RESUMEN
PURPOSE: Short interfering RNA (siRNA) therapy promises a new era in treatment of breast cancers but effective delivery systems are needed for clinical use. Since silencing complementary targets may offer improved efficacy, this study was undertaken to identify non-viral carriers for combinatorial siRNA delivery for more effective therapy. METHODS: A library of lipid-substituted polymers from low molecular weight polyethyleneimine (PEI), linoleic acid (LA) and α-linoleic acid (αLA) with amide or thioester linkages was prepared and investigated for delivering Mcl-1, survivin and STAT5A siRNAs in breast cancer cells. RESULTS: The effective polymers formed 80-190 nm particles with similar zeta-potentials, but the serum stability was greater for complexes formed with amide-linked lipid conjugates. The LA and αLA substitutions, with the low molecular weight PEI (1.2 kDa and 2.0 kDa) were able to deliver siRNA effectively to cells and retarded the growth of breast cancer cells. The amide-linked lipid substituents showed higher cellular delivery of siRNA as compared to thioester linkages. Upon combinational delivery of siRNAs, growth of MCF-7 cells was inhibited to a greater extent with 2.0PEI-LA9 mediated delivery of Mcl-1 combined survivin siRNAs as compared to individual siRNAs. The qRT-PCR analysis confirmed the decrease in mRNA levels of target genes with specific siRNAs and 2.0PEI-LA9 was the most effective polymer for delivering siRNAs (either single or in combination). CONCLUSIONS: This study yielded effective siRNA carriers for combinational delivery of siRNAs. Careful choice of siRNA combinations will be critical since targeting individual genes might alter the expression of other critical mediators.
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Neoplasias de la Mama/metabolismo , Portadores de Fármacos/química , Marcación de Gen/métodos , Polietileneimina/química , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacología , Línea Celular Tumoral , Femenino , Silenciador del Gen , Humanos , Ácido Linoleico , Lípidos , Células MCF-7 , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Polietileneimina/metabolismo , Polímeros/química , Polímeros/metabolismo , Factor de Transcripción STAT5/metabolismo , Survivin/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Mentha piperita essential oils (MPEO) were loaded into chitosan nanogel to use as antibiofilm agent against Streptococcus mutans and to protect its dental plaque. Chitosan nanoparticles (CsNPs) were prepared by sol-gel method using linking bridge of tripolyphosphate (TPP). Physiological properties of MPEO-CNs were assessed by FTIR, SEM/EDX, DLS and zeta potential. Release kinetics, MIC and MBC were determined for MPEO-CNs. Expression of biofilm-associated genes including 8 genes: grfB, C and D, brpA, spaP, gbpB, relA and vicR was investigated at the presence of sub-MIC of MPEO-CNs. Most abundant bioactive compounds of MPEO were l-menthol (45.05%) and l-menthal (17.53%). SEM/EDX exhibited successful entrapment of MPEO into CsNPs followed by the changes in abundance of elemental peaks. A signal at 1737 cm-1 on chitosan spectrum was attributed to the carboxylic (CO) groups overlapped by MPEO incorporation. A new signal at 2361 cm-1 was assigned to electrostatic interactions of amine groups in chitosan with phosphoric units of TPP within the MPEO-chitosan. MPEO incorporation into porous nanogel decreased monodispersity of the nanoparticles and then raises z-average. Maximum release of MPEO was about 50% during 360 h in a hydroalcoholic solvent at ambient temperature. The adherence of bacterial cells showed high sensitivity to the nanoformulation of MPEO compared with unloaded chitosan-nanogel. Antibiofilm inhibition of S. mutans occurred in 50 and 400 µg/mL for MPEO-CNs and unloaded-nanogel, respectively. Among biofilm synthesis genes, gtfB, gtfC, gtfD were slightly affected by MPEO-CNs treatment, while gbpB, spaP, brpA, relA, and vicR genes underwent significant down-regulation in the presence of both unloaded-nanogel and MPEO-loaded-nanogel. This study demonstrated that the MPEO-CNs promised an efficient nanoformulation with the greatest inhibitory action against some glycosyltransferase genes (gtfB, C and D) as important enzymes involved in extracellular polymers. Finally, the results concluded that MPEO-CNs have a potential use as antibiofilm agent in toothpaste or mouth washing formulations.
