透明质酸修饰的核壳纳米粒对视网膜色素上皮细胞基因转染效率的研究

In vitro study on gene transfection efficiency of hyaluronic acid modified core-shell liponanoparticles in human retinal pigment epithelium cells

本文旨在制备透明质酸(hyaluronic acid,HA)修饰的核壳纳米粒(pHA-LCS-NPs)作为基因药物传递载体,并对视网膜色素上皮细胞(human retinal pigment epithelium,ARPE-19)基因转染效率进行研究。采用薄膜分散水化-挤膜法制备核壳纳米粒(LCS-NPs),通过HA与二油酰磷脂酰乙醇胺(dioleoyl phosphatidylethanolamine,DOPE)发生酰胺反应,对脂质膜进行修饰,制得pHA-LCS-NPs。本研究对纳米粒微观形态、粒径和zeta电位进行考察;采用XTT法测定载体的细胞毒性;以绿色荧光蛋白(green fluorescent protein,pEGFP)基因为报告基因,探讨不同含量HA修饰的pHA-LCS-NPs对ARPE-19细胞基因转染效率的影响。结果表明:纳米粒呈现清晰的核壳结构,平均粒径为(214.9±7.2)nm,zeta电位为(-35±3.7)mV;pHA-LCS-NPs组24 h基因的累积释放率低于30%;48 h的细胞转染效率是壳聚糖纳米粒(CS-NPs)组的1.81倍、裸质粒组的3.75倍,且无明显细胞毒性。以上结果显示,pHA-LCS-NPs有潜力成为一种高效低毒的非病毒型基因药物传递载体。

The aim of this study is to prepare hyaluronic acid （HA） modified core-shell liponanoparticles （pHA-LCS-NPs） as gene delivery system and investigate its gene transfection efficiency in human retinal pigment epithelium （ARPE-19） cells in vitro. The pHA-LCS-NPs was prepared by firstly hydrating dry lipid film with CS-NPs suspension to get LCS-NPs, then modifying the lipid bilayer with HA by amidation reaction between HA and dioleoyl phosphatidylethanolamine （DOPE）. Its morphology, particle size and zeta potential were investigated. XTT assay was used to evaluate the cell safety of different vectors in vitro. The gene transfection efficiency of pHA-LCS-NPs modified with different contents of HA was investigated in ARPE-19 cells with green fluorescent protein （pEGFP） as the reporter gene. The results showed that the obtained pHA-LCS-NPs exhibited a clear core-shell structure with the average particles size of （214.9 ±7.2） nm and zeta potential of （-35±3.7） mV. The 24 h cumulative release of gene from pHA-LCS-NPs was less than 30%. After 48 h incubation, gene transfection efficiency of pHA-LCS-NPs/pEGFP was 1.81 times and 3.75 times higher than that of CS-NPs/pEGFP and naked pEGFP, respectively. Also no obvious cytotoxicity was observed on pHA-LCS-NPs. It suggested that the pHA-LCS-NPs might be promising non-viral gene delivery systems with high efficiency and low cytotoxicity.