Microbubble-endothelial cell interactions in 3D: Internalization of microbubbles and pore or tunnel formation for drug delivery

Abstract

Ultrasound-activated microbubbles can locally enhance vascular drug delivery, but fully understanding the mechanism requires further investigation. The aims of this in vitro study were to (1) assess the initial single microbubble-endothelial cell (n = 301) 3D morphology, (2) determine whether the ligand type on the targeted microbubble affected this morphology, and (3) investigate the morphology’s influence on microbubble oscillation and drug delivery outcome, all using high-resolution 3D confocal microscopy in combination with ultra-high-speed imaging (∼17 Mfps). Non-targeted and IgG1-κ control microbubbles were not internalized by endothelial cells, while targeted microbubbles were internalized. The internalization depth was ligand-dependent, since microbubbles having αvβ3 as target were significantly more internalized than those with CD31. Although the internalized microbubbles (n = 246) had a damped oscillation upon ultrasound insonification (2 MHz, 200 kPa, 10 cycles), their ability to sonoporate cells (i.e., PI uptake) was increased. Upon sonoporation (n = 230), either pores or tunnels (i.e. transcellular perforation) were formed in the cell membrane for respectively intracellular or transcellular delivery. Fewer transcellular perforations and smaller pore areas were observed for internalized microbubbles. In conclusion, receptor-mediated microbubble internalization, its effect on microbubble oscillation, and resulting membrane perforation were revealed using a state-of-the-art imaging system, thereby providing novel insights.