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Biopelículas/efectos de los fármacos , Quitosano/administración & dosificación , Aceites de Plantas/administración & dosificación , Polietilenglicoles/administración & dosificación , Polietileneimina/administración & dosificación , Streptococcus mutans/efectos de los fármacos , Diente/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Quitosano/metabolismo , Caries Dental/tratamiento farmacológico , Caries Dental/microbiología , Placa Dental/tratamiento farmacológico , Placa Dental/microbiología , Humanos , Mentha piperita , Nanogeles , Aceites de Plantas/aislamiento & purificación , Aceites de Plantas/metabolismo , Polietilenglicoles/metabolismo , Polietileneimina/metabolismo , Streptococcus mutans/crecimiento & desarrollo , Diente/microbiologíaRESUMEN
In the present study, a pH/redox-responsive cationic polymer dot (CD) was successfully prepared for a near-infrared (NIR)-mediated, simultaneously controllable photothermal temperature guided imaging off/on system to monitor therapeutic delivery. Carbonized disulfide cross-linked branched polyethyleneimine (bPEI) was conjugated with folic acid (FA) as a targeting moiety and partially formed an ionic complex with anionic indocyanine green (ICG) to afford a bPEI-based CD (ICG-CD). This was responsive to mild reductive (glutathione, GSH) and acidic tumor conditions, which enabled the simultaneous biodegradation of those hydrophobic and complex sites. The ICG-CD internalized readily into the cytoplasm of cancer cells by a FA receptor and cationic-mediated endocytosis in the off state, whereas if ICG-CD met intracellular GSH at high concentrations, GSH contributed partially to the recovery of fluorescence and was then internalized into acidic endosomes to induce complete restoration of fluorescence. This tumor-sensitive degradability of the CD not only facilitated ICG release in the tumor location but also allowed controllable photothermal therapy effects of nanoparticles under NIR irradiation, which resulted in improved cancer therapy. Taken together, the results indicate great potential in tumor targeting, intracellular imaging, and controllable therapeutic delivery through a fluorescence off/on assay under the pH/redox conditions of cancer cells.
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Antineoplásicos/farmacología , Colorantes Fluorescentes/farmacología , Verde de Indocianina/farmacología , Puntos Cuánticos/química , Animales , Antineoplásicos/química , Carbono/química , Línea Celular Tumoral , Perros , Endocitosis/fisiología , Endosomas/metabolismo , Fluorescencia , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Receptores de Folato Anclados a GPI/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Glutatión/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hipertermia Inducida/métodos , Verde de Indocianina/química , Verde de Indocianina/metabolismo , Rayos Infrarrojos , Células de Riñón Canino Madin Darby , Oxidación-Reducción , Fototerapia/métodos , Polietilenglicoles/química , Polietilenglicoles/metabolismo , Polietileneimina/química , Polietileneimina/metabolismo , Puntos Cuánticos/efectos de la radiación , Nanomedicina Teranóstica/métodosRESUMEN
Nanosized polyelectrolyte complexes are attractive delivery vehicles for the transfer of therapeutic genes to diseased cells. Here we report the application of self-assembled ternary complexes constructed with plasmid DNA, branched polyethylenimine and hyaluronic acid-green tea catechin conjugates for targeted gene delivery. These conjugates not only stabilize plasmid DNA/polyethylenimine complexes via the strong DNA-binding affinity of green tea catechin, but also facilitate their transport into CD44-overexpressing cells via receptor-mediated endocytosis. The hydrodynamic size, surface charge and physical stability of the complexes are characterized. We demonstrate that the stabilized ternary complexes display enhanced resistance to nuclease attack and polyanion-induced dissociation. Moreover, the ternary complexes can efficiently transfect the difficult-to-transfect HCT-116 colon cancer cell line even in serum-supplemented media due to their enhanced stability and CD44-targeting ability. Confocal microscopic analysis demonstrates that the stabilized ternary complexes are able to promote the nuclear transport of plasmid DNA more effectively than binary complexes and hyaluronic acid-coated ternary complexes. The present study suggests that the ternary complexes stabilized with hyaluronic acid-green tea catechin conjugates can be widely utilized for CD44-targeted delivery of nucleic acid-based therapeutics.
