encapsulated ID4 protein as a therapeutic. Mechanistically, ID4 may assimilate multiple regulatory pathways for example epigenetic re-programming, integration of multiple AR co-regulators or signaling pathways resulting in tumor suppressor activity of ID4. PMID:27487149

  12. Function and frustration of multi-drug ABC exporter protein and design of model proteins for drug delivery using protein hydration thermodynamics.

    PubMed

    Urry, Dan W; Urry, Kelley D; Szaflarski, Witold; Nowicki, Michal; Zabel, Maciej

    2009-01-01

    The mechanism is presented whereby simultaneous hydrolysis of two molecules of ATP in the ATP-binding cassette (ABC) exporter protein, Sav 1866, opens a transmembrane channel to pump drug out of the cell and confers drug resistance, e.g., gives rise to methicillin resistant Staphylococcus aureus, MRSA. The proposed mechanism suggests pharmaceutical design strategies for overloading the capacity of two molecules of ATP to open access to the channel for export. Structural homology of Staphylococcus aureus, Sav 1866, to human P-glyco-protein and MRP2, suggests a similar mechanism could be relevant to human carcinoma cells. The transport mechanism utilizes two thermodynamic quantities -DeltaG(HA), the change in Gibbs free energy for hydrophobic association, and DeltaG(ap), an apolar-polar repulsive free energy for hydration, derived from studies on designed elastic-contractile model proteins (ECMPs). These quantities also allow design of remarkably biocompatible ECMPs as drug delivery vehicles with remarkable control of release profiles and of ECMPs that provide the means of developing pharmaceuticals for blocking multi-drug resistance.

  13. Noncationic Rigid and Anisotropic Coiled-Coil Proteins Exhibit Cell-Penetration Activity.

    PubMed

    Nakayama, Norihisa; Hagiwara, Kyoji; Ito, Yoshihiro; Ijiro, Kuniharu; Osada, Yoshihito; Sano, Ken-ichi

    2015-08-04

    Numerous cationic peptides that penetrate cells have been studied intensively as drug delivery system carriers for cellular delivery. However, cationic molecules tend to be cytotoxic and cause inflammation, and their stability in the blood is usually low. We have previously demonstrated that a rigid and fibrous cationic coiled-coil protein exhibited cell-penetrating ability superior to that of previously reported cell-penetrating peptides. Making use of structural properties, here we describe the cell-penetrating activity of a rigid and fibrous coiled-coil protein with a noncationic surface. A fibrous coiled-coil protein of pI 6.5 penetrated 100% of the cells tested in vitro at a concentration of 500 nM, which is comparable to that of previously reported cell-penetrating peptides. We also investigated the effect of cell-strain dependency and short-term cytotoxicity.

  14. Activated protein C: biased for translation

    PubMed Central

    Zlokovic, Berislav V.; Mosnier, Laurent O.

    2015-01-01

    The homeostatic blood protease, activated protein C (APC), can function as (1) an antithrombotic on the basis of inactivation of clotting factors Va and VIIIa; (2) a cytoprotective on the basis of endothelial barrier stabilization and anti-inflammatory and antiapoptotic actions; and (3) a regenerative on the basis of stimulation of neurogenesis, angiogenesis, and wound healing. Pharmacologic therapies using recombinant human and murine APCs indicate that APC provides effective acute or chronic therapies for a strikingly diverse range of preclinical injury models. APC reduces the damage caused by the following: ischemia/reperfusion in brain, heart, and kidney; pulmonary, kidney, and gastrointestinal inflammation; sepsis; Ebola virus; diabetes; and total lethal body radiation. For these beneficial effects, APC alters cell signaling networks and gene expression profiles by activating protease-activated receptors 1 and 3. APC’s activation of these G protein–coupled receptors differs completely from thrombin’s activation mechanism due to biased signaling via either G proteins or β-arrestin-2. To reduce APC-associated bleeding risk, APC variants were engineered to lack >90% anticoagulant activity but retain normal cell signaling. Such a neuroprotective variant, 3K3A-APC (Lys191-193Ala), has advanced to clinical trials for ischemic stroke. A rich data set of preclinical knowledge provides a solid foundation for potential translation of APC variants to future novel therapies. PMID:25824691

  15. LASER ENGINEERED DISSOLVING MICRONEEDLES FOR ACTIVE TRANSDERMAL DELIVERY OF NADROPARIN CALCIUM

    PubMed Central

    Gomaa, Yasmine A.; Garland, Martin J.; McInnes, Fiona; El-Khordagui, Labiba K.; Wilson, Clive; Donnelly, Ryan F.

    2014-01-01

    There is an urgent need to replace the injection currently used for low molecular weight heparin (LMWH) multi-dose therapy with a non-invasive delivery device. In this study, laser-engineered dissolving microneedle (DMN) arrays fabricated from aqueous blends of 15% w/w poly (methylvinylether co maleic anhydride) have been fabricated as a potential device for the active transdermal delivery of nadroparin calcium (NC) as a model LMWH. An array loading of 630 IU of NC was achieved without compromising the array mechanical strength or the drug bioactivity. Application of NC-DMNs to dermatomed human skin (DHS) using the single step “poke and release” approach allowed permeation of approximately 10.6 % of the total NC load over a 48 h-study period. The cumulative amount of NC that permeated DHS at 24 h and 48 h attained 12.28 ± 4.23 IU/cm2 and 164.84 ± 8.47 IU/cm2, respectively. Skin permeation of NC could be modulated by controlling the DMN array variables, such as MN length and array density as well as application force to meet various clinical requirements including adjustment for body mass and renal function. NC-loaded DMN offer potentials as a relatively low cost functional delivery system for the transdermal delivery of LMWH and other macromolecules. PMID:22836025

  16. Toxin delivery by the coat protein of an aphid-vectored plant virus provides plant resistance to aphids.

    PubMed

    Bonning, Bryony C; Pal, Narinder; Liu, Sijun; Wang, Zhaohui; Sivakumar, S; Dixon, Philip M; King, Glenn F; Miller, W Allen

    2014-01-01

    The sap-sucking insects (order Hemiptera), including aphids, planthoppers, whiteflies and stink bugs, present one of the greatest challenges for pest management in global agriculture. Insect neurotoxins offer an alternative to chemical insecticides for controlling these pests, but require delivery into the insect hemocoel. Here we use the coat protein of a luteovirus, an aphid-vectored plant virus, to deliver a spider-derived, insect-specific toxin that acts within the hemocoel. The luteovirid coat protein is sufficient for delivery of fused proteins into the hemocoel of pea aphids, Acyrthosiphon pisum, without virion assembly. We show that when four aphid pest species-A. pisum, Rhopalosiphum padi, Aphis glycines and Myzus persicae-feed on a recombinant coat protein-toxin fusion, either in an experimental membrane sachet or in transgenic Arabidopsis plants, they experience significant mortality. Aphids fed on these fusion proteins showed signs of neurotoxin-induced paralysis. Luteovirid coat protein-insect neurotoxin fusions represent a promising strategy for transgenic control of aphids and potentially other hemipteran pests.

  17. Synaptic Vesicle Proteins and Active Zone Plasticity

    PubMed Central

    Kittel, Robert J.; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention. PMID:27148040

  18. Production of bioinspired and rationally designed polymer hydrogels for controlled delivery of therapeutic proteins

    NASA Astrophysics Data System (ADS)

    Kim, Sung Hye

    patterns of functional groups. However, heterogeneity in the composition and in the polydispersity of heparin has been problematic in controlled delivery system and thus motivated the development of homogeneous heparin mimics. Peptides of appropriate sequence and chemical function have therefore recently emerged as potential replacements for heparin in select applications. Studied was the assessment of the binding affinities of multiple sulfated peptides (SPs) for a set of heparin-binding peptides (HBPs) and for VEGF; these binding partners have application in the selective immobilization of proteins and in hydrogel formation through non-covalent interactions. Sulfated peptides were produced via solid-phase methods, and their affinity for the HBPs and VEGF was assessed via affinity liquid chromatography (ALC), surface plasmon resonance (SPR), and in select cases, isothermal titration calorimetry (ITC). The shortest peptide, SPa, showed the highest affinity binding of HBPs and VEGF165 in both ALC and SPR measurements, with slight exceptions. Of the investigated HBPs, a peptide based on the heparin-binding domain of human platelet factor 4 showed greatest binding affinities toward all of the SPs, consistent with its stronger binding to heparin. The affinity between SPa and PF4ZIP was indicated via SPR ( KD = 5.27 muM) and confirmed via ITC (KD = 8.09 muM). The binding by SPa of both VEGF and HBPs suggests its use as a binding partner to multiple species, and the use of these interactions in assembly of materials. Given that the peptide sequences can be varied to control binding affinity and selectivity, opportunities are also suggested for the production of a wider array of matrices with selective binding and release properties useful for biomaterials applications. Hydrogel consisting of SPa was formed via a covalent Michael Addition reaction between maleimide- and thiol-terminated multi-arm PEGs and Cys-SPa. The mechanical property of hydrogel was tunable from ca. 186 to

  19. Lipid Dependent Mechanisms of Protein Pump Activity

    DTIC Science & Technology

    1989-05-23

    properties which result form the colligative interactions of many lipid molecules. Important materials properties include . . . i I I II II I i I 1 the...d identify by olock number) *This project is aime at investigating if a lipid elastic property , known as the spontaneous radius of curvature Ro’, is...a regulated membrane property and if its value modulates membrane protein activity. Specific aims reported on here include: 1) Correlation of ion pump

  20. Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells.

    PubMed

    Xiao, Yuhong; Kwon, Kwang-Chul; Hoffman, Brad E; Kamesh, Aditya; Jones, Noah T; Herzog, Roland W; Daniell, Henry

    2016-02-01

    Targeted oral delivery of GFP fused with a GM1 receptor binding protein (CTB) or human cell penetrating peptide (PTD) or dendritic cell peptide (DCpep) was investigated. Presence of GFP(+) intact plant cells between villi of ileum confirm their protection in the digestive system from acids/enzymes. Efficient delivery of GFP to gut-epithelial cells by PTD or CTB and to M cells by all these fusion tags confirm uptake of GFP in the small intestine. PTD fusion delivered GFP more efficiently to most tissues or organs than the other two tags. GFP was efficiently delivered to the liver by all fusion tags, likely through the gut-liver axis. In confocal imaging studies of human cell lines using purified GFP fused with different tags, GFP signal of DCpep-GFP was only detected within dendritic cells. PTD-GFP was only detected within kidney or pancreatic cells but not in immune modulatory cells (macrophages, dendritic, T, B, or mast cells). In contrast, CTB-GFP was detected in all tested cell types, confirming ubiquitous presence of GM1 receptors. Such low-cost oral delivery of protein drugs to sera, immune system or non-immune cells should dramatically lower their cost by elimination of prohibitively expensive fermentation, protein purification cold storage/transportation and increase patient compliance.

  1. Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells

    PubMed Central

    Xiao, Yuhong; Kwon, Kwang-Chul; Hoffman, Brad E.; Kamesh, Aditya; Jones, Noah T.; Herzog, Roland W.; Daniell, Henry

    2016-01-01

    Targeted oral delivery of GFP fused with a GM1 receptor binding protein (CTB) or human cell penetrating peptide (PTD) or dendritic cell peptide (DCpep) was investigated. Presence of GFP+ intact plant cells between villi of ileum confirm their protection in the digestive system from acids/enzymes. Efficient delivery of GFP to gut-epithelial cells by PTD or CTB and to M cells by all these fusion tags confirm uptake of GFP in the small intestine. PTD fusion delivered GFP more efficiently to most tissues or organs than other two tags. GFP was efficiently delivered to the liver by all fusion tags, likely through the gut-liver axis. In confocal imaging studies of human cell lines using purified GFP fused with different tags, GFP signal of DCpep-GFP was only detected within dendritic cells. PTD-GFP was only detected within kidney or pancreatic cells but not in immune modulatory cells (macrophages, dendritic, T, B, or mast cells). In contrast, CTB-GFP was detected in all tested cell types, confirming ubiquitous presence of GM1 receptors. Such low-cost oral delivery of protein drugs to sera, immune system or non-immune cells should dramatically lower their cost by elimination of prohibitively expensive fermentation, protein purification cold storage/transportation and increase patient compliance. PMID:26706477

  2. Development of novel protein-Ag nanocomposite for drug delivery and inactivation of bacterial applications.

    PubMed

    Vimala, Kanikireddy; Varaprasad, Kokkarachedu; Sadiku, Rotimi; Ramam, Koduri; Kanny, Krishnan

    2014-02-01

    The potential applications, in the biomedical fields, of curcumin loaded silver nanocomposite were studied by using bovine serum albumin (protein) and acrylamide. The design and development of silver nanoparticles with small size and adequate stability are very important, in addition to their applicability, particularly in bio-medicine. In this study, silver nanoparticles were prepared by chemical reduction method, employing sodium borohydride as the reducing agent for silver nanoparticles. The properties of the protein hydrogels formed were characterized via Fourier transform infrared spectroscopy and X-ray diffraction analyses. The size and its distribution, and formation of metal nanoparticles were confirmed by transmission electron microscopy indicating the diameter of the silver nanoparticles in the range of 3-8 nm. The thermal study of curcumin-silver nanocomposite hydrogels was determined by thermo-gravimetric analysis. In order to increase the antibacterial activity of theses inorganic nanomaterials, natural biological curcumin was incorporated into the protein hydrogel. The main emphasis in this investigation is to increase the antibacterial activity of the hydrogels by loading curcumin, for advanced medical application and as a model drug.

  3. Synthesis and physicochemical characterization of a novel amphiphilic polylactic acid-hyperbranched polyglycerol conjugate for protein delivery.

    PubMed

    Gao, Xiujun; Zhang, Xinge; Wu, Zhongming; Zhang, Xuejiao; Wang, Zhen; Li, Chaoxing

    2009-12-03

    Amphiphilic copolymers with polylactic acid (PLA) chains grafted onto hyperbranched polyglycerol (HPG) have been synthesized and characterized. The copolymer nanoparticles with corona and core structure were formed by self-assembly in aqueous solution. The loading capacity and association efficiency were up to 23% and 86%, respectively. Protein release profiles with different copolymer compositions and BSA concentrations all showed a burst effect followed by a continuous release phase. The released BSA from the copolymer nanoparticles remained in its original structure over a period of 4 days, as testified by circular dichroism spectroscopy. Furthermore, cell viability research suggested good biocompatibility of the copolymer nanoparticles, which have a promising potential for protein delivery system.

  4. Design of cytotoxic ribonucleases by cationization to enhance intracellular protein delivery.

    PubMed

    Futami, Junichiro; Yamada, Hidenori

    2008-06-01

    The cytotoxic properties of naturally occurring or engineered RNases correlate well with their efficiency of cellular internalization and digestion level of cellular RNA. Cationized RNases are considered to adsorb to the anionic cellular surface by Coulombic interactions, and then become efficiently internalized into cells by an endocytosis-like pathway. The design of cytotoxic RNases by chemical modification of surface carboxylic residues is one of the powerful strategies for enhancing cellular internalization and is accompanied with a decreased sensitivity for the cytoplasmic RNase inhibitor. Although chemically modified cationized RNases showed decreased ribonucleolytic activity, improved endocytosis and decreased affinity to the endogenous RNase inhibitor conclusively contribute to their ability to digest cellular RNA. Furthermore, the cytotoxicity of cationized RNases can be drastically enhanced by co-endocytosis with an endosome-destabilizing peptide. Since efficient cellular internalization of proteins into living cells is an important technology for biotechnology, studies concerning the design of cytotoxic RNases provided general perceptions for protein-based drug design.

  5. Production of recombinant human growth hormone conjugated with a transcytotic peptide in Pichia pastoris for effective oral protein delivery.

    PubMed

    Lee, Jun-Yeong; Kang, Sang-Kee; Li, Hui-Shan; Choi, Chang-Yun; Park, Tae-Eun; Bok, Jin-Duck; Lee, Seung-Ho; Cho, Chong-Su; Choi, Yun-Jaie

    2015-05-01

    Among the possible delivery routes, the oral administration of a protein is simple and achieves high patient compliance without pain. However, the low bioavailability of a protein drug in the intestine due to the physical barriers of the intestinal epithelia is the most critical problem that needs to be solved. To overcome the low bioavailability of a protein drug in the intestine, we aimed to construct a recombinant Pichia pastoris expressing a human growth hormone (hGH) fusion protein conjugated with a transcytotic peptide (TP) that was screened through peroral phage display to target goblet cells in the intestinal epithelia. The TP-conjugated hGH was successfully produced in P. pastoris in a secreted form at concentrations of up to 0.79 g/l. The function of the TP-conjugated hGH was validated by in vitro and in vivo assays. The transcytotic function of the TP through the intestinal epithelia was verified only in the C terminus conjugated hGH, which demonstrated the induction of IGF-1 in a HepG2 cell culture assay, a higher translocation of recombinant hGH into the ileal villi after oral administration in rats and both IGF-1 induction and higher body weight gain in rats after oral administration. The present study introduces the possibility for the development of an effective oral protein delivery system in the pharmaceutical and animal industries through the introduction of an effective TP into hGH.

  6. Comparison of Metalloproteinase Protein and Activity Profiling

    PubMed Central

    Giricz, Orsi; Lauer, Janelle L.; Fields, Gregg B.

    2010-01-01

    Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The present study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer and one normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media, both in amount and in variety. TIMP-1 was produced in all cell lines. MMP activity assays with three different FRET substrates were then utilized to determine if protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was only confirmed in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays. PMID:20920458

  7. Effect of penetration modifiers on the dermal and transdermal delivery of drugs and cosmetic active ingredients.

    PubMed

    Otto, A; Wiechers, J W; Kelly, C L; Hadgraft, J; du Plessis, J

    2008-01-01

    In this study the effect of 2 penetration modifiers, dimethyl isosorbide (DMI) and diethylene glycol monoethyl ether (DGME) on the skin delivery of hydroquinone (HQ), salicylic acid (SA) and octadecenedioic acid (DIOIC) was investigated. Ten percent DMI and DGME were separately formulated into oil-in-water emulsions containing 1.8% HQ, SA and DIOIC, respectively. Skin delivery and the flux across split-thickness human skin of the active ingredients were determined using Franz diffusion cells. An emulsion with 10% water incorporated instead of the water-soluble penetration modifiers served as a control. The study showed that neither 10% DMI nor 10% DGME significantly enhanced the skin permeation of the various lipophilic active ingredients or the uptake into the skin. It was hypothesized that the addition of the penetration modifiers to the emulsions not only enhanced the solubility of the various active ingredients in the skin but also in the formulation, resulting in a reduced thermodynamic activity and hence a weaker driving force for penetration. Therefore, the effect of DMI and DGME on the solubility of the active ingredients in the skin was counteracted by a simultaneous reduction in the thermodynamic activity in the formulation.

  8. Assembly of HE800 exopolysaccharide produced by a deep-sea hydrothermal bacterium into microgels for protein delivery applications.

    PubMed

    Zykwinska, Agata; Marquis, Mélanie; Sinquin, Corinne; Cuenot, Stéphane; Colliec-Jouault, Sylvia

    2016-05-20

    Assembly of biopolymers into microgels is an elegant strategy for bioencapsulation with various potential biomedical applications. Such biocompatible and biodegradable microassemblies are developed not only to protect the encapsulated molecule but also to ensure its sustained local delivery. The present study describes the fabrication of microassemblies from a marine HE800 exopolysaccharide (EPS), which displays a glycosaminoglycan (GAG)-like structure and biological properties. HE800 EPS was assembled, through physical cross-linking with divalent ions, into microgel particles and microfibers using microfluidics. The microparticle morphology was highly affected by the polysaccharide concentration and its molecular weight. A model protein, namely Bovine Serum Albumin (BSA) was subsequently encapsulated within HE800 microparticles in one-step process using microfluidics. The protein release was tuned by the microparticle morphology with a lower protein amount released from the most homogeneous structures. Our findings demonstrate the high potential of HE800 EPS based microassemblies as innovative protein microcarriers for further biomedical applications.

  9. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  10. GNeosomes: Highly Lysosomotropic Nanoassemblies for Lysosomal Delivery.

    PubMed

    Wexselblatt, Ezequiel; Esko, Jeffrey D; Tor, Yitzhak

    2015-01-01

    GNeosomes, lysosomotropic lipid vesicles decorated with guanidinoneomycin, can encapsulate and facilitate the cellular internalization and lysosomal delivery of cargo ranging from small molecules to high molecular weight proteins, in a process that is exclusively dependent on cell surface glycosaminoglycans. Their cellular uptake mechanism and co-localization with lysosomes, as well as the delivery, release, and activity of internalized cargo, are quantified. GNeosomes are proposed as a universal platform for lysosomal delivery with potential as a basic research tool and a therapeutic vehicle.

  11. Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles.

    PubMed

    Lainšček, Duško; Lebar, Tina; Jerala, Roman

    2017-02-26

    Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators.

  12. Improved mucoadhesion and cell uptake of chitosan and chitosan oligosaccharide surface-modified polymer nanoparticles for mucosal delivery of proteins.

    PubMed

    Dyawanapelly, Sathish; Koli, Uday; Dharamdasani, Vimisha; Jain, Ratnesh; Dandekar, Prajakta

    2016-08-01

    The main aim of the present study was to compare mucoadhesion and cellular uptake efficiency of chitosan (CS) and chitosan oligosaccharide (COS) surface-modified polymer nanoparticles (NPs) for mucosal delivery of proteins. We have developed poly (D, L-lactide-co-glycolide) (PLGA) NPs, surface-modified COS-PLGA NPs and CS-PLGA NPs, by using double emulsion solvent evaporation method, for encapsulating bovine serum albumin (BSA) as a model protein. Surface modification of NPs was confirmed using physicochemical characterization methods such as particle size and zeta potential, SEM, TEM and FTIR analysis. Both surface-modified PLGA NPs displayed a slow release of protein compared to PLGA NPs. Furthermore, we have explored the mucoadhesive property of COS as a material for modifying the surface of polymeric NPs. During in vitro mucoadhesion test, positively charged COS-PLGA NPs and CS-PLGA NPs exhibited enhanced mucoadhesion, compared to negatively charged PLGA NPs. This interaction was anticipated to improve the cell interaction and uptake of NPs, which is an important requirement for mucosal delivery of proteins. All nanoformulations were found to be safe for cellular delivery when evaluated in A549 cells. Moreover, intracellular uptake behaviour of FITC-BSA loaded NPs was extensively investigated by confocal laser scanning microscopy and flow cytometry. As we hypothesized, positively charged COS-PLGA NPs and CS-PLGA NPs displayed enhanced intracellular uptake compared to negatively charged PLGA NPs. Our results demonstrated that CS- and COS-modified polymer NPs could be promising carriers for proteins, drugs and nucleic acids via nasal, oral, buccal, ocular and vaginal mucosal routes.

  13. pH/sugar dual responsive core-cross-linked PIC micelles for enhanced intracellular protein delivery.

    PubMed

    Ren, Jie; Zhang, Yanxin; Zhang, Ju; Gao, Hongjun; Liu, Gan; Ma, Rujiang; An, Yingli; Kong, Deling; Shi, Linqi

    2013-10-14

    Herein, a series of biocompatible, robust, pH/sugar-sensitive, core-cross-linked, polyion complex (PIC) micelles based on phenylboronic acid-catechol interaction were developed for protein intracellular delivery. The rationally designed poly(ethylene glycol)-b-poly(glutamic acid-co-glutamicamidophenylboronic acid) (PEG-b-P(Glu-co-GluPBA)) and poly(ethylene glycol)-b-poly(l-lysine-co-ε-3,4-dihydroxyphenylcarboxyl-L-lysine) (PEG-b-P(Lys-co-LysCA)) copolymers were successfully synthesized and self-assembled under neutral aqueous condition to form uniform micelles. These micelles possessed a distinct core-cross-linked core-shell structure comprised of the PEG outer shell and the PGlu/PLys polyion complex core bearing boronate ester cross-linking bonds. The cross-linked micelles displayed superior physiological stabilities compared with their non-cross-linked counterparts while swelling and disassembling in the presence of excess fructose or at endosomal pH. Notably, either negatively or positively charged proteins can be encapsulated into the micelles efficiently under mild conditions. The in vitro release studies showed that the release of protein cargoes under physiological conditions was minimized, while a burst release occurred in response to excess fructose or endosomal pH. The cytotoxicity of micelles was determined by cck-8 assay in HepG2 cells. The cytochrome C loaded micelles could efficiently delivery proteins into HepG2 cells and exhibited enhanced apoptosis ability. Hence, this type of core-cross-linked PIC micelles has opened a new avenue to intracellular protein delivery.

