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目的评价靶向人晶状体上皮细胞免疫载药纳米微球HILE6-PLA(5-FU)-NP的免疫学特征及体外对靶细胞的特异性结合和杀伤作用。方法采用碳二亚胺法将5-氟尿嘧啶(5-FU)聚乳酸纳米微球PLA(5-FU)-NP与抗人晶状体上皮细胞单克隆抗体HILE6连接,制备免疫载药纳米微球。测定其中5-FU与HILE6物质的量之比;ELISA法检测连接后单克隆抗体HILE6的活性变化。设免疫纳米微球组、HILE6与载药纳米微球混合组、载药纳米微球组、空白对照组,MTT法检测对晶状体上皮细胞增殖的抑制作用。间接免疫荧光法观察单克隆抗体HILE6、免疫纳米微球和载药纳米微球对兔晶状体上皮细胞的附着、内化过程。结果免疫纳米微球中5-FU与HILE6物质的量之比约为1809∶1,单克隆抗体HILE6保留免疫活性可达原抗体的84%。免疫纳米微球对晶状体上皮细胞作用2h的半数抑制剂量IC50为5.0μg/ml,抑制作用较载药纳米微球组、单克隆抗体与微球混合组有明显提高。免疫纳米微球在30min内可识别并附着于兔晶状体上皮细胞;2h进入细胞质均匀分布;4h后集中于细胞核区。结论免疫纳米微球HILE6-PLA(5-FU)-NP可携带大量药物,保持特异的免疫活性,在体外识别并进入晶状体上皮细胞有效抑制其增殖。
OBJECTIVE: To evaluate the immunological characteristics of HILE6-PLA (5-FU) -NP targeting human lens epithelial cells and the specific binding and killing effect on target cells in vitro. Methods 5-Fluorouracil (5-FU) PLA-5P was conjugated with HILE6 monoclonal antibody to human lens epithelial cells by carbodiimide method to prepare the drug-loaded nanospheres. The ratio of 5-FU and HILE6 was measured. The activity of HILE6 was detected by ELISA. The immunosuppressive microspheres group, HILE6 and drug-loaded nano-microspheres mixed group, drug-loaded nano-microspheres group, blank control group and MTT assay were used to detect the inhibitory effects on the proliferation of lens epithelial cells. Indirect immunofluorescence method was used to observe the attachment and internalization process of rabbit lens epithelial cells by monoclonal antibody HILE6, immunized nanospheres and drug loaded nanospheres. Results The ratio of 5-FU to HILE6 in immunized nanospheres was about 1809:1. The monoclonal antibody HILE6 retained the immunogenicity up to 84% of the original antibody. The IC50 of IC50 of immunized nanospheres on lens epithelial cells for 2h was 5.0μg / ml, which was significantly higher than that of drug-loaded nanospheres and monoclonal antibody mixed with microspheres. Immune nanospheres could be identified and attached to rabbit lens epithelial cells within 30 min; 2 h into the cytoplasm evenly distributed; 4 h after the focus on the nucleus. CONCLUSION: HILE6-PLA (5-FU) -NP immunized with a large number of drugs can maintain specific immunological activity and recognize and enter into lens epithelial cells in vitro to effectively inhibit proliferation.