Abstract
Targeted therapy and molecular imaging using ultrasound have been widely explored using microbubble contrast agents, and more recently, activatable droplet contrast agents that vaporize when exposed to focused ultrasound have been explored. These droplets are coated with a stabilizing, functionalizable shell, typically comprised of fully saturated phospholipids. While the shedding of the lipid shell under ultrasound exposure is a well-studied phenomenon in microbubbles, it has not been fully explored in droplet-based contrast agents, particularly in those that undergo a reversible phase change and recondense following vaporization. Here, we investigate the retention of the lipid shell following repeated transient vaporization events. Two separate fluorescent markers were used to track individual lipid subpopulations: PEGylated lipids, to which targeting ligands are typically bound, and non-PEGylated lipids, which primarily contribute to droplet stability. Following confirmation of the homogeneous surface distribution of each subpopulation of shell lipids using confocal microscopy, high-speed optical imaging provided visual evidence of the ability to repeatedly induce vaporization and recondensation in micron-scale droplets using 5.208 MHz, 3.17 MPa focused ultrasound pulses transmitted from an imaging transducer. Flow cytometry analysis indicated that while PEGylated lipids were fully retained following repeated transient phase change events, 20% of the bulk lipids were shed. While this likely contributed to an observed significant reduction in the average droplet diameter, the selective binding capabilities of droplets functionalized with an RGD peptide, targeted to the integrin αvβ3, were not affected. These results indicate that repeated droplet activation may promote shifts in the droplet size distribution but will not influence the accuracy of targeting for therapy or molecular imaging.