  14. Mussel inspired protein-mediated surface functionalization of electrospun nanofibers for pH-responsive drug delivery

    PubMed Central

    Jiang, Jiang; Xie, Jingwei; Ma, Bing; Bartlett, David E.; Xu, An; Wang, Chi-Hwa

    2014-01-01

    pH-responsive drug delivery systems could mediate drug releasing rate by changing pH values at specific time as per the pathophysiological need of the disease. Herein, we demonstrated a mussel inspired protein polydopamine coating can tune the loading and releasing rate of charged molecules from electrospun poly (ε-caprolactone) (PCL) nanofibers in solutions with different pH values. In vitro release profiles showed that the positive charged molecules released significantly faster in acidic than those in neutral and basic environments within the same incubation time. The results of fluorescein diacetate staining and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed the viability of cancer cells after treatment with doxorubicin released media at different pH values qualitatively and quantitatively, indicating the media contained doxorubicin which was released in solutions at low pH values could kill significantly higher number of cells than that released in solutions at high pH values. Together, the pH-responsive drug delivery systems based on polydopamine-coated PCL nanofibers could have potential applications in oral delivery of anticancer drugs for treating gastric cancer and vaginal delivery of anti-viral drugs or anti-inflammatory drugs, which could raise their efficacy, deliver them to the specific target, and minimize their toxic side effects. PMID:24287161

  15. Folic acid-conjugated amphiphilic alternating copolymer as a new active tumor targeting drug delivery platform

    PubMed Central

    Li, Xia; Szewczuk, Myron R; Malardier-Jugroot, Cecile

    2016-01-01

    Targeted drug delivery using polymeric nanostructures is an emerging cancer research area, engineered for safer, more efficient, and effective use of chemotherapeutic drugs. A pH-responsive, active targeting delivery system was designed using folic acid functionalized amphiphilic alternating copolymer poly(styrene-alt-maleic anhydride) (FA-DABA-SMA) via a biodegradable linker 2,4-diaminobutyric acid (DABA). The polymeric template is pH responsive, forming amphiphilic nanostructures at pH 7, allowing the encapsulation of hydrophobic drugs on its interior. Moreover, the structure is stable only at neutral pH and collapses in the acidic tumor microenvironment, releasing drugs on-site from its core. The delivery vehicle is investigated using human pancreatic PANC-1 cancer cells and RAW-Blue™ mouse macrophage reporter cell line, both of which have overly expression of folic acid receptors. To trace the cellular uptake by both cell lines, curcumin was selected as a dye and drug mimic owing to its fluorescence nature and hydrophobic properties. Fluorescent microscopy of FA-DABA-SMA loaded with curcumin revealed a significant internalization of the dye by human pancreatic PANC-1 cancer cells compared to those with unfunctionalized polymers (SMA). Moreover, the FA-DABA-SMA polymers exhibit rodlike association specific to the cells. Both empty SMA and FA-DABA-SMA show little toxicity to PANC-1 cells as characterized by WST-1 cell proliferation assay. These results clearly indicate that FA-DABA-SMA polymers show potential as an active tumor targeting drug delivery system with the ability to internalize hydrophobic chemotherapeutics after they specifically attach to cancer cells. PMID:28008233

  16. In vivo delivery of bovine viral diahorrea virus, E2 protein using hollow mesoporous silica nanoparticles

    NASA Astrophysics Data System (ADS)

    Mahony, D.; Cavallaro, A. S.; Mody, K. T.; Xiong, L.; Mahony, T. J.; Qiao, S. Z.; Mitter, N.

    2014-05-01

    Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 μg Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 μg dose of E2 adsorbed to 250 μg HMSA was compared to immunisation with Opti-E2 (50 μg) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 μg). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine.Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral

  17. Activation and activities of the p53 tumour suppressor protein

    PubMed Central

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pathways that regulate p53 and the pathways that are induced by p53, as well as their implications for cancer therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11747320

  18. Formation and stabilization of nanoemulsion-based vitamin E delivery systems using natural biopolymers: Whey protein isolate and gum arabic.

    PubMed

    Ozturk, Bengu; Argin, Sanem; Ozilgen, Mustafa; McClements, David Julian

    2015-12-01

    Natural biopolymers, whey protein isolate (WPI) and gum arabic (GA), were used to fabricate emulsion-based delivery systems for vitamin E-acetate. Stable delivery systems could be formed when vitamin E-acetate was mixed with sufficient orange oil prior to high pressure homogenization. WPI (d32=0.11 μm, 1% emulsifier) was better than GA (d32=0.38 μm, 10% emulsifier) at producing small droplets at low emulsifier concentrations. However, WPI-stabilized nanoemulsions were unstable to flocculation near the protein isoelectric point (pH 5.0), at high ionic strength (>100mM), and at elevated temperatures (>60 °C), whereas GA-stabilized emulsions were stable. This difference was attributed to differences in emulsifier stabilization mechanisms: WPI by electrostatic repulsion; GA by steric repulsion. These results provide useful information about the emulsifying and stabilizing capacities of natural biopolymers for forming food-grade vitamin-enriched delivery systems.

  19. Maurer's clefts of Plasmodium falciparum are secretory organelles that concentrate virulence protein reporters for delivery to the host erythrocyte

    PubMed Central

    Bhattacharjee, Souvik; van Ooij, Christiaan; Balu, Bharath; Adams, John H.

    2008-01-01

    In blood-stage infection by the human malaria parasite Plasmodium falciparum, export of proteins from the intracellular parasite to the erythrocyte is key to virulence. This export is mediated by a host-targeting (HT) signal present on a “secretome” of hundreds of parasite proteins engaged in remodeling the erythrocyte. However, the route of HT-mediated export is poorly understood. Here we show that minimal soluble and membrane protein reporters that contain the HT motif and mimic export of endogenous P falciparum proteins are detected in the lumen of “cleft” structures synthesized by the pathogen. Clefts are efficiently targeted by the HT signal. Furthermore, the HT signal does not directly translocate across the parasitophorous vacuolar membrane (PVM) surrounding the parasite to deliver protein to the erythrocyte cytoplasm, as suggested by current models of parasite protein trafficking to the erythrocyte. Rather, it is a lumenal signal that sorts protein into clefts, which then are exported beyond the PVM. These data suggest that Maurer's clefts, which are unique to the virulent P falciparum species, are pathogen-induced secretory organelles that concentrate HT-containing soluble and membrane parasite proteins in their lumen for delivery to the host erythrocyte. PMID:18057226

  20. Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins

    PubMed Central

    Jia, Jingyue; Bai, Fang; Jin, Yongxin; Santostefano, Katherine E.; Ha, Un-Hwan; Wu, Donghai

    2015-01-01

    The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies. Significance The present study describes a novel and powerful tool for the delivery of the genome editing enzyme transcription activator-like effector nuclease (TALEN) directly into pluripotent stem cells (PSCs), achieving desired base changes on the genomes of PSCs with high efficiency. This novel approach uses bacteria as a protein delivery

  1. Interaction of Protein Inhibitor of Activated STAT (PIAS) Proteins with the TATA-binding Protein, TBP*

    PubMed Central

    Prigge, Justin R.; Schmidt, Edward E.

    2007-01-01

    Transcription activators often recruit promoter-targeted assembly of a pre-initiation complex; many repressors antagonize recruitment. These activities can involve direct interactions with proteins in the pre-initiation complex. We used an optimized yeast two-hybrid system to screen mouse pregnancy-associated libraries for proteins that interact with TATA-binding protein (TBP). Screens revealed an interaction between TBP and a single member of the zinc finger family of transcription factors, ZFP523. Two members of the protein inhibitor of activated STAT (PIAS) family, PIAS1 and PIAS3, also interacted with TBP in screens. Endogenous PIAS1 and TBP co-immunoprecipitated from nuclear extracts, suggesting the interaction occurred in vivo. In vitro-translated PIAS1 and TBP coimmunopreciptated, which indicated that other nuclear proteins were not required for the interaction. Deletion analysis mapped the PIAS-interacting domain of TBP to the conserved TBPCORE and the TBP-interacting domain on PIAS1 to a 39-amino acid C-terminal region. Mammals issue seven known PIAS proteins from four pias genes, pias1, pias3, piasx, and piasy, each with different cell type-specific expression patterns; the TBP-interacting domain reported here is the only part of the PIAS C-terminal region shared by all seven PIAS proteins. Direct analyses indicated that PIASx and PIASy also interacted with TBP. Our results suggest that all PIAS proteins might mediate situation-specific regulatory signaling at the TBP interface and that previously unknown levels of complexity could exist in the gene regulatory interplay between TBP, PIAS proteins, ZFP523, and other transcription factors. PMID:16522640

  2. Three-dimensionally presented anti-fouling zwitterionic motifs sequester and enable high-efficiency delivery of therapeutic proteins

    PubMed Central

    Liu, Pingsheng; Skelly, Jordan D.; Song, Jie

    2014-01-01

    Zwitterions are well known for their anti-biofouling properties. Past investigations of zwitterionic materials for biomedical uses have been centered on exploiting their ability to inhibit non-specific adsorption of proteins. Here, we report that zwitterionic motifs, when 3-dimensionally (3D) presented (e.g. in crosslinked hydrogels), could effectively sequester osteogenic bone morphogenetic protein-2 (rhBMP-2). The ionic interactions between rhBMP-2 and the 3D zwitterionic network enabled dynamic sequestering and sustained release of the protein with preserved bioactivity. We further demonstrated that the zwitterionic hydrogel confers high-efficiency in vivo local delivery of rhBMP-2. It can template the functional healing of critical-size femoral segmental defects in rats with rhBMP-2 at a loading dose substantially lower than those required for current natural or synthetic polymeric carriers. These findings reveal a novel function of zwitterionic materials beyond their commonly perceived anti-biofouling property, and may establish 3D zwitterionic matrices as novel high-efficiency vehicles for protein/ionic drug therapeutic delivery. PMID:24956565

  3. Three-dimensionally presented anti-fouling zwitterionic motifs sequester and enable high-efficiency delivery of therapeutic proteins.

    PubMed

    Liu, Pingsheng; Skelly, Jordan D; Song, Jie

    2014-10-01

    Zwitterions are well known for their anti-biofouling properties. Past investigations of zwitterionic materials for biomedical uses have been centered on exploiting their ability to inhibit non-specific adsorption of proteins. Here, we report that zwitterionic motifs, when presented in three dimensions (e.g. in crosslinked hydrogels), could effectively sequester osteogenic bone morphogenetic protein-2 (rhBMP-2). The ionic interactions between rhBMP-2 and the 3-D zwitterionic network enabled dynamic sequestering and sustained release of the protein with preserved bioactivity. We further demonstrated that the zwitterionic hydrogel confers high-efficiency in vivo local delivery of rhBMP-2. It can template the functional healing of critical-size femoral segmental defects in rats with rhBMP-2 at a loading dose substantially lower than those required for current natural or synthetic polymeric carriers. These findings reveal a novel function of zwitterionic materials beyond their commonly perceived anti-biofouling property, and may establish 3-D zwitterionic matrices as novel high-efficiency vehicles for protein/ionic drug therapeutic delivery.

  4. Bioactive polysaccharide-based pH-sensitive polymers for cytoplasmic delivery of antigen and activation of antigen-specific immunity.

    PubMed

    Yuba, Eiji; Yamaguchi, Ayaka; Yoshizaki, Yuta; Harada, Atsushi; Kono, Kenji

    2017-03-01

    For establishment of cancer immunotherapy, antigen carriers are needed which have functions not only to deliver antigen into cytosol of dendritic cells (DCs), which induces antigen-specific cellular immune responses, but also to activate DCs. We previously reported cytoplasmic delivery of antigen using liposomes modified with pH-sensitive polymers such as carboxylated poly(glycidol)s or dextran. Modification using these polymers provides stable liposomes with pH-sensitive fusogenic/membrane-disruptive ability. For this study, bioactive polysaccharide-based pH-sensitive polymers were constructed to achieve not only cytoplasmic delivery of antigen but also activation of DCs. Curdlan and mannan were used as bioactive polysaccharides because they are known to activate DCs via their respective interactions with Dectin-1 and Dectin-2. Carboxylated curdlan and mannan promoted Th1 cytokine production from DCs, indicating the activation of DCs by these polysaccharide derivatives. These polymer-modified liposomes released their contents at weakly acidic pH and delivered model antigenic proteins into cytosol of DCs. Subcutaneous administration of curdlan derivative-modified or mannan derivative-modified liposomes induced strong antigen-specific immune responses and stronger antitumor effects than those of liposomes modified with dextran derivative. Therefore, bioactive polysaccharide-modified liposomes that achieve both cytoplasmic delivery of antigen and activation of DCs are promising for cancer immunotherapy.

  5. Biodistribution and Efficacy of Targeted Pulmonary Delivery of a Protein Kinase C-δ Inhibitory Peptide: Impact on Indirect Lung Injury

    PubMed Central

    Mondrinos, Mark J.; Knight, Linda C.; Kennedy, Paul A.; Wu, Jichuan; Kauffman, Matthew; Baker, Sandy T.; Wolfson, Marla R.

    2015-01-01

    Sepsis and sepsis-induced lung injury remain a leading cause of death in intensive care units. We identified protein kinase C-δ (PKCδ) as a critical regulator of the acute inflammatory response and demonstrated that PKCδ inhibition was lung-protective in a rodent sepsis model, suggesting that targeting PKCδ is a potential strategy for preserving pulmonary function in the setting of indirect lung injury. In this study, whole-body organ biodistribution and pulmonary cellular distribution of a transactivator of transcription (TAT)–conjugated PKCδ inhibitory peptide (PKCδ-TAT) was determined following intratracheal (IT) delivery in control and septic [cecal ligation and puncture (CLP)] rats to ascertain the impact of disease pathology on biodistribution and efficacy. There was negligible lung uptake of radiolabeled peptide upon intravenous delivery [<1% initial dose (ID)], whereas IT administration resulted in lung retention of >65% ID with minimal uptake in liver or kidney (<2% ID). IT delivery of a fluorescent-tagged (tetramethylrhodamine-PKCδ-TAT) peptide demonstrated uniform spatial distribution and cellular uptake throughout the peripheral lung. IT delivery of PKCδ-TAT at the time of CLP surgery significantly reduced PKCδ activation (tyrosine phosphorylation, nuclear translocation and cleavage) and acute lung inflammation, resulting in improved lung function and gas exchange. Importantly, peptide efficacy was similar when delivered at 4 hours post-CLP, demonstrating therapeutic relevance. Conversely, spatial lung distribution and efficacy were significantly impaired at 8 hours post-CLP, which corresponded to marked histopathological progression of lung injury. These studies establish a functional connection between peptide spatial distribution, inflammatory histopathology in the lung, and efficacy of this anti-inflammatory peptide. PMID:26243739

  6. Activated air produced by shielded sliding discharge plasma mediates plasmid DNA delivery to mammalian cells.

    PubMed

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Heller, Richard

    2015-12-01

    Cold plasma is emerging as a potential method for medical applications. The current study assessed the efficacy of a novel cold plasma reactor based on shielded sliding discharge producing cathode-directed streamers generated in ambient air for the delivery of plasmid DNA. Experiments were performed with mouse melanoma cells (B16F10) and human keratinocyte cells (HaCaT) inoculated with plasmid DNA encoding luciferase. Quantitative results measured over a 72-h period displayed luciferase expression levels as high as 5-fold greater in cells exposed to plasma-activated air (PAA) than levels obtained from the inoculation of plasmid DNA alone (P < 0.05, P < 0.01). No effect on cell viability was observed. Delivery of plasmid encoding GFP to HaCaT cells seeded on polycaprolactone (PCL) scaffolds was confirmed by immunostaining. The use of cold plasma for DNA delivery is attractive as it provides a non-viral, non-invasive method where the electrode or the plasma itself never directly contacts the exposed site. The current device design provides localized DNA transfer using a novel technology. Our report suggests PAA warrants further exploration as an alternative or supplemental approach for DNA transfer.

  7. Cationic Polymethacrylate-Modified Liposomes Significantly Enhanced Doxorubicin Delivery and Antitumor Activity

    PubMed Central

    Wang, Wenxi; Shao, Anna; Zhang, Nan; Fang, Jinzhang; Ruan, Jennifer Jin; Ruan, Benfang Helen

    2017-01-01

    Liposome (LP) encapsulation of doxorubicin (DOX) is a clinically validated method for cancer drug delivery, but its cellular uptake is actually lower than the free DOX. Therefore, we modified DOX-LP with a cationic polymer (Eudragit RL100; ER) to improve its cellular uptake and antitumor activity. The resulting DOX-ERLP was a 190 nm nanoparticle that was absorbed efficiently and caused cancer cell death in 5 hrs. Growth as measured by the MTT assay or microscopic imaging demonstrated that DOX-ERLP has at least a two-fold greater potency than the free DOX in inhibiting the growth of a DOX resistant (MCF7/adr) cell and an aggressive liver cancer H22 cell. Further, its in vivo efficacy was tested in H22-bearing mice, where four injections of DOX-ERLP reduced the tumor growth by more than 60% and caused an average of 60% tumor necrosis, which was significantly better than the DOX and DOX-LP treated groups. Our work represents the first use of polymethacrylate derivatives for DOX liposomal delivery, demonstrating the great potential of cationic polymethacrylate modified liposomes for improving cancer drug delivery. PMID:28225062

  8. Alginate-whey protein dry powder optimized for target delivery of essential oils to the intestine of chickens.

    PubMed

    Zhang, Y; Gong, J; Yu, H; Guo, Q; Defelice, C; Hernandez, M; Yin, Y; Wang, Q

    2014-10-01

    In poultry production, there is a lack of effective and convenient approaches to deliver bioactive compounds such as some essential oils, which have been proposed as alternatives to antibiotic growth promoters. The objective of this research was to develop a method for target delivery of essential oils in feed to the lower intestines of chickens. Carvacrol was used as a model essential oil, and 2 food-grade biopolymers, alginate and whey protein, were selected to encapsulate carvacrol in microparticles. The effects of a medium molecular weight alginate, a low molecular weight alginate (LBA), and whey protein concentrations on the properties of carvacrol-loaded microparticles were investigated using response surface methodology. The encapsulation efficiencies for all the tested formulations were ≥ 98% and carvacrol content in the dry microparticles was 72 ± 2% (wt/wt). The microparticles showed good gastric resistance and rapid intestinal release under simulated gastrointestinal conditions. Alginate concentrations had the strongest influence on the gastric resistance of microparticles, whereas whey protein was the dominant parameter in controlling the intestinal release. The concentration of LBA was found to be the critical factor affecting the mechanical strength of the microparticles. A predicted optimum formulation from in vitro optimization was tested in chickens. It was found that a negligible amount of carvacrol was detected in the intestines of chickens fed with unencapsulated carvacrol. Microparticles of predicted optimum formulation delivered a remarkably higher concentration of carvacrol to the jejunum and ileum regions. The high concentration was sustained for more than 3 h after oral administration. The in vivo release of carvacrol from the microparticles appeared faster than release from in vitro simulation. Nonetheless, the in vitro simulation provided good indications of the in vivo performance, and thus may serve as a useful tool for formula

  9. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  10. Tuning the activity of mitochondria-penetrating peptides for delivery or disruption.

    PubMed

    Horton, Kristin L; Pereira, Mark P; Stewart, Kelly M; Fonseca, Sonali B; Kelley, Shana O

    2012-02-13

    Mitochondrially targeted agents have the capacity to be both vehicles for the delivery of bioactive agents and mitochondrial disrupters and show promise for the treatment of various diseases. Engineering these agents to specifically accumulate or disrupt the mitochondrion is challenging, as there is a fine line between characteristics of the molecules that accomplish each task. Here, we assess the physicochemical properties governing mitochondrial matrix accumulation or membrane disruption caused by mitochondria-penetrating peptides. Increases in peptide length and hydrophobicity were uncovered as the dominant factors in deriving membrane disruptive activity. Shorter, less hydrophobic peptides did not disrupt the mitochondrial membrane, but rather accumulated in the mitochondrial matrix without interfering with cellular activity. These shorter peptides, however, can trigger cytochrome c release through activation of the permeability transition pore complex (PTPC), but only at very high concentrations. This study illustrates that the activity of a mitochondria-localizing agent can be controlled through alterations in peptide hydrophobicity and dosing concentrations.

  11. Heat dissipation guides activation in signaling proteins

    PubMed Central

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein–coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  12. Thermogelling Biodegradable Copolymer Aqueous Solutions for Injectable Protein Delivery and Tissue Engineering

    SciTech Connect

    Jeong, Byeongmoon; Lee, Kyeonghee M.; Gutowska, Anna; An, Yuehuei H.

    2002-07-01

    This paper reports on the thermogelling, biodegradable polymer formulations based on poly(DL-lactic acid-co-glycolic acid)(polyethylene glycol) graft copolymers for in-vivo biomedical applications. The description includes diabetic control by sustained insulin delivery and cartilage repair by chondrocyte cell delivery. With one injection of the formula, the blood glucose level could be controlled from 5 to 16 days in diabetic rats by varying the polymer composition. Promising cartilage repair was observed using chondrocyte suspension in the thermogelling biodegradable depot.

  13. Activated Protein C action in inflammation

    PubMed Central

    Sarangi, Pranita P.; Lee, Hyun-wook; Kim, Minsoo

    2010-01-01

    Summary Activated protein C (APC) is a natural anticoagulant that plays an important role in coagulation homeostasis by inactivating the procoagulation factor Va and VIIIa. In addition to its anticoagulation functions, APC also has cytoprotective effects such as anti-inflammatory, anti-apoptotic, and endothelial barrier protection. Recently, a recombinant form of human APC (rhAPC or drotrecogin alfa activated; known commercially as “Xigris”) was approved by the US Federal Drug Administration for treatment of severe sepsis associated with a high risk of mortality. Sepsis, also known as systemic inflammatory response syndrome (SIRS) resulting from infection, is a serious medical condition in critical care patients. In sepsis, hyperactive and dysregulated inflammatory responses lead to secretion of pro- and anti-inflammatory cytokines, activation and migration of leucocytes, activation of coagulation, inhibition of fibrinolysis, and increased apoptosis. Although initial hypotheses focused on antithrombotic and profibrinolytic functions of APC in sepsis, other agents with more potent anticoagulation functions were not effective in treating severe sepsis. Furthermore, APC therapy is also associated with the risk of severe bleeding in treated patients. Therefore, the cytoprotective effects, rather than the anticoagulant effect of APC are postulated to be responsible for the therapeutic benefit of APC in the treatment of severe sepsis. PMID:19995397

  14. Delivery systems and adjuvants for oral vaccines.

    PubMed

    Lavelle, Ed C; O'Hagan, D T

    2006-11-01

    The oral route is the ideal means of delivering prophylactic and therapeutic vaccines, offering significant advantages over systemic delivery. Most notably, oral delivery is associated with simple administration and improved safety. In addition, unlike systemic immunisation, oral delivery can induce mucosal immune responses. However, the oral route of vaccine delivery is the most difficult because of the numerous barriers posed by the gastrointestinal tract. To facilitate effective immunisation with peptide and protein vaccines, antigens must be protected, uptake enhanced and the innate immune response activated. Numerous delivery systems and adjuvants have been evaluated for oral vaccine delivery, including live vectors, inert particles and bacterial toxins. Although developments in oral vaccines have been disappointing so far, in terms of the generation of products, the availability of a range of novel delivery systems offers much greater hope for the future development of improved oral vaccines.

  15. Enzymatic crosslinking and degradation of gelatin as a switch for bone morphogenetic protein-2 activity.

    PubMed

    Kuwahara, Kenrick; Fang, Josephine Y; Yang, Zhi; Han, Bo

    2011-12-01

    Current therapies for tissue regeneration rely on the presence or direct delivery of growth factors to sites of repair. Bone morphogenetic protein-2 (BMP-2), combined with a carrier (usually collagen), is clinically proven to induce new bone formation during spinal fusion and nonunion repair. However, due to BMP-2's short half-life and its diffusive properties, orders of magnitude above physiological levels are required to ensure effectiveness. In addition, a high dose of this multifunctional growth factor is known to induce adverse effects in patients. To circumvent these challenges, we proposed and tested a new approach for BMP-2 delivery, by controlling BMP activity via carrier binding and localized proteolysis. BMP-2 was covalently bound to gelatin through site-specific enzymatic crosslinking using a microbial transglutaminase. Binding of BMP-2 to gelatin can completely switch off BMP-2 activity, as evidenced by loss of its transdifferentiating ability toward C2C12 promyoblasts. When gelatin sequestered BMP-2 is incubated with either microbial collagenase or tissue-derived matrix metalloproteinases, BMP-2 activity is fully restored. The activity of released BMP-2 correlates with the protease activity in a dose- and time-dependent manner. This observation suggests a novel way of delivering BMP-2 and controlling its activity. This improved delivery method, which relies on a physiological feedback, should enhance the known potential of this and other growth factors for tissue repair and regeneration.

  16. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    PubMed

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  17. Temporal Changes of Protein Composition in Breast Milk of Chinese Urban Mothers and Impact of Caesarean Section Delivery.

    PubMed

    Affolter, Michael; Garcia-Rodenas, Clara L; Vinyes-Pares, Gerard; Jenni, Rosemarie; Roggero, Iris; Avanti-Nigro, Ornella; de Castro, Carlos Antonio; Zhao, Ai; Zhang, Yumei; Wang, Peiyu; Thakkar, Sagar K; Favre, Laurent

    2016-08-17

    Human breast milk (BM) protein composition may be impacted by lactation stage or factors related to geographical location. The present study aimed at assessing the temporal changes of BM major proteins over lactation stages and the impact of mode of delivery on immune factors, in a large cohort of urban mothers in China. 450 BM samples, collected in three Chinese cities, covering 8 months of lactation were analyzed for α-lactalbumin, lactoferrin, serum albumin, total caseins, immunoglobulins (IgA, IgM and IgG) and transforming growth factor (TGF) β1 and β2 content by microfluidic chip- or ELISA-based quantitative methods. Concentrations and changes over lactation were aligned with previous reports. α-lactalbumin, lactoferrin, IgA, IgM and TGF-β1 contents followed similar variations characterized by highest concentrations in early lactation that rapidly decreased before remaining stable up to end of lactation. TGF-β2 content displayed same early dynamics before increasing again. Total caseins followed a different pattern, showing initial increase before decreasing back to starting values. Serum albumin and IgG levels appeared stable throughout lactation. In conclusion, BM content in major proteins of urban mothers in China was comparable with previous studies carried out in other parts of the world and C-section delivery had only very limited impact on BM immune factors.

