靶向EGFRvⅢ包裹液态氟碳的高分子纳米球的制备及其体外靶向能力的评价

Preparation of EGFRvⅢ-targeted high molecular polymer nanoparticles with liquid perfluorocarbon and its targeting ability evaluation in vitro

目的制备靶向表皮细胞生长因子受体突变体Ⅲ(EGFRvⅢ)并包裹液态氟碳高分子纳米球,评价其性质和体外靶向结合能力。方法采用Western Blot和流式细胞术验证抗EGFRvⅢ单克隆抗体(4G1)的特异性;双乳化溶剂挥发法制备包裹液态氟碳的高分子纳米球(P-NPs),光镜和扫描电镜观察其形态及分布;激光粒径仪检测其粒径及电位;碳二亚胺法耦联4G1制备P-NPs-4G1,流式细胞术观察二者连接情况;超声观察其体外经低强度聚焦超声(LIFU)致相变后的表现;流式细胞术和共聚焦显微镜检测P-NPs-4G1体外靶向结合能力。结果 P-NPs-4G1粒径为(563.8±60.3)nm,Zeta电位为(-32.4±3.37)mV;4G1与P-NPs的连接率为(97.37±1.57)%;P-NPs-4G1在体外经LIFU辐照后可增强其显像效果;体外靶向实验证实P-NPs-4G1可与F98__(npEGFRvⅢ)(EGFR-/EGFRvⅢ+)细胞特异性结合,且不与F98EGFR(EGFR+/EGFRvⅢ-)细胞结合。结论本研究成功制备P-NPs-4G1,经体外LIFU辐照后可显著增强超声显像效果,同时可与F98npEGFRvⅢ细胞特异性结合。

Objective To prepare an epidermal growth factor receptor variant Ⅲ （EGFRvⅢ）-targeted high molecular polymer nanoparticles with liquid perfluorocarbon, in order to evaluate its physical properties and targeting ability in vitro. Methods The specificity of EGFRvⅢ monoclonal antibody （4G1） was confirmed by Western Blot and flow cytometry （FACS）. The nanoparticles of macromolecule material with liquid fluorocarbon （P-NPs） were prepared by a double emulsion solvent evaporation method. The properties and distribution of P-NPs were observed by light microscope and scanning electron microscope, and its size and potential were determined by laser particle size instrument. The P-NPs were conjugated with 4G1 by EDC/NHS to prepare a targeted ultrasound contrast agent （P-NPs-4G1）, which binding rate was detected by FACS. The ultrasound imaging was observed the transition performances after irradiation by low intensity focused ultrasound （LIFU）. The targeting ability of P-NPs-4G1 was evaluated by FACS and confocal microscope in vitro. Results The average size and Zeta of P-NPs-4G1 were （563.8＋60.3）nm and （-32.4±3.37）mV. The binding rate of P-NPs-4G1 were （97.37±1.57）%. After irradiation by LIFU in vitro, the imaging effect of P-NPs-4G1 was enhanced; P-NPs-4G1 can bind to F98npEGFRvⅢ cells （EGFR-/EGFRvⅢ＋）, while could not bind to F98EGFR cells （EGFR＋/EGFRvⅢ-）. Conclusion P-NPs-4G1 prepared in this study, can specificity bind to EGFRvⅢ positive tumor cells. After irradiation by LIFU, the imaging effect of P-NPs-4G1 is enhanced in vitro.