RESUMO
Induction of Hypoxia Inducible Factor (HIF) as a direct consequence of oxygen deficiency in tumor tissues is a potent stimulus of CD73 (ecto-5'-nucleotidase) expression. Hypoxic environment and CD73 overexpression are associated with altered metabolism, elevated cancer cell proliferation, and tumor vascularization. Herein, a delivery system was developed for silencing CD73 and HIF-1α gene using siRNA-loaded Superparamagnetic iron oxide (SPION) nanocarriers for cancer treatment. SPIONs were encapsulated with thiolated chitosan (TC) and trimethyl chitosan (TMC) for improving their stabilization and functionalization. The nanoparticles (NPs) were about 133 nm in size, spherical, and non-toxic, and the addition of TAT peptide (derived from HIV-1 TAT protein) to TMC-TC-SPIONs significantly increased their cellular uptake by cancer cells. The produced NPs could efficiently accumulate in the tumor site, indicating their stability and targeting ability in reaching the tumor region. TAT-conjugated TMC-TC-SPIONs containing siRNAs could significantly reduce the HIF-1α and CD73 expression levels in cancer cells. Following transfection, cancer cells showed a significant reduction in migration and proliferation. Moreover, siRNA-loaded NPs could effectively reduce tumor growth and angiogenesis, as investigated by the chick chorioallantoic membrane (CAM) assay. This study suggested that TAT-TMC-TC-SPIONs can be potential nanocarrier for gene transfection in cancer therapy. Moreover, the co-silencing of CD73 and HIF-1α can be assumed as a novel anti-cancer treatment strategy with high tumor suppression potential.
Assuntos
5'-Nucleotidase/genética , Quitosana/administração & dosagem , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Nanopartículas Magnéticas de Óxido de Ferro/administração & dosagem , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem , 5'-Nucleotidase/metabolismo , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quitosana/química , Quitosana/farmacocinética , Progressão da Doença , Liberação Controlada de Fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Nanopartículas Magnéticas de Óxido de Ferro/química , Camundongos Endogâmicos BALB C , Neoplasias/genética , Neoplasias/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
Recently, the emergence of cell-penetrating peptides (CPPs) like TAT has greatly improved the efficiency of cancer therapy by enhancing cellular uptake of nanomaterials. Here, we designed a near-infrared (NIR) triggered TAT-based targeted nanoplatform (cRGD@TAT-DINPs), which co-delivered anticancer drug doxorubicin (DOX) and biocompatible dye indocyanine green (ICG) to realize combined chemo/photothermal/photodynamic therapy of cancer in vitro. The resulting nanoparticles showed favorable monodispersity and colloidal stability. Impressively, the DOX could be released in a promoted manner once the nanoparticles were exposed to NIR light. Confocal laser scanning microscopy (CLSM) and flow cytometry analysis demonstrated an immensely enhanced cellular accumulation of DOX after the simultaneous introduction of targeted ligand cRGD and CPP TAT. In addition, the obtained nanoparticles exhibited explosive temperature elevation and reactive oxygen species (ROS) generation mediated by encapsulated ICG under NIR irradiation, and in vitro cytotoxicity assay confirmed the cRGD@TAT-DINPs had an increasing cytotoxicity and excellent synergistic inhibition capacity. Thus, TAT-based nanosystems provide a high-efficient drug delivery strategy for optimizing combined therapy efficiency of cancer.
