抗ICAM-1液态氟碳纳米微球靶向损伤心肌细胞的体外实验及其细胞毒性作用研究

Cytotoxicity of PFOB Nanoparticle Coupled with ICAM-1 Antibody on Cardiomyocytes and its Targeted Adhesion to Injured Cardiomyocytes in vitro

目的制备一种耦联细胞间黏附分子-1(ICAM-1)单抗的液态氟碳(PFOB)纳米微球,检测其基本理化特性与细胞毒性作用并实现其与体外培养损伤心肌细胞的靶向结合。方法将生物素化ICAM-1单抗与普通(对照组)及生物素化(实验组)PFOB纳米微球耦联,免疫荧光技术检测抗体与微球的耦联情况;MTT法检测微球对心肌细胞的细胞毒性作用;将两组微球分别加入非肿瘤坏死因子-α(TNF-α)损伤和TNF-α损伤后大鼠乳鼠心肌细胞中,观察并半定量计算各组微球与心肌细胞的结合情况。结果 ICAM-1单抗与实验组微球成功耦联,耦联率95%,共聚焦显微镜下微球呈绿色荧光,其粒径、电位、浓度分别为(385.3±88.9)nm,-(60.3±6.11)mV,7.0×108/mL,而对照组微球不见或仅见微弱荧光;不同浓度、不同作用时间下实验组微球对心肌细胞无毒性作用;无论心肌细胞是否暴露于TNF-α,对照组微球均未见附着在心肌细胞膜周,而TNF-α损伤下实验组微球与心肌细胞的附着量是非损伤下的10倍(P<0.01)。结论成功制备出耦联ICAM-1单抗的PFOB纳米微球,该靶向微球无细胞毒性作用且与体外高表达ICAM-1心肌细胞有较强的结合力。

Objecitve To synthesize perfluorooctylbromide （PFOB） nanoparticle coupled with intercellular adhesion molecule-1 （ICAM-1） monoclonal antibody, and to investigate the characteristics of the nonoparticle and its cytotoxcity on and targeted adhesion to injured cardiomocytes in vitro. Methods PFOB nanoparticle （control group） biotinylated PFOB nanoparticle （experimental group） were coupled with biotinylated ICAM-1 antibody, The combination of ICAM-1 antibody and the nanoparticle was detected by immunofluorescent assay. The cytotoxicity of the nanoparticle on rat cardiomyocytes was determined with MTT assay in vitro. The adhesion of the nanoparticle to normal and TNF-α injured cardiomyocytes were observed and semiquantified with optical microscope. Results ICAM-1 antibody was successfully coupled with biotinylated PFOB nanoparticle at a rate around 95~, which showed green fluorescence under the laser Confocal Scanning Microscope, with （385.3＋88.9） nm in size, --（60.3 ~6.11） mV in electric potential, and 7.0 3〈 108/mL in concentrations. No fluorescence was observed with the nonoparticle in the control group. No cytotoxicity of the nonoparticle on rat cardiomyocytes was found. There was limited adhesion of the nanoparticle in the control group to cardiomyocytes, normal or injured. A 10-fold increase in adhesion was detected when the nanopaticle in the experimental group was exposed to the injured cardiomyocytes compared with those exposed to the normal cardiomyocytes [（5.1 ±0. 22） vs. （0. 5±0. 3） nanopaticle per cell, P〈0.05）. Conclusion ICAM-1 monoclonal antibody is successfully coupled with PFOB nanoparticle, which can effectively bind to the cardiomyocytes overexpressing ICAM-1 without showing ctytotoxicity in vitro.