冻干法制备VEGFR2靶向超声微泡体外寻靶及显影实验

Preparation of VEGFR2 Targeted Ultrasound Microbubbles by Freeze-drying and Targeting Study in vitro

目的采用冻干法制备血管内皮生长因子受体2(VEGFR2)靶向超声造影剂,并评价其体外寻靶能力及超声显影效果。方法冻干法制备生物素化微泡,将链霉亲和素及VEGFR2单克隆抗体依次连接至生物素化微泡,构建VEGFR2靶向超声微泡。采用免疫荧光染色法验证链霉亲和素及大鼠抗小鼠VEGFR2单克隆抗体与微泡的结合情况。观察及检测VEGFR2靶向超声微泡的形态、大小及分布。体外培养人脐静脉内皮细胞(HUVEC)至对数期,分3组,分别为非靶向超声微泡组(加入非靶向微泡1×107个)、抗体预饱和+VEGFR2靶向微泡组(加入过量的VEGFR2抗体孵育后,再加入VEGFR2靶向超声微泡1×107个)及VEGFR2靶向超声微泡组(加入VEGFR2靶向超声微泡1×107个),比较各组微泡与细胞的结合情况。采用自制体外循环装置并采集超声图像,检测VEGFR2靶向超声微泡体外显影能力。结果通过免疫荧光染色法的验证,链霉亲和素及大鼠抗小鼠VEGFR2单克隆抗体可与生物素化微泡结合构建VEGFR2靶向超声微泡。VEGFR2靶向微泡光学显微镜下呈圆形,大小一致且分布均匀,平均直径为(1.31±0.93)μm。显微镜下见非靶向微泡组HUVEC周围可见少量微泡结构,抗体预饱和+VEGFR2靶向超声微泡组HUVEC周围几乎无微泡结构,VEGFR2靶向超声微泡组HUVEC周围可见较多微泡结构存在。在超声增强模式下自制微泡显影效果良好,随造影时间延长无明显衰减。结论冻干法可制备VEGFR2靶向超声造影剂,在体外实验中具有良好的寻靶能力且超声辐照下可显影。

Objective To prepare vascular endothelial growth factor receptor2 （VEGFR2） targeted ultrasound contrast agent （microbubbles, MBs） by freeze-dried method and to evaluate its contrast enhanced effect and targeting capability throughin vitro experiments. Methods Targeted MBs were prepared using the biotin-avidin linkage to conjugate rat anti-mouse VEGFR2 monoclonal antibody to the surface of biotinylated MBs. Morphology, size and distribution of MBs were assessed. The binding of streptavidin （FITC marker） and VEGFR2 monoclonal antibody （PE labeled rabbit IgG） to MBs was verified by immunofluorescence staining.in vitro targeting experiments were performed with human umbilical vein endothelial cells （HUVECs）. The binding capacity of MBs to HUVECs were detected by three groups including untargeted MBs group （adding 1×10^7 untargeted MBs）, antibody presaturation added VEGFR2 targeted MBs group （after being incubated with excess VEGFR2 antibody, 1×10^7 VEGFR2 targeted ultrasound MBs were added） and VEGFR2 targeted MBs group （adding 1×10^7 VEGFR2 targeted ultrasound MBs）. Contrast enhanced effects of VEGFR2 targeted MBs were preliminarily examined using an ultrasound imaging system and a home-made extracorporeal circulating device. Results The monoclonal antibody of streptomycin and rat anti-mouse VEGFR2 can be combined with the biotinized MBs to construct the targeted ultrasound MBs of VEGFR2 by immunofluorescence staining. Under the microscope, VEGFR2 targeted MBs were round, uniform in size and uniform in distribution, with a mean diameter of （1.31±0.93） μm. Microscopy showed a small number of MBs around HUVECs in non-targeted MBs group, almost noMBs around HUVECs of antibody presaturation＋VEGFR2 targeted ultrasound MBs group, and many MBs around ]HUVECs of VEGFR2 targeted ultrasound MBs group. The binding capacity was significantly higher than that of untargeted MBs. The self-made MBs developed well and no significant attenuation was observed as time extension in the mode of enhanced ultrasonography. Conclusion The freeze-drying method can be used to prepare VEGFR2 targeted ultrasound contrast agent, which has goodin vitro targeting ability and contrast enhanced effects for ultrasound molecular imaging.