Assuntos
Peptídeos Penetradores de Células/farmacocinética , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinética , Células A549 , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Corantes/administração & dosagem , Doxorrubicina/farmacocinética , Liberação Controlada de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Verde de Indocianina/administração & dosagem , Luz , Nanopartículas/uso terapêutico , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Fotoquimioterapia/métodos , Espécies Reativas de Oxigênio/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
Peptides represent a promising modality for the design of novel therapeutics that can potentially modulate traditionally non-druggable targets. Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are two large families that are being explored extensively as drug delivery vehicles, imaging reagents, or therapeutic treatments for various diseases. Many CPPs and AMPs are cationic among which a significant portion is extremely basic and hydrophilic (e.g., nona-arginine). Despite their attractive therapeutic potential, it remains challenging to directly analyze and quantify these super cationic peptides from biological matrices due to their poor chromatographic behavior and MS response. Herein, we describe a generic method that combines solid phase extraction and LC-MS/MS for analysis of these peptides. As demonstrated, using a dozen strongly basic peptides, low µM concentration of perfluoropentanoic acid (PFPeA) in the mobile phase enabled excellent compound chromatographic retention, thus avoiding co-elution with solvent front ion suppressants. PFPeA also had a charge reduction effect that allowed the selection of parent/ion fragment pairs in the higher m/z region to further reduce potential low molecular weight interferences. When the method was coupled to the optimized sample extraction process, we routinely achieved low digit ng/ml sensitivity for peptides in plasma/tissue. The method allowed an efficient evaluation of plasma stability of CPPs/AMPs without fluorescence derivatization or other tagging methods. Importantly, using the widely studied HIV-TAT CPP as an example, the method enabled us to directly assess its pharmacokinetics and tissue distribution in preclinical animal models.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Ácidos Pentanoicos/química , Peptídeos/análise , Peptídeos/farmacocinética , Animais , Peptídeos Catiônicos Antimicrobianos/análise , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Penetradores de Células/análise , Precipitação Química , Estabilidade de Medicamentos , Fluorocarbonos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Peptídeos/química , Ratos Wistar , Extração em Fase Sólida , Distribuição Tecidual , Ácido Tricloroacético/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/análise , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
As the first in a new class of non-opioid drugs, ω-Conotoxin MVIIA was approved for the management of severe chronic pains in patients who are unresponsive to opioid therapy. Unfortunately, clinical application of MVIIA is severely limited due to its poor ability to penetrate the blood-brain barrier (BBB), reaching the central nervous system (CNS). In the present study, we have attempted to increase MVIIA's ability to cross the BBB via a fusion protein strategy. Our results showed that when the TAT-transducing domain was fused to the MVIIA C-terminal with a linker of varied numbers of glycine, the MVIIA-TAT fusion peptide exhibited remarkable ability to cross the bio-membranes. Most importantly, both intravenous and intranasal administrations of MVIIA-TAT in vivo showed therapeutic efficacy of analgesia. Compared to the analgesic effects of intracerebral administration of the nascent MVIIA, these systemic administrations of MVIIA-TAT require higher doses, but have much prolonged effects. Taken together, our results showed that TAT conjugation of MVIIA not only enables its peripheral administration, but also maintains its analgesic efficiency with a prolonged effective time window. Intranasal administration also rendered the MVIIA-TAT advantages of easy applications with potentially reduced side effects. Our results may present an alternative strategy to improve the CNS accessibility for neural active peptides.
Assuntos
Analgésicos/farmacocinética , Barreira Hematoencefálica/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Proteínas Recombinantes de Fusão/farmacocinética , ômega-Conotoxinas/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinética , Analgésicos/administração & dosagem , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Feminino , Masculino , Camundongos , Dor/tratamento farmacológico , Dor/metabolismo , Medição da Dor/efeitos dos fármacos , Peptídeos/administração & dosagem , Peptídeos/química , Proteína FUS de Ligação a RNA , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/biossíntese , Tremor/tratamento farmacológico , Tremor/metabolismo , ômega-Conotoxinas/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagemRESUMO
Considerable efforts have been devoted to enhancing the cell penetration of nanoparticles by coating cell-penetrating peptides (CPPs) on the surface. However, the internalization mechanism for a CPP at different concentrations varies a lot. It is acknowledged that the mechanism is restricted to endocytic pathway at relatively low concentrations; however, direct translocation becomes dominant at high concentrations. This raises an interesting question on how the surface Tat coating density of the nanoparticles would influence their cell-membrane interaction and the consequent internalization behavior. This study systematically investigates the effect of Tat peptides on the internalization behavior of polymeric micelles by tuning surface Tat coating density, incubation concentrations, incubation time, and other factors using poly(ethylene glycol)-poly(ε-caprolactone) copolymer (PEG-PCL) micelles. It is found that both energy-dependent and energy-independent pathways are involved in the cellular uptake process, and the Tat-conjugated polymeric micelles strongly accumulated on the cell surface at initial stage. The membrane-anchoring and internalization rate increase with the increasing Tat coating density. Furthermore, the increasing of Tat coating density accelerates the energy-independent pathway. It is envisioned that this finding will further shed light on the surface modification of nanoparticles for enhanced cell penetration and direct translocation into cell cytoplasm.
