Dual-Targeted Microbubbles Specific to Integrin αVβ3 and Vascular Endothelial Growth Factor Receptor 2 for Ultrasonography Evaluation of Tumor Angiogenesis

Aggressive tumors are characterized by angiogenesis that promotes the migration and dissemination of tumor cells. Our aim was to develop a dual-targeted microbubble system for non-invasive evaluation of tumor angiogenesis in ultrasound. Avidinylated microbubbles were conjugated with biotinylated arginylglycylaspartic acid and vascular endothelial growth factor receptor 2 (VEGFR2) antibodies. Subcutaneous MHCC-97H liver carcinoma models were established. Non-targeted, αvβ3-targeted, VEGFR2-targeted and dual-targeted microbubbles was intravenously injected in series while acquiring ultrasound images of the tumor. The microbubbles were destroyed by a high-mechanical-index pulse 4 min after the injection. Peak intensity (PI) before and after the destructive pulse was recorded to compare contrast enhancement by different microbubbles. The targeting rates of the integrin-targeted, VEGFR2-targeted and dual-targeted groups were 95.02%, 96.04% and 94.23%, respectively, with no significant differences. Tumors in all groups were significantly enhanced. The time-intensity curve indicated no significant differences in arrival time, PI, area under the curve, amplitude and mean transit time. The difference in ultrasound signal intensity before and after the destructive pulse (⊿PI) for all targeted microbubble groups was significantly greater than that for the non-targeted microbubble group (all p values < 0.05), and the difference for the dual-targeted microbubble group was significantly greater than those of both mono-targeted groups (p <0.05).