穿膜肽GGPFV修饰的柔红霉素/薯蓣皂苷脂质体的处方优化及细胞毒性研究

Formulation Optimization and Cytotoxicity Study of GGPFV-modified Daunorubicin/dioscin Liposomes

目的:制备穿膜肽GGPFV修饰的柔红霉素/薯蓣皂苷脂质体,对其处方进行优化,并初步评价其对乳腺癌细胞的体外毒性。方法:采用薄膜分散法和硫酸铵水化法,将柔红霉素和薯蓣皂苷包载其中,在表面以聚乙二醇-二硬脂酰基磷脂酰乙醇胺2000(DSPE-PEG_(2000))-GGPFV修饰,制备GGPFV修饰的柔红霉素/薯蓣皂苷脂质体。以包封率为指标,采用Box-Behnken响应面法优化处方中水化体积、胆固醇用量和柔红霉素用量;测定按最优处方制备的3批脂质体的包封率。比较柔红霉素/薯蓣皂苷脂质体、GGPFV修饰的柔红霉素/薯蓣皂苷脂质体和空白脂质体作用后对人乳腺癌MDA-MB-435S细胞存活率的影响。结果:最优处方为水化体积5 mL、胆固醇4 mg、蛋黄卵磷脂22 mg、柔红霉素0.55 mg、薯蓣皂苷0.85 mg、DSPE-PEG_(2000)3.5 mg、DSPE-PEG_(2000)-GGPFV 2 mg。所制3批脂质体中,柔红霉素的包封率为(96.21±1.54)%,薯蓣皂苷的包封率为(95.39±2.48)%。体外细胞毒性试验显示,GGPFV修饰的柔红霉素/薯蓣皂苷脂质体对MDA-MB-435S细胞的抑制作用显著强于柔红霉素/薯蓣皂苷脂质体(P<0.05),膜材无细胞毒性。结论:成功制得GGPFV修饰的柔红霉素/薯蓣皂苷脂质体,其对人乳腺癌MDA-MB-435S细胞的体外抑制作用明显增强。

OBJECTIVE:To prepare GGPFV-modified Daunorubicin/dioscin liposomes,and to optimize their formulation and to preliminarily evaluate their cytotoxicity to breast cancer cells in vitro.METHODS:Daunorubicin and diosgenin were wrapped by thin film dispersion method and ammonium sulfate hydration method;the surface was modified with DSPE-PEG2000-GGPFV to prepare GGPFV-modified Daunorubicin/dioscin liposomes.Taking encapsulation rate as index,Box-Behnken response surface methodology was used to optimize the film hydration volume,cholesterol amount and daunorubicin amount in the formulation.The entrapment efficiency of 3 batches of liposomes prepared according to the optimal formulation was determined.The effects of Daunorubicin/dioscin liposomes,GGPFV-modified Daunorubicin/dioscin liposomes and blank liposomes on the survival rate of human breast cancer MDA-MB-435S cells were compared.RESULTS:The optimal formulation was as film hydration volume of 5 mL,cholesterol of 4 mg,yolk lecithin of 22 mg,daunorubicin of 0.55 mg,dioscin of 0.85 mg,DSPE-PEG2000 of 3.5 mg,DSPE-PEG2000-GGPFV of 2 mg.The encapsulation rate of daunorubicin was(96.21±1.54)%and that of dioscin was(95.39±2.48)%in the 3 batches of liposomes prepared.The in vitro cytotoxicity tests showed that the inhibition effect of GGPFV-modified Daunorubicin/dioscin liposome on MDA-MB-435 S cells was significantly stronger than that of Daunorubicin/dioscin liposome(P<0.05).There was no cytotoxicity in the membrane.CONCLUSIONS:GGPFV-modified Daunorubicin/dioscin liposomes are successfully prepared,and its inhibitory effect on human breast cancer MDA-MB-435S cells in vitro was significantly enhanced.