目的：制备131 I标记的、抗西妥昔单克隆抗体（ C225）修饰的免疫纳米脂质体131 I⁃C225⁃BSA⁃PCL，探讨其体外抑制EGFR过表达肿瘤细胞生长的作用。方法分别构建EGFR靶向性和非靶向性纳米脂质体C225⁃BSA⁃PCL和BSA⁃PCL，并行透射电镜和动态光散射分析；使用流式细胞仪和激光扫描共聚焦显微镜观察上述纳米脂质体在EGFR过表达肿瘤细胞中的靶向性结合及内吞情况。用氯胺T直接标记法对纳米脂质体进行131 I标记，MTT法检测131 I标记纳米脂质体的细胞杀伤活性，并观察其细胞摄取及胞内存留情况。采用独立样本t检验对数据进行分析。结果成功构建EGFR靶向性和非靶向性纳米脂质体C225⁃BSA⁃PCL和BSA⁃PCL，其有效直径约为130～180 nm。流式细胞仪检测和共聚焦显微镜观察结果均显示C225⁃BSA⁃PCL能被EGFR过表达的肿瘤细胞摄取，而BSA⁃PCL的摄取相对较弱。 MTT检测结果显示，靶向性核素纳米脂质体131 I⁃C225⁃BSA⁃PCL较非靶向性的131 I⁃BSA⁃PCL具有更强的细胞杀伤效果，IC50值分别为0．03～1．32和0．25～12．19。细胞摄碘实验结果显示，131 I⁃C225⁃BSA⁃PCL能被细胞快速摄取，并于4 h达摄取峰值，其细胞摄取明显高于131 I⁃BSA⁃PCL（ t＝3．03～16．86，均P＜0．05）。结论 EGFR靶向性纳米脂质体C225⁃BSA⁃PCL较BSA⁃PCL具有更强的与EGFR过表达肿瘤细胞结合并被其摄取的能力。131 I⁃C225⁃BSA⁃PCL可在EGFR过表达的肿瘤细胞中存留较长时间并表现出明显的靶向性杀伤效果，能有效抑制肿瘤细胞的生长。%Objective To construct 131 I labeled anti⁃EGFR immunoliposome nanoparticle ( 131 I⁃Cetuaximab ( C225)⁃BSA⁃PCL) , and investigate its inhibitory effect on EGFR⁃overexpressing cancer cells in vitro. Methods Anti⁃EGFR liposome nanoparticle C225⁃BSA⁃PCL and non⁃targeted liposomes BSA⁃PCL were constructed. The products were observed with transmission electron microscopy and dynamic light scat⁃tering. The EGFR⁃targeted binding and cellular uptake in EGFR⁃overexpressing cancer cells were observed with flow cytometry and confocal microscopy. Anti⁃EGFR and non⁃targeted liposomes were labeled with 131 I using the chloramine⁃T method. The targeted cell killing effects of 131 I labeled liposomes were analyzed using MTT assay. The time⁃dependent cellular uptake analysis was used to evaluate the slow⁃release effects of the 131 I labeled liposomes. The independent⁃samples t test was used for data analysis. Results The EG⁃FR⁃targeted liposome C225⁃BSA⁃PCL and non⁃targeted liposome BSA⁃PCL were successfully constructed, and the effective diameters were approximately 130-180 nm. Flow cytometry and confocal microscopy re⁃vealed significant uptake of C225⁃BSA⁃PCL in EGFR⁃overexpressing tumor cells. BSA⁃PCL could also bind to cells with minimal and weak tumor retention. The EGFR⁃targeted radioactive liposome 131I⁃C225⁃BSA⁃PCL showed greater targeted cell killing effect than non⁃targeted liposome 131I⁃BSA⁃PCL,the IC50 values of 131I⁃C225⁃BSA⁃PCL and 131 I⁃BSA⁃PCL were 0. 03-1. 32 and 0. 25-12. 19, respectively. The uptakes of 131 I⁃C225⁃BSA⁃PCL was higher than that of 131 I⁃BSA⁃PCL ( t=3.03-16.86, all P<0.05) and reached the maxi⁃mal level at 4 h after incubation. Conclusions The EGFR⁃targeted liposome C225⁃BSA⁃PCL demonstrated superior cellular binding and uptake on EGFR⁃overexpressing cancer cells compared with BSA⁃PCL. The EGFR⁃targeted radioactive liposome 131 I⁃C225⁃BSA⁃PCL had favorable intracellular retention and excellent targeted cell killing effect, and could effectively suppress the growth of EGFR⁃overexpressing cancer cells.
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