RESUMEN
A new poly(aminoester) (EPAE-FA) containing folic acid and amino groups in the backbone and side chain was synthesized. EPAE-FA self-assembled readily with the plasmid DNA (pCMV-ßgal) in HEPES buffer and was characterized by dynamic light scattering, zeta potential, fluorescence images, and XTT cell viability assays. To evaluate the transfection effect of graft ratio of FA on the EPAE system, EPAE-FA polymers with two different graft ratios (EPAE-FA12k and EPAE-FA14k) were also prepared. This study found that all EPAE-FA polymers were able to bind plasmid DNA and yielded positively charged complexes with nano-sized particles (< 200 nm). To assess the transfection efficiency mediated by EPAE and EPAE-FA polymers, we performed in vitro transfection activity assays using FR-negative (COS-7) and FR-positive (HeLa) cells. The EPAE-FA12k/DNA and EPAE-FA14k/DNA complexes were able to transfect HeLa cell in vitro with higher transfection efficiency than PEI25k/DNA at the similar weight ratio. These results demonstrated that the introduction of FA into EPAE system had a significant effect on transferring ability for FR-positive cells (HeLa). Examination of the cytotoxicity of PEI25k and EPAE-FA system revealed that EPAE-FA system had lower cytotoxicity. In this paper, EPAE-FA seemed to be a novel cationic poly(aminoester) for gene delivery and an interesting candidate for further study.
Asunto(s)
Ácido Fólico/análogos & derivados , Ácido Fólico/síntesis química , Indicadores y Reactivos/síntesis química , Poliaminas/síntesis química , Transfección , Animales , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , ADN/química , ADN/genética , Electroforesis en Gel de Agar , Ácido Fólico/toxicidad , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Indicadores y Reactivos/toxicidad , Tamaño de la Partícula , Plásmidos/química , Plásmidos/genética , Poliaminas/toxicidad , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , VolumetríaRESUMEN
Two new urethane-based acrylates (UAA and PEG-UAA) were synthesized as polymer blocks. The chemical composition of the two monomers was confirmed by IR and NMR. After cross-linking these blockers by radical polymerization, "hexamethylene PU" [poly(hexamethylene-urethane)] and "PEG-hexamethylene PU" [PEG-poly(hexa-methylene-urethane)] were obtained. The platelet adhesion and platelet activation of these polymers were evaluated in the presence of Platelet Rich Plasma (PRP) blood. The relative blood clotting indexes of the polymers were determined to measure their capability of reducing thrombogenicity. The hemolysis of red blood cells was also assessed to examine the haemocompatibility of the polymers. The hexamethylene PU and PEG-hexamethylene PU showed less platelet adhesion, platelet activation, blood clotting and hemolysis than a commercial PU (Tecoflex). The liquid crystal molecule, cholesteryl oleyl carbonate (COC), showed further improved biocompatibility to human blood, after COC was embedded in the PU polymers. PEG-hexamethylene PU + 10% COC demonstrated the best activity in reducing thrombogenicity and the best haemocompatibility. The inclusion of PEG segments into the PEG-UAA structure increased its hydrophilicity. The methylene bis(cyclohexyl) segments in Tecoflex PU decreased haemocompatibility. These observations are in good agreement with performed contact angle measurements. The PEG-hexamethylene PU loaded with COC might be a promising material for applications in bioengineering.
