靶向VEGFR2光声/超声双模态造影剂的制备及体外寻靶实验研究

Preparation of VEGFR2-targeted photoacoustic/ultrasound dual-mode contrast agent and its targeting study in vitro

目的制备一种特异性靶向新生血管内皮细胞的光声/超声双模态造影剂,探讨其体外寻靶能力及双模态显影效果。方法采用多步乳化法制备载有印度墨水和液态氟碳的高分子造影剂,用碳二亚胺法将造影剂与抗血管内皮生长因子受体2(VEGFR2)单克隆抗体相偶联,制备出靶向VEGFR2高分子造影剂(Vi-PFH-PLGA)。检测该造影剂的一般特性、体外寻靶能力及双模态显影效果,并与非靶向高分子造影剂进行比较。结果所制备的靶向高分子造影剂的平均粒径为(565.5±15.6)nm,间接免疫荧光法观察到抗体成功的连接到造影剂表面,流式细胞仪测得抗体微球连接率为99.72%,体外寻靶能力实验显示较多的靶向造影剂呈花环状牢固的聚集在人脐静脉内皮细胞HUVEC表面,而非靶向组和抗体干预组未见造影剂与HUVEC的特异性结合。体外光声/超声显影实验显示,经脉冲激光辐照后,靶向造影剂组可检测到明显的光声信号和超声信号,与非靶向造影剂相比差异无统计学意义(P均>0.05)。结论成功制备出靶向VEGFR2的光声/超声双模态造影剂,该造影剂在体外与HUVEC细胞具有较强的靶向结合能力且具备较好的光声/超声双模态显影效果。

Objective To prepare a vascular endothelial growth factor receptor 2 （VEGFR2）-targeted photoacoustic/ultrasound dual-mode contrast agent, investigate its targeting and dual-mode imaging capability in vitro. Methods Targeted agent loaded with India ink and perfluorohexane were prepared by multi-emulsion technology and conjugated with VEGFR2 monoclonal antibody by EDC/NHS. Its physicochemical properties, targeting capability, and photoacoustic/ultrasound imaging were observed, with non-targeted agent acted as controls. Results The nanoparticles were uniform and stabilized with size about （565.5±15.6）nm. The conjugation of nanoparticles with antibody was demonstrated by indirect immun- ofluoreseence assay and its binding rate was reach up to 99.72%. In the in vitro targeting study, it showed that a number of targeted nanoparticles conjugated and crowded around the human umbilical vein endothelial cells （HUVEC） tightly, while no specific binding was found in the non-targeted group and free antibody intervention group. After the pulsed laser irradiation, strong photoacoustic and ultrasound signals were obviously detected, with no statistical significance between the targeted and non-targeted group. Conclusion The VEGFR2-targeted photoacoustic/ultrasound dual-mode contrast was prepared successfully, which could targeted to HUVEC specially and enhanced photoacoustic/ultrasound imaging significantly.