透明质酸-原酸酯聚合物胶束的制备及性能研究

Preparation and characterization of hyaluronic acid-orthoester polymeric micells as drug delivery carrier

目的合成具有pH敏感性的透明质酸-原酸酯(HA-OE)两亲性聚合物,并研究其所制备胶束的性能、pH敏感性及体外抗肿瘤性能。方法以原酸酯为酸敏基团,设计合成了两亲性聚合物HA-OE,并制备非pH敏感性透明质酸-十八烷胺(HA-C18)为参比聚合物,采用核磁共振对聚合物结构进行表征。采用超声法制备具有核-壳结构的HA-OE和HA-C18聚合物胶束,以阿霉素(DOX)为模型药物,制备载药胶束,并对其性能进行考察。结果空白胶束HA-OE和HA-C18的粒径分别为(217.9±2.1)nm和(163.3±1.7)nm,Zeta电位分别为(-19.7±0.1)mV和(-12.3±0.4)mV。HA-C18聚合物胶束在不同pH下的粒径变化不明显,而HA-OE聚合物胶束的粒径随着pH值的降低呈增大趋势。载药胶束DOX-HA-OE和DOX-HA-C18的粒径分别为(268.7±2.5)nm和(191.9±2.3)nm,Zeta电位分别为(-21.7±0.3)mV和(-19.7±0.1)mV,包封率分别为(93.2±1.8)%和(90.4±1.0)%,载药量为(13.8±0.5)%和(12.7±0.7)%。体外药物释放研究表明,pH=7.4时,两种载药胶束药物释放速度和累积释放量均相当;pH=6.5时,DOX-HA-C18胶束的累积释放量变化不明显,而DOX-HA-OE胶束的累积释放量明显增加;pH=5.0时,DOX-HA-C18胶束释放速度和累积释放量变化不明显,而DOX-HA-OE胶束的药物释放速度明显加快,72 h累积释放量接近80%,说明DOX-HA-OE胶束体外释放行为呈现pH敏感性。在人乳腺癌细胞系MCF-7中评估了体外细胞毒性测定和抗肿瘤活性,结果表明,与游离DOX相比,DOX加载的胶束对肿瘤MCF-7细胞的毒性和细胞摄取明显增强。结论载药胶束DOX-HA-OE具有良好的pH响应性,提高了DOX在肿瘤细胞内的摄取量,提升了体外抗肿瘤效果。

Objective To prepare an amphiphilic of hyaluronic acid-orthoester (HA-OE),and to determine the physicochemical properties,pH sensitivity and anti-tumor effect of polymeric micelles in vitro.Methods Orthoester was taken as an acid-sensitive group,and the pH-sensitive HA-OE polymer was synthesized.The polymer of hyaluronic acid-octadecylamine (HA-C18) without the acid-sensitive group was synthesized as a reference polymer.Those graft copolymers were characterized by 1H NMR.The polymeric micelles were prepared with ultrasonic method.Doxorubicin (DOX) was chosen as a model medicine to prepare drug-loaded micelles and relevant experiments were conducted to evaluate the stability of the micelles,the release behaviors in vitro,the cytotoxicity and the cellular uptake.Results The particle sizes of HA-OE and HA-C18 blank micelles were (217.9±2.1) nm and (163.3±1.7) nm,respectively.The Zeta potentials were (-19.7±0.1) mV and (-12.3±0.4) mV.Notable size changes of the HA-OE micelles containing the orthoester group were found after incubation in acidic media,while no size changes were observed in the polymeric micelles without orthoester bonds.The particle sizes of drug-loaded micelles DOX-HA-OE and DOX-HA-C18 were (68.7±2.5) nm and (191.9±2.3) nm,respectively.The Zeta potentials were (-21.7±0.3) mV and (-19.7±0.1) mV.The encapsulation efficiency was (93.2±1.8)% and (90.4±1.0)%.The drug loading of drug-loaded micelles was (13.8±0.5)% and (12.7±0.7)%.In vitro drug release study indicated that the speed and accumulative drug release of DOX-HA-OE and DOX-HA-C18 were similar at pH 7.4;at pH=6.5,the cumulative release of DOX-HA-C18 did not change significantly,but the cumulative release of DOX-HA-OE increased significantly in the drug-loaded micelles;at pH =5.0,the release rate and cumulative release of DOX-HA-C18 micelles remained insignificant,while the drug release rate of DOX-HA-OE micelles accelerated significantly.The cumulative release at 72 h was close to 80%,indicating that the release behaviors of DOX-HA-OE micelles exhibited pH sensitivity in vitro.The in vitro cytotoxicity assays and anti-tumor activity were evaluated in human breast carcinoma cell line MCF-7.The results of cell experiments indicated that DOX-loaded micelles enhanced the cytotoxicity and cellular uptake in MCF-7 cells compared with free DOX.Conclusion DOX-HA-OE micelles have excellent pH sensitivity,which may greatly increase the uptake of DOX in tumor cells and enhance the anti-tumor effect.