Asunto(s)
Materiales Biocompatibles/síntesis química , Ensayo de Materiales , Poliuretanos/síntesis química , Materiales Biocompatibles/química , Bioingeniería , Coagulación Sanguínea , Plaquetas/fisiología , Hemólisis , Humanos , Activación Plaquetaria , Adhesividad Plaquetaria , Poliuretanos/química , Propiedades de SuperficieRESUMEN
To improve the cytotoxicity of PEI25k and the transfection efficiency of poly(beta-amino ester) with DNA, we synthesized a poly(beta-amino ester), PEDP, bearing ester linkages in the backbone and tertiary amines in the backbone and side chain and prepared a binary mixture, PEDP-PEI25k, using physical blending meyhod. Both poly(beta-amino ester) PEDP and binary mixture PEDP-PEI25k, readily self-assembled with plasmid DNA (pCMV-beta gal) in a HEPES buffer, were characterized by dynamic light scattering. The results reveal that PEDP-PEI25k was able to self-assemble plasmid DNA into PEDP-PEI25k/DNA nano-complexes small enough to enter a cell through endocytosis. Titration studies were performed to determine the buffering capacities of PEDP and PEDP-PEI25k. The COS-7 cell viabilities in the presence of PEDP and PEDP-PEI25k were studied. At low mass ratio of PEDP/PEI25k (1/1), it is found that the transfection curve of PEDP-PEI25k/DNA bearing a maximum peak is similar to that of PEI25k/DNA. In addition, the PEDP-PEI25k/DNA complexes were able to transfect COS-7 cells in vitro with a high efficiency comparable to a well-known gene carrier PEI25k/DNA. The results indicate that binary mixture PEDP-PEI25k is an attractive cationic carrier for gene delivery and an interesting candidate for further study.
Asunto(s)
Transfección , Aminas/química , Animales , Células COS , Cationes , Chlorocebus aethiops , ADN/química , ADN/genética , Ésteres , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Humanos , Plásmidos , PolímerosRESUMEN
To improve the self-assembly efficiency of nanoparticles with DNA, we synthesized lysine-based poly(urethane-co-ester) PMMD (6) and polyester PDMA (8) bearing ester linkages in the backbone and tertiary amines in the side chain. Both poly(urethane-co-ester) PMMD (6) and polyester PDMA (8), readily self-assembled with plasmid DNA (pCMV-beta-gal) in HEPES buffer, were characterized by dynamic light scattering and zeta-potential. The results reveal that PMMD (6) and PDMA (8) were able to self-assemble particles with DNA and yield complexes with positive charges of 80-115 nm and 170-180 nm in size at mass ratios (W/W) of 2/1 and 20/1, respectively. The degradation studies indicate that the half-life of PMMD (6) in the HEPES buffer was 20 h at pH 7.4. Titration studies were performed to determine the buffering capacities of the polymers. In addition, the COS-7 cell viabilities in the presence of PMMD/DNA, PDMA/DNA, and PEI/DNA were studied. The results indicate that PMMD (6) is an attractive cationic poly(urethane-co-ester) for gene delivery and an interesting candidate for further study.
Asunto(s)
ADN/química , Lisina/química , Nanopartículas/química , Poliuretanos/síntesis química , Acrilamidas/química , Aminas/química , Animales , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Diseño de Fármacos , Etanol/química , Hidrólisis , Tamaño de la Partícula , Poliuretanos/química , Poliuretanos/toxicidadRESUMEN
New polyurethane 2-diethylaminoethylamine-polyurethane (LGEA-PU) containing poly(ethylene glycol) segments and tertiary amines was synthesized. LGEA-PU self-assembled readily with the plasmid DNA (pCMV-betagal) in HEPES buffer and was characterized by dynamic light scattering, zeta potential, atomic force microscopy, and XTT cell viability assays. To examine the effect of molecular weight of LGEA-PU systems on transfection, LGEA-PU systems of four different molecular weights (LGEA-PU99, LGEA-PU59, LGEA-PU24, and LGEA-PU7) were prepared. This study found that LGEA-PU99, LGEA-PU59, and LGEA-PU24 were able to bind plasmid DNA and yielded positively charged complexes with a nano-sized transfection (<200 nm). The LGEA-PU59/DNA complexes were able to transfect COS-7 cells in vitro with higher transfection efficiency than the other LGEA-PU systems. These results demonstrated that molecular weights of LGEA-PU systems had a significant effect on transferring ability, except for LGEA-PU99, which showed the strongest DNA condensation. Examination of the cytotoxicity of PEI and LGEA-PU systems revealed that LGEA-PU systems had lower cytotoxicity. In this article, LGEA-PU59 seemed to be a novel cationic polyurethane for gene delivery and an interesting candidate for further study.