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Catequina/análogos & derivados , ADN/administración & dosificación , Ácido Hialurónico/metabolismo , Plásmidos/administración & dosificación , Transfección/métodos , Catequina/química , Catequina/metabolismo , ADN/genética , Endocitosis , Proteínas Fluorescentes Verdes/genética , Células HCT116 , Células HEK293 , Humanos , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/química , Plásmidos/genética , Polietileneimina/química , Polietileneimina/metabolismo , Té/química , Té/metabolismoRESUMEN
CONTEXT: Although several formulation strategies have been developed for the treatment of psoriasis, there is an unmet need for optimization of its therapy. OBJECTIVE: The objective was to develop a nanogel composed of methotrexate (MTX)-loaded nanostructured lipid carrier (MTX-NLC) and to evaluate its potential in imiquimod-induced psoriasis model to ameliorate symptoms of psoriasis. MATERIALS AND METHODS: MTX-NLC nanogel was prepared by hot-homogenization method and optimized by Design of Experiments. Particle size, polydispersity index (PDI) and entrapment efficiency were selected as the critical quality attributes. Antipsoriatic potential of MTX-NLC nanogel was evaluated by Psoriatic Area and Severity Index (PASI) score and histopathological examination in the imiquimod-induced psoriasis model. RESULTS AND DISCUSSION: Optimized MTX-NLC exhibited particle size of 278 ± 10 nm, PDI of 0.231 ± 0.05 and EE of 22.29 ± 1.23%. At the end of 48 h, MTX-NLC gel exhibited slow and prolonged release of MTX (47.32 ± 0.94% versus 94.23 ± 0.79%) compared to MTX gel. Furthermore, it significantly reduced the PASI score with recovery of normalcy of the mice's skin, while the MTX gel exhibited signs of hyper and parakeratosis at the end of the study. CONCLUSION: The developed MTX-NLC gel formulation can be a promising alternative to existing MTX formulation in treating psoriasis.
Asunto(s)
Metotrexato/administración & dosificación , Metotrexato/química , Polietilenglicoles/administración & dosificación , Polietilenglicoles/química , Polietileneimina/administración & dosificación , Polietileneimina/química , Psoriasis/tratamiento farmacológico , Absorción Cutánea/efectos de los fármacos , Administración Tópica , Animales , Química Farmacéutica , Evaluación Preclínica de Medicamentos/métodos , Liberación de Fármacos/efectos de los fármacos , Liberación de Fármacos/fisiología , Metotrexato/metabolismo , Ratones , Ratones Endogámicos BALB C , Nanogeles , Polietilenglicoles/metabolismo , Polietileneimina/metabolismo , Psoriasis/metabolismo , Psoriasis/patología , Distribución Aleatoria , Absorción Cutánea/fisiología , Resultado del TratamientoRESUMEN
Novel nano-particles were developed from lysozyme-pectin through self-assembly, and the nanogels could be used as a carrier for the antitumor agent, methotrexate (MTX). The nanogels exhibited spherical with diameters about 109 ± 2 nm and narrow particle size distribution, as well as negative surface charge. Furthermore, the particle size and morphology of the nanogels hardly changed with the incorporation of MTX. The loading capacity of MTX in nanogels could reach 17.58 ± 0.85%. MTX-loaded nanogels were pH-dependent, accelerated release of MTX at a decreasing pH from 7.4 to 5.3. The MTT assay indicated that encapsulated MTX exhibited higher anticancer activity than free MTX. Meanwhile, MTX-loaded nanogels could be effectively endocytosed by HepG2 cells, resulting in enhanced cancer-cell apoptosis comparing to free MTX. It indicated that the nanogels had good biocompatibility and low toxicity. The obtained nanogels had great potential in the development of a new nanocarrier for anti-cancer drug delivery.
