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

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 through in vitro experiments.

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 1×10⁷ untargeted MBs), antibody presaturation added VEGFR2 targeted MBs group (after being incubated with excess VEGFR2 antibody, 1×10⁷ VEGFR2 targeted ultrasound MBs were added) and VEGFR2 targeted MBs group (adding 1×10⁷ 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.

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 no MBs 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.

The freeze-drying method can be used to prepare VEGFR2 targeted ultrasound contrast agent, which has good in vitro targeting ability and contrast enhanced effects for ultrasound molecular imaging.