Asunto(s)
Materiales Biocompatibles/química , Vectores Genéticos/química , Poliuretanos/química , Transfección , Animales , Materiales Biocompatibles/metabolismo , Biodegradación Ambiental , Células COS , Chlorocebus aethiops , Vectores Genéticos/metabolismo , Peso Molecular , Poliuretanos/metabolismoRESUMEN
The aim of this study is to investigate how the presence of liquid crystal, cholesteryl oleyl carbonate, embedded into polymers (PMMA, Eb270, PU) affects the biocompatibility of composite membranes with human blood. The effects of different surface textures of composite membranes on platelet adhesion and platelet activation were evaluated as well. The adhesion and geometric deformation of platelets were demonstrated by SEM. The quantitative assay of platelet activation was determined by measuring the production of P-Selectin, and by measurement of the blood clotting index when PRP blood was incubated with pure polymer films and composite membranes. Moreover, the hemolysis studies on the damage to red blood cells were performed to gain information on the hemocompatibility of these biomaterials. The results showed that inclusion of cholesteryl oleyl carbonate (COC) embedded in composite membranes, improves their biocompatibility with respect to a substantial reduction of platelet adhesion and the controlled decrease of platelet activation. As the COC content of composite membranes was increased, the value of the blood clotting index increased and the production of P-Selectin decreased. The results also showed that the presence of COC resulted in a decrease of hemolysis ratios. Comparing among three different composite membranes, the best biocompatibility is achieved when PU/COC> or ==Eb270/COC>PMMA/COC. The in vitro studies performed in this work suggest that it may be reasonable to use liquid crystal COC as a mean of surface modification to improve the blood compatibility of biopolymers.
Asunto(s)
Resinas Acrílicas/toxicidad , Cristales Líquidos/toxicidad , Membranas Artificiales , Polimetil Metacrilato/toxicidad , Poliuretanos/toxicidad , Resinas Acrílicas/química , Adsorción , Coagulación Sanguínea/efectos de los fármacos , Carbonatos/química , Carbonatos/toxicidad , Células Cultivadas , Eritrocitos/efectos de los fármacos , Eritrocitos/patología , Hemólisis/efectos de los fármacos , Humanos , Cristales Líquidos/química , Ensayo de Materiales , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Polimetil Metacrilato/química , Poliuretanos/químicaRESUMEN
The characteristics of polymer-DNA complexes formed by positively-negatively charged interaction have a great influence on their transfection potential. Since the limit changes in thermal transitions which were hardly measured in conventional calorimetry, now in this study they have been successfully carried out by highly-sensitive differential scanning calorimetry for better understanding the pDMAEMA-plasmid DNA complexing process. Thermal behaviors of plasmid DNA, polymer and their formed complexes were recorded to give insights into their conformational changes when temperature was raised. In results, the supercoiled or open-circular plasmid DNA is not thermal reversible indicated by the decrease of denaturation peak and disappearance of DNA conformational transition related to its twist status at 50-70 degrees C. The cationic polymer is thermally stable by showing reversible transition peaks after two heating processes. For the cationic polymer-plasmid DNA complexes, electrostatic forces lead to a higher denaturation temperature of plasmid DNA and transition temperature of polymer. Also, heat can cause a topological change in plasmid DNA and then change their mutual complexation capacity.
