RESUMEN
[This corrects the article DOI: 10.1016/j.toxrep.2014.11.006.].
RESUMEN
A series of polyethylenimine (PEI) and γ-polyglutamic acid (PGA) nanocomposites (PPGA) was prepared and evaluated in terms of their cell viability and transfection efficiency in vitro and in vivo. On complexion with pDNA, the positively charged PPGA/DNA nanocomposites resulted in a higher level of in vitro reporter gene transfection (2.7-7.9-fold) as compared to native PEI, and selected commercial reagents and >95% cell viability in HEK293, HeLa and HepG2 cell lines. Further, PPGA-5 nanocomposite (the best working system in terms of transfection efficiency among the series) was found to efficiently transfect primary mouse keratinocytes up to 22% above the control level. PPGA-5, when tested for in vivo cytotoxicity in Drosophila, did not induce any stress in the exposed larvae in comparison with control. In vivo gene expression using PPGA-5 showed the highest transfection efficiency in spleen of mouse closely followed by heart tissues after intravenous injection through tail vein. Besides, these nanocomposites also delivered siRNA efficiently into mammalian cells, resulting in ⼠80% suppression of EGFP expression. These results together demonstrated the potential of the projected nanocomposites for in vivo gene delivery.
Asunto(s)
Portadores de Fármacos/química , Técnicas de Transferencia de Gen , Nanocompuestos , Ácido Poliglutámico/química , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , ADN/administración & dosificación , ADN/genética , Drosophila/efectos de los fármacos , Portadores de Fármacos/toxicidad , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Luciferasas de Luciérnaga/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Plásmidos , Polietileneimina/química , Polietileneimina/toxicidad , Ácido Poliglutámico/toxicidad , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Electricidad Estática , TransfecciónRESUMEN
Of the non-viral vectors, a cationic polymer like PEI is an attractive candidate which however, has been negatively impacted due to its marked toxicity. An anionic sugar polymer gelan gum (GG) has been introduced into PEI system to increase transfection efficiency with minimal toxicity. We showed that one of the synthesized (GP1-GP6) GG-PEI nanocomposites (NCs), GP3, exhibited negligible toxicity in in vitro (primary keratinocytes, HEK293, HeLa and HepG2 cells) and in vivo (Drosophila melanogaster) as compared to PEI or lipofectamin. GP3-pDNA complex was found to be transfected efficiently in the above cells as confirmed by FACS analysis (72.0 + 5.5%) while lipofectamine showed only 12.4 + 3.5% efficiency. GP3 mediated GFP specific siRNA delivery resulted in the knockdown of the GFP expression by approximately 77% and JNK (60%). In vivo gene expression studies in mice revealed reporter gene expression in spleen. The study demonstrates that GG blended PEI NCs hold promise for future applications in gene delivery both in vitro and in vivo.
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ADN/administración & dosificación , ADN/farmacocinética , Iminas/química , Nanocápsulas/química , Polietilenos/química , Polisacáridos Bacterianos/química , Transfección/métodos , Animales , ADN/genética , Composición de Medicamentos/métodos , Vectores Genéticos/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , Distribución TisularRESUMEN
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.