RESUMO
Use of cationic polymers as nonviral gene vectors has several limitations such as low transfection efficiency, high toxicity, and inactivation by serum. In this study, varying amounts of low molecular weight branched polyethylenimine 1.8 kDa (bPEI 1.8) were introduced on to a neutral polymer, poly(vinyl alcohol) (PVA), to bring in cationic charge on the resulting PVA-PEI (PP) nanocomposites. We rationalized that by introducing bPEI 1.8, buffering and condensation properties of the proposed nanocomposites would result in improved gene transfer capability. A series of PVA-PEI (PP) nanocomposites was synthesized using well-established epoxide chemistry and characterized by IR and NMR. Particle size of the PP/DNA complexes ranged between 120 to 135 nm, as determined by dynamic light scattering (DLS), and DNA retardation assay revealed efficient binding capability of PP nanocomposites to negatively charged nucleic acids. In vitro transfection of PP/DNA complexes in HEK293, HeLa, and CHO cells revealed that the best working formulation in the synthesized series, PP-3/DNA complex, displayed ~2-50-fold higher transfection efficiency than bPEIs (1.8 and 25 kDa) and commercial transfection reagents. More importantly, the PP/DNA complexes were stable over a period of time, along with their superior transfection efficiency in the presence of serum compared to serum-free conditions, retaining the nontoxic property of low molecular weight bPEI. The in vivo administration of PP-3/DNA complex in Balb/c mice showed maximum gene expression in their spleen. The study demonstrates the potential of PP nanocomposites as promising nonviral gene vectors for in vivo applications.
Assuntos
DNA , Expressão Gênica , Técnicas de Transferência de Genes , Nanocompostos/química , Polietilenoimina , Álcool de Polivinil , Animais , Células CHO , Cricetinae , Cricetulus , DNA/química , DNA/farmacologia , Vetores Genéticos/química , Vetores Genéticos/farmacologia , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoimina/química , Polietilenoimina/farmacologia , Álcool de Polivinil/química , Álcool de Polivinil/farmacologiaRESUMO
Chitosan was partially converted to its chlorohydrin derivative by the reaction with epichlohydrin, which was subsequently reacted with varying amounts of lPEI(2.5 kD) to obtain a series of chitosan-lPEI(2.5 kD) copolymers (CP). These copolymers were then characterized and evaluated in terms of transfection efficiency (in vitro and in vivo), cell viability, DNA release and buffering capacity. The CP-4 copolymer (the best among the CP series) showed enhanced transfection (-2 - 24 folds) in comparison with chitosan, lPEI(2.5 kD), bPEI(25 kD) and Lipofectamine in HEK293, HeLa and CHO cells. The buffering capacity (in the pH range of 3 - 7.5), as shown by confocal microscopy, and DNA-release capability of the CP copolymers, was found to be significantly enhanced over chitosan. Intravenous administration of CP-4/DNA polyplex in mice followed by the reporter gene analysis showed the highest gene expression in spleen. Collectively, these results demonstrate the potential of CP-4 copolymer as a safe and efficient nonviral vector. From the Clinical Editor: Chitosan -PEI (2.5 kD) copolymers (CP) were characterized and their transfection efficiency, DNA release and buffering capacity were studied. The CP-4 copolymer significantly enhanced buffering capacity and provided the highest gene expression levels. The method may be used to enhance DNA transfection.
Assuntos
Quitosana/análogos & derivados , DNA/administração & dosagem , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Polietilenoimina/análogos & derivados , RNA Interferente Pequeno/administração & dosagem , Adsorção/efeitos dos fármacos , Animais , Soluções Tampão , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Quitosana/química , Quitosana/toxicidade , Cricetinae , Cricetulus , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Tamanho da Partícula , Plasmídeos/metabolismo , Polietilenoimina/química , Polietilenoimina/toxicidade , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Eletricidade Estática , TransfecçãoRESUMO
Linear polyethylenimine (lPEI, 25 kDa) nanoparticles' (LPN) series was synthesized by varying percentage of cross-linking with 1,4-butanediol diglycidyl ether (BDE) and their size, surface charge, morphology, pDNA protection/release, cytotoxicity and transfection efficiency were evaluated. Synthesized nanoparticles (NPs) were spherical in shape (size: â¼109 - 235 nm; zeta potential: +38 to +16 mV). These NPs showed increased buffering capacity with increasing percent cross-linking and also exhibited excellent transfection efficiency (i.e., â¼1.3 - 14.7 folds in case of LPN-5) in comparison with lPEI and the commercial transfection agents used in this study. LPN-5 based GFP-specific siRNA delivery resulted in â¼86% suppression of targeted gene expression. These particles were relatively nontoxic in vitro (in cell lines) and in vivo (in Drosophila). In vivo gene expression studies using LPN-5 in Balb/c mice through intravenous injection showed maximum expression of the reporter gene in the spleen. These results together demonstrate the potential of these particles as efficient transfection reagents. FROM THE CLINICAL EDITOR: The authors demonstrate a novel method of synthesizing linear PEI nanoparticles to utilize these as transfection agents.