Asunto(s)
Calor , Plásmidos/química , Polímeros/química , Rastreo Diferencial de Calorimetría , Cationes/química , Sistemas de Liberación de Medicamentos , Estabilidad de Medicamentos , Metacrilatos/química , Desnaturalización de Ácido Nucleico , Nylons/químicaRESUMEN
To improve the transfection efficiency of poly(urethane-co-ester) and the cytotoxicity of PEI25k with DNA, we synthesized a new poly(urethane-co-ester), PUE, bearing ester linkages and amino groups in the backbone and urethane linkages in the side-chain, and then prepared a binary mixture, PUE-PEI25k, using a physical blending method. The structure of PUE was confirmed by FT-IR and NMR spectra. Both poly(urethane-co-ester), PUE, and binary mixture PUE-PEI25k, readily self-assembled with plasmid DNA (pCMV-ßgal) in a HEPES buffer, were characterized by dynamic light scattering. The results revealed that PUE and PUE-PEI25k were able to self-assemble plasmid DNA into PUE/DNA and PUE-PEI25k/DNA nano-complexes small enough to enter a cell through endocytosis. Titration studies were performed to determine the buffering capacities of PUE and PUE-PEI25k. The COS-7 cell viability in the presence of PEI25k, PUE and PUE-PEI25k was studied. At low mass ratio of PUE/PEI25k (150:1), it was found that the PUE-PEI25k/DNA complexes were able to transfect COS-7 cells in vitro with a high efficiency comparable to a well-known gene carrier, PEI25k/DNA. The results indicate that the binary mixture PUE-PEI25k is an attractive cationic carrier for gene delivery and an interesting candidate for further study.
Asunto(s)
Portadores de Fármacos/química , Portadores de Fármacos/síntesis química , Poliésteres/química , Poliésteres/síntesis química , Poliuretanos/química , Poliuretanos/síntesis química , Transfección/métodos , Animales , Células COS , Chlorocebus aethiops , ADN/química , ADN/genética , ADN/metabolismo , Portadores de Fármacos/toxicidad , Hidrólisis , Tamaño de la Partícula , Plásmidos/química , Plásmidos/genética , Plásmidos/metabolismo , Poliésteres/toxicidad , Poliuretanos/toxicidadRESUMEN
A one-step preparation of nanoparticles with poly(lactide-co-glycolide) (PLGA) pre-modified with polyethylenimine (PEI) is better in requirements for DNA delivery compared to those prepared in a two-step process (preformed PLGA nanoparticles and subsequently coated with PEI). The particles were prepared by emulsification of PLGA/ethyl acetate in an aqueous solution of PVA and PEI. DLS, AFM and SEM were used for the size characteristics. The cytotoxicity of PLGA/PEI nanoparticles was detected by MTT assay. The transfection activity of the particles was measured using pEGFP and pß-gal plasmid DNA. Results showed that the PLGA/PEI nanoparticles were spherical and non-porous with a size of about 0.2 µm and a small size distribution. These particles had a positive zeta potential demonstrating that PEI was attached. Interestingly, the zeta potential of the particles (from one-step procedure) was substantially higher than that of two-step process and is ascribed to the conjugation of PEI to PLGA via aminolysis. The PLGA/PEI nanoparticles were able to bind DNA and the formed complexes had a substantially lower cytotoxicity and a higher transfection activity than PEI polyplexes. In conclusion, given their small size, stability, low cytotoxicity and good transfection activity, PLGA/PEI-DNA complexes are attractive gene delivery systems.
Asunto(s)
ADN/metabolismo , Nanopartículas , Polietileneimina/química , Poliglactina 910/química , Transfección/métodos , Acetatos/química , Animales , Células COS , Cationes , Chlorocebus aethiops , ADN/química , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Tamaño de la Partícula , Polietileneimina/toxicidad , Poliglactina 910/toxicidad , Alcohol Polivinílico/química , Propiedades de Superficie , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
New glycidyl-based (epoxide-based) poly(aminoester) (EPAE) containing hydroxyl and amino groups in the backbone and side chain was synthesized. EPAE self-assembled readily with the plasmid DNA(pCMV-betagal) in HEPES buffer and was characterized by dynamic light scattering, Zeta-potential, fluorescence images, and XTT cell viability assays. To evaluate the effect of molecular weight of EPAE system on transfection, EPAE polymers with three different molecular weights (EPAE22k, EPAE18k, and EPAE8k) were also prepared. This study found that all EPAE polymers were able to bind plasmid DNA and yielded positively charged complexes with a nano-sized particles (200 nm). The EPAE22k/DNA and EPAE18k/DNA complexes were able to transfect COS-7 cell in vitro with higher transfection efficiency than other EPAE8k/DNA. These results demonstrated that molecular weight of EPAE system had a significant effect on transferring ability. Examination of the cytotoxicity of PEI25k and EPAEs system revealed that EPAEs system had lower cytotoxicity. In this article, EPAEs seemed to be a novel cationic poly(aminoester) for gene delivery and an interesting candidate for further study.