Asunto(s)
Antimetabolitos Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Metotrexato/administración & dosificación , Muramidasa/química , Pectinas/química , Polietilenglicoles/química , Polietileneimina/química , Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Metotrexato/química , Metotrexato/farmacología , Muramidasa/metabolismo , Nanogeles , Pectinas/metabolismo , Polietilenglicoles/metabolismo , Polietileneimina/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
AIM: Our investigation was aimed to investigate the potential suitability of meloxicam-loaded nanostructured lipid carriers (MLX-NLC) gel for topical application. MAIN METHODS: MLX-NLC gel was prepared and in vivo skin penetration ability of the NLC gel was evaluated using confocal laser scanning microscopy. We studied the effect of MLX-NLC gel on the changes in lipid profile of skin to get an insight into its skin penetration enhancement mechanism. Acetic acid induced writhing test was performed to evaluate the analgesic effect. Drug concentration-time profile of MLX in rat plasma and skin after topical and oral treatment with MLX-NLC gel and oral MLX-solution, respectively, was observed. MLX-NLC gel was subjected to primary skin irritation test, sub-acute dermal toxicity study. Storage stability of MLX-NLC gel was also assessed for 90 days. KEY FINDINGS: NLC gel was effective in permeating Rhodamine 123 to deeper layers of rat skin. Changes in skin lipid prolife were observed in the rat skin on treatment with MLX-NLC gel and the results supported skin lipid extraction as a possible penetration enhancement mechanism. MLX-NLC gel demonstrated sustained pain inhibitory effect. Pharmacokinetics study established that topical application of MLX-NLC gel had the potential to avoid systemic uptake and hence the risk of systemic adverse effects. MLX-NLC gel demonstrated good skin tolerability and biosafety. Excellent physical stability of nanogel was observed at 4 ± 2 °C. SIGNIFICANCE: The study revealed that NLC gel is a promising carrier system for the topical application of MLX without side effects.
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Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Lípidos/química , Polietilenglicoles/química , Polietileneimina/química , Absorción Cutánea/fisiología , Tiazinas/química , Tiazoles/química , Administración Cutánea , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Estabilidad de Medicamentos , Femenino , Lípidos/administración & dosificación , Masculino , Meloxicam , Nanogeles , Polietilenglicoles/administración & dosificación , Polietilenglicoles/metabolismo , Polietileneimina/administración & dosificación , Polietileneimina/metabolismo , Conejos , Ratas , Absorción Cutánea/efectos de los fármacos , Tiazinas/administración & dosificación , Tiazinas/metabolismo , Tiazoles/administración & dosificación , Tiazoles/metabolismoRESUMEN
Reduction-controlled hierarchical unpacking is proposed for the development of virus-mimicking gene carriers. Disulfide-bond-modified hyaluronic acid (HA) is deposited onto the surface of diselenide-conjugated oligoethylenimine/DNA polyplexes to form DNA/OEI-SeSex/HA-SS-COOH (DOS) polyplexes. The cleavage of the disulfide and diselenide bonds is triggered by the gradient GSH level at the tumor site and inside the cells. The transfection efficiency of DOS show significant enhancement over DNA/poly(ethylene imine) (DP) in vitro and in vivo.
