曲妥珠单抗修饰的阿霉素免疫脂质体的制备及体外性质研究

Preparation and Biological Activity in vitro of Trastuzumab-modified Doxorubicin Immunoliposome

目的:制备表面键合曲妥珠单抗(trastuzumab,TMAB)的阿霉素免疫脂质(Doxorubicin-loadedimmunoliposome,DOX-IML),并对其体外性质进行研究。方法:将磷脂酰胆碱、胆固醇、阿霉素、DSPE-MPEG2000以一定比例混合,采用薄膜超声分散法制备阿霉素脂质体,将聚乙二醇衍生物(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinimidyl(polyethylene glycol)-3400]、DSPE-PEG3400-NHS)连接到TMAB;再与阿霉素脂质体连接得到DOX-IML。研究不同浓度的TMAB对DOX-IML入胞能力及细胞毒性的影响;测定免疫脂质体的包封率、载药率、粒径、电荷及稳定性等性质;动态透析法模拟体外释药特性,激光共聚焦观察免疫脂质体对AU565细胞抗体介导的入胞作用;MTT法研究DOX-IML抑制肿瘤细胞的生长。结果:成功制备了表面键合TMAB的阿霉素免疫脂质体,配体载入率分别是53%、75.5%、84%;每毫克DOX-IML中抗体的含量分别是37、83、108μg·mg-1;阿霉素的包封率为76.85%、载药量为8.03%;粒径131.8nm;表面电荷-27mV。抗体含量83μg·mg-1的DOX-IML组的细胞存活率最低,细胞内荧光强度最高,且该免疫脂质体稳定性良好,具有一定缓释作用。DOX-IML具有较强的特异性靶向作用,其入胞能力和细胞毒性均高于阿霉素脂质体。结论:DOX-IML具有较强的特异性靶向作用,其入胞能力和细胞毒性均高于阿霉素脂质体,抗体含量适中时其入胞能力和细胞毒性最强。

Objective： To prepare the doxombicin immunoliposome（DOX IML） which was bonding trastuzumab（TMAB） on the surface and study its characterizations in vitro. Methods： Doxorubicin liposomes was prepared by film dispersion method with phosphatidylcholine, cholestenone, doxombicin, DSPE-MPEG 2000, the polyethylene glycol derivatives （DSPE-PEG-NHS）combined with TMAB; immunoliposomes were prepared by the combination of TMAB-（PEG-DSPE）n and doxorubicin liposome. The properties of doxorubicin-loaded immunoliposome was analyzed such as encapsulation efficiency, the rate of drug loading, and average particle sizeand surface charge. The cellular uptake was assessed by confocal laser scanning microscopy（CLSM） using AU565 cells. The growth of rumor cells which was inhibited by doxombicin were loaded immunoliposome by MTT method. Results： DOX IML was successfully prepared and the binding rate of average ligand were 53 %, 75.5%, and 84 % with the content of antibody on DOX IML being 37, 83, and 108μg·mg^-1 with the entrapment efficiency of DOX liposomesbeing 76.85% and the rate of drug loading being 8.03% and the average particle size being 131.8 nm and the surface charge being -27 mV, the cell survival rate of the DOX IML （83 μg·mg^-1） was the lowest, the intracellular fluorescence intensity of the group was the highest, the immunoliposomeswere stable and had the effect of controlled release. DOX IML had stronger effect of specific target. The endocytosis capacity and toxicity ofDOX IMLwere higher than that of doxorubicin liposomes. Conclusion： DOX IML had strongly specific and targeted effect, both its endocytosis capacity and cytotoxicity were stronger than those of oxorubicin liposomal. When the contentration of antibody on DOX IML was moderate, the endocytosis capacity and cytotoxicity were the strongest.