Assuntos
Membrana Celular/metabolismo , Peptídeos Penetradores de Células , Micelas , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Células A549 , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacocinética , Peptídeos Penetradores de Células/farmacologia , HIV-1 , Células HeLa , Células Hep G2 , Humanos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologiaRESUMO
We herein propose a polymeric nanovehicle system that has the ability to remarkably improve cellular uptake and transdermal delivery. Cell-penetrating peptide-patchy deformable polymeric nanovehicles were fabricated by tailored coassembly of amphiphilic poly(ethylene oxide)- block-poly(ε-caprolactone) (PEO- b-PCL), mannosylerythritol lipid (MEL), and YGRKKRRQRRR-cysteamine (TAT)-linked MEL. Using X-ray diffraction, differential scanning calorimetry, and nuclear magnetic resonance analyses, we revealed that the incorporation of MEL having an asymmetric alkyl chain configuration was responsible for the deformable phase property of the vehicles. We also discovered that the nanovehicles were mutually attracted, exhibiting a gel-like fluid characteristic due to the dipole-dipole interaction between the hydroxyl group of MEL and the methoxy group of PEO- b-PCL. Coassembly of TAT-linked MEL with the deformable nanovehicles significantly enhanced cellular uptake due to macropinocytosis and caveolae-/lipid raft-mediated endocytosis. Furthermore, the in vivo skin penetration test revealed that our TAT-patchy deformable nanovehicles remarkably improved transdermal delivery efficiency.
Assuntos
Glicolipídeos/química , Nanopartículas/química , Fragmentos de Peptídeos/administração & dosagem , Poliésteres/química , Absorção Cutânea , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem , Administração Cutânea , Adulto , Linhagem Celular , Cisteamina/química , Feminino , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
This study was aimed to evaluate the immunogenicity of recombinant plasmid deoxyribonucleic acid (DNA), pBud-H5-green fluorescent protein (GFP)-interferon-regulatory factor (IRF)3 following delivery using polyamidoamine (PAMAM) dendrimer and transactivator of transcription (TAT)-conjugated PAMAM dendrimer as well as the effect of IRF3 as the genetic adjuvant. BALB/c mice were vaccinated transdermally with pBud-H5-GFP, PAMAM/pBud-H5-GFP, TAT-PAMAM/pBud-H5-GFP, and TAT-PAMAM/pBud-H5-GFP-IRF3. The expression analysis of H5 gene from the blood by using quantitative real-time reverse transcriptase polymerase chain reaction confirmed the ability of PAMAM dendrimer as a carrier for gene delivery, as well as the ability of TAT peptide to enhance the delivery efficiency of PAMAM dendrimer. Mice immunized with modified PAMAM by TAT peptide showed higher hemagglutination inhibition titer, and larger CD3+/CD4+ T cells and CD3+/CD8+ T cells population, as well as the production of cytokines, namely, interferon (IFN)-γ, interleukin (IL)-2, IL-15, IL-12, IL-6, and tumor necrosis factor-α compared with those immunized with native PAMAM. These results suggest that the function of TAT peptide as a cell-penetrating peptide is able to enhance the gene delivery, which results in rapid distribution of H5 in the tissues of the immunized mice. Furthermore, pBud-H5-GFP co-expressing IRF3 as a genetic adjuvant demonstrated the highest hemagglutination inhibition titer besides larger CD3+/CD4+ and CD3+/CD8+ T cells population, and strong Th1-like cytokine responses among all the systems tested. In conclusion, TAT-PAMAM dendrimer-based delivery system with IRF3 as a genetic adjuvant is an attractive transdermal DNA vaccine delivery system utilized to evaluate the efficacy of the developed DNA vaccine in inducing protection during challenge with virulent H5N1 virus.
