Ultrasound-activated microbubbles (MB) show great promise for drug delivery¹. However, the mechanism is still unknown. To better predict and control the different drug delivery pathways, underlying biological and physical mechanisms of the MB-cell interaction need to be studied. Much insight into cellular effects has been gained with confocal microscopy²⁻⁴ and into MB behavior with ultra-high speed camera studies⁴⁻⁵. However, to study all aspects of the MB-cell interaction simultaneously, a combined confocal microscope and ultra-high speed camera would be needed. A state-of-the-art optical imaging system was therefore developed by connecting an upright custom built Nikon A1R confocal microscope to the Brandaris 128 ultra-high speed camera (25 Mfps)⁶, thereby achieving the nanometer and nanosecond resolution needed to visualize cellular effects and resolve MB oscillation upon ultrasound insonification.

Confluent endothelial cells were evaluated for opening of cell-cell junctions with CellMask and for sonoporation with Propidium Iodide (PI). The cellular response of single αVβ3-targeted MB (n=168; 2-7 µm in diameter) was monitored up to 4 min after ultrasound insonification (2 MHz, 100-400 kPa, 10-cycles). Cell-cell junction 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 MB excursion was larger when a cell was sonoporated (1.0 µm) versus non-sonoporated (0.47 µm). In conclusion, using the state-of-the-art imaging system we can now elucidate the relationship between MB oscillation behavior and drug delivery pathways.

Refs: ¹Kooiman et al, Adv Drug Del Rev 2014, 72:28; ²Hu et al, Ultrasound Med Biol 2013, 39:2393; ³De Cock et al, J Contr Rel 2015, 197:20; ⁴Helfield et al, Proc Natl Acad Sci USA 2016, 113:9983; ