Rhodamine-Loaded Intercellular Adhesion Molecule–1-targeted Microbubbles for Dual-Modality Imaging Under Controlled Shear Stresses

Abstract

Background— The ability to image incipient atherosclerosis is based on the early events taking place at the endothelial level. We hypothesized that the expression of intercellular adhesion molecule-1 even in vessels with high flow rates can be imaged at the molecular level using 2 complementary imaging techniques: 2-photon laser scanning microscopy and contrast-enhanced ultrasound. Methods and Results— Using 2-photon laser scanning microscopy and contrast-enhanced ultrasound, intercellular adhesion molecule-1–targeted and rhodamine-loaded microbubbles were shown to be specifically bound to tumor necrosis factor-α–stimulated human umbilical vein endothelial cells and murine carotid arteries (44 wild-type mice) at shear stresses ranging from 1.25 to 120 dyn/cm 2 . Intercellular adhesion molecule-1–targeted and rhodamine-loaded microbubbles bound 8× more efficient ( P =0.016) to stimulated human umbilical vein endothelial cells than to unstimulated cells and 14× more than nontargeted microbubbles ( P =0.016). In excised carotids, binding efficiency did not decrease significantly when increasing the flow rate from 0.25 to 0.6 mL/min. Higher flow rates (0.8 and 1 mL/min) showed significantly reduced microbubbles retention, by 38% ( P =0.03) and 55% ( P =0.03), respectively. Ex vivo results were translatable in vivo, confirming that intercellular adhesion molecule-1–targeted and rhodamine-loaded microbubbles are able to bind specifically to the inflamed carotid artery endothelia under physiological flow conditions and to be noninvasively detected using contrast-enhanced ultrasound. Conclusions— Our data provide groundwork for the implementation of molecular ultrasound imaging in vessels with high shear stress and flow rates, as well as for the future development of image-guided therapeutic interventions, and multiphoton microscopy as the appropriate method of validation.