紫杉醇外泌体的载药工艺优化及体外抗肿瘤作用研究

Optimization of Drug Loading Process of Paclitaxel Exosomes and Its Anti-tumor Study in Vitro

目的优化紫杉醇(PTX)外泌体给药系统的载药工艺并评价其有效性和安全性,为开发新型抗肝癌制剂提供依据。方法采用超速离心法提取骨髓间充质干细胞外泌体(BMSCs-Exo);电穿孔法包载紫杉醇,以药物包封率作为评价指标,通过响应面法优化载药工艺;高效液相色谱法测定紫杉醇外泌体(Exo-PTX)的载药量及考察释药行为;CCK-8法检测Exo-PTX对肝癌细胞HepG2增殖的抑制作用;溶血实验评价Exo-PTX的安全性。结果Exo-PTX的最佳电穿孔法载药工艺条件为:PTX药物浓度81.80μg·mL^(-1)、电压0.6 mV、电击次数8次,Exo-PTX的平均包封率为(22.38±0.08)%。同时Exo-PTX具有良好的缓释性能,且在微酸性的肿瘤微环境下可加速释放,在模拟正常体液和肿瘤环境下,48 h时的累计释放度分别为(49.35±0.67)%、(61.94±0.43)%。Exo-PTX对体外HepG2细胞具有显著的抑制作用且无溶血风险。结论在最佳载药工艺条件下制备的Exo-PTX具有较好的体外抗肝癌作用,并呈明显的浓度依赖性,缓释性良好,为PTX制剂的二次开发提供了依据。

Objective To refine the drug-loading process of the paclitaxel exosome delivery system and evaluate its efficacy and safety,thereby furnishing a foundation for the development of novel anti-liver cancer formulations.Methods Bone marrow mesenchymal stem cell-derived exosomes(BMSCs-Exo)were extracted using the ultracentrifugation method.Paclitaxel(PTX)was encapsulated into the exosomes via electroporation,with drug encapsulation efficiency serving as the evaluation criterion.The drug loading process was optimized through response surface method.High performance liquid chromatography was applied to determine the drug loading capacity of paclitaxel-loaded exosomes(Exo-PTX)and investigate the drug release behavior.The inhibitory effect of Exo-PTX on the proliferation of HepG2 was examined using the CCK-8 assay.Additionally,hemolysis test was used to evaluate the safety of Exo-PTX.Results The optimal conditions for drug loading via electroporation for Exo-PTX were as follows:PTX drug concentration of 81.80μg·mL^(-1),voltage of 0.6 mV,and number of pulses of eight.The average encapsulation efficiency of Exo-PTX was(22.38±0.08)%.Meanwhile,Exo-PTX demonstrated excellent sustainedrelease properties and accelerated release in the slightly acidic tumor microenvironment.The cumulative release rates at 48 h in simulated body fluid and tumor environments were(49.35±0.67)%and(61.94±0.43)%at 48 h,respectively.Exo-PTX showed significant inhibitory effects on HepG2 cells in vitro and revealed no risk of hemolysis.Conclusion Exo-PTX,which was prepared under optimal loading conditions,exhibited robust in vitro anti-liver cancer effects in a dose-dependent.Moreover,Exo-PTX showed excellent sustained-release properties.The study provides a basis for the secondary development of PTX formulations.