  18. Temporal Changes of Protein Composition in Breast Milk of Chinese Urban Mothers and Impact of Caesarean Section Delivery

    PubMed Central

    Affolter, Michael; Garcia-Rodenas, Clara L.; Vinyes-Pares, Gerard; Jenni, Rosemarie; Roggero, Iris; Avanti-Nigro, Ornella; de Castro, Carlos Antonio; Zhao, Ai; Zhang, Yumei; Wang, Peiyu; Thakkar, Sagar K.; Favre, Laurent

    2016-01-01

    Human breast milk (BM) protein composition may be impacted by lactation stage or factors related to geographical location. The present study aimed at assessing the temporal changes of BM major proteins over lactation stages and the impact of mode of delivery on immune factors, in a large cohort of urban mothers in China. 450 BM samples, collected in three Chinese cities, covering 8 months of lactation were analyzed for α-lactalbumin, lactoferrin, serum albumin, total caseins, immunoglobulins (IgA, IgM and IgG) and transforming growth factor (TGF) β1 and β2 content by microfluidic chip- or ELISA-based quantitative methods. Concentrations and changes over lactation were aligned with previous reports. α-lactalbumin, lactoferrin, IgA, IgM and TGF-β1 contents followed similar variations characterized by highest concentrations in early lactation that rapidly decreased before remaining stable up to end of lactation. TGF-β2 content displayed same early dynamics before increasing again. Total caseins followed a different pattern, showing initial increase before decreasing back to starting values. Serum albumin and IgG levels appeared stable throughout lactation. In conclusion, BM content in major proteins of urban mothers in China was comparable with previous studies carried out in other parts of the world and C-section delivery had only very limited impact on BM immune factors. PMID:27548208

  19. PLGA based drug delivery systems: Promising carriers for wound healing activity.

    PubMed

    Chereddy, Kiran Kumar; Vandermeulen, Gaëlle; Préat, Véronique

    2016-03-01

    Wound treatment remains one of the most prevalent and economically burdensome healthcare issues in the world. Current treatment options are limited and require repeated administrations which led to the development of new therapeutics to satisfy the unmet clinical needs. Many potent wound healing agents were discovered but most of them are fragile and/or sensitive to in vivo conditions. Poly(lactic-co-glycolic acid) (PLGA) is a widely used biodegradable polymer approved by food and drug administration and European medicines agency as an excipient for parenteral administrations. It is a well-established drug delivery system in various medical applications. The aim of the current review is to elaborate the applications of PLGA based drug delivery systems carrying different wound healing agents and also present PLGA itself as a wound healing promoter. PLGA carriers encapsulating drugs such as antibiotics, anti-inflammatory drugs, proteins/peptides, and nucleic acids targeting various phases/signaling cycles of wound healing, are discussed with examples. The combined therapeutic effects of PLGA and a loaded drug on wound healing are also mentioned.

  20. Recent insights into the biological activities and drug delivery systems of tanshinones

    PubMed Central

    Cai, Yuee; Zhang, Wenji; Chen, Zirong; Shi, Zhi; He, Chengwei; Chen, Meiwan

    2016-01-01

    Tanshinones, the major lipid-soluble pharmacological constituents of the Chinese medicinal herb Tanshen (Salvia miltiorrhiza), have attracted growing scientific attention because of the prospective biomedical applications of these compounds. Numerous pharmacological activities, including anti-inflammatory, anticancer, and cardio-cerebrovascular protection activities, are exhibited by the three primary bioactive constituents among the tanshinones, ie, tanshinone I (TNI), tanshinone IIA (TNIIA), and cryptotanshinone (CPT). However, due to their poor solubility and low dissolution rate, the clinical applications of TNI, TNIIA, and CPT are limited. To solve these problems, many studies have focused on loading tanshinones into liposomes, nanoparticles, microemulsions, cyclodextrin inclusions, solid dispersions, and so on. In this review, we aim to offer an updated summary of the biological activities and drug delivery systems of tanshinones to provide a reference for these constituents in clinical applications. PMID:26792989

  1. Delivery of Molecules into Human Corneal Endothelial Cells by Carbon Nanoparticles Activated by Femtosecond Laser.

    PubMed

    Jumelle, Clotilde; Mauclair, Cyril; Houzet, Julien; Bernard, Aurélien; He, Zhiguo; Forest, Fabien; Peoc'h, Michel; Acquart, Sophie; Gain, Philippe; Thuret, Gilles

    2015-01-01

    Corneal endothelial cells (CECs) form a monolayer at the innermost face of the cornea and are the engine of corneal transparency. Nevertheless, they are a vulnerable population incapable of regeneration in humans, and their diseases are responsible for one third of corneal grafts performed worldwide. Donor corneas are stored in eye banks for security and quality controls, then delivered to surgeons. This period could allow specific interventions to modify the characteristics of CECs in order to increase their proliferative capacity, increase their resistance to apoptosis, or release immunosuppressive molecules. Delivery of molecules specifically into CECs during storage would therefore open up new therapeutic perspectives. For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient. The delivery of molecules into cells by carbon nanoparticles activated by femtosecond laser pulses is a promising recent technique developed on non-adherent cells. The nanoparticles are partly consummated by the reaction releasing CO and H2 gas bubbles responsible for the shockwave at the origin of cell transient permeation. Our aim was to develop an experimental setting to deliver a small molecule (calcein) into the monolayer of adherent CECs. We confirmed that increased laser fluence and time exposure increased uptake efficiency while keeping cell mortality below 5%. We optimized the area covered by the laser beam by using a motorized stage allowing homogeneous scanning of the cell culture surface using a spiral path. Calcein uptake reached median efficiency of 54.5% (range 50.3-57.3) of CECs with low mortality (0.5%, range (0.55-1.0)). After sorting by flow cytometry, CECs having uptaken calcein remained viable and presented normal morphological characteristics. Delivery of molecules into CECs by carbon nanoparticles activated by femtosecond laser could prove useful for future

  2. Hyaluronan microgel as a potential carrier for protein sustained delivery by tailoring the crosslink network.

    PubMed

    Luo, Chunhong; Zhao, Jianhao; Tu, Mei; Zeng, Rong; Rong, Jianhua

    2014-03-01

    Hyaluronan (HA) microgels with different crosslink network, i.e. HGPs-1, HGPs-1.5, HGPs-3, HGPs-6 and HGPs-15, were synthesized using divinyl sulfone (DVS) as the crosslinker in an inverse microemulsion system for controlling the sustained delivery of bovine serum albumin (BSA). With increasing the crosslinker content, the average particle size slightly increased from 1.9 ± 0.3 μm to 3.6 ± 0.5 μm by dynamic laser scattering analysis. However, the crosslinker content had no significant effect on the morphology of HA microgels by scanning and transmission electron microscopes. Fourier transform infrared spectroscopy and elemental analysis proved more sulfur participated in the crosslink reaction when raising the crosslinker amount. The water swelling test confirmed the increasing crosslink density with the crosslinker content by calculating the average molecular weight between two crosslink points to be 8.25 ± 2.51 × 10(5), 1.26 ± 0.43 × 10(5), 0.96 ± 0.09 × 10(5), 0.64 ± 0.03 × 10(5), and 0.11 ± 0.01 × 10(5) respectively. The degradation of HA microgels by hyaluronidase slowed down by enhancing the crosslink density, only about 5% of HGPs-15 was degraded as opposed to over 90% for HGPs-1. BSA loading had no obvious influence on the surface morphology of HA microgels but seemed to induce their aggregation. The increase of crosslink density decreased the BSA loading capacity but facilitated its long-term sustained delivery. When the molar ratio of DVS to repeating unit of HA reached 3 or higher, similar delivery profiles were obtained. Among all these HA microgels, HGPs-3 was the optimal carrier for BSA sustained delivery in this system because it possessed both high BSA loading capacity and long-term delivery profile simultaneously.

  3. Electrokinetic delivery of persulfate to remediate PCBs polluted soils: Effect of different activation methods.

    PubMed

    Fan, Guangping; Cang, Long; Gomes, Helena I; Zhou, Dongmei

    2016-02-01

    Persulfate-based in-situ chemical oxidation (ISCO) for the remediation of organic polluted soils has gained much interest in last decade. However, the transportation of persulfate in low-permeability soil is very low, which limits its efficiency in degrading soil pollutants. Additionally, the oxidation-reduction process of persulfate with organic contaminants takes place slowly, while, the reaction will be greatly accelerated by the production of more powerful radicals once it is activated. Electrokinetic remediation (EK) is a good way for transporting persulfate in low-permeability soil. In this study, different activation methods, using zero-valent iron, citric acid chelated Fe(2+), iron electrode, alkaline pH and peroxide, were evaluated to enhance the activity of persulfate delivered by EK. All the activators and the persulfate were added in the anolyte. The results indicated that zero-valent iron, alkaline, and peroxide enhanced the transportation of persulfate at the first stage of EK test, and the longest delivery distance reached sections S4 or S5 (near the cathode) on the 6th day. The addition of activators accelerated decomposition of persulfate, which resulted in the decreasing soil pH. The mass of persulfate delivered into the soil declined with the continuous decomposition of persulfate by activation. The removal efficiency of PCBs in soil followed the order of alkaline activation > peroxide activation > citric acid chelated Fe(2+) activation > zero-valent iron activation > without activation > iron electrode activation, and the values were 40.5%, 35.6%, 34.1%, 32.4%, 30.8% and 30.5%, respectively. The activation effect was highly dependent on the ratio of activator and persulfate.

  4. Design of an In Situ Cross-Linked Eutectic Tablet for Enhanced Delivery of Gastro-Sensitive Proteins and Peptides.

    PubMed

    Choonara, Bibi F; Choonara, Yahya E; Kumar, Pradeep; du Toit, Lisa C; Pillay, Viness

    2016-07-01

    In the present study, a eutectic platform was designed as an in situ cross-linked eutectic tablet for structural protection, enhanced intestinal permeation, and controlled release of proteins after oral administration. Physicochemical and physicomechanical analyses of the eutectic tablets were undertaken to elucidate the in situ cross-linking mechanism, thermal transitions, crystallinity, ex vivo permeation, and in vitro release of the protein. Following thermal characterization, results revealed successful eutectic formation with a melting point to 37°C. Protein release from the formulation was controlled over 24 h with a maximum fractional release of ±0.8 for all formulations. The release pattern alternated between phases of burst and slow release which was attributed to the combined effects of swelling, surface erosion, and in situ cross-linking. Mathematical modeling of the protein release kinetics corresponded best with the Higuichi model with near zero-order (R(2) ≈ 0.9787) release. The permeation-enhancing effect of menthol contained within the eutectic powder blend was investigated and results showed an enhanced protein flux (0.0576-0.0720 mg·cm(-2) h(-1)) across the intestinal tissue model compared with a control formulation. Extensive in vitro characterization highlighted the successful design of the eutectic tablets as a potential oral delivery system for proteins with structural protection, enhanced intestinal permeation, and controlled release.

  5. Self-reinforcement and protein sustained delivery of hyaluronan hydrogel by tailoring a dually cross-linked network.

    PubMed

    Luo, Chunhong; Xu, Guoguang; Wang, Xinghui; Tu, Mei; Zeng, Rong; Rong, Jianhua; Zhao, Jianhao

    2015-01-01

    A series of self-reinforcing hyaluronan hydrogels were developed to improve mechanical properties and protein sustained delivery thanks to a dually cross-linked network. Hyaluronan gel particles (HGPs, 1-5 μm in diameter) with different cross-linking densities, i.e. HGPs-1.5, HGPs-3 and HGPs-15, were prepared in an inverse emulsion system and used as the reinforcing phase after glycidyl methacrylation, while glycidyl methacrylated hyaluronan with a substitution degree of 45.2% was synthesized as the matrix phase. These two phases were cross-linked under ultraviolet irradiation to form self-reinforcing hyaluronan hydrogels (srHAs) that showed typical cross-linked structure of HGPs connecting the matrix phase by cross-section observation. In comparison to hyaluronan bulk gels and their blends with HGPs, srHAs distinctly enhanced the mechanical properties and BSA long-term sustained delivery, especially srHA-1.5 showed the highest compressive modulus of 220±15 kPa and the slowest BSA delivery (67% release at 14 d). The 3T3 fibroblast cell culture showed that all the srHAs had no cytotoxicity.

  6. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  7. Folate receptor targeted three-layered micelles and hydrogels for gene delivery to activated macrophages.

    PubMed

    Mohammadi, Mariam; Li, Ying; Abebe, Daniel G; Xie, Yuran; Kandil, Rima; Kraus, Teresa; Gomez-Lopez, Nardhy; Fujiwara, Tomoko; Merkel, Olivia M

    2016-12-28

    New folic acid (FA) coupled three layered micelles (3LM) were designed to encapsulate DNA, and their application as delivery system that specifically targets activated macrophages was investigated for new treatment options in rheumatoid arthritis (RA). FA coupled poly(l-lactide)-b-poly(ethylene glycol) (FA-PEG-PLLA) was synthesized via the NHS-ester activated/amine coupling method. Fluorescein labeled folic acid was used for flow cytometric detection of the expression of functional folic receptor β in LPS-activated and resting macrophages. FA coupled 3LM were formulated in a two-step procedure and characterized regarding hydrodynamic diameters and zeta potentials. The presence of the targeting ligand was shown not to increase the size of the 3LM compared to their non-targeted counterparts. Targeted and non-targeted 3LM were used in vitro to optimize uptake conditions in the RAW 264.7 macrophage cell line. The amount of FA coupled polymer in the final formulation was found to be optimal at 75% FA-PEG-PLLA and 25% PLLA-PEG-PLLA. Subsequently, transgene expression in vitro in RAW 264.7 cells and ex vivo in primary activated and resting mouse macrophages was determined as a function of FR-mediated internalization of 3LM encapsulating GFP expressing plasmid. FR-overexpressing activated cells, as successfully identified by internalization of FA-fluorescein, showed significantly higher GFP expression in vitro and ex vivo than resting macrophages with only a basal level of FR expression. Lastly, injectable hydrogels as depot formulation were formed by stereocomplexation, and their degradation, DNA release profiles, and dissociation into intact 3LM were found to be beneficial for potential in vivo application. Our findings confirm that FA-3LM are taken up by activated macrophages via folate receptor mediated endocytosis and that their hydrogels release intact 3LM for efficient transfection of primary macrophages. Therefore, FA-3LM could become a promising delivery system

  8. PolyMetformin combines carrier and anticancer activities for in vivo siRNA delivery

    PubMed Central

    Zhao, Yi; Wang, Wei; Guo, Shutao; Wang, Yuhua; Miao, Lei; Xiong, Yang; Huang, Leaf

    2016-01-01

    Metformin, a widely implemented anti-diabetic drug, exhibits potent anticancer efficacies. Herein a polymeric construction of Metformin, PolyMetformin (PolyMet) is successfully synthesized through conjugation of linear polyethylenimine (PEI) with dicyandiamide. The delocalization of cationic charges in the biguanide groups of PolyMet reduces the toxicity of PEI both in vitro and in vivo. Furthermore, the polycationic properties of PolyMet permits capture of siRNA into a core-membrane structured lipid-polycation-hyaluronic acid (LPH) nanoparticle for systemic gene delivery. Advances herein permit LPH-PolyMet nanoparticles to facilitate VEGF siRNA delivery for VEGF knockdown in a human lung cancer xenograft, leading to enhanced tumour suppressive efficacy. Even in the absence of RNAi, LPH-PolyMet nanoparticles act similarly to Metformin and induce antitumour efficacy through activation of the AMPK and inhibition of the mTOR. In essence, PolyMet successfully combines the intrinsic anticancer efficacy of Metformin with the capacity to carry siRNA to enhance the therapeutic activity of an anticancer gene therapy. PMID:27264609

  9. A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

    PubMed Central

    Steer, Beatrix; Adler, Barbara; Jonjic, Stipan; Stewart, James P.; Adler, Heiko

    2010-01-01

    Background Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. Methodology/Principal Findings Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. Conclusions/Significance In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein. PMID:20657771

  10. Intracellular Delivery of Proteins into Mouse Müller Glia Cells in vitro and in vivo Using Pep-1 Transfection Reagent

    PubMed Central

    Wang, Minhua H.; Frishman, Laura J.

    2009-01-01

    Direct protein transfection is a potentially valuable tool for studying protein function in basic and clinical research. A major challenge is to enable a sufficiently large amount of protein to penetrate the plasma membrane of the transfected cells. Pep-1, a protein transfection reagent, was evaluated for its ability and efficiency in delivering proteins and antibodies into mouse Müller cells in vitro and in vivo. Pep-1 delivered active beta-galactosidase enzyme and antibodies (non-specific IgG and Cy3 conjugated anti-vimentin) into cultured Müller cells with high efficiency. Transfection efficiency increased with increasing concentration of the protein in the complex and with incubation time. Following intravitreal injection of Pep-1/IgG complexes in vivo, retinal histology was preserved and immunostaining showed that the antibodies were distributed widely across the retinal surface, with the most intense staining located near the retino-vitreal border. For complexes using non-specific IgG, double staining with anti-glutamine synthetase identified many IgG-positive cells as Müller glia. IgG immunoreactivity was also detected in the cytoplasm and occasionally in the nuclei of inner retinal neurons. Dark-adapted flash electroretinogram (ERG) recordings from injected eyes were nearly identical to ERG recordings from control eyes, suggesting that injection of Pep-1/IgG complex has minimal effects on retinal function. Therefore, Pep-1 is a useful tool for intracellular delivery of antibodies to study the role of proteins in living cells. PMID:19056421

  11. Intracellular delivery of proteins into mouse Müller glia cells in vitro and in vivo using Pep-1 transfection reagent.

    PubMed

    Wang, Minhua H; Frishman, Laura J; Otteson, Deborah C

    2009-03-15

    Direct protein transfection is a potentially valuable tool for studying protein function in basic and clinical research. A major challenge is to enable a sufficiently large amount of protein to penetrate the plasma membrane of the transfected cells. Pep-1, a protein transfection reagent, was evaluated for its ability and efficiency in delivering proteins and antibodies into mouse Müller cells in vitro and in vivo. Pep-1 delivered active beta-galactosidase enzyme and antibodies (non-specific IgG and Cy3-conjugated anti-vimentin) into cultured Müller cells with high efficiency. Transfection efficiency increased with increasing concentration of the protein in the complex and with incubation time. Following intravitreal injection of Pep-1/IgG complexes in vivo, retinal histology was preserved and immunostaining showed that the antibodies were distributed widely across the retinal surface, with the most intense staining located near the retino-vitreal border. For complexes using non-specific IgG, double staining with anti-glutamine synthetase identified many IgG-positive cells as Müller glia. IgG immunoreactivity was also detected in the cytoplasm and occasionally in the nuclei of inner retinal neurons. Dark-adapted flash electroretinogram (ERG) recordings from injected eyes were nearly identical to ERG recordings from control eyes, suggesting that injection of Pep-1/IgG complex has minimal effects on retinal function. Therefore, Pep-1 is a useful tool for intracellular delivery of antibodies to study the role of proteins in living cells.

  12. Intranuclear Delivery of a Novel Antibody-Derived Radiosensitizer Targeting the DNA-Dependent Protein Kinase Catalytic Subunit

    SciTech Connect

    Xiong Hairong; Lee, Robert J.; Haura, Eric B.; Edwards, John G.; Dynan, William S.; Li Shuyi

    2012-07-01

    Purpose: To inhibit DNA double-strand break repair in tumor cells by delivery of a single-chain antibody variable region fragment (ScFv 18-2) to the cell nucleus. ScFv 18-2 binds to a regulatory region of the DNA-dependent protein kinase (DNA-PK), an essential enzyme in the nonhomologous end-joining pathway, and inhibits DNA end-joining in a cell-free system and when microinjected into single cells. Development as a radiosensitizer has been limited by the lack of a method for intranuclear delivery to target cells. We investigated a delivery method based on folate receptor-mediated endocytosis. Methods and Materials: A recombinant ScFv 18-2 derivative was conjugated to folate via a scissile disulfide linker. Folate-ScFv 18-2 was characterized for its ability to be internalized by tumor cells and to influence the behavior of ionizing radiation-induced repair foci. Radiosensitization was measured in a clonogenic survival assay. Survival curves were fitted to a linear-quadratic model, and between-group differences were evaluated by an F test. Sensitization ratios were determined based on mean inhibitory dose. Results: Human KB and NCI-H292 lung cancer cells treated with folate-conjugated ScFv 18-2 showed significant radiosensitization (p < 0.001). Sensitization enhancement ratios were 1.92 {+-} 0.42 for KB cells and 1.63 {+-} 0.13 for NCI-H292 cells. Studies suggest that treatment inhibits repair of radiation-induced DSBs, as evidenced by the persistence of {gamma}-H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056. Conclusions: Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.

  13. Integrin Targeting and Toxicological Assessment of Peptide-Conjugated Liposome Delivery Systems to Activated Endothelial Cells.

    PubMed

    Kermanizadeh, Ali; Villadsen, Klaus; Østrem, Ragnhild G; Jensen, Knud J; Møller, Peter; Loft, Steffen

    2017-04-01

    Utilization of functionalized liposomes as the means of targeted delivery of therapeutics may enhance specific transport of biologically active drugs to target tissues, while avoiding or reducing undesired side effects. In the present investigation, peptide-conjugated cationic liposomes were constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide-conjugated liposomes induced only cytotoxicity at the highest concentration in non-activated or activated endothelial cells, as well as in co-culture of endothelial cells and macrophages. There was unaltered secretion of cytokines after exposure of peptide-conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5β1) were more effective in targeting of activated endothelial cells, as compared to a liposome with a peptide that targeted both the fibronectin and vitronectin receptors, as well as liposomes with a control peptide. The liposome targeted to the fibronectin receptor also displayed uptake in endothelial cells in co-culture with activated macrophages. Therefore, this study demonstrates the feasibility of constructing a peptide-conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells.

  14. Oxidized alginate hydrogels for bone morphogenetic protein-2 delivery in long bone defects.

    PubMed

    Priddy, Lauren B; Chaudhuri, Ovijit; Stevens, Hazel Y; Krishnan, Laxminarayanan; Uhrig, Brent A; Willett, Nick J; Guldberg, Robert E

    2014-10-01

    Autograft treatment of large bone defects and fracture non-unions is complicated by limited tissue availability and donor site morbidity. Polymeric biomaterials such as alginate hydrogels provide an attractive tissue engineering alternative due to their biocompatibility, injectability, and tunable degradation rates. Irradiated RGD-alginate hydrogels have been used to deliver proteins such as bone morphogenetic protein-2 (BMP-2), to promote bone regeneration and restoration of function in a critically sized rat femoral defect model. However, slow degradation of irradiated alginate hydrogels may impede integration and remodeling of the regenerated bone to its native architecture. Oxidation of alginate has been used to promote degradation of alginate matrices. The objective of this study was to evaluate the effects of alginate oxidation on BMP-2 release and bone regeneration. We hypothesized that oxidized-irradiated alginate hydrogels would elicit an accelerated release of BMP-2, but degrade faster in vivo, facilitating the formation of higher quality, more mature bone compared to irradiated alginate. Indeed, oxidation of irradiated alginate did accelerate in vitro BMP-2 release. Notably, the BMP-2 retained within both constructs was bioactive at 26days, as observed by induction of alkaline phosphatase activity and positive Alizarin Red S staining of MC3T3-E1 cells. From the in vivo study, robust bone regeneration was observed in both groups through 12weeks by radiography, micro-computed tomography analyses, and biomechanical testing. Bone mineral density was significantly greater for the oxidized-irradiated alginate group at 8weeks. Histological analyses of bone defects revealed enhanced degradation of oxidized-irradiated alginate and suggested the presence of more mature bone after 12weeks of healing.

  15. New biodegradable dextran-based hydrogels for protein delivery: Synthesis and characterization.

    PubMed

    Pacelli, Settimio; Paolicelli, Patrizia; Casadei, Maria Antonietta

    2015-08-01

    A new derivative of dextran grafted with polyethylene glycol methacrylate through a carbonate bond (DEX-PEG-MA) has been synthesized and characterized. The photo-crosslinking reaction of DEX-PEG-MA allowed the obtainment of biodegradable networks tested for their mechanical and release properties. The new hydrogels were compared with those made of dextran methacrylate (DEX-MA), often employed as drug delivery systems of small molecules. The inclusion of PEG as a spacer created additional interactions among the polymeric chains improving the extreme fragility and lack of hardness typical of gels made of DEX-MA. Moreover, the different behavior in terms of swelling and degradability of the networks was able to affect the release of a model macromolecule over time, making DEX-PEG-MA matrices suitable candidates for the delivery of high molecular weight peptides. Interestingly, the combination of the two dextran derivatives showed intermediate ability to modulate the release of high molecular weight macromolecules.

  16. Alk5 inhibition increases delivery of macromolecular and protein-bound contrast agents to tumors

    PubMed Central

    Daldrup-Link, Heike E.; Mohanty, Suchismita; Ansari, Celina; Ito, Ken; Hong, Su Hyun; Hoffmann, Matthias; Pisani, Laura; Boudreau, Nancy; Gambhir, Sanjiv Sam; Coussens, Lisa M.

