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

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.