负载多柔比星的EGFR靶向光敏脂质体对胃癌细胞的杀伤作用研究

Study on the anti-tumor efficacy of a DOX-loading EGFR-targeting UV-responsive liposomal drug delivery system

目的:构建靶向EGFR的光敏脂质体,提高化疗药物对胃癌细胞系的杀伤效果,并降低相关毒副反应。方法:以联乙炔基甘油磷脂酰胆碱(Diacetylenic glycerophosphatidylcholine,PC)(以下简称PC)为原料,采用薄膜分散法制备光反应性脂质体,表面修饰表皮生长因子受体(Epidermal growth factor receptor,EGFR)抗体Certuximab的Fab’片段,交联脂质体,制备靶向光敏脂质体,增强其在肿瘤组织内部的富集以及肿瘤细胞对脂质体药物的摄取。透射电镜观察脂质体的形态,动态光散射仪测定脂质体的粒径和血液学稳定性,倒置荧光显微镜和流式细胞术检测人体胃腺癌细胞N87对脂质体药物的摄取能力,CCK-8试剂盒(Cell Counting Kit-8)比较脂质体与游离药物对人体胃癌细胞N87细胞的体外杀伤效应,荷瘤小鼠皮下肿瘤抑制实验比较脂质体药物与游离药物对N87细胞的体内杀伤效应。结果:制备出的对紫外线敏感的EGFR靶向脂质体具有规整的球状结构、合适的粒径大小和很强的血液学稳定性。胃癌细胞N87对靶向脂质体(PC-Dox-Fab,PDF)的摄取能力较非靶向脂质体(PC-Dox-BSA,PDB)和游离DOX有显著增强(P<0.05)。PDF对N87细胞的半数抑制浓度(Half inhibitory concentration,IC50)较PDB及游离DOX有显著降低(P<0.05)。体内研究结果显示,PDF能够显著抑制荷瘤小鼠的皮下肿瘤生长(P<0.05)。结论:本研究制备的靶向EGFR的光敏脂质体拥有很好的稳定性,在体内外研究中对胃癌细胞具有很强的杀伤效果,具有广阔的临床应用前景。

Objective:To construct photosensitive liposome targeting EGFR,improve the killing effect of chemotherapeutics drugs on gastric cancer cell lines,and reduce related toxic and side effects.Methods:Diacetylenic glycerophosphatidylcholine(PC)was used as raw material to prepare photoreactive liposomes by thin-film dispersion method.The Fab'fragment of epidermal growth factor receptor(EGFR)antibody was modified on the surface of the liposomes,and then cross-linked liposomes were used to prepare targeted photosensitive liposomes,so as to enhance their accumulation in tumor tissues and the uptake of liposomes by tumor cells.The morphology of liposomes was observed by transmission electron microscope.The particle size and hematological stability of liposomes were determined by dynamic light scattering.The uptake of liposomes by human gastric adenocarcinoma cell line n87 was detected by inverted fluorescence microscope and flow cytometry.The killing effect of liposome and free drug on human gastric cancer cell line n87 in vitro was compared,and the killing effect of liposome and free drug on human gastric cancer cell line n87 in vivo was compared in subcutaneous tumor inhibition test of tumor bearing mice.Results:The prepared EGFR-targeted liposomes sensitive to ultraviolet rays have fine spherical structure,suitable particle size,and strong hematological stability.The uptake capacity of gastric cancer cell N87 for Fab’-navigated liposome(PC-DOX-Fab,PDF)was significantly enhanced compared with that of non-targeting liposomes(PC-DOX-BSA,PDB)and free DOX(P=0.004).The inhibitory concentration 50(IC50)of targeted liposome drugs on N87 cells was significantly lower compared with that of non-targeted liposome drugs and free DOX(P<0.05).In vivo studies demonstrated that PDF can significantly inhibit the subcutaneous tumor growth of tumor-bearing mice(P<0.05).Conclusion:In this study,we have identified a novel liposomal drug delivery system for improved chemotherapy efficacy of stomach cancer,which owns a fine spherical structure and excellent serum stability.The in vitro and in vivo experimental results clearly suggested that this Fab’navigated UV-responsive liposome exhibits potent NHL suppressing activity,which deserves further investigation for clinical application.