Assuntos
Dendrímeros/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/imunologia , Vacinas de DNA/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Administração Cutânea , Animais , Peptídeos Penetradores de Células , Citocinas/metabolismo , Dendrímeros/química , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Testes de Inibição da Hemaglutinação , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Fator Regulador 3 de Interferon/genética , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de DNA/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
PURPOSE: In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed. METHODS: First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry. RESULTS: TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-). In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine. The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05). CONCLUSIONS: The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Assuntos
Dendrímeros/química , Dendrímeros/farmacocinética , Técnicas de Transferência de Genes , Peptídeos/química , Vacinas de DNA/química , Vacinas de DNA/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Animais , Chlorocebus aethiops , Peptídeos/farmacocinética , Poliaminas/química , Vacinas de DNA/genética , Células Vero , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
In this study, a dual-ligand polymer-lipid hybrid nanoparticle drug delivery vehicle comprised of an anti-HER2/neu peptide (AHNP) mimic with a modified HIV-1 Tat (mTAT) was established for the targeted treatment of Her2/neu-overexpressing cells. The resultant dual-ligand hybrid nanoparticles (NPs) consisted of a poly(lactide-co-glycolide) core, a near 90% surface coverage of the lipid monolayer, and a 5.7 nm hydrated polyethylene glycol shell. Ligand density optimization study revealed that cellular uptake efficiency of the hybrid NPs could be manipulated by controlling the surface-ligand densities. Furthermore, the cell uptake kinetics and mechanism studies showed that the dual-ligand modifications of hybrid NPs altered the cellular uptake pathway from caveolae-mediated endocytosis (CvME) to the multiple endocytic pathways, which would significantly enhance the NP internalization. Upon the systemic investigation of the cellular uptake behavior of dual-ligand hybrid NPs, docetaxel (DTX), a hydrophobic anticancer drug, was successfully encapsulated into dual-ligand hybrid NPs with high drug loading for Her2/neu-overexpressing SK-BR-3 breast cancer cell treatment. The DTX-loaded dual-ligand hybrid NPs showed a decreased burst release and a more gradual sustained drug release property. Because of the synergistic effect of dual-ligand modification, DTX-loaded dual-ligand hybrid NPs exerted substantially better therapeutic potency against SK-BR-3 cancer cells than other NP formulations and free DTX drugs. These results demonstrate that the dual-ligand hybrid NPs could be a promising vehicle for targeted drug delivery to treat breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Nanocápsulas/química , Receptor ErbB-2/metabolismo , Taxoides/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinética , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Difusão , Docetaxel , Humanos , Ligantes , Lipídeos/química , Nanocápsulas/ultraestrutura , Polímeros/química , Taxoides/química , Resultado do Tratamento , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic molecules, yet the identity of their uptake routes remained unclear and is still under debate. In this study we provide new insights into CPP entry routes by quantitatively measuring the intracellular uptake of FAM-labeled Tat-peptide under rigorous kinetic and thermal conditions. The uptake of Tat-peptide between 4 and 15°C corresponds to Q10=1.1, proceeding through a prompt (<5 min), temperature-independent process, suggesting direct membrane translocation. At longer durations, Tat rate of uptake shows linear dependence on temperature with Q10=1.44, accompanied by activation energy Ea=4.45 Kcal/mole. These values are significantly lower than those we found for the macropinocytosis probe dextran (Q10=2.2 and Ea=7.2 Kcal/mole) which possesses an exponential dependence on temperature, characteristic of endocytosis processes. Tat-peptide and dextran do not interfere with each other's uptake rate and the ratio of Tat-peptide uptake to its extracellular concentration is ~15 times higher than that for dextran. In addition, Phloretin, a modulator of cell membrane dipole potential, is shown to increase dextran uptake but to reduce that of Tat. We conclude that the uptake of Tat differs from that of dextran in all parameters. Tat uptake proceeds by dual entry routes which differ by their energy dependence.
Assuntos
Endocitose/fisiologia , Pinocitose/fisiologia , Temperatura , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinética , Citoesqueleto de Actina/metabolismo , Algoritmos , Sequência de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Dextranos/farmacocinética , Polarização de Fluorescência , Humanos , Fluidez de Membrana , Microscopia de Fluorescência , Floretina/farmacologia , Fatores de TempoRESUMO
Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs could not actively target the cell nuclei, which is contrary to previous observations based on fixed cell results. More importantly, the inheritance of TGNPs to the daughter cells through mitosis was found to be the major route to metabolize TGNPs by HeLa cells. These understandings on the cellular uptake mechanism and intracellular fate of nanocargos in living cells would provide deep insight on how to improve and controllably manipulate their translocation efficiency for targeted drug delivery.