Asunto(s)
ADN/química , Compuestos Epoxi/síntesis química , Metacrilatos/síntesis química , Plásmidos , Polímeros/síntesis química , Transfección/métodos , Animales , Tampones (Química) , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Cromatografía en Gel , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Compuestos Epoxi/toxicidad , Células HeLa , Humanos , Hidrólisis , Luz , Espectroscopía de Resonancia Magnética , Metacrilatos/toxicidad , Estructura Molecular , Peso Molecular , Nanopartículas , Polímeros/toxicidad , Dispersión de Radiación , Espectroscopía Infrarroja por Transformada de Fourier , Factores de TiempoRESUMEN
To improve the transfection efficiency of polycations with DNA, we synthesized poly(ester-co-urethane)(PEU-g-PEI800) with short chain PEI800 in the side chain, and poly(ester-co-urethane)(PEU) without short chain PEI800. Both PEU-g-PEI800 and PEU, readily self-assembled with plasmid DNA (pCMV-betagal) in a HEPES buffer, were characterized by dynamic light scattering and zeta-potential. The results reveal that PEU-g-PEI800 and PEU were able to self-assemble particles with DNA and yield nano-sized complexes (<200nm) with positive charge at N/P ratios of 20/1 and 120/1, respectively. The degradation studies indicate that the half-life of PEU-g-PEI800 and PEU in the HEPES buffer were 14 and 35h at pH 7.4, respectively. Titration studies were performed to determine the buffering capacities of the polymers. The COS-7 cell viabilities in the presence of PEU-g-PEI800/DNA, PEU/DNA, and PEI25k/DNA were studied. In addition, The PEU-g-PEI800/DNA complexes were able to transfect COS-7 cells in vitro with a high efficiency comparable to a well-known gene carrier PEI25k. The results indicate that PEU-g-PEI800 is an attractive cationic poly (ester-co-urethane) for gene delivery and an interesting candidate for further study.
Asunto(s)
ADN/administración & dosificación , Poliésteres/química , Polietileneimina/química , Poliuretanos/química , Transfección , Cationes , ADN/química , ADN/genética , Hidrólisis , Luz , Espectroscopía de Resonancia Magnética , Microscopía de Fuerza Atómica , Plásmidos , Dispersión de RadiaciónRESUMEN
Polyurethanes (PUs) are a class of biodegradable polymers that have been applied as tissue-engineering materials with minimum toxicity. In our study, a new series of cationic PUs containing tertiary amines in the backbone and primary, secondary and tertiary amines in the side chains (PU1, PU2 and PU3, respectively) was synthesized and used as nonviral vectors for gene delivery. In addition, we introduced glycidol into the structure of PU for greater solubility and biocompatibility and grafted various amines in the side chains (PUg1, PUg2, PUg3). The structural characteristics of PUs and the physicochemical properties of their formed complexes with DNA were determined and correlated with their transfection efficiency. The results reveal that PU1, PU3, PUg1 and PUg3 could bind with DNA and yielded positively charged complexes with a condensed size required for transfection. These PUs are considered to be noncytotoxic (hundred times less) compared to polyethylenimine (PEI) or poly(2-dimethylaminoethyl methacrylate), (PDMAEMA). The hydrolytical degradation studies indicate that PU-glycidyl systems (PUg1 and PUg3) can be degraded in 20 mM HEPES buffer at pH 7.4 in approximately 8 h but that PU1 and PU3 lasted much longer. PUg1 and PUg3 are the best amongst cationic PUs to transfect DNA into COS-7 cells with an efficacy comparable to the well-known gene carrier PDMAEMA. In addition, PUg1 and PUg3 possess greater biocompatibility and biodegradability. A new way to prepare cationic polymers without cytotoxicity but with highly transfecting activity could be very helpful to the in vivo gene transfection where large amounts of cationic polymers are required.
