【摘要】：大多數(shù)陽離子化高分子載體在血清環(huán)境中會大量吸附血清蛋白而導致轉染效率低下,因而難以應用于臨床。設計合成羧基化葡聚糖(Dex-COOH)和羧基化葡聚糖修飾富勒烯(C60-Dex-COOH)兩種陰離子化高分子,并將其包覆在精胺化普魯蘭(Ps)與siRNA形成的復合物(PS/siRNA)外層,以減少復合物對血清蛋白的吸附。采用DLS/ELS檢測陰離子包覆前后復合物的顆粒粒徑及Zeta電位;用QCM-D驗證陰離子包覆層的形成,并評價包覆層對牛血清白蛋白(BSA)吸附的影響;同時應用cck-8試劑、流式細胞術,檢測包覆前后及不同包覆比例下復合物的細胞毒性、進入細胞的情況及對Hela-EGFP細胞的干擾效率。結果表明,Dex-COOH與C60-Dex-COOH能夠在PS/siRNA外部形成穩(wěn)定的負電性包覆層,有效阻止BSA的吸附,并在無血清的條件下顯著降低復合物的細胞毒性;在有血清的環(huán)境中,表面包覆了陰離子化高分子的復合物可以不受血清蛋白的影響而被細胞大量攝取。對包覆了C60-Dex-COOH的PS/siRNA復合物轉染組施加光照,可促使復合物從溶酶體中逃逸,將血清條件下PS/siRNA的干擾效率提高20%。該策略可有效提高陽離子化高分子載體介導的RNA干擾效率。
[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.
【作者單位】： 中國醫(yī)學科學院基礎醫(yī)學研究所北京協(xié)和醫(yī)學院基礎學院;
【基金】：國家自然科學基金(81271688) 國家重大科學研究計劃(2011CB933504)
【分類號】：R450
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本文編號：2303815