两亲性嵌段聚合物PLGA-b-(PEI-co-PEG)的合成及其自组装电正性胶束的表征

Synthesis of amphiphilic block copolymer of PLGA-b-(PEI-co-PEG) and characterization of the self-assembled cationic micelles

目的合成一种生物可降解与低细胞毒性的两亲性嵌段共聚物PLGA-b-(PEI-co-PEG),并研究其胶束化行为。方法采用开环聚合法合成PLGA;使用低相对分子质量的聚乙烯亚胺(PEI1800)与聚乙二醇(PEG2000)相互交联合成水溶性PEI-co-PEG共聚物;采用脱水缩合法,合成PLGA-b-(PEI-co-PEG)。根据PEI-co-PEG在37℃PBS中孵育不同时间的相对分子质量变化情况,评估其生物降解性。通过MTT法测定PLGA-b-(PEI-co-PEG)与PEI-co-PEG对MCF-7的细胞毒性。采用标准透析法制备电正性PLGA-b-(PEI-co-PEG)胶束,使用马尔文激光粒度分析仪测定其粒径分布与Zeta电位;采用简单混合法制备PLGA-b-(PEIco-PEG)胶束/胰岛素复合物;使用透射电镜表征胶束及胶束/胰岛素复合物的形貌。采用荧光猝灭法,测定胶束/胰岛素复合物在不同浓度盐离子溶液中的稳定性。结果成功合成了两亲性嵌段共聚物PLGA-b-(PEI-co-PEG)。PEI-co-PEG在37℃PBS溶液中的降解半衰期约为48 h。PLGA-b-(PEI-co-PEG)与PEI-co-PEG对MCF-7的半数抑制浓度(IC50)分别为1375.7μg/m L与425.1μg/m L。PLGA-b-(PEI-co-PEG)胶束(粒径:99.5±2.61 nm,Zeta电位:52.9±2.38 m V)可与胰岛素形成纳米尺寸的胶束/胰岛素复合物;胶束/胰岛素复合物在150 mmol/L Na Cl溶液中的解离率为27.6%。结论 PEI-co-PEG在体外条件下展现了较好的降解性。PLGA-b-(PEI-co-PEG)的细胞毒性显著低于PEI-co-PEG(P<0.05)。PLGA-b-(PEI-co-PEG)胶束/胰岛素复合物在生理条件下具有良好的盐离子稳定性。

Objective To synthesize a biodegradable and minimally cytotoxic amphiphilic block copolymer of PLGA-b-（PEI-coPEG） and study its micellization behavior. Methods PLGA was synthesized by ring-opening polymerization. The cross-linked copolymer of PEI-co-PEG was synthesized from the low-molecular-weight polyethyleneimine（PEI, 1800 D） and hydrophilic poly（ethylene glycol）（PEG, 2000 D）. PLGA-b-（PEI-co-PEG） was synthesized by dehydration condensation reaction of PLGA and water soluble PEI-co-PEG. The biodegradability of PEI-co-PEG was evaluated according to the molecular weight change after incubation at 37 ℃ for different time. The cytotoxicity of PLGA-b-（PEI-co-PEG） and PEI-co-PEG in MCF-7 cells was determined by MTT assay. The cationic PLGA-b-（PEI-co-PEG） micelles were prepared by standard dialysis method. The particle size and Zeta potential of the micelles were measured by a Malvern laser particle size analyzer. Micelle/insulin complexes were prepared by simple mixing method and their morphology were characterized by transmission electron microscopy（TEM）. The fluorescence quenching method was used to determine the stability of the micelle/insulin complexes at different salt concentrations. Results Amphiphilic block copolymer of PLGA-b-（PEI-co-PEG） was successfully synthesized. The half-life of PEI-co-PEG degradation in PBS at 37 ℃ was about 48 h. The 50% cell inhibiting concentration（IC50） of PLGA-b-（PEIco-PEG） and PEI-co-PEG in MCF-7 cells were 1375.7 μg/m L and 425.1 μg/m L, respectively. The micelles of PLGA-b-（PEI-coPEG）（particle size： 99.5±2.61 nm, Zeta potential： 52.9±2.38 m V） were complexed with insulin via electrostatic interaction and formed nanoscale micelle/insulin complexes. The dissociation rate of micelle/insulin complexes in 150 mmol/L Na Cl solution was 27.6%. Conclusion The synthesized PEI-co-PEG shows good degradability in vitro. The cytotoxicity of PLGA-b-（PEI-coPEG） is significantly lower than PEI-co-PEG, and PLGA-b-（PEI-co-PEG） micelle/insulin complexes have good salt-resistant stability in physiological condition.