Assuntos
Nanopartículas Metálicas/química , Nanocápsulas/química , Frações Subcelulares/química , Frações Subcelulares/ultraestrutura , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinética , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacocinética , Ouro/química , Células HeLa , Humanos , Teste de Materiais , Taxa de Depuração Metabólica , Nanopartículas Metálicas/ultraestrutura , Nanocápsulas/ultraestrutura , Análise Espaço-TemporalRESUMO
Therapeutic application of many drugs is often hampered by poor or denied access to intracellular targets. A case in point is miltefosine (MT), an orally active antiparasitic drug, which becomes ineffective when parasites develop dysfunctional uptake systems. We report here the synthesis of a fluorescent BODIPY-embedding MT analogue with appropriate thiol functionalization allowing linkage to the cell-penetrating Tat(48-60) peptide through disulfide or thioether linkages. The resulting constructs are efficiently internalized into the otherwise MT-invulnerable R40 Leishmania strain, resulting in fast parasite killing, and hence successful avoidance of the resistance. In the disulfide-linked conjugate, an additional fluoro tag on the Tat moiety allows to monitor its reductive cleavage within the cytoplasm. Terminally differentiated cells such as peritoneal macrophages, impervious to MT unless infected by Leishmania, can uptake the drug in its Tat-conjugated form. The results afford proof-of-principle for using CPP vectors to avert drug resistance in parasites, and/or for tackling leishmaniasis by modulating macrophage uptake.
Assuntos
Anti-Helmínticos/síntese química , Anti-Helmínticos/farmacocinética , Compostos de Boro/química , Compostos de Boro/farmacocinética , Peptídeos Penetradores de Células/química , Leishmania/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Anti-Helmínticos/química , Linhagem Celular , Peptídeos Penetradores de Células/farmacocinética , Desenho de Fármacos , Humanos , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/parasitologia , Estrutura Molecular , Fosforilcolina/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
Peptide and protein drugs have become the new generation of therapeutics, yet most of them are only available as injections, and reports on oral local intestinal delivery of peptides and proteins are quite limited. The aim of this work was to develop and evaluate a water-in-oil (w/o) microemulsion system in vitro and in vivo for local intestinal delivery of water-soluble peptides after oral administration. A fluorescent labeled peptide, 5-(and-6)-carboxytetramethylrhodamine labeled HIV transactivator protein TAT (TAMRA-TAT), was used as a model peptide. Water-in-oil microemulsions consisting of Miglyol 812, Capmul MCM, Tween 80, and water were developed and characterized in terms of appearance, viscosity, conductivity, morphology, and particle size analysis. TAMRA-TAT was loaded and its enzymatic stability was assessed in modified simulated intestinal fluid (MSIF) in vitro. In in vivo studies, TAMRA-TAT intestinal distribution was evaluated using fluorescence microscopy after TAMRA-TAT microemulsion, TAMRA-TAT solution, and placebo microemulsion were orally gavaged to mice. The half-life of TAMRA-TAT in microemulsion was enhanced nearly three-fold compared to that in the water solution when challenged by MSIF. The treatment with TAMRA-TAT microemulsion after oral administration resulted in greater fluorescence intensity in all intestine sections (duodenum, jejunum, ileum, and colon) compared to TAMRA-TAT solution or placebo microemulsion. The in vitro and in vivo studies together suggested TAMRA-TAT was better protected in the w/o microemulsion in an enzyme-containing environment, suggesting that the w/o microemulsions developed in this study may serve as a potential delivery vehicle for local intestinal delivery of peptides or proteins after oral administration.
Assuntos
Sistemas de Liberação de Medicamentos , Mucosa Intestinal/metabolismo , Peptídeos/administração & dosagem , Animais , Estabilidade de Medicamentos , Condutividade Elétrica , Emulsões , Feminino , Absorção Intestinal , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/farmacocinética , Rodaminas/administração & dosagem , Viscosidade , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
Herein we report on the development of a novel method of constraining a cell-penetrating peptide, which can be used to trigger transport of liposomes into cells upon in this case radiation with UV-light. A cell-penetrating peptide, which was modified on both termini with an alkyl chain, was anchored to the liposomal surface in a constrained and deactivated form. Since one of the two alkyl chains was connected to the peptide via a UV-cleavable linker, disconnection of this alkyl chain upon irradiation led to the exposure of the cell-penetrating peptide, and mediated the transport of the entire liposome particle into cells.