Assuntos
Butileno Glicóis/farmacologia , Nanopartículas/efeitos adversos , Polietilenoimina/farmacologia , Transfecção/métodos , Animais , Butileno Glicóis/química , DNA/química , DNA/genética , Drosophila , Portadores de Fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Nanopartículas/uso terapêutico , Polietilenoimina/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
The main aim of the current study is to compare the physicochemical properties, cytotoxicity and gene-transfer ability of electrostatically and covalently linked nanocomposites of polyethylenimine (PEI) and polyacrylic acid (PAA) on mammalian cells. Two series of nanocomposites, ionic PEI-PAA (iPP) and covalent PEI-PAA (cPP), were synthesized by varying the amounts of polyacrylic acid (PAA). Physicochemical characterization revealed that iPP nanopcomposites were of bigger sized than cPP nanocomposites with zeta potential almost comparable. Nucleic acid binding assay displayed that iPP and cPP nanocomposites, having sufficient cationic charge, efficiently interacted with plasmid DNA and completely retarded its electrophoretic mobility on agarose gel. In vitro MTT assay showed slightly higher cell viability of cPP/pDNA complexes over their ionic counterparts. Both the series of nanocomposite/pDNA complexes exhibited considerably higher transfection efficacy compared to pDNA complexes of native bPEI and the standard transfection reagent, Lipofectamine, with cPP/pDNA complexes performed much better than iPP/pDNA complexes. Flow cytometry further confirmed these findings where cPP-4/pDNA complex showed transfection in â¼ 85% HEK293 cells, while iPP-2/pDNA complex transfected â¼ 67% HEK293 cells. Lipofectamine/pDNA and bPEI/pDNA complexes could transfect just â¼ 35% and â¼ 26% HEK293 cells. All these results demonstrate the superiority of covalently linked nanocomposites (cPP) which could be used as efficient carriers for nucleic acids in future gene delivery applications.
Assuntos
Resinas Acrílicas/química , Nanocompostos/química , Plasmídeos/genética , Polietilenoimina/química , Animais , Células CHO , Sobrevivência Celular/genética , Cricetulus , DNA/química , DNA/genética , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência , Estrutura Molecular , Tamanho da Partícula , Plasmídeos/química , Espectrometria de Fluorescência , Transfecção/métodosRESUMO
Recently, polyethylenimines (PEIs) have emerged as efficient vectors for nucleic acids delivery. However, inherent cytotoxicity has limited their in vivo applications. To address this concern as well as to incorporate hydrophobic domains for improving interactions with the lipid bilayers in the cell membranes, we have tethered varying amounts of amphiphilic pyridoxyl moieties onto bPEI to generate a small series of pyridoxyl-PEI (PyP) polymers. Spectroscopic characterization confirms the formation of PyP polymers, which subsequently form stable complexes with pDNA in nanometric range with positive surface charge. The projected modification not only accounts for a decrease in the density of 1° amines but also allows formation of relatively loose complexes with pDNA (cf. bPEI). Alleviation of the cytotoxicity, efficient interaction with cell membranes and easy disassembly of the pDNA complexes have led to the remarkable enhancement in the transfection efficiency of PyP/pDNA complexes in mammalian cells with one of the formulations, PyP-3/pDNA complex, showing transfection in â¼68% cells compared to â¼16% cells by Lipofectamine/pDNA complex. Further, the efficacy of PyP-3 vector has been established by delivering GFP-specific siRNA resulting in â¼88% suppression of the target gene expression. These results demonstrate the efficacy of the projected carriers that can be used in future gene therapy applications.
Assuntos
Aminas/química , Materiais Biocompatíveis/farmacologia , Técnicas de Transferência de Genes , Polietilenoimina/farmacologia , Soluções Tampão , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Hemólise/efeitos dos fármacos , Heparina/metabolismo , Humanos , Ligantes , Lipídeos/química , Células MCF-7 , Ensaios de Proteção de Nucleases , Tamanho da Partícula , Plasmídeos/metabolismo , Polietilenoimina/síntese química , Polietilenoimina/química , RNA Interferente Pequeno/metabolismo , Eletricidade Estática , TitulometriaRESUMO
A series of electrostatically crosslinked nanoparticles, N-(2-hydroxyethyl)-polyethylenimine-PEG600 (HePP), was prepared by allowing N-(2-hydroxyethyl)-polyethylenimine (HeP) to interact with polyethyleneglycol (600) dicarboxylic acid (HOOC-PEG600-COOH, PEG600dc), they were then evaluated for their capability to transfect cells in vitro and in vivo. DLS studies revealed the size of the HePP nanoparticles in the range 106-170 nm, which efficiently condensed nucleic acids and provided sufficient protection against nuclease degradation. HePP-pDNA complexes exhibited a considerably higher transfection efficiency and cell viability in various mammalian cell lines, with HePP-3-pDNA displaying the highest gene expression, which outperformed HeP and the commercially available transfection reagent, Lipofectamine™. Also, HePP-3 mediated sequential delivery of GFP specific siRNA resulted in â¼76% suppression of the target gene. Intravenous administration of HePP-3-pDNA complex to mice, followed by monitoring of the reporter gene analysis post 7d, revealed the highest gene expression occurred in the spleen. Together, these results advocate the potential of HePP nanoparticles as efficient vectors for gene delivery in vitro and in vivo.
