目的 制备Angiopep-2（Ang）修饰的白藜芦醇（resveratrol，Res）脂质体（Ang-Lip/Res），并对其进行处方优化和体外脑靶向性研究。方法 采用薄膜分散法制备Ang-Lip/Res，利用Box-Benhken响应面法对影响其包封率的3因素（磷脂的质量浓度、磷脂与药物的质量比、磷脂与胆固醇的质量比）进行优化处方最佳制备工艺并验证脂质体的包封率；采用磺酰罗丹明B蛋白法考察脂质体对小鼠神经细胞的毒性；采用流式细胞仪和荧光显微镜探究细胞摄取情况；构建体外血脑屏障模型，比较白藜芦醇脂质体（Res-Lip）跨越血脑屏障的能力。结果 Ang-Lip/Res的最优处方：磷脂的质量浓度为4.4mg/mL，磷脂与白藜芦醇的质量比为11∶1，磷脂与胆固醇的质量比为5.5∶1。按照最佳处方，以磷脂、胆固醇、DSPE-PEG₂₀₀₀及DSPE-PEG₂₀₀₀-Ang为膜材制备的Ang-Lip/Res包封率为（93.28±1.35）%。细胞毒实验结果表明，50μmol/L浓度以内，AngLip/Res对小鼠神经细胞N2a和bEnd.3无明显毒性；体外细胞摄取实验结果表明，与非靶向Res-Lip相比，Ang的修饰明显增加了bEnd.3细胞对脂质体的摄取，提高了脂质体体外BBB渗透率。结论 成功制备Ang-Lip/Res，可进一步应用于防治脑部疾病的研究。

Objective To prepare and optimize the prescription of Angiopep-2 (Ang) modified resveratrol (Res) liposomes (Ang-Lip/ Res) and perform in vitro brain targeting study. Methods The Ang-Lip/Res were prepared by thin film dispersion method, and the three factors [the egg yolk phosphatidylcholine (EPC) concentration, the mass ratio of EPC to cholesterol, and the mass ratio of to Res] affecting liposomes encapsulation rate were optimized using Box-Benhken response surface methodology, and the encapsulation rates of the liposomes were verified; The antiproliferation effect of liposomes on N2a and bEnd.3 cells was investigated by SRB assay. Flow cytometry and microscope were utilized for exploring the cellular uptake. An in vitro model of the blood-brain barrier was constructed to compare the ability of resveratrol liposomes to cross the blood-brain barrier. Results The optimized preparation process and formulation were as follow: EPC concentration of 4.4 mg/mL, EPC to cholesterol mass ratio of 11:1, EPC to resveratrol mass ratio of 5.5:1. According to the optimal prescription, the encapsulation efficiency of Ang-Lip/Res prepared with EPC, cholesterol, DSPEPEG₂₀₀₀ and DSPE-PEG₂₀₀₀-Ang as membrane materials were (93.28 ±1.35)%. The results of cytotoxicity assay showed that AngLip/Res within 50 μmol/L concentration range was not significantly toxic to mouse neuronal cells N2a and bEnd.3 cells. The in vitro cell uptake results showed that modification of Ang significantly increased uptake efficiency of Ang-Lip/Res by bEnd.3 cells compared to non-targeted Res-Lip and improved its BBB penetration in vitro. Conclusion The Ang-Lip/Res is successfully prepared, which can be used for further research of prevention and treatment of brain diseases.

R283.6

国家自然科学基金项目(81874347);辽宁中医药大学高层次引进人才科研启动资金资助项目(2100221005);辽宁中医药大学2021年博士后启动资金(21601A2100)