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
发布时间：2018-11-01 11:40
【摘要】：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。
[Abstract]:Most cationic polymer carriers adsorb serum proteins in the serum environment, which leads to low transfection efficiency, so it is difficult to be used in clinic. Two kinds of anionic polymers, carboxylated dextran (Dex-COOH) and carboxylated dextran modified fullerene (C60-Dex-COOH), were designed and synthesized and coated in the outer layer of the complex (PS/siRNA) formed by (Ps) and siRNA. In order to reduce the complex to the serum protein adsorption. The particle size and Zeta potential of the composite were measured by DLS/ELS, the formation of anion coating layer was verified by QCM-D, and the influence of the coating layer on the adsorption of bovine serum albumin (BSA) was evaluated. At the same time, cck-8 reagent and flow cytometry were used to detect the cytotoxicity of the complex before and after the coating and the different coating ratio, and to detect the cell entry and the interference efficiency of the complex to the Hela-EGFP cells. The results showed that Dex-COOH and C60-Dex-COOH could form a stable negative coating layer on the outside of PS/siRNA, effectively prevent the adsorption of BSA, and significantly reduce the cytotoxicity of the complex under the condition of no serum. In the presence of serum, complexes coated with anionic polymers can be absorbed in large quantities by cells without the influence of serum proteins. Applying light to the transfected group of PS/siRNA complex coated with C60-Dex-COOH could induce the complex to escape from lysosome and increase the interference efficiency of PS/siRNA in serum by 20%. This strategy can effectively improve the efficiency of cationic polymer carrier mediated RNA interference.
【作者单位】： 中国医学科学院基础医学研究所北京协和医学院基础学院;
【基金】：国家自然科学基金(81271688) 国家重大科学研究计划(2011CB933504)
【分类号】：R450
，
本文编号：2303815
[Abstract]:Most cationic polymer carriers adsorb serum proteins in the serum environment, which leads to low transfection efficiency, so it is difficult to be used in clinic. Two kinds of anionic polymers, carboxylated dextran (Dex-COOH) and carboxylated dextran modified fullerene (C60-Dex-COOH), were designed and synthesized and coated in the outer layer of the complex (PS/siRNA) formed by (Ps) and siRNA. In order to reduce the complex to the serum protein adsorption. The particle size and Zeta potential of the composite were measured by DLS/ELS, the formation of anion coating layer was verified by QCM-D, and the influence of the coating layer on the adsorption of bovine serum albumin (BSA) was evaluated. At the same time, cck-8 reagent and flow cytometry were used to detect the cytotoxicity of the complex before and after the coating and the different coating ratio, and to detect the cell entry and the interference efficiency of the complex to the Hela-EGFP cells. The results showed that Dex-COOH and C60-Dex-COOH could form a stable negative coating layer on the outside of PS/siRNA, effectively prevent the adsorption of BSA, and significantly reduce the cytotoxicity of the complex under the condition of no serum. In the presence of serum, complexes coated with anionic polymers can be absorbed in large quantities by cells without the influence of serum proteins. Applying light to the transfected group of PS/siRNA complex coated with C60-Dex-COOH could induce the complex to escape from lysosome and increase the interference efficiency of PS/siRNA in serum by 20%. This strategy can effectively improve the efficiency of cationic polymer carrier mediated RNA interference.
【作者单位】： 中国医学科学院基础医学研究所北京协和医学院基础学院;
【基金】：国家自然科学基金(81271688) 国家重大科学研究计划(2011CB933504)
【分类号】：R450
，
本文编号：2303815
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