阴离子化高分子包覆非病毒载体提高有血清环境下RNA干扰效率的研究

大多数阳离子化高分子载体在血清环境中会大量吸附血清蛋白而导致转染效率低下,因而难以应用于临床。设计合成羧基化葡聚糖(Dex-COOH)和羧基化葡聚糖修饰富勒烯(C60-Dex-COOH)两种阴离子化高分子,并将其包覆在精胺化普鲁兰(Ps)与siRNA形成的复合物(PS/siRNA)外层,以减少复合物对血清蛋白的吸附。采用DLS/ELS检测阴离子包覆前后复合物的颗粒粒径及Zeta电位;用QCM-D验证阴离子包覆层的形成,并评价包覆层对牛血清白蛋白(BSA)吸附的影响;同时应用cck-8试剂、流式细胞术,检测包覆前后及不同包覆比例下复合物的细胞毒性、进入细胞的情况及对Hela-EGFP细胞的干扰效率。结果表明,Dex-COOH与C60-Dex-COOH能够在PS/siRNA外部形成稳定的负电性包覆层,有效阻止BSA的吸附,并在无血清的条件下显著降低复合物的细胞毒性;在有血清的环境中,表面包覆了阴离子化高分子的复合物可以不受血清蛋白的影响而被细胞大量摄取。对包覆了C60-Dex-COOH的PS/siRNA复合物转染组施加光照,可促使复合物从溶酶体中逃逸,将血清条件下PS/siRNA的干扰效率提高20%。该策略可有效提高阳离子化高分子载体介导的RNA干扰效率。

Anionic Polymer Coating Enhance the RNAi Efficiency of Non-Viral Carriers in Serum Containing Environment

The clinical application of cationic polymer carriers was limited because the adsorption of protein in serum environment led to low transfection efficiency. In this work, we synthesized carboxylic Dextran (Dex-COOH) and water-soluble carboxylic fullerene (C60-Dex-COOH) anionic polymers, coated on the pullulan-spermine and siRNA complex (PS/siRNA) to reduce the adsorption of serum protein. The particle size and Zeta potential were analyzed by DLS/ELS, respectively. The formation of anion coating and bovine serum albumin (BSA) adsorption of complexes were tested by QCM-D. Cellular uptake, cytotoxicity on the Hela cells and RNAi efficiency on the Hela-EGFP cells were examined by cck-8 reagent and flow cytometry. The results showed that Dex-COOH and C60-Dex-COOH could coat on PS/siRNA to form stable electronegative complexes, prevent the adsorption of serum effectively. Cytotoxicity of complexes was reduced in the absence of serum. On the other hand, in the serum environment, anionic polymer coated complexes could help complexes enter cells and PS/siRNA@C60-Dex-COOH with visible light irradiation would escape from the lysosome, increased the interference efficiency by 20% compared with PS/siRNA. In summary, this coating strategy provided an effective solution of increasing RNAi efficiency mediated by cationic polymers.