    2016-01-01

    Limited transendothelial permeability across tumor microvessels represents a significant bottleneck in the development of tumor-specific diagnostic agents and theranostic drugs. Here, we show an approach to increase transendothelial permeability of macromolecular and nanoparticle-based contrast agents via inhibition of the type I TGF-β receptor, activin-like kinase 5 (Alk5), in tumors. Alk5 inhibition significantly increased tumor contrast agent delivery and enhancement on imaging studies, while healthy organs remained relatively unaffected. Imaging data correlated with significantly decreased tumor interstitial fluid pressure, while tumor vascular density remained unchanged. This immediately clinically translatable concept involving Alk5 inhibitor pretreatment prior to an imaging study could be leveraged for improved tumor delivery of macromolecular and nanoparticle-based imaging probes and, thereby, facilitate development of more sensitive imaging tests for cancer diagnosis, enhanced tumor characterization, and personalized, image-guided therapies. PMID:27182558

  17. Potent Delivery of Functional Proteins into Mammalian Cells in Vitro and in Vivo Using a Supercharged Protein

    PubMed Central

    2010-01-01

    The inability of proteins to potently penetrate mammalian cells limits their usefulness as tools and therapeutics. When fused to superpositively charged GFP, proteins rapidly (within minutes) entered five different types of mammalian cells with potency up to ∼100-fold greater than that of corresponding fusions with known protein transduction domains (PTDs) including Tat, oligoarginine, and penetratin. Ubiquitin-fused supercharged GFP when incubated with human cells was partially deubiquitinated, suggesting that proteins delivered with supercharged GFP can access the cytosol. Likewise, supercharged GFP delivered functional, nonendosomal recombinase enzyme with greater efficiencies than PTDs in vitro and also delivered functional recombinase enzyme to the retinae of mice when injected in vivo. PMID:20545362

  18. Chitosan coated nanostructured lipid carriers for brain delivery of proteins by intranasal administration.

    PubMed

    Gartziandia, Oihane; Herran, Enara; Pedraz, Jose Luis; Carro, Eva; Igartua, Manoli; Hernandez, Rosa Maria

    2015-10-01

    The remarkable increase in the prevalence of neurodegenerative diseases has become a serious public health problem. Considering the lack of effective treatments to address these diseases and the difficulties in accessing the brain due to the blood-brain barrier (BBB), to attain a successful strategy to improve drug delivery to the brain, the administration route becomes a point of interest. The intranasal route provides a non-invasive method to bypass the BBB. Moreover, the development of new technologies for the protection and delivery of peptides is an interesting approach to consider. Thus, in this work, a suitable chitosan coated nanostructured lipid carrier (CS-NLC) formulation with the capacity to reach the brain after being intranasally administered was successfully developed and optimized. The optimal formulation displayed a particle size of 114 nm with a positive surface charge of +28 mV. The in vitro assays demonstrated the biocompatibility of the nanocarrier and its cellular uptake by 16HBE14o- cells. Furthermore, no haemagglutination or haemolysis processes were observed when the particles were incubated with erythrocytes, and no toxicity signals appeared in the nasal mucosa of mice after the administration of CS-NLCs. Finally, the biodistribution study of CS-NLC-DiR demonstrated an efficient brain delivery of the particles after intranasal administration. In conclusion, CS-NLC can be considered to be a safe and effective nanocarrier for nose-to-brain drug delivery; however, to obtain a higher concentration of the drug in the brain following intranasal administration, further modifications are warranted in the CS-NLC formulation.

  19. Tobacco mosaic virus-based protein nanoparticles and nanorods for chemotherapy delivery targeting breast cancer

    PubMed Central

    Bruckman, Michael A.; Czapar, Anna E.; VanMeter, Allen; Randolph, Lauren N.; Steinmetz, Nicole F.

    2016-01-01

    Drug delivery systems are required for drug targeting to avoid adverse effects associated with chemotherapy treatment regimes. Our approach is focused on the study and development of plant virus-based materials as drug delivery systems; specifically, this work focuses on the tobacco mosaic virus (TMV). Native TMV forms a hollow, high aspect-ratio nanotube measuring 300 × 18 nm with a 4 nm-wide central channel. Heat-transformation can be applied to TMV yielding spherical nanoparticles (SNPs) measuring ~50 nm in size. While bioconjugate chemistries have been established to modify the TMV rod, such methods have not yet been described for the SNP platform. In this work, we probed the reactivity of SNPs toward bioconjugate reactions targeting lysine, glutamine/aspartic acid, and cysteine residues. We demonstrate functionalization of SNPs using these chemistries yielding efficient payload conjugation. In addition to covalent labeling techniques, we developed encapsulation techniques, where the cargo is loaded into the SNP during heat-transition from rod-to-sphere. Finally, we developed TMV and SNP formulations loaded with the chemotherapeutic doxorubicin, and we demonstrate the application of TMV rods and spheres for chemotherapy delivery targeting breast cancer. PMID:26941034

  20. Tobacco mosaic virus-based protein nanoparticles and nanorods for chemotherapy delivery targeting breast cancer.

    PubMed

    Bruckman, Michael A; Czapar, Anna E; VanMeter, Allen; Randolph, Lauren N; Steinmetz, Nicole F

    2016-06-10

    Drug delivery systems are required for drug targeting to avoid adverse effects associated with chemotherapy treatment regimes. Our approach is focused on the study and development of plant virus-based materials as drug delivery systems; specifically, this work focuses on the tobacco mosaic virus (TMV). Native TMV forms a hollow, high aspect-ratio nanotube measuring 300×18nm with a 4nm-wide central channel. Heat-transformation can be applied to TMV yielding spherical nanoparticles (SNPs) measuring ~50nm in size. While bioconjugate chemistries have been established to modify the TMV rod, such methods have not yet been described for the SNP platform. In this work, we probed the reactivity of SNPs toward bioconjugate reactions targeting lysine, glutamine/aspartic acid, and cysteine residues. We demonstrate functionalization of SNPs using these chemistries yielding efficient payload conjugation. In addition to covalent labeling techniques, we developed encapsulation techniques, where the cargo is loaded into the SNP during heat-transition from rod-to-sphere. Finally, we developed TMV and SNP formulations loaded with the chemotherapeutic doxorubicin, and we demonstrate the application of TMV rods and spheres for chemotherapy delivery targeting breast cancer.

  1. Herpes simplex virus type 1 VP22-mediated intercellular delivery of PTEN increases the antitumor activity of PTEN in esophageal squamous cell carcinoma cells in vitro and in vivo.

    PubMed

    Yu, Xian; Li, Tingting; Xia, Yifan; Lei, Jun; Wang, Yan; Zhang, Lijuan

    2016-05-01

    In the past decade, studies have revealed that the phosphatase and tensin homolog (PTEN) protein, a tumor suppressor, comprises a potential biological marker and therapeutic target for esophageal squamous cell carcinoma (ESCC). As such, the delivery of the PTEN gene represents a powerful strategy for ESCC therapy. The tegument protein VP22 of herpes simplex virus type 1 (HSV-1) has been reported to act as a transporter of heterologous proteins across the host cell membrane, thereby enhancing the biological functions of these proteins. In the present study, the intercellular delivery and antitumor activity of the fusion protein PTEN-VP22 were examined in the esophageal squamous cell carcinoma cell line Eca109 both in vitro and in vivo. VP22-mediated PTEN intercellular delivery was confirmed in the Eca109 cells by western blot analysis and by quantitation of immunofluorescence. VP22 alone did not exert antiproliferative effects or induce cell cycle arrest, induction of apoptosis, blockage of the Akt and focal adhesion kinase (FAK) pathways, tumor growth inhibition, or antiangiogenic effects in Eca109 cells. However, compared with PTEN alone, PTEN-VP22 exerted significantly higher antiproliferative effects and induced cell cycle arrest at G1 stage, apoptosis and antiangiogenic effects in Eca109 cells. Together, our findings demonstrate that VP22 alone does not exert antitumor activity directly; however, this protein mediates the intercellular delivery of PTEN and thereby increases its intracellular concentration to achieve a therapeutic steady state, leading to an overall increase in the antitumor activity of PTEN. This study provides further experimental data to confirm the potential of VP22-based intercellular delivery strategies for enhancing the efficacy of gene therapy for cancer treatment.

  2. [Gene delivery system based on low molecular weight polyethylenimine and its transfection activity in the skin tissue].

    PubMed

    Liu, Yong Jun; Li, Ya Ping; Li, Jun; Jia, Jing Fen; Liu, Lei

    2004-04-01

    Low molecular weight polyethylenimine (LMW-PEI) was linked to an expressing plasmid contains a green fluorescence protein (GFP) reporter gene and effective gene transfer was observed in CM7221 cell line tested. We examined the relationship among the molecular weight, structure of PEI and their transfection activity and cytotoxicity on CM7221 cell line. We also examined the position and continuance time of the GFP reporter gene expressed in the skin tissue of mouse. Results showed that LMW-PEI/DNA complexes led to high levels of expression in the CM7221 cell line (about 55%). However, with the increasing of PEI molecular weight, the transfection activity of PEI was decreasing. There was an increasing cytotoxicity with the larger PEI molecules. Further research showed that LMW-PEI induced a significant and long-lasting (7 days) expression of the GFP reporter gene in the hair vesicle, sweat, gland, sebaceous gland in the mouse skin tissues. The LMW-PEI described here is a new, highly efficient and non-cytotoxic vector. It would be a useful non-viral vector for gene delivery technology, particular useful as simple skin-specific vehicles of therapeutic genes.

  3. Protein-Protein Interactions Suggest Novel Activities of Human Cytomegalovirus Tegument Protein pUL103

    PubMed Central

    Ortiz, Daniel A.; Glassbrook, James E.

    2016-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is an enveloped double-stranded DNA virus that causes severe disease in newborns and immunocompromised patients. During infection, the host cell endosecretory system is remodeled to form the cytoplasmic virion assembly complex (cVAC). We and others previously identified the conserved, multifunctional HCMV virion tegument protein pUL103 as important for cVAC biogenesis and efficient secondary envelopment. To help define its mechanisms of action and predict additional functions, we used two complementary methods, coimmunoprecipitation (co-IP) and proximity biotinylation (BioID), to identify viral and cellular proteins that interact with pUL103. By using the two methods in parallel and applying stringent selection criteria, we identified potentially high-value interactions of pUL103 with 13 HCMV and 18 cellular proteins. Detection of the previously identified pUL103-pUL71 interaction, as well as verification of several interactions by reverse co-IP, supports the specificity of our screening process. As might be expected for a tegument protein, interactions were identified that suggest distinct roles for pUL103 across the arc of lytic infection, including interactions with proteins involved in cellular antiviral responses, nuclear activities, and biogenesis and transport of cytoplasmic vesicles. Further analysis of some of these interactions expands our understanding of the multifunctional repertoire of pUL103: we detected HCMV pUL103 in nuclei of infected cells and identified an ALIX-binding domain within the pUL103 sequence. IMPORTANCE Human cytomegalovirus (HCMV) is able to reconfigure the host cell machinery to establish a virion production factory, the cytoplasmic virion assembly complex (cVAC). cVAC biogenesis and operation represent targets for development of novel HCMV antivirals. We previously showed that the HCMV tegument protein pUL103 is required for cVAC biogenesis. Using pUL103 as bait, we investigated viral and

  4. Systemic protein delivery by muscle-gene transfer is limited by a local immune response

    PubMed Central

    Wang, Lixin; Dobrzynski, Eric; Schlachterman, Alexander; Cao, Ou; Herzog, Roland W.

    2005-01-01

    Adeno-associated viral (AAV) vectors have been successfully used for therapeutic expression of systemic transgene products (such as factor IX or erythropoietin) following in vivo administration to skeletal muscle of animal models of inherited hematologic disorders. However, an immune response may be initiated if the transgene product represents a neoantigen. Here, we use ovalbumin (OVA) as a model antigen and demonstrate immune-mediated elimination of expression on muscle-directed AAV-2 gene transfer. Administration to immune competent mice resulted in transient systemic OVA expression. Within 10 days, OVA-specific T-helper cells had been activated in draining lymph nodes, an inflammatory immune response ensued, and OVA-expressing muscle fibers were destroyed by a cytotoxic CD8+ T-cell response. Use of a muscle-specific promoter did not prevent this immune response. Adoptively transferred CD4+ cells transgenic for a T-cell receptor specific to OVA peptide-major histocompatibility complex class II showed antigen-specific, vector dose-dependent proliferation confined to the draining lymph nodes of AAV-OVA–transduced muscle within 5 days after gene transfer and subsequently participated in lymphocytic infiltration of transduced muscle. This study documents that a local immune response limits sustained expression of a secreted protein in muscle gene transfer, a finding that may have consequences for design of clinical protocols. PMID:15713796

  5. 78 FR 9719 - Agency Information Collection Activities: Entry and Immediate Delivery Application and Simplified...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-11

    ... Delivery Application and Simplified Entry AGENCY: U.S. Customs and Border Protection (CBP), Department of... concerning the Entry and Immediate Delivery Application (Forms 3461 and 3461 ALT) and Simplified Entry. This... concerning the following information collection: Title: Entry and Immediate Delivery Application...

  6. 76 FR 82315 - Agency Information Collection Activities: Entry/Immediate Delivery Application and Simplified Entry

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-30

    ... Delivery Application and Simplified Entry AGENCY: U.S. Customs and Border Protection, Department of... Delivery Application (Forms 3461 and 3461 ALT) and Simplified Entry. This is a proposed revision and... forms of information. Title: Entry/Immediate Delivery Application and Simplified Entry. OMB Number:...

  7. 76 FR 66740 - Agency Information Collection Activities: Entry and Immediate Delivery Application

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-27

    ... Delivery Application AGENCY: U.S. Customs and Border Protection (CBP), Department of Homeland Security... concerning the Entry and Immediate Delivery Application (Forms 3461 and 3461 ALT). This request for comment...: Title: Entry and Immediate Delivery Application. OMB Number: 1651-0024. Form Number: CBP Form 3461...

  8. Active packaging for topical cosmetic/drug products: a hot-melt extruded preservative delivery device.

    PubMed

    Zema, L; Sangalli, M E; Maroni, A; Foppoli, A; Bettero, A; Gazzaniga, A

    2010-06-01

    A delivery device intended for the prolonged release of antimicrobial agents, able to enhance the stability profile of liquid/semi-solid cosmetic/pharmaceutical products for topical application, was proposed in the present study. With the aid of a simulation program based on compartment models, the relevant kinetic and formulation parameters were defined using dehydroacetic acid sodium salt (DHA.Na, Prevan) as the model preservative. Indeed, the overall DHA.Na degradation rate is increased in the presence of formaldehyde releasers that are often employed as co-preservatives. Inert matrices (3 g weight and 18 mm diameter) based on high-density polyethylene (HDPE), possibly consistent with the design of an active packaging meant for preservative delivery, were prepared by hot-melt extrusion. Units with satisfactory physical-technological properties could be obtained up to 50%w/w loads of antimicrobial agent. In an attempt to modify the relevant Fickian release profiles by varying the area exposed to the medium, matrix systems coated with an impermeable film except for one base (CMs) or for the inner surface of a central drilled hole (PCMs) were investigated. On the basis of the n exponent of power equation and the outcome of linear fitting, PCMs were proven able to yield the zero-order release behaviour needed to ensure constant DHA.Na levels over a predetermined time period, as indicated by the simulation process.

  9. Efficient gene delivery with osmotically active and hyperbranched poly(ester amine)s.

    PubMed

    Arote, Rohidas B; Lee, Eun-Sun; Jiang, Hu-Lin; Kim, You-Kyoung; Choi, Yun-Jaie; Cho, Myung-Haing; Cho, Chong-Su

    2009-12-01

    Degradable and hyperbranched poly (ester amine)s (PEAs) were successfully synthesized by Michael addition reaction between hydrophilic glycerol triacrylate (GTA) and low-molecular-weight polyethylenimine (LMW-PEI) and evaluated as nonviral gene carriers. PEAs effectively condensed DNA with particle sizes below 200 nm and suitable surface charges (15-45 mV), suitable for intracellular delivery. PEAs degraded in a controlled fashion showing half-lives of more than 12 days and were essentially nontoxic in three different cell lines. Elevated transfection levels by luciferase assay revealed the superiority of PEAs over PEI 25K and Lipofectamine. PEAs synthesized using 1:4 mol ratio of GTA to PEI [GTA/PEI-1.2(1:4)] showed highest transfection efficiency in HepG2 cells. PEAs showed significant gene expression in vitro as well as in vivo through aerosol administration. Reduction in packed cell volume (PCV) of cells when treated with polyplexes supported the hyperosmotic effect of PEAs. Effect of bafilomycin A1 on transfection efficiency of PEAs on 293T cells indicated its endosomal buffering capacity. High transfection efficiency was attributed to the synergism from hyperosmotic glycerol backbone in the PEAs and endosomal buffering capacity of PEI amine groups. Therefore, this convergence of osmotically active biodegradable PEAs suggests their potential as a safe and efficient gene delivery vector.

  10. Mesoporous silica nanoparticles functionalized with folic acid/methionine for active targeted delivery of docetaxel

    PubMed Central

    Khosravian, Pegah; Shafiee Ardestani, Mehdi; Khoobi, Mehdi; Ostad, Seyed Naser; Dorkoosh, Farid Abedin; Akbari Javar, Hamid; Amanlou, Massoud

    2016-01-01

    Mesoporous silica nanoparticles (MSNs) are known as carriers with high loading capacity and large functionalizable surface area for target-directed delivery. In this study, a series of docetaxel-loaded folic acid- or methionine-functionalized mesoporous silica nanoparticles (DTX/MSN-FA or DTX/MSN-Met) with large pores and amine groups at inner pore surface properties were prepared. The results showed that the MSNs were successfully synthesized, having good pay load and pH-sensitive drug release kinetics. The cellular investigation on MCF-7 cells showed better performance of cytotoxicity and cell apoptosis and an increase in cellular uptake of targeted nanoparticles. In vivo fluorescent imaging on healthy BALB/c mice proved that bare MSN-NH2 are mostly accumulated in the liver but MSN-FA or MSN-Met are more concentrated in the kidney. Importantly, ex vivo fluorescent images of tumor-induced BALB/c mice organs revealed the ability of MSN-FA to reach the tumor tissues. In conclusion, DTX/MSNs exhibited a good anticancer activity and enhanced the possibility of targeted drug delivery for breast cancer. PMID:27980423

  11. Recent Advances in Anticancer Activities and Drug Delivery Systems of Tannins.

    PubMed

    Cai, Yuee; Zhang, Jinming; Chen, Nelson G; Shi, Zhi; Qiu, Jiange; He, Chengwei; Chen, Meiwan

    2016-12-22

    Tannins, polyphenols in medicinal plants, have been divided into two groups of hydrolysable and condensed tannins, including gallotannins, ellagitannins, and (-)-epigallocatechin-3-gallate (EGCG). Potent anticancer activities have been observed in tannins (especially EGCG) with multiple mechanisms, such as apoptosis, cell cycle arrest, and inhibition of invasion and metastases. Furthermore, the combinational effects of tannins and anticancer drugs have been demonstrated in this review, including chemoprotective, chemosensitive, and antagonizing effects accompanying with anticancer effect. However, the applications of tannins have been hindered due to their poor liposolubility, low bioavailability, off-taste, and shorter half-life time in human body, such as EGCG, gallic acid, and ellagic acid. To tackle these obstacles, novel drug delivery systems have been employed to deliver tannins with the aim of improving their applications, such as gelatin nanoparticles, micelles, nanogold, liposomes, and so on. In this review, the chemical characteristics, anticancer properties, and drug delivery systems of tannins were discussed with an attempt to provide a systemic reference to promote the development of tannins as anticancer agents.

  12. Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis.

    PubMed

    Takiar, Vinita; Nishio, Saori; Seo-Mayer, Patricia; King, J Darwin; Li, Hui; Zhang, Li; Karihaloo, Anil; Hallows, Kenneth R; Somlo, Stefan; Caplan, Michael J

    2011-02-08

    Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD.

  13. Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

    PubMed

    Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C; Shi, Chong-Shan; Srinivasan, Lalitha; McQuiston, Travis J; Hwang, Il-Young; Yeh, Anthony J; Blumer, Joe B; Briken, Volker; Williamson, Peter R; Otto, Michael; Fraser, Iain D C; Kehrl, John H

    2016-01-15

    Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

  14. Human carotid atherosclerotic plaque protein(s) change HDL protein(s) composition and impair HDL anti-oxidant activity.

    PubMed

    Cohen, Elad; Aviram, Michael; Khatib, Soliman; Volkova, Nina; Vaya, Jacob

    2016-01-01

    High density lipoprotein (HDL) anti-atherogenic functions are closely associated with cardiovascular disease risk factor, and are dictated by its composition, which is often affected by environmental factors. The present study investigates the effects of the human carotid plaque constituents on HDL composition and biological functions. To this end, human carotid plaques were homogenized and incubated with HDL. Results showed that after incubation, most of the apolipoprotein A1 (Apo A1) protein was released from the HDL, and HDL diameter increased by an average of approximately 2 nm. In parallel, HDL antioxidant activity was impaired. In response to homogenate treatment HDL could not prevent the accelerated oxidation of LDL caused by the homogenate. Boiling of the homogenate prior to its incubation with HDL abolished its effects on HDL composition changes. Moreover, tryptophan fluorescence quenching assay revealed an interaction between plaque component(s) and HDL, an interaction that was reduced by 50% upon using pre-boiled homogenate. These results led to hypothesize that plaque protein(s) interacted with HDL-associated Apo A1 and altered the HDL composition. Immuno-precipitation of Apo A1 that was released from the HDL after its incubation with the homogenate revealed a co-precipitation of three isomers of actin. However, beta-actin alone did not significantly affect the HDL composition, and yet the active protein within the plaque was elusive. In conclusion then, protein(s) in the homogenate interact with HDL protein(s), leading to release of Apo A1 from the HDL particle, a process that was associated with an increase in HDL diameter and with impaired HDL anti-oxidant activity.

  15. Design, in vitro stability, and ocular hypotensive activity of t-butalone chemical delivery systems.

    PubMed

    Reddy, I K; Vaithiyalingam, S R; Khan, M A; Bodor, N S

    2001-08-01

    The objective of this work was to synthesize two bioreversible diacyl derivatives of t-butalone (chemical delivery systems), determine their chemical and in vitro stability, and investigate their potential use as topical antiglaucoma agents. The stability of these compounds was determined in isotonic phosphate buffers (pH range 5-8) and in selected biological media, including human whole blood, rabbit and rat blood, and the anterior segment tissues of rabbit. The ocular hypotensive activity of these compounds in unrestrained, normotensive albino rabbits was determined with a pneumatonometer. The two compounds were stable at lower pH. The stability decreased as the pH increased, suggesting their lability to base-catalyzed hydrolysis. These compounds exhibited significant differences in the hydrolytic rates in the whole blood among species examined (rat > rabbit > human). The observed rates of disappearance in different ocular tissues were indicative of relative enzyme activity in these media (iris-ciliary body > cornea > aqueous humor). The two compounds exhibited a significant ocular hypotensive activity (P < 0.01) at 2% dose level. The peak activity was found between 2 and 4 h, and the activity was maintained for 4.5 to 7 h. The dipivalyl derivative of t-butalone exhibited more pronounced decrease in intraocular pressure than that of diisovaleryl derivative. The present study suggests the possible use of diacyl derivatives of t-butalone as ocular hypotensive agents.

  16. The Prediabetes Detection and Physical Activity Intervention Delivery (PRE-PAID) program.

    PubMed

    Rowan, Chip P; Riddell, Michael C; Jamnik, Veronica K

    2013-12-01

    Inspired by increases in the prevalence and incidence of prediabetes, the Pre-diabetes Detection and Physical Activity Intervention Delivery Project (PRE-PAID) is a multiphasic program that identifies persons at high risk for developing type 2 diabetes, provides an opportunity for culturally appropriate, community-based physical activity and facilitates training of qualified exercise professionals on diabetes screening as well as prediabetes-specific training recommendations. This article provides an overview of the PRE-PAID project and includes some preliminary screening data, as well as lessons learned from the implementation of community-based physical activity programs that target specific, high-risk ethnicities. Recommendations and special considerations involving physical activity that targets persons with prediabetes also are discussed. A total of 691 individuals have undergone the PRE-PAID risk-identification process, which involves a brief questionnaire and point-of-care finger-prick hemoglobin A1C testing. The mean hemoglobin A1C level was 6.0±0.90% (mean ± standard deviation). Questionnaire scores showed that, on average, the individuals screened had 3 to 5 typical risk factors for type 2 diabetes, such as high body mass index, waist circumference, physical inactivity or family history of diabetes. Community-specific breakdowns of these results also are presented in this article. Sharing experiences from the PRE-PAID project can help formulate a framework for future prediabetes screening and physical activity interventions that are community based, target persons with prediabetes and are culturally appropriate.

  17. Parenteral and mucosal delivery of a novel multi-epitope M protein-based group A streptococcal vaccine construct: investigation of immunogenicity in mice.

    PubMed

    Dunn, Linda A; McMillan, David J; Batzloff, Michael; Zeng, Weiguang; Jackson, David C J; Upcroft, Jacqueline A; Upcroft, Peter; Olive, Colleen

    2002-06-21

    Primary vaccine strategies against group A streptococci (GAS) have focused on the M protein--the target of opsonic antibodies important for protective immunity. We have previously reported protection of mice against GAS infection following parenteral delivery of a multi-epitope vaccine construct, referred to as a heteropolymer. This current report has assessed mucosal (intranasal (i.n.) and oral) delivery of the heteropolymer in mice with regard to the induction and specificity of mucosal and systemic antibody responses, and compared this to parenteral delivery. GAS-specific IgA responses were detected in saliva and gut upon i.n. and oral delivery of the heteropolymer co-administered with cholera toxin B subunit, respectively. High titre serum IgG responses were elicited to the heteropolymer following all routes of delivery when administered with adjuvant. Moreover, as with parenteral delivery, serum IgG antibodies were detected to the individual heteropolymer peptides following i.n. but not oral delivery. These data support the potential of the i.n. route in the mucosal delivery of a GAS vaccine.

