胆甾醇修饰的PEG-P[Asp(DET)]/miRNA复合物的制备、理化性能及细胞摄取能力

Cholesterol modified PEG-P[Asp(DET)]/miRNA complex:preparation,physicochemical properties,and cellular uptake

目的制备疏水基修饰的聚阳离子高分子基因载体PEG-P[Asp(DET)]-10%胆甾醇,对其复合miRNA的理化性质和细胞摄取能力进行研究。方法开环聚合合成PEG-P[Asp(DET)],再采用氯甲酸胆甾醇基进行疏水修饰,核磁共振法验证其结构;使其与hsa-miR-15a形成胶束复合物,对该胶束复合物的粒径、Zeta电位、稳定性、包封率以及细胞毒性进行考察;以白血病细胞K562细胞系为模型细胞进行体外细胞实验对其摄取进行考察。结果合成的PEG-P[Asp(DET)]-10%胆甾醇具有良好的溶解性,可与miRNA形成稳定的胶束复合物,当氮磷比(N/P)=20,基因浓度在5μmol/mL时,形成的胶束复合物粒径为(192.4±10.8)nm,Zeta电位为(6.9±0.9)mV,包封率为(90.5±3.2)%。该复合物在实验条件下表现出了相当的稳定性,细胞对该复合物的摄取能力明显强于商品化试剂脂质体lipo2000。结论PEG-P[Asp(DET)]-10%胆甾醇是一种优良的高分子基因传递系统载体,在体外实验中可有效实现细胞对于miRNA的稳定摄取。

Objective To synthesize PEG P[Asp(DET)]with 10G cholesterol chloroforinate modified on its side chain and to study the physicochemical properties and cellular uptake of polymer complex PEG P[Asp(DET)]chole/hsa miR 15a.Methods PEG P[Asp(DET)]was synthesized by ring opening polymerization and modification with cholesterol chloroformate to acquire hydrophobicity,and MRI was used to verify its structure.The particle size,Zeta potential,stability,encapsulation efficiency,and cytotoxicity of the polymer complex PEG P[Asp(DET)]10%chole/hsa miR 15a were examined.Finally,in vilro cellular uptake experiment was carried out with the leukemia cell line K562.Results The synthesized polymer PEG P[Asp(DET)]10%chole had fine solubility and could form into stable polymer complex PEG P[Asp(DET)]chole/hsa miR 15a.When nitrogen phosphorus ratio(N/P)was at 20 and concentration of the miRNA was 5μmol/mL,the particle size was(192.4±10.8)nm,Zeta potential was(6.9±0.9)mV,and encapsulation efficiency was(90.5±3.2)%.The complex displayed good stability under experimental condition.In vitro cellular uptake experimental indicated that the uptake capacity of PEG P[Asp(DET)]10%chole/hsa miR 15a was higher than that of the commercial agent lipo2000.Conclusion PEG P[Asp(DET)]10%chole is a fine gene polymer carrier for miRNA and can achieve stable cellular uptake of miRNA.