【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(dǎo)致轉(zhuǎn)染效率低下,因而難以應(yīng)用于臨床。設(shè)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(fù)合物(PS/siRNA)外層,以減少復(fù)合物對血清蛋白的吸附。采用DLS/ELS檢測陰離子包覆前后復(fù)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(yīng)用cck-8試劑、流式細(xì)胞術(shù),檢測包覆前后及不同包覆比例下復(fù)合物的細(xì)胞毒性、進(jìn)入細(xì)胞的情況及對Hela-EGFP細(xì)胞的干擾效率。結(jié)果表明,Dex-COOH與C60-Dex-COOH能夠在PS/siRNA外部形成穩(wěn)定的負(fù)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(fù)合物的細(xì)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(fù)合物可以不受血清蛋白的影響而被細(xì)胞大量攝取。對包覆了C60-Dex-COOH的PS/siRNA復(fù)合物轉(zhuǎn)染組施加光照,可促使復(fù)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(dǎo)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(xué)科學(xué)院基礎(chǔ)醫(yī)學(xué)研究所北京協(xié)和醫(yī)學(xué)院基礎(chǔ)學(xué)院;
【基金】：國家自然科學(xué)基金(81271688) 國家重大科學(xué)研究計劃(2011CB933504)
【分類號】：R450
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本文編號：2303815