  18. Transcription activation by the adenovirus E1a protein

    NASA Astrophysics Data System (ADS)

    Lillie, James W.; Green, Michael R.

    1989-03-01

    The adenovirus Ela protein stimulates transcription of a wide variety of viral and cellular genes. It is shown here that Ela has the two functions characteristic of a typical cellular activator: one direct Ela to the promoter, perhaps by interacting with a DMA-bound protein, and the other, an activating region, enables the bound activator to stimulate transcription.

  19. Oral delivery system for two-pulse colonic release of protein drugs and protease inhibitor/absorption enhancer compounds.

    PubMed

    Del Curto, Maria Dorly; Maroni, Alessandra; Palugan, Luca; Zema, Lucia; Gazzaniga, Andrea; Sangalli, Maria Edvige

    2011-08-01

    It is well known that the intestinal stability and absorption of protein drugs are improved when enzyme inhibitors/permeation enhancers are coadministered. Recently, it was hypothesized that an increased effectiveness of these adjuvants might be achieved by timing their release prior to that of the protein, so that a more favorable environment would be established in advance. Therefore, an oral system was proposed for two-pulse colonic release of insulin and the protease inhibitor camostat mesilate/absorption enhancer sodium glycocholate. The device consisted of a drug-containing core, an inner swellable/erodible low-viscosity hydroxypropyl methylcellulose (HPMC) coating, an intermediate adjuvant layer, and an additional outer HPMC coating. HPMC coats and camostat mesilate/sodium glycocholate films with differing thicknesses were applied to immediate-release tablet cores by aqueous spray coating. The obtained units were characterized for weight, thickness, breaking force, and release performance. All systems showed satisfactory technological properties and the pursued pulsatile delivery behavior, with programmable delay phases preceding inhibitor/enhancer release and elapsing between inhibitor/enhancer and protein release, respectively. Indeed, both lag times linearly correlated with the relevant HPMC coating level. The system was thus proven suitable for yielding two-pulse release profiles, in which lag phases could be modulated to provide convenient concentration patterns for proteins and adjuvants.

  20. DNA helicase activity in purified human RECQL4 protein.

    PubMed

    Suzuki, Takahiro; Kohno, Toshiyuki; Ishimi, Yukio

    2009-09-01

    Human RECQL4 protein was expressed in insect cells using a baculovirus protein expression system and it was purified to near homogeneity. The protein sedimented at a position between catalase (230 kDa) and ferritin (440 kDa) in glycerol gradient centrifugation, suggesting that it forms homo-multimers. Activity to displace annealed 17-mer oligonucleotide in the presence of ATP was co-sedimented with hRECQL4 protein. In ion-exchange chromatography, both DNA helicase activity and single-stranded DNA-dependent ATPase activity were co-eluted with hRECQL4 protein. The requirements of ATP and Mg for the helicase activity were different from those for the ATPase activity. The data suggest that the helicase migrates on single-stranded DNA in a 3'-5' direction. These results suggest that the hRECQL4 protein exhibits DNA helicase activity.

  1. Using online computer tailoring to promote physical activity: a randomized trial of text, video, and combined intervention delivery modes.

    PubMed

    Soetens, Katja C M; Vandelanotte, Corneel; de Vries, Hein; Mummery, Kerry W

    2014-12-01

    Website-delivered interventions are increasingly used to deliver physical activity interventions, yet problems with engagement and retention result in reduced effectiveness. Hence, alternative modes of online intervention delivery need to be explored. Therefore, this study aimed to evaluate the acceptability and effectiveness of a computer-tailored physical activity intervention delivered on the Internet in 3 delivery modes: video, text, or both. Australian adults (N = 803), recruited through e-mail, were randomized into the three delivery modes and received personal physical activity advice. Intervention content was identical across groups. Repeated measures analyses of variance were used to compare the three groups regarding acceptability, website usability, and physical activity. Participants in the video group accepted the content of the physical activity advice significantly better (F = 5.59; p < .01), and spent significantly more time on the website (F = 21.19; p < .001) compared with the text and combination groups. Total physical activity improved significantly over time in all groups (F = 3.95; p < .01). Although the combination group increased physical activity the most, few significant differences between groups were observed. Providing video-tailored feedback has advantages over the conventional text-tailored interventions; however, this study revealed few behavioral differences. More studies, examining alternative delivery modes, that can overcome the limitations of the present study, are needed.

  2. Ion-activated in situ gelling systems for sustained ophthalmic delivery of ciprofloxacin hydrochloride.

    PubMed

    Balasubramaniam, J; Pandit, J K

    2003-01-01

    The poor bioavailability and therapeutic response exhibited by the conventional ophthalmic solutions due to precorneal elimination of the drug may be overcome by the use of in situ gel forming systems that are instilled as drops into the eye and undergo a sol-gel transition in the cul-de-sac. Our present work describes the formulation and evaluation of an ophthalmic delivery system of an antibacterial agent, CPH, based on the concept of ion-activated in situ gelation. Gelrite gellan gum, a novel ophthalmic vehicle that gels in the presence of mono or divalent cations, present in the lacrimal fluid was used alone and in combinations with sodium alginate as the gelling agent. The developed formulations were therapeutically efficacious and provided sustained release of the drug over an 8-hr period in vitro.

  3. Synthesis, characterization, biodegradability and biocompatibility of a temperature-sensitive PBLA-PEG-PBLA hydrogel as protein delivery system with low critical gelation concentration.

    PubMed

    Xu, Yourui; Shen, Yan; Xiong, Yerong; Li, Chang; Sun, Chunmeng; Ouahab, Ammar; Tu, Jiasheng

    2014-09-01

    Temperature-sensitive hydrogels were designed using a series of A-B-A triblock copolymers consisting of poly (ethylene glycol) (PEG) with different molecular weights as the hydrophilic block B and poly (β-butyrolactone-co-lactic acid)(PBLA) with varying block lengths and composition as the hydrophobic block A. The triblock copolymers were synthesized by ring-opening polymerization (ROP) of β-BL and LA in bulk using PEG as an initiator and Sn(Oct)2 as the catalyst. Their chemical structure and molecular characteristics were determined by NMR, GPC and DSC, and the relationship between structure and phase behaviors in aqueous solutions was investigated as well. It was found that the phase behaviors in aqueous solutions including critical micelle concentration (CMC), sol-gel-sedimentation phase transition temperature, gel window width and critical gelation concentration (CGC) are largely dependent on the molecular weight and block length ratio of PEG/PBLA. Most importantly, they show a very low CGC ranging from 4 to 8 wt% because of the introduction of β-BL. Furthermore, the biodegradability and biocompatibility of the hydrogels were evaluated. Finally, lysozyme as a model protein was used to evaluate the ability to deliver protein drugs in a sustained release manner and biologically active form. All results demonstrated that the temperature-sensitive in situ forming hydrogel has a promising potential as sustained delivery system for protein drugs.

  4. Trithorax group proteins: switching genes on and keeping them active.

    PubMed

    Schuettengruber, Bernd; Martinez, Anne-Marie; Iovino, Nicola; Cavalli, Giacomo

    2011-11-23

    Cellular memory is provided by two counteracting groups of chromatin proteins termed Trithorax group (TrxG) and Polycomb group (PcG) proteins. TrxG proteins activate transcription and are perhaps best known because of the involvement of the TrxG protein MLL in leukaemia. However, in terms of molecular analysis, they have lived in the shadow of their more famous counterparts, the PcG proteins. Recent advances have improved our understanding of TrxG protein function and demonstrated that the heterogeneous group of TrxG proteins is of critical importance in the epigenetic regulation of the cell cycle, senescence, DNA damage and stem cell biology.

  5. Targeted delivery of chemotherapy using HSP90 inhibitor drug conjugates is highly active against pancreatic cancer models.

    PubMed

    Bobrov, Egor; Skobeleva, Natalia; Restifo, Diana; Beglyarova, Natalya; Cai, Kathy Q; Handorf, Elizabeth; Campbell, Kerry; Proia, David A; Khazak, Vladimir; Golemis, Erica A; Astsaturov, Igor

    2017-01-17

    The lack of effective treatment modalities is a major problem in pancreatic cancer (PCa), a devastating malignancy that is nearly universally driven by the "undruggable" KRAS and TP53 cancer genes. Poor tumor tissue penetration is the major source of resistance in pancreatic cancer where chemotherapy is the mainstay of treatment. In this study we exploited the selective tumor-targeting properties of the heat shock 90 protein inhibitors as the vehicle for drug delivery to pancreatic tumor tissues. STA-12-8666 is a novel esterase-cleavable conjugate of an HSP90i and a topoisomerase I inhibitor, SN-38. STA-12-8666 selectively binds activated HSP90 and releases its cytotoxic payload resulting in drug accumulation in pancreatic cancer cells in vivo. We investigated the preclinical activity of STA-12-8666 in patient derived xenograft and genetic models of pancreatic cancer.Treatment with STA-12-8666 of the KPC mice (knock-in alleles of LSL-KrasG12D, Tp53fl/fl and Pdx1-Cre transgene) at the advanced stages of pancreatic tumors doubled their survival (49 days vs. 74 days, p=0.008). STA-12-8666 also demonstrated dramatically superior activity in comparison to equimolar doses of irinotecan against 5 patient-derived pancreatic adenocarcinoma xenografts with prolonged remissions in some tumors. Analysis of activity of STA-12-8666 against tumor tissues and matched cell lines demonstrated prolonged accumulation and release of cytotoxic payload in the tumor leading to DNA damage response and cell cycle arrest.Our results provide a proof-of-principle validation that HSP90i-based drug conjugates can overcome the notorious treatment resistance by utilizing the inherently high affinity of pancreatic cancer cells to HSP90 antagonists.

  6. Nanoparticles for intracellular-targeted drug delivery

    NASA Astrophysics Data System (ADS)

    Paulo, Cristiana S. O.; Pires das Neves, Ricardo; Ferreira, Lino S.

    2011-12-01

    Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

  7. Effects of protein molecular weight on the intrinsic material properties and release kinetics of wet spun polymeric microfiber delivery systems.

    PubMed

    Lavin, Danya M; Zhang, Linda; Furtado, Stacia; Hopkins, Richard A; Mathiowitz, Edith

    2013-01-01

    Wet spun microfibers have great potential for the design of multifunctional controlled release scaffolds. Understanding aspects of drug delivery and mechanical strength, specific to protein molecular weight, may aid in the optimization and development of wet spun fiber platforms. This study investigated the intrinsic material properties and release kinetics of poly(l-lactic acid) (PLLA) and poly(lactic-co-glycolic acid) (PLGA) wet spun microfibers encapsulating proteins with varying molecular weights. A cryogenic emulsion technique developed in our laboratory was used to encapsulate insulin (5.8 kDa), lysozyme (14.3 kDa) and bovine serum albumin (BSA, 66.0 kDa) within wet spun microfibers (~100 μm). Protein loading was found to significantly influence mechanical strength and drug release kinetics of PLGA and PLLA microfibers in a molecular-weight-dependent manner. BSA encapsulation resulted in the most significant decrease in strength and ductility for both PLGA and PLLA microfibers. Interestingly, BSA-loaded PLGA microfibers had a twofold increase (8±2 MPa to 16±1 MPa) in tensile strength and a fourfold increase (3±1% to 12±6%) in elongation until failure in comparison to PLLA microfibers. PLGA and PLLA microfibers exhibited prolonged protein release up to 63 days in vitro. Further analysis with the Korsmeyer-Peppas kinetic model determined that the mechanism of protein release was dependent on Fickian diffusion. These results emphasize the critical role protein molecular weight has on the properties of wet spun filaments, highlighting the importance of designing small molecular analogues to replace growth factors with large molecular weights.

  8. Delivery of orally supplemented alpha-tocotrienol to vital organs of rats and tocopherol-transport protein deficient mice.

    PubMed

    Khanna, Savita; Patel, Viren; Rink, Cameron; Roy, Sashwati; Sen, Chandan K

    2005-11-15

    The natural vitamin E tocotrienol (TCT) possesses biological properties not shared by tocopherols (TCP). Nanomolar alpha-TCT, not alpha-TCP, is potently neuroprotective (JBC 275:13049; 278:43508). Tocopherol-transport protein (TTP) represents the primary mechanism for maintaining normal alpha-TCP concentrations in plasma and extrahepatic tissues. TTP primarily transports alpha-TCP and has low affinity for alpha-TCT. There are no studies that have investigated tissue delivery of alpha-TCT when orally gavaged on a long-term basis. A long-term study was conducted to examine the effects of alpha-TCT or alpha-TCP supplementation, either alone or in combination, on tissue levels. Rats were maintained on a vitamin E-deficient diet and gavaged with alpha-TCT or alpha-TCP alone or in combination. Five generations of rats were studied over 60 weeks. TTP-deficient mice were supplemented with TCT and bred to examine tissue delivery of oral alpha-TCT. Orally supplemented alpha-TCT was effectively delivered to most tissues over time. When co-supplemented, alpha-TCP outcompeted alpha-TCT for transport systems delivering vitamin E to tissues. To evaluate the significance of TTP in alpha-TCT delivery to tissues, tissue levels of alpha-TCT in supplemented TTP-deficient mice were studied. alpha-TCT was transported to several vital organs in TTP-deficient mice. alpha-TCT restored fertility in TTP-deficient mice. In sum, orally supplemented alpha-TCT was successfully delivered to several vital organs. The transport efficiency of alpha-TCT to tissues may be maximized by eliminating the co-presence of alpha-TCP in the oral supplement. Examination of whether alpha-TCT may benefit humans suffering from neurological disorders because of congenital TTP deficiency is warranted.

  9. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    PubMed Central

    Suh, Jin Sook; Lee, Jue Yeon; Choi, Yoon Jung; You, Hyung Keun; Hong, Seong-Doo; Chung, Chong Pyoung; Park, Yoon Jeong

    2014-01-01

    Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue

  10. [Increased fibrinolytic activity during cardiopulmonary bypass is caused by activated protein C system].

    PubMed

    Gando, S; Tedo, I; Masio, H; Goda, Y; Kawahigashi, H

    1994-06-01

    To examine the hypothesis that activated protein C system during cardiopulmonary bypass surgery may increase fibrinolytic activity during cardiopulmonary bypass, protein C activity, protein C antigen and thrombomodulin of sixteen patients undergoing elective cardiopulmonary bypass surgery were investigated after induction of anesthesia, before and after cardiopulmonary bypass, and at the end of operation. Protein C activity decreased and thrombomodulin increased significantly after the cardiopulmonary bypass. There were no significant correlations of thrombomodulin with protein C activity and protein C antigen. In conclusion, we have demonstrated that protein C system is activated and circulating thrombomodulin appears in the systemic circulation during cardiopulmonary bypass surgery and this enhanced activation of protein C system is possibly related to the reported increase of fibrinolytic activity during cardiopulmonary bypass.

  11. A Perspective on CRN Proteins in the Genomics Age: Evolution, Classification, Delivery and Function Revisited

    PubMed Central

    Amaro, Tiago M. M. M.; Thilliez, Gaëtan J. A.; Motion, Graham B.; Huitema, Edgar

    2017-01-01

    Plant associated microbes rely on secreted virulence factors (effectors) to modulate host immunity and ensure progressive infection. Amongst the secreted protein repertoires defined and studied in pathogens to date, the CRNs (for CRinkling and Necrosis) have emerged as one of only a few highly conserved protein families, spread across several kingdoms. CRN proteins were first identified in plant pathogenic oomycetes where they were found to be modular factors that are secreted and translocated inside host cells by means of a conserved N-terminal domain. Subsequent localization and functional studies have led to the view that CRN C-termini execute their presumed effector function in the host nucleus, targeting processes required for immunity. These findings have led to great interest in this large protein family and driven the identification of additional CRN-like proteins in other organisms. The identification of CRN proteins and subsequent functional studies have markedly increased the number of candidate CRN protein sequences, expanded the range of phenotypes tentatively associated with function and revealed some of their molecular functions toward virulence. The increased number of characterized CRNs also has presented a set of challenges that may impede significant progress in the future. Here, we summarize our current understanding of the CRNs and re-assess some basic assumptions regarding this protein family. We will discuss the latest findings on CRN biology and highlight exciting new hypotheses that have emanated from the field. Finally, we will discuss new approaches to study CRN functions that would lead to a better understanding of CRN effector biology as well as the processes that lead to host susceptibility and immunity. PMID:28217133

  12. Intracellular Delivery of Proteins and Nucleic Acids to Mammalian Cells With Invaplex Isolated From Shigella flexneri

    DTIC Science & Technology

    2004-12-01

    Kopecko, 1987: Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping...Oaks, E. V., C. K. Stover, and R. M. Rice, 1987: Molecular cloning and expression of Rickettsia tsutsugamushi genes for two major protein antigens...kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta56 gene and precise identification of

  13. Delivery of Prolamins to the Protein Storage Vacuole in Maize Aleurone Cells[W

    PubMed Central

    Reyes, Francisca C.; Chung, Taijoon; Holding, David; Jung, Rudolf; Vierstra, Richard; Otegui, Marisa S.

    2011-01-01

    Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals. PMID:21343414

  14. Delivery of a Protease-Activated Cytolytic Peptide Prodrug by Perfluorocarbon Nanoparticles.

    PubMed

    Jallouk, Andrew P; Palekar, Rohun U; Marsh, Jon N; Pan, Hua; Pham, Christine T N; Schlesinger, Paul H; Wickline, Samuel A

    2015-08-19

    Melittin is a cytolytic peptide derived from bee venom that inserts into lipid membranes and oligomerizes to form membrane pores. Although this peptide is an attractive candidate for treatment of cancers and infectious processes, its nonspecific cytotoxicity and hemolytic activity have limited its therapeutic applications. Several groups have reported the development of cytolytic peptide prodrugs that only exhibit cytotoxicity following activation by site-specific proteases. However, systemic administration of these constructs has proven difficult because of their poor pharmacokinetic properties. Here, we present a platform for the design of protease-activated melittin derivatives that may be used in conjunction with a perfluorocarbon nanoparticle delivery system. Although native melittin was substantially hemolytic (HD50: 1.9 μM) and cytotoxic (IC50: 2.4 μM), the prodrug exhibited 2 orders of magnitude less hemolytic activity (HD50: > 100 μM) and cytotoxicity (IC50: > 100 μM). Incubation with matrix metalloproteinase-9 (MMP-9) led to cleavage of the prodrug at the expected site and restoration of hemolytic activity (HD50: 3.4 μM) and cytotoxicity (IC50: 8.1 μM). Incubation of the prodrug with perfluorocarbon nanoparticles led to stable loading of 10,250 peptides per nanoparticle. Nanoparticle-bound prodrug was also cleaved and activated by MMP-9, albeit at a fourfold slower rate. Intravenous administration of prodrug-loaded nanoparticles in a mouse model of melanoma significantly decreased tumor growth rate (p = 0.01). Because MMPs and other proteases play a key role in cancer invasion and metastasis, this platform holds promise for the development of personalized cancer therapies directed toward a patient's individual protease expression profile.

  15. Regulated exocytosis contributes to protein kinase C potentiation of vanilloid receptor activity.

    PubMed

    Morenilla-Palao, Cruz; Planells-Cases, Rosa; García-Sanz, Nuria; Ferrer-Montiel, Antonio

    2004-06-11

    The vanilloid receptor-1 (TRPV1) plays a key role in the perception of peripheral thermal and inflammatory pain. TRPV1 expression and channel activity are notably up-regulated by proalgesic agents. The transduction pathways involved in TRPV1 sensitization are still elusive. We have used a yeast two-hybrid screen to identify proteins that associate with the N terminus of TRPV1. We report that two vesicular proteins, Snapin and synaptotagmin IX (Syt IX), strongly interact in vitro and in vivo with the TRPV1 N-terminal domain. In primary dorsal root ganglion neurons, TRPV1 co-distributes in vesicles with Syt IX and the vesicular protein synaptobrevin. Neither Snapin nor Syt IX affected channel function, but they notably inhibited protein kinase C (PKC)-induced potentiation of TRPV1 channel activity with a potency that rivaled the blockade evoked by botulinum neurotoxin A, a potent blocker of neuronal exocytosis. Noteworthily, we found that PKC activation induced a rapid delivery of functional TRPV1 channels to the plasma membrane. Botulinum neurotoxin A blocked the TRPV1 membrane translocation induced by PKC that was activated with a phorbol ester or the metabotropic glutamate receptor mGluR5. Therefore, our results indicate that PKC signaling promotes at least in part the SNARE-dependent exocytosis of TRPV1 to the cell surface. Taken together, these findings imply that activity-dependent delivery of channels to the neuronal surface may contribute to the buildup and maintenance of thermal inflammatory hyperalgesia in peripheral nociceptor terminals.

  16. Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae.

    PubMed

    Ioannou, Petros; Andrianaki, Aggeliki; Akoumianaki, Tonia; Kyrmizi, Irene; Albert, Nathaniel; Perlin, David; Samonis, George; Kontoyiannis, Dimitrios P; Chamilos, Georgios

    2015-12-07

    The modest in vitro activity of echinocandins against Aspergillus implies that host-related factors augment the action of these antifungal agents in vivo. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against various Aspergillus species under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (P = 0. 0005). Importantly, the enhanced activity of caspofungin against Aspergillus spp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinating Aspergillus hyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin with Aspergillus hyphae (P < 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery to Aspergillus hyphae.

  17. Linking Nontraditional Physical Activity and Preterm Delivery in Urban African-American Women

    PubMed Central

    Sealy-Jefferson, Shawnita; Hegner, Kristy; Misra, Dawn P.

    2015-01-01

    Background Traditional risk factors for preterm delivery (PTD) do not account for the disparate rates among African-American women. Physical activity during pregnancy may protect women from PTD, but few studies exist in African Americans. Our objective was to examine the relationships between PTD and intensity and duration of leisure time physical activity (LTPA) as well as non-LTPA such as stair climbing and walking for a purpose during pregnancy. Methods Data were from a hybrid retrospective/prospective cohort study of urban low-income African-American women enrolled from 2001 to 2004 in the Baltimore PTD Study (n = 832). PTD was defined as birth before 37 completed weeks of gestation. Study participants reported physical activity during prenatal (n = 456) and post-partum (n = 376) interviews. Findings The rate of PTD was 16.7%. In unadjusted log-binomial regression models, we found no significant associations. However, in models adjusted for illicit drug use, locus of control, and a validated family resources scale, we found a significant decrease in prevalence of PTD for women who walked for a purpose more than 30 min/d (prevalence ratio, 0.64; 95% CI, 0.43–0.94), compared with women who walked less than or equal to 30 min/d. Conclusions These results suggest that walking for a purpose during pregnancy may confer protection against PTD among urban low-income African Americans. PMID:24981398

  18. Preparation of albumin based nanoparticles for delivery of fisetin and evaluation of its cytotoxic activity.

    PubMed

    Ghosh, Pooja; Singha Roy, Atanu; Chaudhury, Susmitnarayan; Jana, Saikat Kumar; Chaudhury, Koel; Dasgupta, Swagata

    2016-05-01

    Fisetin is a well known flavonoid that shows several properties such as antioxidant, antiviral and anticancer activities. Its use in the pharmaceutical field is limited due to its poor aqueous solubility which results in poor bioavailability and poor permeability. The aim of our present study is to prepare fisetin loaded human serum albumin nanoparticles to improve its bioavailability. The nanoparticles were prepared by a desolvation method and characterized by spectroscopic and microscopic techniques. The particles were smooth and spherical in nature with an average size of 220 ± 8 nm. The encapsulation efficiency was found to be 84%. The in vitro release profile showed a biphasic pattern and the release rate increases with increase in ionic strength of solution. We have also confirmed the antioxidant activity of the prepared nanoparticles by a DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. Further its anticancer activity was evaluated using MCF-7 breast cancer cell lines. Our findings suggest that fisetin loaded HSA nanoparticles could be used to transfer fisetin to target areas under specific conditions and thus may find use as a delivery vehicle for the flavonoid.

  19. Synthesis of a novel, sequentially active-targeted drug delivery nanoplatform for breast cancer therapy.

    PubMed

    Satsangi, Arpan; Roy, Sudipa S; Satsangi, Rajiv K; Tolcher, Anthony W; Vadlamudi, Ratna K; Goins, Beth; Ong, Joo L

    2015-08-01

    Breast cancer is the leading cause of cancer deaths among women. Paclitaxel (PTX), an important breast cancer medicine, exhibits reduced bioavailability and therapeutic index due to high hydrophobicity and indiscriminate cytotoxicity. PTX encapsulation in one-level active targeting overcomes such barriers, but enhances toxicity to normal tissues with cancer-similar expression profiles. This research attempted to overcome this challenge by increasing selectivity of cancer cell targeting while maintaining an ability to overcome traditional pharmacological barriers. Thus, a multi-core, multi-targeting construct for tumor specific delivery of PTX was fabricated with (i) an inner-core prodrug targeting the cancer-overexpressed cathepsin B through a cathepsin B-cleavable tetrapeptide that conjugates PTX to a poly(amidoamine) dendrimer, and (ii) the encapsulation of this prodrug (PGD) in an outer core of a RES-evading, folate receptor (FR)-targeting liposome. Compared to traditional FR-targeting PTX liposomes, this sequentially active-targeted dendrosome demonstrated better prodrug retention, an increased cytotoxicity to cancer cells (latter being true when FR and cathepsin B activities were both at moderate-to-high levels) and higher tumor reduction. This research may eventually evolve a product platform with reduced systemic toxicity inherent with traditional chemotherapy and localized toxicity inherent to single-target nanoplatforms, thereby allowing for better tolerance of higher therapeutic load in advanced disease states.

