Human serum albumin nanoparticles for efficient delivery of Cu, Zn superoxide dismutase gene
Human serum albumin nanoparticles for efficient delivery of Cu, Zn superoxide dismutase gene
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发表时间：
2007-05
期刊：
影响因子：
2.2
通讯作者：
Yun Mo;Micheal E. Barnett;D. Takemoto;H. Davidson;U. Kompella
中科院分区：
文献类型：
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作者：
Yun Mo;Micheal E. Barnett;D. Takemoto;H. Davidson;U. Kompella
目的探讨人血清白蛋白纳米粒（HSA NP）作为铜锌超氧化物歧化酶（SOD 1）基因眼部非病毒载体的可行性。方法以人血清白蛋白（HSA）为载体，采用去溶剂交联法制备Cu，Zn超氧化物歧化酶（SOD 1）基因纳米粒。评价了pSOD负载的HSA NP的体外释放特性、对DNA酶I和玻璃体液降解的稳定性、细胞毒性、细胞摄取机制、体外转染效率和体内基因表达。体外研究采用培养的人视网膜色素上皮（ARPE-19）细胞，体内研究采用小鼠模型。对于细胞摄取分析，使用异硫氰酸荧光素（FITC）标记的人血清白蛋白（HSA）。结果采用去溶剂交联法将SOD 1基因包裹于人血清白蛋白中。基因负载的HSA NP具有120 nm的平均尺寸、-44 mV的ζ电位和84%的质粒包封效率。在高交联度下，HSA NP使质粒在体外持续释放超过6天，并稳定质粒DNA抵抗DNase I和玻璃体液降解。在用浓度高达5 mg/ml的空白HSA NP处理96小时的ARPE 19细胞中未观察到细胞毒性。HSA NP的细胞摄取是通过受体介导的内吞作用，主要涉及小窝途径。共聚焦分析表明HSA NP的快速内/溶酶体逃逸。此外，共聚焦研究表明，HSA容易进入细胞核。在体外，pSOD-HSA NP在ARPE-19细胞中导致超过80%的转染效率，这比Lipofectamine高5倍。HSA NP转染的细胞表现出增强的SOD 1活性，比未处理的细胞高5倍，表明功能基因的过表达。以130 ng质粒的剂量将HSA NP玻璃体内注射到小鼠眼睛中，与对照动物中不可检测的表达相比，在48 h时产生可检测水平的融合蛋白表达。结论本研究中开发的HSA NP为非病毒基因递送到视网膜提供了一种非常有前途的方法。
Purpose To assess the potential of human serum albumin nanoparticles (HSA NP) as a nonviral vector for ocular delivery of Cu, Zn superoxide dismutase (SOD1) gene. Methods Cu, Zn superoxide dismutase (SOD1) gene-encapsulated nanoparticles (NP) were developed using human serum albumin (HSA), an endogenous protein, by a desolvation-crosslinking method. The pSOD-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 in vivo gene expression. In vitro studies employed cultured human retinal pigment epithelial (ARPE-19) cells and in vivo studies employed a mouse model. For cell uptake analysis, fluorescein isothiocyanate (FITC)-labeled human serum albumin (HSA) was used. Results Plasmid containing SOD1 gene was encapsulated in HSA by a desolvation-crosslinking method. Gene-loaded HSA NP has a mean size of 120 nm, zeta potential of -44 mV, and plasmid encapsulation efficiency of 84%. At high crosslinking degree, HSA NP sustained the in vitro release of plasmid over 6 days, and stabilized plasmid DNA against DNase I and vitreous humor degradation. No cytotoxicity was observed in ARPE 19 cells treated with blank HSA NP at concentrations up to 5 mg/ml for 96 h. Cellular uptake of HSA NP was via receptor-mediated endocytosis that involves primarily caveolae-pathways. Confocal analysis indicated rapid endo/lysosomal escape of HSA NP. Further, confocal studies indicated that HSA readily enters the cell nucleus. In vitro, pSOD-HSA NP resulted in more than 80% transfection efficiency in ARPE-19 cells, which was 5 fold higher than Lipofectamine. HSA NP-transfected cells exhibited enhanced SOD1 activity that was 5 fold higher than untreated cells, indicating the overexpression of the functional gene. Intravitreal injection of HSA NP to the mouse eye at a dose of 130 ng of plasmid 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 very promising approach for nonviral gene delivery to the retina.