Human serum albumin nanoparticles for efficient delivery of Cu,Zn superoxide dismutase gene

[摘要] Purpose: To assess the potential of human serum albuminnanoparticles (HSA NP) as a nonviral vector for ocular delivery of Cu,Zn superoxide dismutase (SOD1) gene.Methods: Cu, Zn superoxide dismutase (SOD1) gene-encapsulatednanoparticles (NP) were developed using human serum albumin (HSA), anendogenous protein, by a desolvation-crosslinking method. ThepSOD-loaded HSA NP was evaluated for in vitro release characteristics,stability against DNase I and vitreous humor degradation, cytotoxicity,cellular uptake mechanisms, in vitro transfection efficiency, and invivo gene expression. In vitro studies employed cultured human retinalpigment epithelial (ARPE-19) cells and in vivo studies employed a mousemodel. For cell uptake analysis, fluorescein isothiocyanate(FITC)-labeled human serum albumin (HSA) was used.Results: Plasmid containing SOD1 gene was encapsulated in HSA bya desolvation-crosslinking method. Gene-loaded HSA NP has a mean size of120 nm, zeta potential of -44 mV, and plasmid encapsulation efficiencyof 84%. At high crosslinking degree, HSA NP sustained the in vitrorelease of plasmid over 6 days, and stabilized plasmid DNA against DNaseI and vitreous humor degradation. No cytotoxicity was observed in ARPE19 cells treated with blank HSA NP at concentrations up to 5 mg/ml for96 h. Cellular uptake of HSA NP was via receptor-mediated endocytosisthat involves primarily caveolae-pathways. Confocal analysis indicatedrapid endo/lysosomal escape of HSA NP. Further, confocal studiesindicated that HSA readily enters the cell nucleus. In vitro, pSOD-HSANP resulted in more than 80% transfection efficiency in ARPE-19 cells,which was 5 fold higher than Lipofectamine. HSA NP-transfected cellsexhibited enhanced SOD1 activity that was 5 fold higher than untreatedcells, indicating the overexpression of the functional gene.Intravitreal injection of HSA NP to the mouse eye at a dose of 130 ng ofplasmid produced detectable level of fusion protein expression at 48 h,compared to non-detectable expression in control animals.Conclusions: The HSA NP developed in this study offers a verypromising approach for nonviral gene delivery to the retina.

[学科分类] 生物化学/生物物理