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Materiales Biomiméticos/química , ADN/genética , ADN/metabolismo , Portadores de Fármacos/química , Polietileneimina/química , Transfección/métodos , Virus/química , Animales , Materiales Biomiméticos/metabolismo , Portadores de Fármacos/metabolismo , Endocitosis , Células Hep G2 , Humanos , Ratones , Oxidación-Reducción , Polietileneimina/metabolismo , Selenio/química , Virus/metabolismoRESUMEN
Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA. They recognize specific cellular nucleic acid sequences and form stable complexes with complementary DNA or RNA. Here, we designed an oligo-aspartic acid-PNA conjugate and showed its enhanced delivery into cells with high gene correction efficiency using conventional cationic carriers, such as polyethylenimine (PEI) and Lipofectamine 2000. The negatively charged oligo-aspartic acid-PNA (Asp(n)-PNA) formed complexes with PEI and Lipofectamine, and the resulting Asp(n)-PNA/PEI and Asp(n)-PNA/Lipofectamine complexes were introduced into cells. We observed significantly enhanced cellular uptake of Asp(n)-PNA by cationic carriers and detected an active splicing correction effect even at nanomolar concentrations. We found that the splicing correction efficiency of the complex depended on the kind of the cationic carriers and on the number of repeating aspartic acid units. By enhancing the cellular uptake efficiency of PNAs, these results may provide a novel platform technology of PNAs as bioactive substances for their biological and therapeutic applications.
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Ácido Aspártico/análogos & derivados , Ácidos Nucleicos de Péptidos/administración & dosificación , Ácidos Nucleicos de Péptidos/química , Ácido Aspártico/metabolismo , Cationes/metabolismo , Portadores de Fármacos/metabolismo , Células HeLa , Humanos , Lípidos/análisis , Ácidos Nucleicos de Péptidos/genética , Ácidos Nucleicos de Péptidos/metabolismo , Polietileneimina/metabolismo , Empalme del ARN , TransfecciónRESUMEN
The adeno-associated virus (AAV) is one of the most useful viral vectors for gene delivery for both in vivo and in vitro applications. A variety of methods have been established to produce and characterize recombinant AAV (rAAV) vectors; however most methods are quite cumbersome and obtaining consistently high titer can be problematic. This protocol describes a triple-plasmid co-transfection approach with 25 kDa linear polyethylenimine (PEI) in 293 T cells for the production of AAV serotype 2. Seventy-two hours post-transfection, supernatant and cells were harvested and purified by a discontinuous iodixanol density gradient ultracentrifugation, then dialyzed and concentrated with an Amicon 15 100,000 MWCO concentration unit. To optimize the protocol for AAV2 production using PEI, various N/P ratios and DNA amounts were compared. We found that an N/P ratio of 40 coupled with 1.05 µg DNA per ml of media (21 µg DNA/15 cm dish) was found to produce the highest yields for viral replication and assembly measured multiple ways. The infectious units, as determined by serial dilution, were between 1×10(8) and 2×10(9) IU/ml. The genomic titer of the viral stock was determined by qPCR and ranged from 2×10(12) to 6×10(13) VG/ml. These viral vectors showed high expression both in vivo within the brain and in vitro in cell culture. The use of linear 25 kDa polyethylenamine PEI as a transfection reagent is a simple, more cost-effective, and stable means of high-throughput production of high-titer AAV serotype 2. The use of PEI also eliminates the need to change cell medium post-transfection, lowering cost and workload, while producing high-titer, efficacious AAV2 vectors for routine gene transfer.