  20. Encapsulation, protection, and release of hydrophilic active components: potential and limitations of colloidal delivery systems.

    PubMed

    McClements, David Julian

    2015-05-01

    There have been major advances in the development of edible colloidal delivery systems for hydrophobic bioactives in recent years. However, there are still many challenges associated with the development of effective delivery systems for hydrophilic bioactives. This review highlights the major challenges associated with developing colloidal delivery systems for hydrophilic bioactive components that can be utilized in foods, pharmaceuticals, and other products intended for oral ingestion. Special emphasis is given to the fundamental physicochemical phenomena associated with encapsulation, stabilization, and release of these bioactive components, such as solubility, partitioning, barriers, and mass transport processes. Delivery systems suitable for encapsulating hydrophilic bioactive components are then reviewed, including liposomes, multiple emulsions, solid fat particles, multiple emulsions, biopolymer particles, cubosomes, and biologically-derived systems. The advantages and limitations of each of these delivery systems are highlighted. This information should facilitate the rational selection of the most appropriate colloidal delivery systems for particular applications in the food and other industries.

  1. Nanoscale Drug Delivery Platforms Overcome Platinum-Based Resistance in Cancer Cells Due to Abnormal Membrane Protein Trafficking

    PubMed Central

    Xue, Xue; Hall, Matthew D.; Zhang, Qiang; Wang, Paul C.; Gottesman, Michael M.; Liang, Xing-Jie

    2014-01-01

    The development of cellular resistance to platinum-based chemotherapies is often associated with reduced intracellular platinum concentrations. In some models, this reduction is due to abnormal membrane protein trafficking, resulting in reduced uptake by transporters at the cell surface. Given the central role of platinum drugs in the clinic, it is critical to overcome cisplatin resistance by bypassing the plasma membrane barrier to significantly increase the intracellular cisplatin concentration enough to inhibit the proliferation of cisplatin-resistant cells. Therefore, rational design of appropriate nanoscale drug delivery platforms (nDDPs) loaded with cisplatin or other platinum analogs as payloads is a possible strategy to solve this problem. This review will focus on the known mechanism of membrane trafficking in cisplatin-resistant cells, and the development and employment of nDDPs to improve cell uptake of cisplatin. PMID:24219825

  2. Nanoscale drug delivery platforms overcome platinum-based resistance in cancer cells due to abnormal membrane protein trafficking.

    PubMed

    Xue, Xue; Hall, Matthew D; Zhang, Qiang; Wang, Paul C; Gottesman, Michael M; Liang, Xing-Jie

    2013-12-23

    The development of cellular resistance to platinum-based chemotherapies is often associated with reduced intracellular platinum concentrations. In some models, this reduction is due to abnormal membrane protein trafficking, resulting in reduced uptake by transporters at the cell surface. Given the central role of platinum drugs in the clinic, it is critical to overcome cisplatin resistance by bypassing the plasma membrane barrier to significantly increase the intracellular cisplatin concentration enough to inhibit the proliferation of cisplatin-resistant cells. Therefore, rational design of appropriate nanoscale drug delivery platforms (nDDPs) loaded with cisplatin or other platinum analogues as payloads is a possible strategy to solve this problem. This review will focus on the known mechanism of membrane trafficking in cisplatin-resistant cells and the development and employment of nDDPs to improve cell uptake of cisplatin.

  3. Co-delivery and controlled release of stromal cell-derived factor-1α chemically conjugated on collagen scaffolds enhances bone morphogenetic protein-2-driven osteogenesis in rats

    PubMed Central

    SUN, HAIPENG; WANG, JINMING; DENG, FEILONG; LIU, YUN; ZHUANG, XIUMEI; XU, JIAYUN; LI, LONG

    2016-01-01

    There has been considerable focus in investigations on the delivery systems and clinical applications of bone morphogenetic protein-2 (BMP-2) for novel bone formation. However, current delivery systems require high levels of BMP-2 to exert a biological function. There are several concerns in using of high levels of BMP-2, including safety and the high cost of treatment. Therefore, the development of strategies to decrease the levels of BMP-2 required in these delivery systems is required. In our previous studies, a controlled-release system was developed, which used Traut's reagent and the cross-linker, 4-(N-maleimi-domethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC), to chemically conjugate BMP-2 directly on collagen discs. In the current study, retention efficiency and release kinetics of stromal cell-derived factor-1α (SDF-1α) cross-linked on collagen scaffolds were detected. In addition, the osteogenic activity of SDF-1α and suboptimal doses of BMP-2 cross-linked on collagen discs following subcutaneous implantation in rats were evaluated. Independent two-tailed t-tests and one-way analysis of variance were used for analysis. In the present study, the controlled release of SDF-1α chemically conjugated on collagen scaffolds was demonstrated. By optimizing the concentrations of Traut's reagent and the Sulfo-SMCC cross-linker, a significantly higher level of SDF-1α was covalently retained on the collagen scaffold, compared with that retained using a physical adsorption method. Mesenchymal stem cell homing indicated that the biological function of the SDF-1α cross-linked on the collagen scaffolds remained intact. In rats, co-treatment with SDF-1α and a suboptimal dose of BMP-2 cross-linked on collagen scaffolds using this chemically conjugated method induced higher levels of ectopic bone formation, compared with the physical adsorption method. No ectopic bone formation was observed following treatment with a

  4. In situ synthesis of new magnetite chitosan/carrageenan nanocomposites by electrostatic interactions for protein delivery applications.

    PubMed

    Long, Jie; Yu, Xiaoqin; Xu, Enbo; Wu, Zhengzong; Xu, Xueming; Jin, Zhengyu; Jiao, Aiquan

    2015-10-20

    We present a simple method to develop magnetite chitosan/carrageenan nanocomposites by in situ synthesis under mild conditions, and then their potential for controlled release of macromolecules was also evaluated. The structural, morphological and magnetic properties of the as-prepared materials were studied by vibrating sample magnetometer, X-ray diffractometer, Fourier transform infrared spectroscopy, thermogravimetric analyzer and transmission electron microscopy. With the varying mass ratio (chitosan to Fe3O4-carrageenan nanocomposite), the developed nanocarriers presented sizes within 73-355nm and zeta potentials of -42-32mV. Using bovine serum albumin as model protein, the adsorption and release behaviors were investigated. Nanocarriers evidenced excellent loading capacity of 181mgg(-1) at protein concentration of 0.2mgmL(-1), and demonstrated capacity to provide a sustained release up to 85% of adsorbed protein in 30min in intestinal medium rather than acidic medium. These results suggest that the developed magnetite chitosan/carrageenan nanocomposites are promising in the application of magnetically targeted delivery of therapeutic macromolecules.

  5. Identification of highly active flocculant proteins in bovine blood.

    PubMed

    Piazza, George J; Nuñez, Alberto; Garcia, Rafael A

    2012-03-01

    Synthetic polymeric flocculants are used extensively for wastewater remediation, soil stabilization, and reduction in water leakage from unlined canals. Sources of highly active, inexpensive, renewable flocculants are needed to replace synthetic flocculants. High kaolin flocculant activity was documented for bovine blood (BB) and blood plasma with several anticoagulant treatments. BB serum also had high flocculant activity. To address the hypothesis that some blood proteins have strong flocculating activity, the BB proteins were separated by SEC. Then, the major proteins of the flocculant-active fractions were separated by SDS-PAGE. Identity of the major protein components was determined by tryptic digestion and peptide analysis by MALDI TOF MS. The sequence of selected peptides was confirmed using TOF/TOF-MS/MS fragmentation. Hemoglobin dimer (subunits α and β) was identified as the major protein component of the active fraction in BB; its high flocculation activity was confirmed by testing a commercial sample of hemoglobin. In the same manner, three proteins from blood plasma (fibrinogen, γ-globulin, α-2-macroglobulin) were found to be highly active flocculants, but bovine serum albumin, α-globulin, and β-globulin were not flocculants. On a mass basis, hemoglobin, γ-globulin, α-2-macroglobulin were as effective as anionic polyacrylamide (PAM), a widely used synthetic flocculant. The blood proteins acted faster than PAM, and unlike PAM, the blood proteins flocculants did not require calcium salts for their activity.

  6. Role of chitosan co-formulation in enhancing interleukin-12 delivery and antitumor activity

    PubMed Central

    Yang, Lirong; Zaharoff, David A.

    2013-01-01

    Local delivery systems that provide sustained, high concentrations of antitumor cytokines in the tumor microenvironment while minimizing systemic dissemination are needed to realize the potential of cytokine-based immunotherapies. Recently, co-formulations of cytokines with chitosan solutions have been shown to increase local cytokine retention and bioactivity. In particular, intratumoral (i.t.) injections of chitosan/IL-12 can eliminate established tumors and generate tumor-specific immune responses. In the present study, we explored the mechanisms by which chitosan potentiated IL-12’s antitumor activity. The location of chitosan/IL-12 injection was found to be critical for optimal cytokine delivery. I.t. injections eliminated 9 of 10 MC38 adenocarcinomas while contralateral and peritumoral injections delayed tumor growth but could not eliminate tumors. Microdosing studies demonstrated that IL-12 depots, simulated through daily i.t. injections with IL-12 alone, were not as effective as weekly i.t. chitosan/IL-12. 50–75% of mice receiving daily IL-12 microdoses and 87.5% of mice receiving weekly chitosan/IL-12 were cured of MC38 tumors. Chitosan was found to increase IL-12-mediated leukocytic expansion in tumors and tumor-draining lymph nodes (TDLNs) by 40% and 100%, respectively. Immunophenotyping studies demonstrated that chitosan co-formulation amplified IL-12-induced increases in important effector populations, such as CD8+IFN-γ+ and NKT cells, in tumors and dendritic cell populations in TDLNs. Remarkable increases in Gr-1+CD11b+ tumor infiltrates were also observed in mice receiving chitosan or chitosan/IL-12. This population does not appear be suppressive and may facilitate the local antitumor response. Presented data suggest that chitosan-mediated depot formation and enhanced local cytokine retention is significantly, but not entirely, responsible for increased cytokine bioactivity. PMID:23453060

  7. Hydrophobic drug-triggered self-assembly of nanoparticles from silk-elastin-like protein polymers for drug delivery.

    PubMed

    Xia, Xiao-Xia; Wang, Ming; Lin, Yinan; Xu, Qiaobing; Kaplan, David L

    2014-03-10

    Silk-elastin-like protein polymers (SELPs) combine the mechanical and biological properties of silk and elastin. These properties have led to the development of various SELP-based materials for drug delivery. However, SELPs have rarely been developed into nanoparticles, partially due to the complicated fabrication procedures, nor assessed for potential as an anticancer drug delivery system. We have recently constructed a series of SELPs (SE8Y, S2E8Y, and S4E8Y) with various ratios of silk to elastin blocks and described their capacity to form micellar-like nanoparticles upon thermal triggering. In this study, we demonstrate that doxorubicin, a hydrophobic antitumor drug, can efficiently trigger the self-assembly of SE8Y (SELPs with silk to elastin ratio of 1:8) into uniform micellar-like nanoparticles. The drug can be loaded in the SE8Y nanoparticles with an efficiency around 6.5% (65 ng doxorubicin/μg SE8Y), S2E8Y with 6%, and S4E8Y with 4%, respectively. In vitro studies with HeLa cell lines demonstrate that the protein polymers are not cytotoxic (IC50 > 200 μg/mL), while the doxorubicin-loaded SE8Y nanoparticles showed a 1.8-fold higher cytotoxicity than the free drug. Confocal laser scanning microscopy (CLSM) and flow cytometry indicate significant uptake of the SE8Y nanoparticles by the cells and suggest internalization of the nanoparticles through endocytosis. This study provides an all-aqueous, facile method to prepare nanoscale, drug-loaded SELPs packages with potential for tumor cell treatments.

  8. Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides

    PubMed Central

    Bilichak, Andriy; Luu, Justin; Eudes, François

    2015-01-01

    Microspores are specialized generative cells with haploid genome that demonstrate the amenability toward embryogenesis under certain conditions. The induced microspore culture technique is largely exploited by the breeding programs of wheat and other crops due to its high efficiency for generation of the large number of haploid plants in the relatively short period of time. The ability to produce mature double haploid plant from a single cell has also attracted attention of the plant biotechnologists in the past few years. More importantly, the possibility to deliver proteins for improvement of embryogenesis and the genome modification purposes holds great potential for transgene-free wheat biotechnology. In the present study, we examined the ability of cationic and amphipathic cell penetrating peptides (CPPs) to convey a covalently-linked mCherry protein inside the viable microspores. We demonstrate that the affinity of CPPs to the microspore cells dependents on their charge with the highest efficiency of CPP-mCherry binding to the cells achieved by cationic CPPs (penetratin and R9). Additionally, due to overall negative charge of the microspore cell wall, the successful uptake of the protein cargo by live microspore cells is attained by utilization of a reversible disulfide bond between the R9 CPP and mCherry protein. Overall, the approach proposed herein can be applied by the other biotechnology groups for the fast and efficient screening of the different CPP candidates for their ability to deliver proteins inside the viable plant cells. PMID:26379691

  9. Global Analysis of Protein Activities Using Proteome Chips

    NASA Astrophysics Data System (ADS)

    Zhu, Heng; Bilgin, Metin; Bangham, Rhonda; Hall, David; Casamayor, Antonio; Bertone, Paul; Lan, Ning; Jansen, Ronald; Bidlingmaier, Scott; Houfek, Thomas; Mitchell, Tom; Miller, Perry; Dean, Ralph A.; Gerstein, Mark; Snyder, Michael

    2001-09-01

    To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

  10. Canine parvovirus NS1 protein exhibits anti-tumor activity in a mouse mammary tumor model.

    PubMed

    Gupta, Shishir Kumar; Yadav, Pavan Kumar; Gandham, Ravi Kumar; Sahoo, A P; Harish, D R; Singh, Arvind Kumar; Tiwari, A K

    2016-02-02

    Many viral proteins have the ability to kill tumor cells specifically without harming the normal cells. These proteins, on ectopic expression, cause lysis or induction of apoptosis in the target tumor cells. Parvovirus NS1 is one of such proteins, which is known to kill high proliferating tumor cells. In the present study, we assessed the apoptosis inducing ability of canine parvovirus type 2 NS1 protein (CPV2.NS1) in vitro in 4T1 cells, and found it to cause significant cell death due to induction of apoptosis through intrinsic or mitochondrial pathway. Further, we also evaluated the oncolytic activity of CPV2.NS1 protein in a mouse mammary tumor model. The results suggested that CPV2.NS1 was able to inhibit the growth of 4T1 induced mouse mammary tumor as indicated by significantly reduced tumor volume, mitotic, AgNOR and PCNA indices. Further, inhibition of tumor growth was found to be because of induction of apoptosis in the tumor cells, which was evident by a significant increase in the number of TUNEL positive cells. Further, CPV2.NS1 was also able to stimulate the immune cells against the tumor antigens as indicated by the increased CD4+ and CD8+ counts in the blood of CVP2.NS1 treated mice. Further optimization of the delivery of NS1 protein and use of an adjuvant may further enhance its anti-tumor activity.

  11. A Near-Infrared Laser-Activated "Nanobomb" for Breaking the Barriers to MicroRNA Delivery.

    PubMed

    Wang, Hai; Agarwal, Pranay; Zhao, Shuting; Yu, Jianhua; Lu, Xiongbin; He, Xiaoming

    2016-01-13

    A near-infrared laser-activated "nanobomb" is synthesized using lipid and multiple polymers to break the extra-cellular and intracellular barriers to cytosolic delivery of microRNAs. The nanobomb can be used to effectively destroy tumors and cancer stem-like cells in vitro and in vivo with minimal side effects.

  12. Dual subcellular compartment delivery of doxorubicin to overcome drug resistant and enhance antitumor activity

    PubMed Central

    Song, Yan-feng; Liu, Dao-zhou; Cheng, Ying; Liu, Miao; Ye, Wei-liang; Zhang, Bang-le; Liu, Xin-you; Zhou, Si-yuan

    2015-01-01

    In order to overcome drug resistant and enhance antitumor activity of DOX, a new pH-sensitive micelle (DOX/DQA-DOX@DSPE-hyd-PEG-AA) was prepared to simultaneously deliver DOX to nucleus and mitochondria. Drug released from DOX/DQA-DOX@DSPE-hyd-PEG-AA showed a pH-dependent manner. DOX/DQA-DOX@DSPE-hyd-PEG-AA induced the depolarization of mitochondria and apoptosis in MDA-MB-231/ADR cells and A549 cells, which resulted in the high cytotoxicity of DOX/DQA-DOX@DSPE-hyd-PEG-AA against MDA-MB-231/ADR cells and A549 cells. Confocal microscopy confirmed that DOX/DQA-DOX@DSPE-hyd-PEG-AA simultaneously delivered DQA-DOX and DOX to the mitochondria and nucleus of tumor cell. After DOX/DQA-DOX@DSPE-hyd-PEG-AA was injected to the tumor-bearing nude mice by the tail vein, DOX was mainly found in tumor tissue. But DOX was widely distributed in the whole body after the administration of free DOX. Compared with free DOX, the same dose of DOX/DQA-DOX@DSPE-hyd-PEG-AA significantly inhibited the growth of DOX-resistant tumor in tumor-bearing mice without obvious systemic toxicity. Therefore, dual subcellular compartment delivery of DOX greatly enhanced the antitumor activity of DOX on DOX-resistant tumor. DOX/DQA-DOX@DSPE-hyd-PEG-AA has the potential in target therapy for DOX-resistant tumor. PMID:26530454

  13. Targeted antigen delivery and activation of dendritic cells in vivo: steps towards cost effective vaccines.

    PubMed

    Tacken, Paul J; Figdor, Carl G

    2011-02-01

    During the past decade, the immunotherapeutic potential of ex vivo generated professional antigen presenting dendritic cells (DCs) has been explored in the clinic. Albeit safe, clinical results have thus far been limited. A major disadvantage of current cell-based dendritic cell (DC) therapies, preventing universal implementation of this form of immunotherapy, is the requirement that vaccines need to be tailor made for each individual. Targeted delivery of antigens to DC surface receptors in vivo would circumvent this laborious and expensive ex vivo culturing steps involved with these cell-based therapies. In addition, the opportunity to target natural and often rare DC subsets in vivo might have advantages over loading more artificial ex vivo cultured DCs. Preclinical studies show targeting antigens to DCs effectively induces humoral responses, while cellular responses are induced provided a DC maturation or activation stimulus is co-administered. Here, we discuss strategies to target antigens to distinct DC subsets and to simultaneously employ adjuvants to activate these cells to induce immunity.

  14. Sustained ocular delivery of brimonidine tartrate using ion activated in situ gelling system.

    PubMed

    Geethalakshmi, A; Karki, Roopa; Jha, Sajal Kumar; Venkatesh, D P; Nikunj, B

    2012-03-01

    The poor bioavailability and therapeutic response exhibited by conventional eye drops due to rapid precorneal elimination of the drug may be overcome by the use of an in situ gelling systems that are instilled as drops into the eye and undergo a sol-to-gel transition in the cul-de-sac which improves patient compliance as the dosage regimen is one drop of the dosage form twice a day. The loss of drug overcomes due to the immediate gel formation between the eye membrane and the drug being entrapped simultaneously in sol-gel transition in the cul de sac. The present work describes the formulation and evaluation of an ophthalmic delivery system of an antiglaucomal agent, brimonidine tartrate based on the concept of ion-activated in situ gelation. Gelrite was used as the gelling agent, which gels in the presence of mono or divalent cations present in the lacrimal fluid. The formulations were evaluated for clarity, pH measurement, gelling capacity, drug content estimation, rheological study, in-vitro diffusion study, antibacterial activity, isotonicity testing, eye irritation testing. In the developed formulations Gelrite Brimonidine-3 (GB3) exhibited sustained release of drug from formulation over a period of 8 hrs thus increasing residence time of the drug, non-irritating with no ocular damage or abnormal clinical signs to the cornea, iris or conjunctiva, stable and sterile. These results demonstrate that the developed system is an alternative to conventional ophthalmic drops, with better patient compliance, and is industrially oriented and economical.

  15. Delivery of methoxymorpholinyl doxorubicin by interleukin 2-activated NK cells: effect in mice bearing hepatic metastases

    PubMed Central

    Quintieri, L; Rosato, A; Amboldi, N; Vizler, C; Ballinari, D; Zanovello, P; Collavo, D

    1999-01-01

    The possibility of using interleukin 2 (IL-2)-activated natural killer cells (A-NK) to carry methoxymorpholinyl doxorubicin (MMDX; PNU 152243) to liver-infiltrating tumours was explored in mice bearing 2-day established M5076 reticulum cell sarcoma hepatic metastases. In vitro, MMDX was 5.5-fold more potent than doxorubicin against M5076 tumour cells. MMDX uptake by A-NK cells correlated linearly with drug concentration in the incubation medium [correlation coefficient (r) = 0.999]; furthermore, as MMDX incorporation was readily reproducible in different experiments, the amount of drug delivered by A-NK cells could be modulated. In vivo experiments showed that intravenous (i.v.) injection of MMDX-loaded A-NK cells exerted a greater therapeutic effect than equivalent or even higher doses of free drug. The increase in lifespan (ILS) following A-NK cell delivery of 53 μg kg−1 MMDX, a dosage that is ineffective when administered in free form, was similar to that observed in response to 92 μg kg−1 free drug, a dosage close to the 10% lethal dose (ILS 42% vs. 38% respectively). These results correlated with pharmacokinetic studies showing that MMDX encapsulation in A-NK cells strongly modifies its organ distribution and targets it to tissues in which IL-2 activated lymphocytes are preferentially entrapped after i.v. injection. © 1999 Cancer Research Campaign PMID:10098738

  16. Development of a long-acting, protein-loaded, redox-active, injectable gel formed by a polyion complex for local protein therapeutics.

    PubMed

    Ishii, Shiro; Kaneko, Junya; Nagasaki, Yukio

    2016-04-01

    Although cancer immunotherapies are attracting much attention, it is difficult to develop bioactive proteins owing to the severe systemic toxicity. To overcome the issue, we designed new local protein delivery system by using a protein-loaded, redox-active, injectable gel (RIG), which is formed by a polyion complex (PIC) comprising three components, viz., cationic polyamine-poly(ethylene glycol)-polyamine triblock copolymer possessing ROS-scavenging moieties as side chains; anionic poly(acrylic acid); and a protein. The mixture formed the protein-loaded PIC flower micelles at room temperature, which immediately converted to a gel with high mechanical strength upon exposure to physiological conditions. Because the protein electrostatically interacts with the PIC gel network, RIG provided a sustained release of the protein without a significant initial burst, regardless of the types of proteins in vitro, and much longer retention of the protein at the local injection site in mice than that of the naked protein. Subcutaneous injections of IL-12@RIG in the vicinity of tumor tissue showed remarkable tumor growth inhibition in tumor-bearing mice, compared to that observed with injection of IL-12 alone, suppressing adverse events caused by IL-12-induced ROS. Our results indicate that RIG has potential as a platform technology for an injectable sustained-release carrier for proteins.

  17. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal po...

  18. Controlled intra- and transdermal protein delivery using a minimally invasive Erbium:YAG fractional laser ablation technology.