Assuntos
DNA/química , Técnicas de Transferência de Genes , Nanopartículas/química , Polietilenoglicóis/química , Polietilenoimina/química , RNA Interferente Pequeno/química , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Nanopartículas/administração & dosagem , Nanopartículas/toxicidade , Tamanho da PartículaRESUMO
Branched polyethylenimine (bPEI, 25 kDa) has been widely used as an efficient delivery vector for nucleic acids in vitro. However, its charge-associated toxicity has limited its in vivo applications. In an attempt to control its toxicity, it was reacted with varying amounts of glycidol (2,3-epoxy-1-propanol) to obtain a small series of hydrophilic polymers, 2,3-dihydroxypropyl-grafted-polyethylenimines (DHP-g-P). The resulting polymers were characterized by (1)H-NMR and subjected to interaction with negatively charged pDNA, which yielded complexes in the size range of ~171-190 nm with a zeta potential of â¼+33-39 mV. Acid-base titration revealed no effect of substitution on the buffering capacity of the modified polymers. Grafting of 2,3-dihydroxypropyl groups on bPEI significantly improved the cell viability (i.e. almost non-toxic) as well as the DNA release properties of these modified polymers compared to native bPEI. Formation of a relatively loose DHP-g-P25/pDNA complex (the best working system in terms of transfection efficiency) resulted in the efficient nuclear release of pDNA for transcription, a prerequisite for efficient transfection. Subsequently, upon evaluation of their ability to transfer nucleic acids in vitro, the DHP-g-P/pDNA complexes exhibited higher gene transfection efficiency with one of the formulations, DHP-g-P25/DNA complex, displaying ~2.7 folds higher GFP expression than bPEI and ~2.3-3.5 folds higher than the selected commercial transfection reagents used in this study. Further to quantify the extent of GFP positive cells, FACS analysis was performed, which revealed DHP-g-P25/DNA mediated gene expression in ~51% cells outcompeting bPEI, Superfect™, Fugene™ and Lipofectamine™. Sequential delivery of GFP-specific siRNA resulted in ~78% suppression of the target gene compared to ~49% achieved by Fugene™. All these results demonstrate the potential of these polymers for in vivo gene delivery.
Assuntos
Vetores Genéticos/genética , Ácidos Nucleicos/metabolismo , Polietilenoimina/síntese química , Polietilenoimina/metabolismo , Animais , Soluções Tampão , Células CHO , Sobrevivência Celular , Cricetinae , Cricetulus , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Microscopia Confocal , Ensaios de Proteção de Nucleases , Plasmídeos/metabolismo , Polietilenoimina/química , Eletricidade Estática , TransfecçãoRESUMO
Development of efficient and safe nucleic acid carriers (vectors) is one of the essential requirements for the success of gene therapy. Here, we have evaluated the gene transfer capability of chitosan-PEI (CP) conjugates prepared by conjugating low molecular weight branched polyethylenimine (LMWP) with depolymerized chitosans (7 and 10 kDa) via their terminal aldehyde/keto groups. The CP conjugates interacted efficiently with nucleic acids and also showed higher cellular uptake. These conjugates on complexation with DNA yielded nanoparticles in the size range of 100-130 nm (in case of C7P) and 115-160 nm (in case of C10P), which exhibited significantly higher transfection efficiency (~2-42 folds) in vitro compared to chitosans (high and low mol. wt.) and the commercially available transfection reagents retaining cell viability almost comparable to the native chitosan. Of the two CP conjugates, chitosan 7 kDa-LMWP (C7P) displayed higher gene transfer ability in the presence and absence of serum. Luciferase reporter gene analysis in male Balb/c mice receiving intravenous administration of C7P3/DNA polyplex showed the maximum expression in their spleen. Further, tuftsin, a known macrophage targeting molecule, was tethered to C7P3 and the resulting complex, i.e., C7P3-T/DNA, exhibited significantly higher gene expression in cultured mouse peritoneal macrophages as compared to unmodified C7P3/DNA complex without any cytotoxicity demonstrating the suitability of the conjugate for targeted applications. Conclusively, the study demonstrates the potential of the projected conjugates for gene delivery for wider biomedical applications.