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Dependovirus/crecimiento & desarrollo , Dependovirus/aislamiento & purificación , Polietileneimina/química , Transfección , Línea Celular , Centrifugación por Gradiente de Densidad , Diálisis , Humanos , Nitrógeno/análisis , Fósforo/análisis , Polietileneimina/metabolismo , Carga Viral , Cultivo de Virus/métodosRESUMEN
Branched Polyethylenimine, 25 kDa (PEI), was blended with gellan gum, an anionic heteropolysaccharide, for partial neutralization of its excess positive charge to form gellan gum-polyethylenimine (GP) nanocomposites (NCs). Subsequently, we manipulated the amount of gellan gum for obtaining a series of NCs and characterized them for their size, charge and morphology. Among all the NCs, one member, named GP3, showed the best transfection efficiency in tested cell lines in comparison with the rest of the series, PEI, Lipofectamine and other commercial transfection agents and also exhibited minimum cytotoxicity. It was found to transfect primary cells of mouse skin with better efficiency than PEI and Lipofectamine and was able to protect the plasmid DNA from nucleases and serum proteins present in the blood. GP3 exhibited efficient intracellular delivery of plasmid as revealed by confocal studies while its intracellular presence was also confirmed by the knockdown of GFP expression (using GFP specific siRNA) and JNKII by quantifying proteins in cell lysates and by western blotting and hybridization, respectively. In vivo cytotoxicity studies in Drosophila showed lack of induction of stress response in the exposed organisms. Further, exposed organisms did not show any developmental delay or mortality and no morphological defects were observed in the emerged flies. In vivo gene expression studies in Balb/c mice revealed maximum expression of luciferase enzyme in spleen. The study suggests that GP3 may act as an efficient non-viral gene carrier with diverse biomedical applications.
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Sistemas de Liberación de Medicamentos , Terapia Genética/métodos , Nanocompuestos/química , Polietileneimina/química , Polisacáridos Bacterianos/química , Animales , Técnicas de Cultivo de Célula , ADN/análisis , ADN/metabolismo , Evaluación Preclínica de Medicamentos , Excipientes/química , Femenino , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Nanocompuestos/análisis , Plásmidos , Polietileneimina/metabolismo , Polisacáridos Bacterianos/metabolismo , ARN Interferente Pequeño/genética , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Thermoregulatory neurons in the preoptic area of the anterior hypothalamus (POA) form synaptic networks, which affect responses that regulate body temperature. To characterize these pathways of activation, projections to effector control areas, like the dorsomedial hypothalamus (DMH), require labeling in live tissue slices. Traditional fluorescent dyes label axon terminals near an injection site, but unfortunately, also that of nearby fibers of passage. Here, we describe a novel methodology for retrograde labeling of neurons in vitro, which will allow for further electrophysiological recording. To determine if POA neurons project to the DMH, we have used nanometer-sized, gold nanoprobes, which provide for specific neuronal entry, via synapses in close proximity to the injection site. Upon neuronal entry, these nanoprobe complexes diffuse to the soma, where they are readily visualized and quantified. We found that conjugation of these gold nanoprobes with VGLUT-2 antibodies and polyethyleneimine (PEI) facilitates neuronal entry and a high level of labeling efficacy. This novel method, adapted from emerging cancer therapy technologies, is highly specific for determining axon terminal projections within particular neuronal populations, while maintaining neuronal viability for targeted live cell electrophysiological recording.