    PubMed

    Bachhav, Y G; Heinrich, A; Kalia, Y N

    2013-06-01

    The aim of the study was (i) to investigate the feasibility of using fractional laser ablation to create micropore arrays in order to deliver proteins into and across the skin and (ii) to demonstrate how transport rates could be controlled by variation of poration and formulation conditions. Four proteins with very different structures and properties were investigated - equine heart cytochrome c (Cyt c; 12.4 kDa), recombinant human growth hormone expressed in Escherichia coli (hGH; 22 kDa), urinary follicle stimulating hormone (FSH; 30 kDa) and FITC-labelled bovine serum albumin (FITC-BSA; 70 kDa). The transport experiments were performed using a scanning Er:YAG diode pumped laser (P.L.E.A.S.E.®; Precise Laser Epidermal System). The distribution of FITC-BSA in the micropores following P.L.E.A.S.E.® poration was visualised by using confocal laser scanning microscopy (CLSM). Porcine skin was used for the device parameter and CLSM studies; its validity as a model was confirmed by subsequent comparison with transport of Cyt c and FITC-BSA across P.L.E.A.S.E.® porated human skin. No protein transport (deposition or permeation) was observed across intact skin; however, P.L.E.A.S.E.® poration enabled total delivery after 24h of 48.2±8.9, 8.1±4.2, 0.2±0.1 and 273.3±30.6 μg/cm(2) for Cyt c, hGH, FSH and FITC-BSA, respectively, using 900 pores/135.9 cm(2). Calculation of permeability coefficients showed that there was no linear dependence of transport on molecular weight ((1.6±0.3), (0.1±0.05), (0.08±0.03) and (0.9±0.1)×10(-3) cm/h, for Cyt c, hGH, FSH and FITC-BSA, respectively); indeed, a U-shaped curve was observed. This suggested that molecular weight was not a sufficiently sensitive descriptor and that transport was more likely to be determined by the surface properties of the respective proteins since these would govern interactions with the local microenvironment. Increasing pore density (i.e. the number of micropores per unit area) had a statistically

  19. TAT-Mediated Delivery of Tousled Protein to Salivary Glands Protects Against Radiation-Induced Hypofunction

    SciTech Connect

    Sunavala-Dossabhoy, Gulshan; Palaniyandi, Senthilnathan; Richardson, Charles; De Benedetti, Arrigo; Schrott, Lisa; Caldito, Gloria

    2012-09-01

    Purpose: Patients treated with radiotherapy for head-and-neck cancer invariably suffer its deleterious side effect, xerostomia. Salivary hypofunction ensuing from the irreversible destruction of glands is the most common and debilitating oral complication affecting patients undergoing regional radiotherapy. Given that the current management of xerostomia is palliative and ineffective, efforts are now directed toward preventive measures to preserve gland function. The human homolog of Tousled protein, TLK1B, facilitates chromatin remodeling at DNA repair sites and improves cell survival against ionizing radiation (IR). Therefore, we wanted to determine whether a direct transfer of TLK1B protein to rat salivary glands could protect against IR-induced salivary hypofunction. Methods: The cell-permeable TAT-TLK1B fusion protein was generated. Rat acinar cell line and rat salivary glands were pretreated with TAT peptide or TAT-TLK1B before IR. The acinar cell survival in vitro and salivary function in vivo were assessed after radiation. Results: We demonstrated that rat acinar cells transduced with TAT-TLK1B were more resistant to radiation (D{sub 0} = 4.13 {+-} 1.0 Gy; {alpha}/{beta} = 0 Gy) compared with cells transduced with the TAT peptide (D{sub 0} = 4.91 {+-} 1.0 Gy; {alpha}/{beta} = 20.2 Gy). Correspondingly, retroductal instillation of TAT-TLK1B in rat submandibular glands better preserved salivary flow after IR (89%) compared with animals pretreated with Opti-MEM or TAT peptide (31% and 39%, respectively; p < 0.01). Conclusions: The results demonstrate that a direct transfer of TLK1B protein to the salivary glands effectively attenuates radiation-mediated gland dysfunction. Prophylactic TLK1B-protein therapy could benefit patients undergoing radiotherapy for head-and-neck cancer.

  20. Chemically modified inulin microparticles serving dual function as a protein antigen delivery vehicle and immunostimulatory adjuvant.

    PubMed

    Gallovic, Matthew D; Montjoy, Douglas G; Collier, Michael A; Do, Clement; Wyslouzil, Barbara E; Bachelder, Eric M; Ainslie, Kristy M

    2016-03-01

    To develop a new subunit vaccine adjuvant, we chemically modified a naturally-occurring, immunostimulatory inulin polysaccharide to produce an acid-sensitive biopolymer (acetalated inulin, Ace-IN). Various hydrophobic Ace-IN polymers were formed into microparticles (MPs) by oil-in-water emulsions followed by solvent evaporation These Ace-IN MPs possessed tunable degradation characteristics that, unlike polyesters used in FDA-approved microparticulate formulations, had only pH-neutral hydrolytic byproducts. Macrophages were passively targeted with cytocompatible Ace-IN MPs. TNF-α production by macrophages treated with Ace-IN MPs could be altered by adjusting the polymers' chemistry. Mice immunized with Ace-IN MPs encapsulating a model ovalbumin (OVA) antigen showed higher production of anti-OVA IgG antibody levels relative to soluble antigen. The antibody titers were also comparable to an alum-based formulation. This proof-of-concept establishes the potential for chemically-modified inulin MPs to simultaneously enable dual functionality as a stimuli-controlled antigen delivery vehicle and immunostimulatory adjuvant.

  1. 78 FR 23945 - Agency Information Collection Activities: Entry and Immediate Delivery Application and Simplified...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-23

    ... Delivery Application and Simplified Entry AGENCY: U.S. Customs and Border Protection, Department of... Delivery Application (Forms 3461 and 3461 ALT) and Simplified Entry. This is a proposed extension of an..., or other technological techniques or other forms of information. Title: Entry and Immediate...

  2. CRISPRs for Optimal Targeting: Delivery of CRISPR Components as DNA, RNA, and Protein into Cultured Cells and Single-Cell Embryos

    PubMed Central

    Kouranova, Evguenia; Forbes, Kevin; Zhao, Guojun; Warren, Joe; Bartels, Angela; Wu, Yumei; Cui, Xiaoxia

    2016-01-01

    The rapid development of CRISPR technology greatly impacts the field of genetic engineering. The simplicity in design and generation of highly efficient CRISPR reagents allows more and more researchers to take on genome editing in different model systems in their own labs, even for those who found it daunting before. An active CRISPR complex contains a protein component (Cas9) and an RNA component (small guide RNA [sgRNA]), which can be delivered into cells in various formats. Cas9 can be introduced as a DNA expression plasmid, in vitro transcripts, or as a recombinant protein bound to the RNA portion in a ribonucleoprotein particle (RNP), whereas the sgRNA can be delivered either expressed as a DNA plasmid or as an in vitro transcript. Here we compared the different delivery methods in cultured cell lines as well as mouse and rat single-cell embryos and view the RNPs as the most convenient and efficient to use. We also report the detection of limited off-targeting in cells and embryos and discuss approaches to lower that chance. We hope that researchers new to CRISPR find our results helpful to their adaptation of the technology for optimal gene editing. PMID:27094534

  3. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells.

    PubMed

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-10-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants.

  4. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    PubMed

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

  5. Microfluidics-assisted engineering of polymeric microcapsules with high encapsulation efficiency for protein drug delivery.

    PubMed

    Pessi, Jenni; Santos, Hélder A; Miroshnyk, Inna; JoukoYliruusi; Weitz, David A; Mirza, Sabiruddin

    2014-09-10

    In this study, microfluidic technology was employed to develop protein formulations. The microcapsules were produced with a biphasic flow to create water-oil-water (W/O/W) double emulsion droplets with ultrathin shells. Optimized microcapsule formulations containing 1% (w/w) bovine serum albumin (BSA) in the inner phase were prepared with poly(vinyl alcohol), polycaprolactone and polyethylene glycol. All the particles were found to be intact and with a particle size of 23-47 μm. Furthermore, the particles were monodisperse, non-porous and stable up to 4 weeks. The encapsulation efficiency of BSA in the microcapsules was 84%. The microcapsules released 30% of their content within 168 h. This study demonstrates that microfluidics is a powerful technique for engineering formulations for therapeutic proteins.

  6. AKAP-Lbc nucleates a protein kinase D activation scaffold.

    PubMed

    Carnegie, Graeme K; Smith, F Donelson; McConnachie, George; Langeberg, Lorene K; Scott, John D

    2004-09-24

    The transmission of cellular signals often proceeds through multiprotein complexes where enzymes are positioned in proximity to their upstream activators and downstream substrates. In this report we demonstrate that the A-kinase anchoring protein AKAP-Lbc assembles an activation complex for the lipid-dependent enzyme protein kinase D (PKD). Using a combination of biochemical, enzymatic, and immunofluorescence techniques, we show that the anchoring protein contributes to PKD activation in two ways: it recruits an upstream kinase PKCeta and coordinates PKA phosphorylation events that release activated protein kinase D. Thus, AKAP-Lbc synchronizes PKA and PKC activities in a manner that leads to the activation of a third kinase. This configuration illustrates the utility of kinase anchoring as a mechanism to constrain the action of broad-spectrum enzymes.

  7. Regulation of the activity of protein kinases by endogenous heat stable protein inhibitors.

    PubMed

    Szmigielski, A

    1985-01-01

    Protein kinase activities are regulated by endogenous thermostable protein inhibitors. Type I inhibitor is a protein of MW 22,000-24,000 which inhibits specifically cyclic AMP-(cAMP) dependent protein kinase (APK) as a competitive inhibitor of catalytic subunits of the enzyme. Type I inhibitor activity changes inversely according to the activation of adenylate cyclase and the changes in cAMP content in tissues. It seems that type I inhibitor serves as a factor preventing spontaneous cAMP-dependent phosphorylation in unstimulated cell. The other thermostable protein which inhibits APK activity has been found in Sertoli cell-enriched testis (testis inhibitor). Physiological role of the testis inhibitor is unknown. Type II inhibitor is a protein of MW 15,000 which blocks phosphorylation mediated by cAMP and cyclic GMP (cGMP) dependent (APK and GPK) and cyclic nucleotide independent protein kinases as a competitive inhibitor of substrate proteins. Activity of this inhibitor specifically changes in reciprocal manner to the changes in cGMP content. It seems that type II inhibitor serves as a factor preventing the phosphorylation catalyzed by GPK when cGMP content is low. Stimulation of guanylate cyclase and activation of GPK is followed by a decrease of type II inhibitor activity. This change in relationship between activities of GPK and type II inhibitor allows for effective phosphorylation catalyzed by this enzyme when cGMP content is increased.

  8. Liquid filled nanoparticles as a drug delivery tool for protein therapeutics.

    PubMed

    Venkatesan, Natarajan; Yoshimitsu, Junichiro; Ito, Yukako; Shibata, Nobuhito; Takada, Kanji

    2005-12-01

    In the present study, an attempt was made to study the feasibility of nanoparticulate adsorbents in the presence of an absorption enhancer, as a drug delivery tool for the administration of erythropoietin (EPO) to the small intestine. Liquid filled nano- and micro-particles (LFNPS/LFMPS) were prepared using solid adsorbents such as porous silicon dioxide (Sylysia 550), carbon nanotubes (CNTs), carbon nanohorns, fullerene, charcoal and bamboo charcoal. Surfactants such as a saturated polyglycolysed C8-C18 glyceride (Gelucire 44/14), PEG-8 capryl/caprylic acid glycerides (Labrasol) and polyoxyethylene hydrogenated castor oil derivative (HCO-60) were used as an absorption enhancer at 50mg/kg along with casein/lactoferrin as enzyme inhibitors. The absorption of EPO was studied by measuring serum EPO levels by an ELISA method after small intestinal administration of EPO-LFNPS preparation to rats at the EPO dose level of 100 IU/kg. Among the adsorbents studied, CNTs showed the highest serum EPO level of 62.7 +/- 11.6 mIU/ml. In addition, with the use of casein, EPO absorption was improved, C(max) 143.1 +/- 15.2 mIU/ml. Labrasol showed the highest absorption enhancing effect after intra-jejunum administration than Gelucire 44/14 and HCO-60, 25.6 +/- 3.2 and 22.2 +/- 3.6 mIU/ml, respectively. Jejunum was found to be the best absorption site for the absorption of EPO from LFNPS. The use of CNTs as LFNPS, improved the bioavailability of EPO to 11.5% following intra-small intestinal administration.

  9. Mussel-Inspired Protein Nanoparticles Containing Iron(III)-DOPA Complexes for pH-Responsive Drug Delivery.

    PubMed

    Kim, Bum Jin; Cheong, Hogyun; Hwang, Byeong Hee; Cha, Hyung Joon

    2015-06-15

    A novel bioinspired strategy for protein nanoparticle (NP) synthesis to achieve pH-responsive drug release exploits the pH-dependent changes in the coordination stoichiometry of iron(III)-3,4-dihydroxyphenylalanine (DOPA) complexes, which play a major cross-linking role in mussel byssal threads. Doxorubicin-loaded polymeric NPs that are based on Fe(III)-DOPA complexation were thus synthesized with a DOPA-modified recombinant mussel adhesive protein through a co-electrospraying process. The release of doxorubicin was found to be predominantly governed by a change in the structure of the Fe(III)-DOPA complexes induced by an acidic pH value. It was also demonstrated that the fabricated NPs exhibited effective cytotoxicity towards cancer cells through efficient cellular uptake and cytosolic release. Therefore, it is anticipated that Fe(III)-DOPA complexation can be successfully utilized as a new design principle for pH-responsive NPs for diverse controlled drug-delivery applications.

  10. Safety and efficacy of self-assembling bubble carriers stabilized with sodium dodecyl sulfate for oral delivery of therapeutic proteins.

    PubMed

    Lin, Po-Yen; Chuang, Er-Yuan; Chiu, Yi-Hsuan; Chen, Hsin-Lung; Lin, Kun-Ju; Juang, Jyuhn-Huarng; Chiang, Ching-Hua; Mi, Fwu-Long; Sung, Hsing-Wen

    2016-12-20

    Sodium dodecyl sulfate (SDS) is generally regarded as a potent permeability enhancer in oral formulations; however, one concern related to the use of any permeation enhancer is its possible absorption of unwanted toxins during the period of epithelial permeability enhancement. In this work, the safety and efficacy of an SDS-containing bubble carrier system that is developed from an orally administered enteric-coated capsule are evaluated. The bubble carriers comprise diethylene triamine pentaacetic acid (DTPA) dianhydride, sodium bicarbonate (SBC), SDS, and insulin. Upon exposure to the intestinal fluid, DTPA dianhydride hydrolyzes to yield acids, and SBC rapidly reacts with these acids to generate CO2, producing bubble carriers, each containing a self-assembling water film. The hydrophilic insulin is entrapped in the self-assembled water film, which is stabilized by SDS. The SDS in the bubble carrier system can act as a dissolution enhancer in the dispersion of insulin molecules, as a surfactant that stabilizes the bubble carriers, as a protease inhibitor that protects the protein drug, and as a permeation enhancer that augments its oral bioavailability. Hence, a significant increase in the plasma insulin level and an excellent blood glucose-lowering response in diabetic rats are effectively achieved. Moreover, the enhancement of epithelial permeation by this SDS-containing formulation does not promote the absorption of intestinal endotoxins. The above facts indicate that the bubble carrier system that is stabilized by SDS can be used as a safe and potent carrier in the oral delivery of therapeutic proteins.

  11. Delivery of endocytosed proteins to the cell-division plane requires change of pathway from recycling to secretion.

    PubMed

    Richter, Sandra; Kientz, Marika; Brumm, Sabine; Nielsen, Mads Eggert; Park, Misoon; Gavidia, Richard; Krause, Cornelia; Voss, Ute; Beckmann, Hauke; Mayer, Ulrike; Stierhof, York-Dieter; Jürgens, Gerd

    2014-04-08

    Membrane trafficking is essential to fundamental processes in eukaryotic life, including cell growth and division. In plant cytokinesis, post-Golgi trafficking mediates a massive flow of vesicles that form the partitioning membrane but its regulation remains poorly understood. Here, we identify functionally redundant Arabidopsis ARF guanine-nucleotide exchange factors (ARF-GEFs) BIG1-BIG4 as regulators of post-Golgi trafficking, mediating late secretion from the trans-Golgi network but not recycling of endocytosed proteins to the plasma membrane, although the TGN also functions as an early endosome in plants. In contrast, BIG1-4 are absolutely required for trafficking of both endocytosed and newly synthesized proteins to the cell-division plane during cytokinesis, counteracting recycling to the plasma membrane. This change from recycling to secretory trafficking pathway mediated by ARF-GEFs confers specificity of cargo delivery to the division plane and might thus ensure that the partitioning membrane is completed on time in the absence of a cytokinesis-interphase checkpoint. DOI: http://dx.doi.org/10.7554/eLife.02131.001.

  12. Self-assembled multicompartment liquid crystalline lipid carriers for protein, peptide, and nucleic acid drug delivery.

    PubMed

    Angelova, Angelina; Angelov, Borislav; Mutafchieva, Rada; Lesieur, Sylviane; Couvreur, Patrick

    2011-02-15

    Lipids and lipopolymers self-assembled into biocompatible nano- and mesostructured functional materials offer many potential applications in medicine and diagnostics. In this Account, we demonstrate how high-resolution structural investigations of bicontinuous cubic templates made from lyotropic thermosensitive liquid-crystalline (LC) materials have initiated the development of innovative lipidopolymeric self-assembled nanocarriers. Such structures have tunable nanochannel sizes, morphologies, and hierarchical inner organizations and provide potential vehicles for the predictable loading and release of therapeutic proteins, peptides, or nucleic acids. This Account shows that structural studies of swelling of bicontinuous cubic lipid/water phases are essential for overcoming the nanoscale constraints for encapsulation of large therapeutic molecules in multicompartment lipid carriers. For the systems described here, we have employed time-resolved small-angle X-ray scattering (SAXS) and high-resolution freeze-fracture electronic microscopy (FF-EM) to study the morphology and the dynamic topological transitions of these nanostructured multicomponent amphiphilic assemblies. Quasi-elastic light scattering and circular dichroism spectroscopy can provide additional information at the nanoscale about the behavior of lipid/protein self-assemblies under conditions that approximate physiological hydration. We wanted to generalize these findings to control the stability and the hydration of the water nanochannels in liquid-crystalline lipid nanovehicles and confine therapeutic biomolecules within these structures. Therefore we analyzed the influence of amphiphilic and soluble additives (e.g. poly(ethylene glycol)monooleate (MO-PEG), octyl glucoside (OG), proteins) on the nanochannels' size in a diamond (D)-type bicontinuous cubic phase of the lipid glycerol monooleate (MO). At body temperature, we can stabilize long-living swollen states, corresponding to a diamond cubic phase

  13. The specific activation of TRPC4 by Gi protein subtype.

    PubMed

    Jeon, Jae-Pyo; Lee, Kyu Pil; Park, Eun Jung; Sung, Tae Sik; Kim, Byung Joo; Jeon, Ju-Hong; So, Insuk

    2008-12-12

    The classical type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channels in mammalian cells. Especially, TRPC4 has the similar properties to Ca(2+)-permeable nonselective cation channels (NSCCs) activated by muscarinic stimulation in visceral smooth muscles. In visceral smooth muscles, NSCCs activated by muscarinic stimulation were blocked by anti-Galphai/o antibodies. However, there is still no report which Galpha proteins are involved in the activation process of TRPC4. Among Galpha proteins, only Galphai protein can activate TRPC4 channel. The activation effect of Galphai was specific for TRPC4 because Galphai has no activation effect on TRPC5, TRPC6 and TRPV6. Coexpression with muscarinic receptor M2 induced TRPC4 current activation by muscarinic stimulation with carbachol, which was inhibited by pertussis toxin. These results suggest that Galphai is involved specifically in the activation of TRPC4.

  14. Delivery of the ribosome-inactivating protein, gelonin, to lymphoma cells via CD22 and CD38 using bispecific antibodies.

    PubMed Central

    French, R. R.; Penney, C. A.; Browning, A. C.; Stirpe, F.; George, A. J.; Glennie, M. J.

    1995-01-01

    It is well established that bispecific antibodies (BsAbs) can be used effectively in targeting the ribosome-inactivating protein (RIP), saporin, against neoplastic B cells. We have now extended this delivery system for use with gelonin. By measuring antigen-binding characteristics and epitope mapping a panel of anti-gelonin MAbs using the IAsys resonant mirror bisensor, we were able to rapidly select the most suitable for making BaAbs. The Fab' fragments from these MAbs were chemically conjugated with Fab' from either anti-CD22 or anti-CD38. Cytotoxicity assays showed that BsAbs were highly efficient at delivering gelonin to cultured Daudi cells and achieved levels of toxicity which correlated closely with the affinity of the BsAbs. Using pairs of anti-CD22 BsAbs we were able to generate bivalent BsAb-gelonin complexes which achieved IC50 values of 2 x 10(-11) M gelonin, a potency which is equivalent to that reached by saporin in this targeting system. However, because gelonin is 5-10 times less toxic than saporin, the therapeutic ratio for gelonin is superior, making it potentially a more useful agent for human treatment. Cytotoxicity assays and kinetic analysis showed that targeting gelonin via CD38 was 2-5 times less effective than delivery through CD22. However, with a pair of BsAbs designed to co-target gelonin via CD22 and CD38, the cytotoxicity achieved equalled that obtained with a pair of anti-CD22 BsAbs (IC50 = 1 x 10(-11) M). This important result suggests that the anti-CD38 helps bind the gelonin to the cell and is then 'dragged' or 'piggy-backed' into the cell by the anti-CD22 BsAb. The implication of these findings for cancer therapy is discussed. PMID:7734325

  15. Interfacial engineering using mixed protein systems: emulsion-based delivery systems for encapsulation and stabilization of β-carotene.

    PubMed

    Mao, Yingyi; Dubot, Marie; Xiao, Hang; McClements, David Julian

    2013-05-29

    Emulsion-based delivery systems are needed to encapsulate, protect, and deliver lipophilic bioactive components in the food, personal care, and pharmaceutical industries. The functional performance of these systems can be controlled by engineering the composition and structure of the interfacial layer coating the lipid droplets. In this study, interfacial properties were controlled using two globular proteins with widely differing isoelectric points: lactoferrin (LF: pI ≈ 8.5) and β-lactoglobulin (BLG: pI ≈ 5). Oil-in-water emulsions were prepared with different interfacial properties: [LF]-only; [BLG]-only; [LF]-[BLG]-(laminated); [BLG]-[LF]-(laminated); and [BLG/LF]-(mixed). The influence of pH, ionic strength, and temperature on the physical stability of β-carotene-enriched emulsions was investigated. [LF]-emulsions were stable to droplet aggregation from pH 2 to 9 (0 mM NaCl), but all other emulsions aggregated at intermediate pH values. [BLG]-emulsions aggregated at high salt levels (≥50 mM NaCl), but all other emulsions were stable (0 to 300 mM NaCl). [BLG/LF]-emulsions were unstable to heating (≥60 °C), but all other emulsions were stable (30 to 90 °C). Color fading due to β-carotene degradation occurred relatively quickly in [BLG]-emulsions (37 °C) but was considerably lower in all other emulsions, which was attributed to the ability of LF to bind iron or interact with β-carotene. This study provides useful information for designing emulsion-based delivery systems to encapsulate and protect bioactive lipids, such as carotenoids.

  16. Lanthanide-Doped Upconversion Nanoparticles: Emerging Intelligent Light-Activated Drug Delivery Systems.

    PubMed

    Bagheri, Ali; Arandiyan, Hamidreza; Boyer, Cyrille; Lim, May

    2016-07-01

    The development of drug delivery systems (DDSs) using near infrared (NIR) light and upconversion nanoparticles (UCNPs) has generated intensive interest over the past five years. These NIR-initiated DDSs not only offer a high degree of spatial and temporal determination of therapeutic release but also provide precise control over the released dosage. Furthermore, these nanoplatforms confer several advantages over conventional light-based DDSs-NIR offers better tissue penetration depth and a reduced risk of cellular photo-damage caused by exposure to light at high-energy wavelengths (e.g., ultraviolet light, <400 nm). The development of DDSs that can be activated by low intensity NIR illumination is highly desirable to avoid exposing living tissues to excessive heat that can limit the in vivo application of these DDSs. This encompasses research in three directions: (i) enhancing the quantum yield of the UCNPs; (ii) incorporation of photo-responsive materials with red-shifted absorptions into the UCNPs; and (iii) tuning the UCNPs excitation wavelength. This review focuses on recent advances in the development of NIR-initiated DDS, with emphasis on the use of photo-responsive compounds and polymeric materials conjugated onto UCNPs. The challenges that limit UCNPs clinical applications, alongside with the aforementioned techniques that have emerged to overcome these limitations, are highlighted.

  17. Nanosuspension delivery of paclitaxel to xenograft mice can alter drug disposition and anti-tumor activity

    NASA Astrophysics Data System (ADS)

    Chiang, Po-Chang; Gould, Stephen; Nannini, Michelle; Qin, Ann; Deng, Yuzhong; Arrazate, Alfonso; Kam, Kimberly R.; Ran, Yingqing; Wong, Harvey

    2014-04-01

    Paclitaxel is a common chemotherapeutic agent that is effective against various cancers. The poor aqueous solubility of paclitaxel necessitates a large percentage of Cremophor EL:ethanol (USP) in its commercial formulation which leads to hypersensitivity reactions in patients. We evaluate the use of a crystalline nanosuspension versus the USP formulation to deliver paclitaxel to tumor-bearing xenograft mice. Anti-tumor efficacy was assessed following intravenous administration of three 20 mg/kg doses of paclitaxel. Paclitaxel pharmacokinetics and tissue distribution were evaluated, and differences were observed between the two formulations. Plasma clearance and tissue to plasma ratio of mice that were dosed with the nanosuspension are approximately 33- and 11-fold higher compared to those of mice that were given the USP formulation. Despite a higher tumor to plasma ratio for the nanosuspension treatment group, absolute paclitaxel tumor exposure was higher for the USP group. Accordingly, a higher anti-tumor effect was observed in the xenograft mice that were dosed with the USP formulation (90% versus 42% tumor growth inhibition). This reduction in activity of nanoparticle formulation appeared to result from a slower than anticipated dissolution in vivo. This study illustrates a need for careful consideration of both dose and systemic solubility prior utilizing nanosuspension as a mode of intravenous delivery.

  18. Nanosuspension delivery of paclitaxel to xenograft mice can alter drug disposition and anti-tumor activity.

    PubMed

    Chiang, Po-Chang; Gould, Stephen; Nannini, Michelle; Qin, Ann; Deng, Yuzhong; Arrazate, Alfonso; Kam, Kimberly R; Ran, Yingqing; Wong, Harvey

    2014-04-01

    Paclitaxel is a common chemotherapeutic agent that is effective against various cancers. The poor aqueous solubility of paclitaxel necessitates a large percentage of Cremophor EL:ethanol (USP) in its commercial formulation which leads to hypersensitivity reactions in patients. We evaluate the use of a crystalline nanosuspension versus the USP formulation to deliver paclitaxel to tumor-bearing xenograft mice. Anti-tumor efficacy was assessed following intravenous administration of three 20 mg/kg doses of paclitaxel. Paclitaxel pharmacokinetics and tissue distribution were evaluated, and differences were observed between the two formulations. Plasma clearance and tissue to plasma ratio of mice that were dosed with the nanosuspension are approximately 33- and 11-fold higher compared to those of mice that were given the USP formulation. Despite a higher tumor to plasma ratio for the nanosuspension treatment group, absolute paclitaxel tumor exposure was higher for the USP group. Accordingly, a higher anti-tumor effect was observed in the xenograft mice that were dosed with the USP formulation (90% versus 42% tumor growth inhibition). This reduction in activity of nanoparticle formulation appeared to result from a slower than anticipated dissolution in vivo. This study illustrates a need for careful consideration of both dose and systemic solubility prior utilizing nanosuspension as a mode of intravenous delivery.

