Abstract 2482: Fate of Targeted Ultrasound Contrast Agents After Endothelial Adhesion: A Time Course Study

Abstract

Background: Targeted microsphere (μSPH) adhesion to endothelial epitopes forms the basis for ultrasound molecular imaging. The fate of targeted μSPH, once bound to the endothelial target, is unknown. This has limited the optimization of ultrasound imaging strategies; for example, the appropriate time to commence imaging after μSPH injection is uncertain, and in part depends on the time course and duration of μSPH binding. We therefore sought to characterize the behavior of adhered μSPH as a function of time using serial observations of rat cremaster microcirculation after μSPH injection. Methods: Fluorescent nitrogen-encapsulated polymer μSPH (diameter 3.0 ± 1.4 μm) bearing control non-specific IgG (μSPH CTL ) or monoclonal antibody against VCAM (μSPH VCAM ) or ICAM (μSPH ICAM ) were prepared. Inflamed cremaster muscle of 21 anesthetized Wistar rats was exteriorized for intravital microscopy 4 hours after intrascrotal injection of TNF-α (5μg). Each rat received 1 venous injection of a single μSPH species (n=7 rats/μSPH; 10 8 μSPH/injection). An index venule was identified for serial microscopic observation every 5 min starting 10 min after μSPH injection. Results: The cumulative sum of adhered μSPH during 1 hr observation was higher for μSPH ICAM (24±11) and μSPH VCAM (18±1) than μSPH CTL (3±2, p=0.005). The number of μSPH ICAM adherent to the index venule was stable over time (10 min: 2 ± 3; 30 min: 3±2; 80 min: 3 ± 5, ANOVA p=0.9). The number of adhered μSPH VCAM trended downwards (10 min: 5±4; 30 min: 2±2; 80 min: 1±2; ANOVA p=0.08). By 1 hr, adhered μSPH appeared altered in shape; no μSPH transmigration was seen. Conclusions: Nitrogen-filled polymer μSPH targeted to leukocyte adhesion molecules adhere to inflamed endothelium. The extent of μSPH adhesion remains relatively constant over a time period relevant for imaging applications, although beyond 1hr, the μSPH appear morphologically altered and could have different acoustic properties. These data provide a useful and broad time window for effective ultrasound imaging of molecular targets. The time course of μSPH attachment to endothelium is an important consideration when devising strategies for ultrasound molecular imaging and should be characterized in a μSPH- and target- specific manner.