Engineering Responsive Ultrasound Contrast Agents Through Crosslinked Networks on Lipid-Shelled Microbubbles

Ultrasound imaging with contrast agents, especially with lipid-shelled microbubbles, has become a vital tool in clinical diagnostics. Efforts to adapt these agents for molecular imaging have typically focused on targeted binding. More recently, crosslinking the lipid shell to alter its mechanical properties, followed by decrosslinking upon exposure to a stimulus, has been shown as a promising approach for imaging soluble molecular targets. Nevertheless, a systematic study of the influence of crosslinker concentration and structure on the mechanical properties of microbubbles has not been undertaken. An improved understanding of the role of these parameters is necessary to more effectively design contrast agents that detect proteases, an informative class of soluble disease markers. Here, the influence of crosslinker parameters on the acoustic properties of microbubbles, developing a model of crosslinker network formation on microbubble shells that explains the experimental observations, are studied. By incorporating cleavable elements that respond to UV light or proteolysis, kinetically resolved acoustic detection of these stimuli and the relevance of crosslinker design are demonstrated. The framework established in this study can be readily adapted to other protease-cleavable units and provides a basis for the future development of responsive ultrasound contrast agents for molecular imaging of proteolytic activity.