SP94修饰的肝靶向阳离子基因载体的制备及其体外评价

Construction and in vitro evaluation of SP94 modified cationic gene vector targeting hepatocellular carcinoma

目的:研究适配体SP94修饰的阳离子基因载体H_3R_5,合成新型肝靶向纳米复合物SP94-H_3R_5/miR195,增加对肝癌细胞的靶向性,提高基因的转染效率。方法:经半胱氨酸修饰的SP94与H_3R_5末端的半胱氨酸发生氧化反应,组装得到SP94-H_3R_5,利用~1 H-NMR鉴定SP94-H_3R_5载体的结构,通过电位粒度仪测定纳米复合物的电位和粒径,利用琼脂糖凝胶电泳考察载体对miR195的压缩能力。以体外培养的人肝癌SK-Hep-1为研究对象,CCK8法检测SP94-H_3R_5和H_3R_5对细胞增殖的抑制作用,采用激光共聚焦显微镜考察肝癌细胞对纳米复合物的摄取,以pEGFP为报告基因考察基因转染效率,Western blot实验检测SK-Hep-1细胞VEGF的蛋白表达。结果:SP94-H_3R_5具有生物相容性,可以压缩miR195形成稳定的纳米复合物,SP94-H_3R_5/miR195与H_3R_5/miR195相比可以更多地被SK-Hep-1摄取(P<0.01),SP94-H_3R_5转染效率高于非靶向载体H_3R_5,对VEGF的阻滞作用也更高(P<0.01)。结论:SP94-H_3R_5兼具纳米材料的被动靶向作用和适配体的主动靶向作用,有潜力成为肝癌治疗中的新型载体。

OBJECTIVE To investigate aptamer SP94 modified cationic gene vector H3R5, develop a novel liver targeting nanocomposite named SP04-HwR5/miR195 to enhance the targeting to hepatocellular carcinoma （HCC） cells and improve the gene transfection efficiency. METHODS SP94-H3R5 was successfully prepared by the oxidation of the -SH groups of cysteine from SP94 and H3R5, which generated disulfide bonds. The structure of SP94-H3 R5 was identified by 1 H-NMR. The ZetaSizer was applied to estimate the Zeta potential and the size of nanocomposites. Gel electrophoresis was employed to determine the condensation capacity of SP94-H3R5 to miR195. The cytotoxicity, cellular uptake, transfection efficiency of SP94-H3R5 and miR195 interference efficiency of SP94-H3R5/miR195 to VEGF were evaluated using HCC cells SK Hep-1. RESULTS The biocompatible SP94-H3R5 condensed miR195 to become the stable nanocomposites. Compared with H3 R5/miR195, more SP94 H3R5/miR195 could be uptaken by SK-Helyl （P〈0. 01）. The transfection efficiency of SP94-H3R5/miR195 was higher than that of H3R5/miR195 and the blocking effect on VEGF was also higher （P〈0. 01）. CONCLUSION SP94-H3R5 holds the po tential to become an innovative vector targeting HCC by the passive targeting effects of nanomaterials and active targeting effects of aptamer.