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Compuestos de Oro , Hipotálamo/fisiología , Nanopartículas del Metal , Neuronas/fisiología , Área Preóptica/fisiología , Animales , Anticuerpos/metabolismo , Electrofisiología/instrumentación , Electrofisiología/métodos , Hipotálamo/citología , Técnicas In Vitro , Masculino , Nanotecnología , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Trazadores del Tracto Neuronal , Neuronas/citología , Polietileneimina/metabolismo , Área Preóptica/citología , Ratas , Ratas Sprague-Dawley , Sinapsis/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/inmunología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
BACKGROUND: Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo. METHODS: Non-liposomal cationic vectors (FuGene 6), polyethylenimines (ExGen 500), and histidylated polylysine (HPL) were used as non-viral vectors in vitro with secreted alkaline phosphatase (SEAP) as reporter gene. Transfection efficiency was compared in cultured rat, rabbit and human VSMCs and fibroblasts. Zinc chloride (ZnCl2) was added to optimize transfection of rat VSMCs in vitro which were then seeded in vivo. RESULTS: Much higher SEAP levels were obtained in rabbit cells with FuGene 6 (p <0.0001) at day 2 than in equivalent rat and human cells. Rat VSMCs transfected in vitro with FuGene 6 and ExGen 500 expressed higher SEAP levels than with HPL. In rat VSMCs, SEAP secretion was more than doubled by addition of 250 microM ZnCl2 (p <0.0001) for all vectors. Seeding of syngeneic VSMCs transfected under optimized conditions (FuGene 6/pcDNA3-SEAP +250 microM ZnCl2) into healthy Lewis rats using various routes or into post-infarct myocardial scar resulted in a peak of SEAP expression at day 2 and detectable activity in the plasma for at least 8 days. CONCLUSIONS: FuGene 6 is an efficient non-viral transfection reagent for gene transfer in somatic smooth muscle cells in vitro and ZnCl2 enhances its efficiency. This increased expression of the transgene product is maintained after seeding in vivo.
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Terapia Genética , Vectores Genéticos , Fosfatasa Alcalina/metabolismo , Animales , Aorta/metabolismo , Cationes , Supervivencia Celular , Células Cultivadas , Cloruros/química , Cloruros/metabolismo , Cloruros/farmacología , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Genes Reporteros , Humanos , Miocitos del Músculo Liso/citología , Plásmidos/metabolismo , Polietileneimina/metabolismo , Polilisina/química , Conejos , Ratas , Ratas Endogámicas Lew , Factores de Tiempo , Transfección , Zinc/química , Compuestos de Zinc/química , Compuestos de Zinc/metabolismo , Compuestos de Zinc/farmacologíaRESUMEN
Haloalkane dehalogenase from Rhodococcus rhodochrous was covalently immobilized onto a polyethyleneimine impregnated gamma-alumina support. The dehalogenating enzyme was found to retain greater than 40% of its original activity after immobilization, displaying an optimal loading (max. activity/supported protein) of 70 to 75 mg/g with an apparent maximum (max. protein/support) of 156 mg/g. The substrate, 1,2,3-trichloropropane, was found to favorably partition (adsorb) onto the inorganic alumina carrier (10 to 20 mg/g), thereby increasing the local reactant concentration with respect to the catalyst's environment, whereas the product, 2,3-dichloropropan-1-ol, demonstrated no affinity. Additionally, the inorganic alumina support exhibited no adverse effects because of solvent/component incompatibilities or deterioration due to pH variance (pH 7.0 to 10.5). As a result of the large surface area to volume ratio of the support matrix and the accessibility of the bound protein, the immobilized biocatalyst was not subject to internal mass transfer limitations. External diffusional restrictions could be eliminated with simple agitation (mixing speed: 50 rpm; flux: 4.22 cm/min). The pH-dependence of the immobilized dehalogenase was essentially the same as that for the native enzyme. Finally, both the thermostability and resistance toward inactivation by organic solvent were improved by more than an order of magnitude after immobilization.
