目的 制备肝癌细胞Hep1-6外泌体并对化疗药物阿霉素（DOX）进行包载，以期实现对肿瘤细胞更高的靶向活性与杀伤作用。方法 采用梯度离心法对肿瘤细胞Hep1-6来源的外泌体进行制备分离；采用透射电镜技术、表面标记蛋白表征以及纳米颗粒追踪分析技术对外泌体的形态、特征蛋白、粒径分布和浓度进行表征；采用电穿孔方法实现外泌体对DOX的有效包载，制备包载阿霉素外泌体（EXO_DOX）。采用CCK-8法检测EXO_DOX与DOX（0.5、1、2、3、5、10 μg·mL^-1）体外对 Hep1-6细胞增殖的影响，采用激光共聚焦显微镜观察体外 Hep1-6细胞对 EXO_DOX与 DOX（1 μg·mL^-1）的靶向摄取作用。结果 所制备的外泌体具有形态良好 、 粒度均一的特性且具备外泌体特征膜蛋白 CD63、 CD81、 肿瘤易感基因101（TSG101）的表达；在电穿孔条件为150 V和75 μF下外泌体对DOX具备良好的包载特性；相比于单独给药DOX，在同等质量浓度下 EXO_DOX对 Hep1-6细胞增殖抑制作用显著增强（P＜0.05、0.01），同时肿瘤细胞对 EXO_DOX的摄取更具靶向性。结论 制备的EXO_DOX较DOX具有更强的体外细胞毒活性，EXO_DOX表现出对肿瘤细胞更高的靶向特性。

Objective To extract exosomes from hepatoma cells Hep1-6 and carry out effective inclusion of doxorubicin (DOX) as a chemotherapy drug to achieve higher targeting activity and killing effect on tumor cells.Methods Gradient centrifugation was used to prepare and isolate exosomes from tumor cells. The morphology, characteristic marker protein, particle size distribution and concentration of exosomes were characterized by transmission electron microscopy, Western blotting and nanoparticle tracking analysis (NTA). Exosomes were effectively loaded with doxorubicin by electroporation to prepare exosomes-doxorubicin (EXO_DOX). The tumor killing activity and targeted uptake of EXO_DOX and DOX (0.5, 1, 2, 3, 5, 10 μg·mL^-1)were evaluated by CCK-8 and laser confocal assay.Results EXO_DOX with good morphology and uniform particle size were prepared with the expression of the characteristic membrane proteins of exosomes, such as CD63, CD81, and TSG101. The exosomes have good encapsulation characteristics for DOX under 150 V and 75 μF electroporation condition. Compared with DOX alone, EXO_DOX achieve efficient targeted uptake of tumor cells at the same dose, and further improve the killing effect on tumor cells.Conclusions EXO_DOX achieve higher targeting characteristics and stronger cytotoxic activity against tumor cells.

R945；R965

北京市自然科学基金面上项目（7212174）