目的 制备叶酸修饰的克班宁聚乙二醇-聚乳酸羟基乙酸共聚物纳米粒（folic acid modified crebanine polyethylene glycol-polylactic acid hydroxyacetic acid copolymer nanoparticles，FA-Cre@PEG-PLGA NPs），并考察其体外释放及抗肿瘤活性。方法 以克班宁为模型药，用PEG-PLGA共聚物、叶酸等为材料，通过超声乳化-溶剂挥发法制备FA-Cre@PEG-PLGA NPs，采用激光粒度仪、透射电子显微镜和荧光显微镜对FA-Cre@PEG-PLGA NPs的粒径、ζ电位及形态进行表征，紫外光谱法测定并计算克班宁的包封率和载药量，比较FA-Cre@PEG-PLGA NPs和克班宁原料药72 h的体外释放规律，通过溶血实验考察二者生物学特性，CCK-8实验考察两者对正常肝L02细胞存活率的影响及对肝癌Bel-7402细胞体外增殖抑制作用。细胞划痕法考察纳米粒和原料药对肝癌Bel-7402细胞迁移能力的影响。荧光显微镜观察纳米粒在不同时间点对肝癌Bel-7402细胞的摄取情况。结果 FA-Cre@PEG-PLGA NPs的平均粒径为（247.67±2.49）nm，多分散指数（polydispersity index，PDI）为0.139±0.027，ζ电位为（-9.40±0.54）mV，透射电子显微镜下呈圆球状核壳结构，荧光显微镜下观察纳米粒，其气化后明场呈蓝色光点、暗场呈红色光点。FA-Cre@PEG-PLGA NPs中克班宁的包封率为83.78%，载药量为67.78%。FA-Cre@PEG-PLGA NPs与克班宁原料药体外释放均符合Ritger-Peppas模型；安全性评价结果显示，FA-Cre@PEG-PLGA NPs相比于克班宁原料药在50～300 μg/mL内具有良好的生物相容性。FA-Cre@PEG-PLGA NPs对人正常肝L02细胞毒性较低，相比于克班宁原料药有一定的安全性。体外增殖抑制结果显示，两者对Bel-7402肿瘤细胞的增殖抑制率均呈现出剂量依赖性和时间依赖性，且FA-Cre@PEG-PLGA NPs联合超声辐照后对Bel-7402肝癌细胞具有更强的杀伤作用。细胞划痕实验表明，克班宁原料药、FA-Cre@PEG-PLGA NPs对Bel-7402肿瘤细胞迁移均有抑制作用，呈剂量依赖性，且纳米粒联合超声抑制细胞迁移效果更显著。细胞摄取实验表明，相同时间下超声联合纳米粒能有效增强肿瘤细胞的摄取。结论 成功制得FA-Cre@PEG-PLGA NPs，而且体外具有良好的缓释效果和抑瘤作用，为FA-Cre@PEG-PLGA NPs应用于肝癌的治疗研究提供实验依据。

Objective To prepare folic acid modified crebanine polyethylene glycol-polylactic acid hydroxyacetic acid copolymer nanoparticles (FA-Cre@PEG-PLGA NPs) and investigate their in vitro release and anti-tumor activity. Methods FA-Cre@PEG-PLGA NPs were prepared by ultrasonic emulsion-solvent evaporation method with crebanine as a model drug, polyethylene glycol-polylactic acid hydroxyacetic acid (PEG-PLGA) copolymer and folic acid (FA) as materials. The particle size, potential and morphology of FA-Cre@PEG-PLGA NPs were characterized by laser particle size measurement, transmission electron microscopy and fluorescence microscope. The encapsulation rate and drug loading capacity of crebanine were determined and calculated by Ultraviolet spectroscopy. Meanwhile, the in vitro release patterns of FA-Cre@PEG-PLGA NPs and crebanine bulk drug at 72 h were compared. The biological properties were investigated by hemolysis assay. The safety evaluation on L02 cells and the proliferation inhibition on Bel-7402 cells were investigated by CCK-8 assay in vitro. The proliferation of Bel-7402 cells was quantified by cell wound scratch assay. Finally, the uptake capacity of nanoparticles to Bel-7402 cells at different time points was observed by fluorescence microscope. Results The average particle size of FA-Cre@PEG-PLGA NPs was (247.67 ±2.49) nm, with a polydispersity index (PDI) of 0.139 ±0.027 and a ζ potential of (-9.40 ±0.54) mV. The transmission electron microscopy revealed a core-shell structure, while the fluorescence microscopy showed blue dots in the bright field and red dots in the dark area after gasification. The encapsulation rate of crebanine in nanoparticles was 83.78% and the drug loading was 67.78%. The release of FA-Cre@PEG-PLGA NPs and crebanine bulk drug conformed to the Ritger-Peppas model in vitro. The safety evaluation showed that FA-Cre@PEG-PLGA NPs had good biocompatibility within the range of 50-300 μg/mL compared with crebanine bulk drug. The FA-Cre@PEG-PLGA NPs showed lower toxicity to L02 cells than crebanine bulk drug. The proliferation inhibition results showed dose-dependent and time-dependent effects on the proliferation of Bel-7402 cells in vitro. Moreover, the FA-Cre@PEG-PLGA NPs combined with ultrasound irradiation had stronger killing effect on Bel-7402 cells. The cell wound scratch experiments showed that both the crebanine bulk drug and FA-Cre@PEG-PLGA NPs inhibited the migration of Bel-7402 cells in a dose-dependent manner, and the nanoparticles combined with ultrasound irradiation could inhibit the migration of tumor cells more significantly. The cell uptake experiment showed that nanoparticles combined with ultrasound irradiation could effectively enhance the uptake of tumor cells at the same time. Conclusion The FA-Cre@PEG-PLGA NPs were successfully prepared, and exhibited good slow-release effect and antitumor effect in vitro, providing an experimental basis for the application of FA-Cre@PEG-PLGA NPs in the treatment of liver cancer.

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云南省科技厅——云南中医药大学应用基础研究联合专项重点项目（202001AZ070001-008）；云南省科技厅生物医药重大科技专项（202002AA100007）；云南省科技人才平台计划（202105AG070012）；云南省傣医药与彝医药重点实验室开放课题（202210SS2205）；云南省傣医药与彝医药重点实验室开放课题（202210SS2206）；国家中医药管理局“十二五”重点学科——傣药学