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Enzimas Inmovilizadas/metabolismo , Hidrolasas/metabolismo , Propano/análogos & derivados , Rhodococcus/enzimología , Óxido de Aluminio/metabolismo , Catálisis , Materiales Biocompatibles Revestidos , Estabilidad de Enzimas/efectos de los fármacos , Estabilidad de Enzimas/fisiología , Concentración de Iones de Hidrógeno , Cinética , Matemática , Microscopía Electrónica de Rastreo , Polietileneimina/metabolismo , Propano/farmacocinéticaRESUMEN
Triplex-forming oligonucleotides (TFOs) show potential for sequence-specific DNA binding and inhibition of gene expression. We have applied this antigene strategy using a TFO incorporating an Auger-emitting radionucleotide, 125I, to study the production of double-strand breaks (dsb) in the rat aquaporin 5 (rAQP5) cDNA. 125I-TFO bound to the pCMVrAQP5 plasmid in vitro in a dose-dependent manner and formed stable triplexes up to 65 degrees C and in the presence of 140 mM KCl. Further, 125I-TFO resulted in a predictable dsb when analyzed by Southern hybridization. To deliver TFOs to epithelial cells, we employed 125I-TFO-polyethyleneimine-adenovirus (125I-TFO-PEI-Ad) complexes. We hypothesized that these complexes would take advantage of adenoviral characteristics to transfer 125I-TFO to the cell nucleus. Adenovirus-containing complexes brought about greater uptake and nuclear localization of TFOs compared with delivery with 125I-TFO-PEI complexes alone. No significant degradation of 125I-TFO was found after delivery into cells using PEI-Ad complexes and freezing and thawing. We next used PEI-Ad complexes to deliver 125I-TFO and pCMVrAQP5 separately to epithelial cells to determine if triplexes can form de novo within cells, resulting in the specific dsb in the rAQP5 cDNA. After delivery, cell pellets were stored at -80 degrees C for more than 60 days. Thereafter, plasmid DNA was isolated from cells and analyzed for dsb by Southern hybridization. However, none were detected. We conclude that under the experimental conditions employed, effective triplexes, with 125I-TFO and pCMVrAQP5, do not form de novo inside cells.
Asunto(s)
Acuaporinas/metabolismo , ADN Complementario/metabolismo , ADN/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Proteínas de la Membrana , Conformación de Ácido Nucleico , Oligonucleótidos/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Acuaporina 5 , Acuaporinas/genética , Southern Blotting , Línea Celular , Daño del ADN , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Radioisótopos de Yodo/química , Oligonucleótidos/genética , Plásmidos/genética , Plásmidos/metabolismo , Polietileneimina/metabolismo , Ratas , TransfecciónRESUMEN
Graft-copolymers, containing poly(ethylene glycol) (PEG) and polyethyleneimine (PEI) chains have been proposed as carriers for delivery of phosphorothioate oligonucleotides (SODNs). Complexes of such copolymers with SODN self-assemble into particles having a core of neutralized PEI and SODN and a corona of PEG. Transferrin molecules are attached to the PEG corona using avidin/biotin construct. For this purpose, biotin moieties are covalently linked to the free ends of the PEG chains in the PEG-g-PEI copolymer. SODNs are reacted with mixtures of biotinylated and biotin-free PEG-g-PEI copolymers of various compositions to adjust the number of the biotin moieties in the complex. Resulting complexes have small size (ca. 40 nm) and do not aggregate in aqueous solutions for at least several days. To attach transferrin, they are supplemented first with avidin and then with biotin-transferrin conjugate. This increases the effective diameter of the particles to ca. 75-103 nm, depending on the composition of the complex. Cellular accumulation and fluorescence microscopy studies characterize the effects of these modifications on interaction of fluorescently labeled SODNs with KBv cell monolayers. The data suggest significant enhancement of SODN association with cells resulting from modification of the complex with transferrin. SODN complimentary to the site 546-565 of human mdr 1-mRNA was used to inhibit expression of the drug efflux transporter, P-glycoprotein (P-gp), in multiple drug resistant (MDR) cancer cells (KBv, MCF-7 ADR). Accumulation of a P-gp specific probe, rhodamine 123, in the cell monolayers is used to characterize the effects on P-gp efflux system following the treatment of the cells with antisense SODN or its complexes. This study suggests that antisense SODN incorporated in the complexes retain the ability to inhibit P-gp efflux system, while complexes of the randomized control SODN are inactive. Therefore, the antisense SODN is released from the complex and interacts with its intracellular target upon interaction of the complexes with the cells. Furthermore, modification of the complexes with transferrin leads to a significant increase of the effects of the antisense SODN on the P-gp efflux system in the cells. Overall, this study suggests that polyion complex micelles with protein-modified corona are promising tools for the delivery of antisense SODN.