  19. Applicator for in-vitro ultrasound-activated targeted drug delivery

    NASA Astrophysics Data System (ADS)

    Gerold, B.; Gourevich, D.; Volovick, A.; Xu, D.; Arditti, F.; Prentice, P.; Cochran, S.; Gnaim, J.; Medan, Y.; Wang, L.; Melzer, A.

    2012-10-01

    Reducing toxicity and improving uptake of cancer drugs in tumors are important goals of targeted drug delivery (TDD). Ultrasonic drug release from various encapsulants has been a focus of many research groups. However, a single standard ultrasonic device, viable for use by biologists, is not currently present in the market. The device reported here is designed to allow investigation of the impact of ultrasound on cellular uptake and cell viability in-vitro. In it, single-element transducers with different operating frequencies are mounted below a standard 96-well plate. The plate is moved above the transducers, such that each line of wells can be sonicated at a different frequency. To assess the device, 96-well plates were seeded with cells and sonicated using different ultrasonic parameters, with and without doxorubicin. Cell viability was measured by colorimetric MTT assay and the uptake of doxorubicin by cells was also determined. The device proved to be highly viable in preliminary tests; it demonstrated that change in ultrasonic parameters produces different effect on cells. For example, increase in uptake of doxorubicin was demonstrated following ultrasound application. The growing interest in ultrasound-activated TDD emphasizes the need for standardization of the ultrasound device and the one reported here may offer some indications of how that may be achieved. It is planned to further improve the prototype by increasing the number of ultrasonic frequencies and degrees of freedom for each transducer.

  20. Extracellularly activated nanocarriers: A new paradigm of tumor targeted drug delivery

    PubMed Central

    Gullotti, Emily; Yeo, Yoon

    2009-01-01

    One of the main goals of nanomedicine is to develop a nanocarrier that can selectively deliver anti-cancer drugs to the targeted tumors. Extensive efforts have resulted in several tumor-targeted nanocarriers, some of which are approved for clinical use. Most nanocarriers achieve tumor-selective accumulation through the enhanced permeability and retention effect. Targeting molecules such as antibodies, peptides, ligands, or nucleic acids attached to the nanocarriers further enhance their recognition and internalization by the target tissues. While both the stealth and targeting features are important for effective and selective drug delivery to the tumors, achieving both features simultaneously is often found to be difficult. Some of the recent targeting strategies have the potential to overcome this challenge. These strategies utilize the unique extracellular environment of tumors to change the long-circulating nanocarriers to release the drug or interact with cells in a tumor-specific manner. This review discusses the new targeting strategies with recent examples, which utilize the environmental stimuli to activate the nanocarriers. Traditional strategies for tumor-targeted nanocarriers are briefly discussed with an emphasis on their achievements and challenges. PMID:19366234

  1. Lanthanide‐Doped Upconversion Nanoparticles: Emerging Intelligent Light‐Activated Drug Delivery Systems

    PubMed Central

    Bagheri, Ali; Arandiyan, Hamidreza

    2016-01-01

    The development of drug delivery systems (DDSs) using near infrared (NIR) light and upconversion nanoparticles (UCNPs) has generated intensive interest over the past five years. These NIR‐initiated DDSs not only offer a high degree of spatial and temporal determination of therapeutic release but also provide precise control over the released dosage. Furthermore, these nanoplatforms confer several advantages over conventional light‐based DDSs—NIR offers better tissue penetration depth and a reduced risk of cellular photo‐damage caused by exposure to light at high‐energy wavelengths (e.g., ultraviolet light, <400 nm). The development of DDSs that can be activated by low intensity NIR illumination is highly desirable to avoid exposing living tissues to excessive heat that can limit the in vivo application of these DDSs. This encompasses research in three directions: (i) enhancing the quantum yield of the UCNPs; (ii) incorporation of photo‐responsive materials with red‐shifted absorptions into the UCNPs; and (iii) tuning the UCNPs excitation wavelength. This review focuses on recent advances in the development of NIR‐initiated DDS, with emphasis on the use of photo‐responsive compounds and polymeric materials conjugated onto UCNPs. The challenges that limit UCNPs clinical applications, alongside with the aforementioned techniques that have emerged to overcome these limitations, are highlighted. PMID:27818904

  2. Sustained ophthalmic delivery of ofloxacin from an ion-activated in situ gelling system.

    PubMed

    Abraham, Sindhu; Furtado, Sharon; Bharath, S; Basavaraj, B V; Deveswaran, R; Madhavan, V

    2009-04-01

    The poor bioavailability and therapeutic response exhibited by conventional ophthalmic solutions due to rapid pre-corneal elimination of the drug may be overcome by the use of in situ gel forming systems that are instilled as drops into the eye and then undergo a sol-gel transition in the cul-de-sac. The present work describes the formulation and evaluation of an ophthalmic delivery system of an antibacterial agent ofloxacin, based on the concept of ion-activated in situ gelation. Sodium alginate was used as the gelling agent in combination with HPC (Hydroxy Propyl Cellulose) that acted as a viscosity-enhancing agent. In vitro release studies indicated that the alginate/HPC solution retained the drug better than the alginate or HPC solutions alone. The formulations were therapeutically efficacious, sterile, stable and provided sustained release of the drug over a period of time. These results demonstrate that the developed system is an alternative to conventional ophthalmic drops, patient compliance, industrially oriented and economical.

  3. Parameters affecting the transscleral delivery of two positively charged proteins of comparable size.

    PubMed

    Grimaudo, Maria Aurora; Tratta, Elena; Pescina, Silvia; Padula, Cristina; Santi, Patrizia; Nicoli, Sara

    2017-02-20

    Apart from molecular weight and net surface charge, there are other macromolecule-related factors that could, in principle, influence their diffusion across biological tissues, such as shape, conformability, water solubility and surface charge distribution. Lysozyme and cytochrome c, proteins with comparable molecular weight, isoelectric point and net surface charge in physiological conditions (approx. +7.8), are suitable model compounds for comparative studies, in particular to find out if other properties can have a role in the permeation across the sclera. The comparison between lysozyme and cytochrome c permeability was conducted by studying the permeation across the sclera and the choroid-Bruch's membrane and the diffusion across a hyaluronan gel-matrix. Melanin binding tests and the measurement of the electroosmosis flow during transscleral iontophoresis allowed for the evaluation of macromolecules affinity for the ocular tissues. Finally, anodal iontophoresis was applied to further confirm the interaction of the two proteins with the sclera. The data here collected show that two proteins with very similar MW, p Ka and charge can display very different diffusion properties across biological barriers. In particular, these differences can be attributed to a different interaction with specific components of ocular tissues: while the interaction with melanin and collagen fibers is apparently the same for the two molecules, a relevant difference was found in case of hyaluronic acid. Considering also literature evidences, the important parameters that can be responsible for this different affinity are molecular shape (spherical for cytochrome c vs prolate for lysozyme) and a combination of hydrophobic and electrostatic interactions that depends on the surface charge distribution. The interactions between sclera components and lysozyme are relatively strong and were not altered by the application of electric current.

  4. Providing a diet containing only maintenance levels of energy and protein during the latter stages of pregnancy resulted in a prolonged delivery time during parturition in rats.

    PubMed

    Tanaka, Y; Kadokawa, H

    2012-01-01

    In mammals, a prolonged delivery time during parturition is dangerous for both mother and fetus, although the mechanisms that prolong delivery are unclear. To investigate whether nutrition affects delivery time, we administered two feeds containing maintenance (L-feed) or higher (H-feed) levels of energy and protein at different points during the latter half of pregnancy and compared the effects of the various treatments on delivery time in rats. After the rats had been maintained on the L-feed and then copulated on pro-oestrus (Day 0), pregnant females were randomly allocated to one of three groups: (1) the no-improvement group, which was fed L-feed throughout gestation; (2) the early group, which was fed L-feed until Day 11 of gestation and then switched to H-feed; and (3) the late group, which was fed L-feed until Day 16 of gestation and then switched to H-feed. There was no significant difference in the number of pups among the three groups. However, delivery time was significantly longer in the no-improvement group (73.7±5.2 min) than the early (46.9±5.6 min) and late (55.4±5.5 min) groups. Consuming a maintenance diet during the latter half of pregnancy resulted in a prolonged delivery time.

  5. Protein Crystal Growth Activities on STS-42

    NASA Technical Reports Server (NTRS)

    1992-01-01

    The Protein Crystal Growth (PCG) middeck payload is currently manifested to fly on STS-42 in January 1992. This payload is a joint effort between NASA s Office of Commercial Programs (OCP) and Office of Space Science and Applications (OSSA). The PCG experiments are managed by the Center for Macromolecular Crystallography (CMC), a NASA Center for the Commercial Development of Space (CCDS) based at the University of Alabama at Birmingham (UAB). This is the eighth flight of a payload in the PCG program that is jointly sponsored by the OCP and the OSSA. The flight hardware for STS-42 includes six Vapor Diffusion Apparatus (VDA) trays stored in two Refrigerator/Incubator Modules (R/TM s). The VDA trays will simultaneously conduct 120 experiments involving 15 different protein compounds, four of which are sponsored by the OCP, the UAB CCDS, and four co-investigators.

  6. Long-Circulating Heparin-Functionalized Magnetic Nanoparticles for Potential Application as a Protein Drug Delivery Platform

    PubMed Central

    Zhang, Jian; Shin, Meong Cheol; David, Allan E.; Zhou, Jie; Lee, Kyuri; He, Huining; Yang, Victor C.

    2013-01-01

    Starch-coated, PEGylated and heparin-functionalized iron oxide magnetic nanoparticles (DNPH) were successfully synthesized and characterized in detail. The PEGylation (20 kDa) process resulted in an average coating of 430 PEG molecules per nanoparticle. After that, heparin conjugation was carried out to attain the final DNPH platform with 35.4 μg of heparin/mg Fe. Commercially acquired heparin-coated magnetic nanoparticles were also PEGylated (HP) and characterized for comparison. Protamine was selected as a model protein to demonstrate the strong binding affinity and high loading content of DNPH for therapeutically relevant cationic proteins. DNPH showed a maximum loading of 22.9 μg protamine/mg Fe. In the pharmacokinetic study, DNPH displayed a long-circulating half-life of 9.37 h, 37.5-fold longer than that (0.15 h) of H P. This improved plasma stability enabled extended exposure of DNPH to the tumor lesions, as was visually confirmed in a flank 9L-glioma mouse model using magnetic resonance imaging (MRI). Quantitative analysis of the Fe content in excised tumor lesions further demonstrated the superior tumor targeting ability of DNPH, with up to 31.36 μg Fe/g tissue (13.07% injected dose (I.D.)/g tissue) and 7.5-fold improvement over that (4.27 μg Fe/g tissue; 1.78% I.D./g tissue) of HP. Overall, DNPH shed light of the potential to be used as a protein drug delivery platform. PMID:24024964

  7. An isoprenylation and palmitoylation motif promotes intraluminal vesicle delivery of proteins in cells from distant species.

    PubMed

    Oeste, Clara L; Pinar, Mario; Schink, Kay O; Martínez-Turrión, Javier; Stenmark, Harald; Peñalva, Miguel A; Pérez-Sala, Dolores

    2014-01-01

    The C-terminal ends of small GTPases contain hypervariable sequences which may be posttranslationally modified by defined lipid moieties. The diverse structural motifs generated direct proteins towards specific cellular membranes or organelles. However, knowledge on the factors that determine these selective associations is limited. Here we show, using advanced microscopy, that the isoprenylation and palmitoylation motif of human RhoB (-CINCCKVL) targets chimeric proteins to intraluminal vesicles of endolysosomes in human cells, displaying preferential co-localization with components of the late endocytic pathway. Moreover, this distribution is conserved in distant species, including cells from amphibians, insects and fungi. Blocking lipidic modifications results in accumulation of CINCCKVL chimeras in the cytosol, from where they can reach endolysosomes upon release of this block. Remarkably, CINCCKVL constructs are sorted to intraluminal vesicles in a cholesterol-dependent process. In the lower species, neither the C-terminal sequence of RhoB, nor the endosomal distribution of its homologs are conserved; in spite of this, CINCCKVL constructs also reach endolysosomes in Xenopus laevis and insect cells. Strikingly, this behavior is prominent in the filamentous ascomycete fungus Aspergillus nidulans, in which GFP-CINCCKVL is sorted into endosomes and vacuoles in a lipidation-dependent manner and allows monitoring endosomal movement in live fungi. In summary, the isoprenylated and palmitoylated CINCCKVL sequence constitutes a specific structure which delineates an endolysosomal sorting strategy operative in phylogenetically diverse organisms.

  8. Electric field-induced direct delivery of proteins by a nanofountain probe

    PubMed Central

    Loh, Owen Y.; Ho, Andrea M.; Rim, Jee E.; Kohli, Punit; Patankar, Neelesh A.; Espinosa, Horacio D.

    2008-01-01

    We report nanofabrication of protein dot and line patterns using a nanofountain atomic force microscopy probe (NFP). Biomolecules are continuously fed in solution through an integrated microfluidic system, and deposited directly onto a substrate. Deposition is controlled by application of an electric potential of appropriate sign and magnitude between the probe reservoir and substrate. Submicron dot and line molecular patterns were generated with resolution that depended on the magnitude of the applied voltage, dwell time, and writing speed. By using an energetic argument and a Kelvin condensation model, the quasi-equilibrium liquid–air interface at the probe tip was determined. The analysis revealed the origin of the need for electric fields in achieving protein transport to the substrate and confirmed experimental observations suggesting that pattern resolution is controlled by tip sharpness and not overall probe aperture. As such, the NFP combines the high-resolution of dip-pen nanolithography with the efficient continuous liquid feeding of micropipettes while allowing scalability to 1- and 2D probe arrays for high throughput. PMID:18946047

  9. A protein delivery system: biodegradable alginate-chitosan-poly(lactic-co-glycolic acid) composite microspheres.

    PubMed

    Zheng, Cai-Hong; Gao, Jian-Qing; Zhang, Ye-Ping; Liang, Wen-Quan

    2004-10-29

    In the present study we developed alginate-chitosan-poly(lactic-co-glycolic acid) (PLGA) composite microspheres to elevate protein entrapment efficiency and decrease its burst release. Bovine serum albumin (BSA), which used as the model protein, was entrapped into the alginate microcapsules by a modified emulsification method in an isopropyl alcohol-washed way. The rapid drug releases were sustained by chitosan coating. To obtain the desired release properties, the alginate-chitosan microcapsules were further incorporated in the PLGA to form the composite microspheres. The average diameter of the composite microcapsules was 31+/-9microm and the encapsulation efficiency was 81-87%, while that of conventional PLGA microspheres was just 61-65%. Furthermore, the burst releases at 1h of BSA entrapped in composite microspheres which containing PLGA (50:50) and PLGA (70:30) decreased to 24% and 8% in PBS, and further decreased to 5% and 2% in saline. On the contrary, the burst releases of conventional PLGA microspheres were 48% and 52% in PBS, respectively. Moreover, the release profiles could be manipulated by regulating the ratios of poly(lactic acid) to poly(glycolic acid) in the composite microspheres.

  10. An Isoprenylation and Palmitoylation Motif Promotes Intraluminal Vesicle Delivery of Proteins in Cells from Distant Species

    PubMed Central

    Oeste, Clara L.; Pinar, Mario; Schink, Kay O.; Martínez-Turrión, Javier; Stenmark, Harald; Peñalva, Miguel A.; Pérez-Sala, Dolores

    2014-01-01

    The C-terminal ends of small GTPases contain hypervariable sequences which may be posttranslationally modified by defined lipid moieties. The diverse structural motifs generated direct proteins towards specific cellular membranes or organelles. However, knowledge on the factors that determine these selective associations is limited. Here we show, using advanced microscopy, that the isoprenylation and palmitoylation motif of human RhoB (–CINCCKVL) targets chimeric proteins to intraluminal vesicles of endolysosomes in human cells, displaying preferential co-localization with components of the late endocytic pathway. Moreover, this distribution is conserved in distant species, including cells from amphibians, insects and fungi. Blocking lipidic modifications results in accumulation of CINCCKVL chimeras in the cytosol, from where they can reach endolysosomes upon release of this block. Remarkably, CINCCKVL constructs are sorted to intraluminal vesicles in a cholesterol-dependent process. In the lower species, neither the C-terminal sequence of RhoB, nor the endosomal distribution of its homologs are conserved; in spite of this, CINCCKVL constructs also reach endolysosomes in Xenopus laevis and insect cells. Strikingly, this behavior is prominent in the filamentous ascomycete fungus Aspergillus nidulans, in which GFP-CINCCKVL is sorted into endosomes and vacuoles in a lipidation-dependent manner and allows monitoring endosomal movement in live fungi. In summary, the isoprenylated and palmitoylated CINCCKVL sequence constitutes a specific structure which delineates an endolysosomal sorting strategy operative in phylogenetically diverse organisms. PMID:25207810

  11. A logical molecular circuit for programmable and autonomous regulation of protein activity using DNA aptamer-protein interactions.

    PubMed

    Han, Da; Zhu, Zhi; Wu, Cuichen; Peng, Lu; Zhou, Leiji; Gulbakan, Basri; Zhu, Guizhi; Williams, Kathryn R; Tan, Weihong

    2012-12-26

    Researchers increasingly envision an important role for artificial biochemical circuits in biological engineering, much like electrical circuits in electrical engineering. Similar to electrical circuits, which control electromechanical devices, biochemical circuits could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expressions in vivo. (1) As a consequence of their relative robustness and potential applicability for controlling a wide range of in vitro chemistries, synthetic cell-free biochemical circuits promise to be useful in manipulating the functions of biological molecules. Here, we describe the first logical circuit based on DNA-protein interactions with accurate threshold control, enabling autonomous, self-sustained and programmable manipulation of protein activity in vitro. Similar circuits made previously were based primarily on DNA hybridization and strand displacement reactions. This new design uses the diverse nucleic acid interactions with proteins. The circuit can precisely sense the local enzymatic environment, such as the concentration of thrombin, and when it is excessively high, a coagulation inhibitor is automatically released by a concentration-adjusted circuit module. To demonstrate the programmable and autonomous modulation, a molecular circuit with different threshold concentrations of thrombin was tested as a proof of principle. In the future, owing to tunable regulation, design modularity and target specificity, this prototype could lead to the development of novel DNA biochemical circuits to control the delivery of aptamer-based drugs in smart and personalized medicine, providing a more efficient and safer therapeutic strategy.

  12. New constitutive latex osmotin-like proteins lacking antifungal activity.

    PubMed

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events.

  13. On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies.

    PubMed Central

    Smirnov, M D; Triplett, D T; Comp, P C; Esmon, N L; Esmon, C T

    1995-01-01

    Phosphatidylethanolamine (PE) is an important membrane component for supporting activated protein C anticoagulant activity but has little influence on prothrombin activation. This difference constitutes a potential mechanism for selective inhibition of the protein C anticoagulant pathway by lupus anticoagulants and/or antiphospholipid antibodies. In this study, we demonstrate that the presence of PE augments lupus anticoagulant activity. In the plasma of some patients with lupus anticoagulants, activated protein C anticoagulant activity is more potently inhibited than prothrombin activation. As a result, in the presence of activated protein C and PE, these patient plasmas clot faster than normal plasma. Patients with minimal lupus anticoagulant activity are identified whose plasma potently inhibits activated protein C anticoagulant activity. This process is also PE dependent. In three patient plasmas, these phenomena are shown to be due to immunoglobulins. The PE requirement in the expression of activated protein C anticoagulant activity and the PE dependence of some antiphospholipid antibodies provide a mechanistic basis for the selective inhibition of the protein C pathway. Inhibition of activated protein C function may be a common mechanism contributing to increased thrombotic risk in certain patients with antiphospholipid antibodies. PMID:7814631

  14. Expression of proteins and protein kinase activity during germination of aerial spores of Streptomyces granaticolor.

    PubMed

    Mikulík, Karel; Bobek, Jan; Bezousková, Silvia; Benada, Oldrich; Kofronová, Olga

    2002-11-29

    Dormant aerial spores of Streptomyces granaticolor contain pre-existing pool of mRNA and active ribosomes for rapid translation of proteins required for earlier steps of germination. Activated spores were labeled for 30 min with [35S]methionine/cysteine in the presence or absence of rifamycin (400 microg/ml) and resolved by two-dimensional electrophoresis. About 320 proteins were synthesized during the first 30 min of cultivation at the beginning of swelling, before the first DNA replication. Results from nine different experiments performed in the presence of rifamycin revealed 15 protein spots. Transition from dormant spores to swollen spores is not affected by the presence of rifamycin but further development of spores is stopped. To support existence of pre-existing pool of mRNA in spores, cell-free extract of spores (S30 fraction) was used for in vitro protein synthesis. These results indicate that RNA of spores possesses mRNA functionally competent and provides templates for protein synthesis. Cell-free extracts isolated from spores, activated spores, and during spore germination were further examined for in vitro protein phosphorylation. The analyses show that preparation from dormant spores catalyzes phosphorylation of only seven proteins. In the absence of phosphatase inhibitors, several proteins were partially dephosphorylated. The activation of spores leads to a reduction in phosphorylation activity. Results from in vitro phosphorylation reaction indicate that during germination phosphorylation/dephosphorylation of proteins is a complex function of developmental changes.

  15. Adaptor protein Nck1 interacts with p120 Ras GTPase-activating protein and regulates its activity.

    PubMed

    Ger, Marija; Zitkus, Zigmantas; Valius, Mindaugas

    2011-10-01

    Adaptor protein Nck1 binds a number of intracellular proteins and influences various signaling pathways. Here we show that Nck1 directly binds and activates the GTPase-activating protein of Ras (RasGAP), which is responsible for the down-regulation of Ras. The first and the third SH3 domains of Nck1 and the NH(2)-terminal proline-rich sequence of RasGAP contribute most to the complex formation causing direct molecular interaction between the two proteins. Cell adhesion to the substrate is obligatory for the Nck1 and RasGAP association, as cell detachment makes RasGAP incapable of associating with Nck1. This leads to the complex dissipation, decrease of RasGAP activity and the increase of H-Ras-GTP level in the detached cells. Our findings reveal unexpected feature of adaptor protein Nck1 as the regulator of RasGAP activity.

  16. Molecularly imprinted polymeric micro- and nano-particles for the targeted delivery of active molecules.

    PubMed

    Gagliardi, Mariacristina; Mazzolai, Barbara

    2015-01-01

    Molecular imprinting (MI) represents a strategy to introduce a 'molecular memory' in a polymeric system obtaining materials with specific recognition properties. MI particles can be used as drug delivery systems providing a targeted release and thus reducing the side effects. The introduction of molecular recognition properties on a polymeric drug carrier represents a challenge in the development of targeted delivery systems to increase their efficiency. This review will summarize the limited number of drug delivery MI particles described in the literature along with an overview of potential solutions for a larger exploitation of MI particles as targeted drug delivery carriers. Molecularly imprinted drug carriers can be considered interesting candidates to significantly improve the efficiency of a controlled drug treatment.

  17. Size controlled protein nanoemulsions for active targeting of folate receptor positive cells.

    PubMed

    Loureiro, Ana; Nogueira, Eugénia; Azoia, Nuno G; Sárria, Marisa P; Abreu, Ana S; Shimanovich, Ulyana; Rollett, Alexandra; Härmark, Johan; Hebert, Hans; Guebitz, Georg; Bernardes, Gonçalo J L; Preto, Ana; Gomes, Andreia C; Cavaco-Paulo, Artur

    2015-11-01

    Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy.

  18. In situ protein folding and activation in bacterial inclusion bodies.

    PubMed

    Gonzalez-Montalban, Nuria; Natalello, Antonino; García-Fruitós, Elena; Villaverde, Antonio; Doglia, Silvia Maria

    2008-07-01

    Recent observations indicate that bacterial inclusion bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from inclusion bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as inclusion bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in inclusion bodies is negatively regulated by the chaperone DnaK.

  19. Protein stability and enzyme activity at extreme biological temperatures.

    PubMed

    Feller, Georges

    2010-08-18

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  20. Nanomedicine delivery: does protein corona route to the target or off road?

    PubMed

    Maiolo, Daniele; Del Pino, Pablo; Metrangolo, Pierangelo; Parak, Wolfgang J; Baldelli Bombelli, Francesca

    2015-01-01

    Nanomedicine aims to find novel solutions for urgent biomedical needs. Despite this, one of the most challenging hurdles that nanomedicine faces is to successfully target therapeutic nanoparticles to cells of interest in vivo. As for any biomaterials, once in vivo, nanoparticles can interact with plasma biomolecules, forming new entities for which the name protein coronas (PCs) have been coined. The PC can influence the in vivo biological fate of a nanoparticle. Thus for guaranteeing the desired function of an engineered nanomaterial in vivo, it is crucial to dissect its PC in terms of formation and evolution within the body. In this contribution we will review the 'good' and 'bad' sides of the PC, starting from the scientific aspects to the technological applications.