NGR修饰的香豆素-6隐形脂质体的制备及其体外细胞摄取性质

Preparation of NGR-modified coumarin-6 containing stealth liposome and study on its in vitro cell uptake properties

目的:应用NGR导向磷脂化合物修饰香豆素-6隐形脂质体以构建肿瘤新生血管靶向载体。方法:合成NGR导向磷脂化合物并进行了表征。采用薄膜法制备了不同比例NGR修饰的香豆素-6隐形脂质体,以包封率和泄漏率为指标,考察其稳定性;以人脐静脉血管内皮细胞(HUVEC,阳性细胞,CD13高表达)为靶细胞,用流式细胞仪和激光共聚焦显微镜对载体体外细胞摄取性质进行了分析。结果:制得的不同比例NGR修饰的香豆素-6隐形脂质体的包封率均>90.0%,12 h的体外累积泄漏率<9.0%。NGR修饰后的香豆素-6隐形脂质体在HUVEC中的摄取率明显高于未经修饰的隐形脂质体,且以0.64%(摩尔比)的用量摄取效果最好,而人冠状动脉内皮细胞(HCAEC,阴性细胞,CD13无表达)对两者的摄取没有明显差异。激光共聚焦观察表明,脂质体摄取入胞后,香豆素-6主要分布在细胞质中。结论:NGR修饰的隐形脂质体能够实现血管内皮细胞靶向,且用量以0.64%摩尔比为最佳。

OBJECTIVE To prepare tumor angiogenesis targeting carrier by use of NGR-modified coumarin-6 containing stealth liposomes. METHODS NGR-oriented phospholipid compounds were synthesized and characterized. NGR-modified coumarin-6 containing stealth liposomes were prepared by thin film evaporation method. Encapsulation efficiency and leakage versus time profile were used as index to investigate the liposomal stability. Human umbilical vein endothelial cells （HUVEC, positive cell, high expression of CD13） were prepared as model ceils to evaluate uptake efficiency of liposomes by flow cytometry and confocal laser microscopy. RF^ULTS The mean encapsulation efficiency of NGR-modified coumarin-6 containing stealth liposomes was higher than 90. 0% and the in vitro cumulative leakage of NGR-modified coumarin-6 from liposomes were less than 9. 0 % in 12 h. HUVEC uptake rate of NGR-modified liposome was higher than unmodified liposomes, while there was no significant difference between the uptake rate of NGR-modified and unmodified liposomes in human coronary artery endothe- lial cells （HCAEC negative cell, no expression of CD13）. Using confocal laser microscopy, coumarin-6 was found to be mainly distributed in cytoplasm following cellular uptake of NGR modified liposomes. CONCLUSION NGR modified stealth lipo- somes can be applied as vascular endothelial cell targeting carriers for the treatment of tumor. The optimum ratio of NGR DSPE-PEG2000 in lipid composition was 0. 64%.