Targeted microbubble opening of cell-cell junctions for vascular drug delivery elucidated with combined confocal microscopy and Brandaris 128 imaging

Abstract

Ultrasound insonification of microbubbles enhances vascular drug delivery pathways, such as opening of cell-cell junctions and pore formation (sonoporation). However, the underlying mechanism remains unknown. The aim of our study was to elucidate the microbubble-cell interaction using the Brandaris 128 ultra-high speed camera (~17 Mfps), to visualize microbubble oscillation, coupled to a custom-build Nikon confocal microscope, to visualize cellular response. Confluent endothelial cells were evaluated for opening of cell-cell junctions with Cell Mask and for sonoporation with Propidium Iodide (PI). The cellular response of single targeted microbubbles (n = 168) was monitored up to 4 min after ultrasound insonification (2 MHz, 100–400kPa, 10-cycles). Cell-cell junctions opening occurred more often when cells were only partially attached to their neighbors (45%) than when fully attached (15%). Almost all fully attached cells showing cell-cell opening also showed PI uptake (92%). The mean microbubble excursion was larger when a cell was sonoporated (1.0μm) versus non-sonoporated (0.47μm). Additionally, larger microbubble excursion amplitudes correlated with larger pore size coefficients, obtained by fitting the PI uptake profiles to the Fan model [Fan, et al., PNAS, 2012]. In conclusion, using the state-of-the-art imaging system we can now elucidate the relationship between microbubble oscillation behavior and the drug delivery pathways.