Novel anti-VEGFR2 antibody-conjugated nanobubbles for targeted ultrasound molecular imaging in a rabbit VX2 hepatic tumor model†

Abstract

Nanobubbles (NBs), as ultrasound contrast agents, possess the potential for clinical applications in targeted ultrasound molecular imaging due to their small diameters and the specific molecular markers attached. Previous research studies mainly focused on the tumor-specific recruitment capability or drug carriers based on subcutaneous tumor models. In clinical trials, orthotopic tumor models are considered more clinically relevant and better predictive models for assessing drug efficacy compared to standard subcutaneous models. Here, we first prepared uniform-sized NBs with a soft chitosan-lipid membrane containing perfluoropropane gas and then anti-VEGFR2 antibodies were incorporated into NB membranes in order to achieve targeting ability toward tumor angiogenesis. The results of physicochemical characterization (the average size of 260.9 ± 3.3 nm and a PDI of 0.168 ± 0.036, n = 3) indicated that the targeted nanobubbles (tNBs_v) have a spherical morphology and a vacant core. In vitro experiments found that the contrast enhancement abilities of tNBs_v are similar to those of commercial SonoVue. In in vivo experiments, the orthotopic model of the rabbit VX2 hepatic tumor was used to evaluate the targeted binding ability of tNBs_v toward tumor angiogenesis. Ultrasound sonograms revealed that tNBs_v achieved the peak intensity of ultrasound imaging enhancement in the region of peripheral vasculature of VX2 tumors over non-targeted NBs or SonoVue, and the imaging time was longer than that of the other two. Ex vivo fluorescence imaging and examination using a confocal laser scanning microscope further verified that tNBs_v were capable of binding to tumor angiogenesis. These results from our studies suggested that tNBs_v are useful to develop an ultrasound imaging probe to evaluate anti-angiogenic cancer therapy by monitoring tumor angiogenesis.