Long Residence Time of Ultrasound Microbubbles Targeted to Integrin in Murine Tumor Model

Rationale and Objectives

The aim of this study was to evaluate the intratumoral residence time of microbubbles (MBs) targeted to α_vβ₃ integrin expressed in the endothelial cells of mice during the process of tumor angiogenesis.

Materials and Methods

For the preparation of MBs, decafluorobutane gas was sonically dispersed in phosphate buffer saline containing L-A-phosphatidylcholine-distearoyl, polyethylene glycol 40 stearate, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000] in a 77:15:8 molar ratio. Avidin–fluorescein isothiocyanate and biotin–cyclic arginine-glycine-aspartate-D-tyrosine-lysine (cRGD) or biotin–alanine-glycine-aspartate (AGD) conjugates were added to the reaction mixture. Adhesion testing of the targeting MBs was performed for the MS-1 cell line expressing α_vβ₃ integrin in vitro. The in vivo acoustic properties of the MBs were assessed by clinical ultrasound on the HT1080 fibrosarcoma model (n = 8) for 1 hour. Cryosections were stained with hematoxylin and eosin and by immunohistochemical staining to identify expression of α_vβ₃ integrin in the HT1080 tumor.

Results

The adherence of the MBs conjugated to cRGD was significantly greater than the adherence of the MBs conjugated to biotin-AGD (P < .01) for the MS-1 endothelial cell line. The acoustic enhancement on ultrasound was observed as a stable imaging window until 1 hour after injection of the MB conjugates in the mice. The MBs targeted via cRGD preferentially adhered to the vascular endothelium of the HT-1080 tumors. The findings of ultrasound imaging were correlated with immunohistochemical findings for the expression of α_vβ₃ integrin on the vascular endothelium of the tumors.

Conclusions

The prepared MBs conjugated with cRGD demonstrated a sufficient residence time to attach to the target integrin of tumor tissues. This finding suggests that the MBs are a potential molecular contrast agent that enables characterization of tumor angiogenesis and the monitoring of antitumor and antiangiogenic therapy.

Key Words: Angiogenesis, contrast agent, integrin, microbubbles, ultrasonography