Asunto(s)
Aminas/química , ADN/genética , Poliaminas/química , Poliuretanos/química , Transfección/métodos , Animales , Células COS , Catálisis , Supervivencia Celular , Chlorocebus aethiops , ADN/química , ADN/metabolismo , Enzimas de Restricción del ADN/química , Enzimas de Restricción del ADN/metabolismo , Electroforesis en Gel de Agar , Expresión Génica , Hidrólisis , Operón Lac/genética , Estructura Molecular , Tamaño de la Partícula , Plásmidos/química , Plásmidos/genética , Polielectrolitos , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Inefficient release of polymer/DNA complexes from endocytic vesicles into the cytoplasm and the cytotoxic nature of cationic polymers are two of the primary causes of poor gene delivery. EG-polyurethane [poly(ethylene glycol)-PU, Poly 1], EGDM-polyurethane [poly(ethylene glycol), 2-(dimethylamino)ethylamine-PU, Poly 2], and MDEADM-polyurethane [N-methyldiethanolamine, 2-(dimethylamino)ethylamine-PU, Poly 3] were designed in this study to overcome these obstacles. The structural characteristics of polyurethanes and physicochemical properties of their formed complexes with DNA were determined to correlate their transfection efficiency. The results revealed that Poly 2 and Poly 3 could bind with plasmid DNA and yield positively charged complexes with a size required for transfection. Poly 3 showed the best in buffering capacity and its formed complexes with DNA could transfect COS-7 cells better than those of Poly 2 and Poly 1. This study reveals that the amine groups in the polymeric structure and the buffer capacity of a polymeric transfectant would affect its potential in DNA delivery. Also the size and binding properties of DNA and polymeric transfectants can be in correlation to the transfection efficiency of resulting DNA/polymer complexes.
Asunto(s)
Plásmidos/administración & dosificación , Poliuretanos , Transfección/métodos , Animales , Biodegradación Ambiental , Tampones (Química) , Células COS , Chlorocebus aethiops , Estructura Molecular , Plásmidos/farmacocinética , Poliuretanos/síntesis química , Relación Estructura-Actividad , Transfección/normasRESUMEN
A new cationic polymer, N,N-diethylethylenediamine-polyurethane (DEDA-PU), bearing tertiary amines in the backbone and side chains, was synthesized and used as a nonviral vector for gene delivery. The DEDA-PU readily self-assembled with the plasmid DNA (pCMV-betagal) in water and buffer at physiological pH, as determined by agarose gel retardation, dynamic light scattering, zeta potential, atomic force microscopy (AFM), and restriction endonuclease protection assays. The results revealed that DEDA-PU was able to bind with plasmid DNA, yielding positively charged complexes with a size around 100 nm at a DEDA-PU/DNA ratio of 50/1 (w/w). The DEDA-PU/DNA complexes were able to transfect HEK 293 cells in vitro with an efficiency comparable to a well-known gene carrier [poly(2-dimethylaminoethyl methacrylate), PDMAEMA]. The cytotoxicity of DEDA-PU was substantially lower than PDMAEMA. The degradation studies indicated that DEDA-PU degrades hydrolytically in 20 mM HEPES buffer at pH 7.4 with a half-life of approximately 60 h. This study shows that DEDA-PU holds promise as biodegradable polycations for gene delivery and is interesting candidate for further study.