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目的研究携带针对丙型肝炎病毒(hepatitis C virus,HCV)RNA的小干扰RNA(siRNA)的维生素E(Vitamin E)脂质体纳米颗粒在体外靶向抑制HCV复制的作用。方法采取“薄膜水化法”制备Vitamin E脂质体纳米颗粒,应用激光粒度分析仪测量纳米颗粒VE-DC/siRNA的颗粒大小及zeta电位,应用透射电子显微镜观察VE-DC形态及分散性。以Huh7.5.1细胞株作为载体。CCK-8法检测纳米颗粒的细胞毒性,琼脂糖凝胶电泳检测纳米颗粒携带siRNA的血清稳定性,蛋白印迹技术和免疫荧光技术检测该siRNA纳米颗粒的转染效率及对HCV的抑制作用。结果 Vitamin E脂质体纳米颗粒形态呈球形,粒度分布为(125.5±9.0)nm,zeta电位为(39.1±4.8)m V,分散均一。相较同浓度商品化脂质体,VE-DC/siRNA对细胞的毒性相对较小。血清稳定性实验显示,VE-DC/siRNA在24 h后siRNA无降解,较裸siRNA稳定性显著提高。携带siRNA的Vitamin E脂质体纳米颗粒可以较高效率进入Huh7.5.1细胞内,并抑制HCV核心蛋白的表达。结论 Vitamin E脂质体纳米颗粒可以转运siRNA至Huh7.5.1细胞,并抑制HCV核心蛋白的表达。
Objective To investigate the effect of vitamin E liposome nanoparticles carrying small interfering RNA (siRNA) against hepatitis C virus (HCV) RNA in inhibiting the replication of HCV in vitro. Methods Vitamin E liposome nanoparticles were prepared by the “film hydration method”. The particle size and zeta potential of VE-DC / siRNA nanoparticles were measured by laser particle size analyzer. The morphology and dispersion of VE-DC were observed by transmission electron microscopy Sex. Huh7.5.1 cell line as a carrier. The cytotoxicity of nanoparticles was detected by CCK-8 method. The stability of nanoparticles carrying siRNA was detected by agarose gel electrophoresis. Western blotting and immunofluorescence were used to detect the transfection efficiency of siRNA nanoparticles and the inhibition of HCV. Results The morphology of Vitamin E liposome nanoparticles was spherical with a particle size distribution of (125.5 ± 9.0) nm and a zeta potential of (39.1 ± 4.8) mV, with uniform dispersion. Compared with the same concentration of commercial liposomes, VE-DC / siRNA is relatively less toxic to cells. Serum stability experiments showed that VE-DC / siRNA did not degrade after 24 h, which was significantly improved compared with naked siRNA. Vitamin E liposomal nanoparticles carrying siRNA can efficiently enter Huh7.5.1 cells and inhibit HCV core protein expression. Conclusion Vitamin E liposome nanoparticles can transport siRNA to Huh7.5.1 cells and inhibit the expression of HCV core protein.