Assuntos
Peptídeos Penetradores de Células/química , Portadores de Fármacos/química , Raios Ultravioleta , Adesão Celular , Técnicas de Cultura de Células , Peptídeos Penetradores de Células/farmacocinética , Peptídeos Penetradores de Células/efeitos da radiação , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/efeitos da radiação , Endocitose , Citometria de Fluxo , Células HeLa , Humanos , Lipossomos , Microscopia Confocal , Estrutura Molecular , Espalhamento de Radiação , Propriedades de Superfície , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/efeitos da radiaçãoRESUMO
Reactive oxygen species (ROS) contribute to the development of a number of neuronal diseases including ischemia. DJ-1, also known to PARK7, plays an important role in transcriptional regulation, acting as molecular chaperone and antioxidant. In the present study, we investigated whether DJ-1 protein shows a protective effect against oxidative stress-induced neuronal cell death in vitro and in ischemic animal models in vivo. To explore DJ-1 protein's potential role in protecting against ischemic cell death, we constructed cell permeable Tat-DJ-1 fusion proteins. Tat-DJ-1 protein efficiently transduced into neuronal cells in a doseand time-dependent manner. Transduced Tat-DJ-1 protein increased cell survival against hydrogen peroxide (H2O2) toxicity and also reduced intracellular ROS. In addition, Tat-DJ-1 protein inhibited DNA fragmentation induced by H2O2. Furthermore, in animal models, immunohistochemical analysis revealed that Tat-DJ-1 protein prevented neuronal cell death induced by transient forebrain ischemia in the CA1 region of the hippocampus. These results demonstrate that transduced Tat-DJ-1 protein protects against cell death in vitro and in vivo, suggesting that the transduction of Tat-DJ-1 may be useful as a therapeutic agent for ischemic injuries related to oxidative stress.
Assuntos
Isquemia Encefálica/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Proteínas Oncogênicas/administração & dosagem , Estresse Oxidativo , Proteínas Recombinantes de Fusão/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem , Animais , Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/prevenção & controle , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Gerbillinae , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/farmacocinética , Peroxidação de Lipídeos , Malondialdeído/metabolismo , Camundongos , Fármacos Neuroprotetores/farmacocinética , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/farmacocinética , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/metabolismo , Prosencéfalo/patologia , Proteína Desglicase DJ-1 , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/biossíntese , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
Micromolecular agents that block tumor development and metastasis hold great promise as cancer-targeted therapies. Tumor molecular targeted peptide 1 (TMTP1) was previously shown to target primary tumors and metastatic foci specifically. In this study, a group of composite peptides were incorporated to TMPT1. The NF-κB essential modulator-binding domain (NBD), and the trans-activator of transcription (TAT) peptide, were synthesized to enhance the targeted anti-tumor effects of TMTP1. TMTP1-NBD did not exhibit strong affinity to tumor cells as we had expected. Conjugating TAT with TMTP1-NBD ameliorated the poor hydrophilicity and negative charge of TMTP1-NBD. Therefore TMTP1-TAT-NBD displayed strong affinity and anti-tumor effects as we expected in vivo and in vitro. Interestingly cytoplasmic glycogen accumulation as well as apoptosis was observed in TMTP1-TAT-NBD treated PC-3M-1E8 cells. The downstream signaling pathways including AKT, GSK-3ß, IκBα and NF-κB activity were verified to decrease by TMTP1-TAT-NBD. The pharmacokinetics and distribution of TMTP1-TAT-NBD in MDA-MB-231 tumor-bearing mice model provided some evidence for safety of the composite peptide, which showed the fluorescence of the peptide peaked in the tumor 6h after injection, with little fluorescence detected in normal organs except for very weak fluorescence in kidney. In conclusion, TMTP1-TAT-NBD may be a promising targeted anti-tumor agent for primary tumor and metastatic foci, which enhances the anticancer effects through inhibiting the AKT/GSK-3ß/NF-κB pathway comparing with TMTP1.
Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Metástase Neoplásica/tratamento farmacológico , Neoplasias/tratamento farmacológico , Oligopeptídeos/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem , Células 3T3 , Animais , Antineoplásicos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacocinética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/patologia , Oligopeptídeos/farmacocinética , Fragmentos de Peptídeos/farmacocinética , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
Cell-penetrating peptides (CPPs), including TAT-CPP, have been used to deliver exogenous proteins into living cells. Although a number of proteins fused to TAT-CPP can be delivered into various cells, little is known about the proteolytic cleavage of TAT-fusion proteins in cells. In this study, we demonstrate that a small heat shock protein (sHSP), alphaB-crystallin (αB-crystallin), delivered by TAT-CPP is susceptible to proteolytic cleavage by matrix metalloproteinase-1 (MMP-1) in cardiac myoblast H9c2 cells. Recombinant TAT-αB-crystallin was efficiently transduced into H9c2 cells. For a few hours following protein transduction, generation of a 14-kDa fragment, a cleavage band of TAT-αB-crystallin, increased in a time-dependent manner. This fragment was observed only in detergent-insoluble fractions. Interestingly, treatment with MMP inhibitors blocked the cleavage of TAT-αB-crystallin. In test tubes, recombinant MMP-1 processed TAT-αB-crystallin to generate the major cleavage fragment 14-kDa, as observed in the cells treated with TAT-αB-crystallin. The N-terminal sequences of the 14-kDa fragment were identified as Leu-Arg-Ala-Pro-Ser-Trp-Phe, indicating that this fragment is generated by cleavage at Phe54-Leu55 of αB-crystallin. The MMP-1-selective inhibitor abolished the production of 14-kDa fragments in cells. In addition, the cleaved fragment of TAT-αB-crystallin was significantly reduced in cells transfected with MMP-1 siRNA. Moreover, the enzymatic activity of MMP-1 was markedly increased in TAT-αB-crystallin-treated cells. TAT-αB-crystallin has a cytoprotective effect on H9c2 cells under hypoxic insult, moreover, MMP-1-selective inhibitor treatment led to even increased cell viability. These results suggest that MMP-1 is responsible for proteolytic cleavage of TAT-αB-crystallin during its intracellular transduction in H9c2 cells.
Assuntos
Peptídeos Penetradores de Células/farmacocinética , Metaloproteinase 1 da Matriz/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Cadeia B de alfa-Cristalina/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinética , Animais , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/isolamento & purificação , Peptídeos Penetradores de Células/farmacologia , Citoproteção , Sistemas de Liberação de Medicamentos , Ensaios Enzimáticos , Metaloproteinase 1 da Matriz/genética , Mioblastos/efeitos dos fármacos , Mioblastos/enzimologia , Mioblastos/metabolismo , Ratos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Cadeia B de alfa-Cristalina/isolamento & purificação , Cadeia B de alfa-Cristalina/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/isolamento & purificação , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologiaRESUMO
In general, cellular internalization of macromolecular drugs encapsulated in liposomes proceeds via endocytosis. This potentially leads to degradation of the liposome-encapsulated macromolecular content within the endosomal/lysosomal compartment. Therefore, bypassing the endocytic route by conferring a direct plasma membrane translocation property to the liposomes would be very beneficial. Cell penetrating peptides, e.g. TAT-peptide, are exploited in the drug delivery field for their capacity of plasma membrane translocation. Here, we describe the preparation of TAT-peptide modified liposomes and their cellular interaction using live cell flow cytometry and imaging techniques.
Assuntos
Lipossomos/química , Lipossomos/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinética , Linhagem Celular Tumoral , Endocitose , HumanosAssuntos
Peptídeos Penetradores de Células/farmacocinética , Endocitose , RNA Interferente Pequeno/genética , Caveolina 1/fisiologia , Linhagem Celular Tumoral , Clatrina/fisiologia , Endocitose/efeitos dos fármacos , Humanos , Proteínas de Membrana/fisiologia , Transporte Proteico , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
Arginine-rich cell-penetrating peptides are short cationic peptides capable of traversing the plasma membranes of eukaryotic cells. While successful intracellular delivery of many biologically active macromolecules has been accomplished using these peptides, their mechanisms of cell entry are still under investigation. Recent dialogue has centered on a debate over the roles that direct translocation and endocytotic pathways play in internalization of cell-penetrating peptides. In this paper, we review the evidence for the broad range of proposed mechanisms, and show that each distinct process requires negative Gaussian membrane curvature as a necessary condition. Generation of negative Gaussian curvature by cell-penetrating peptides is directly related to their arginine content. We illustrate these concepts using HIV TAT as an example.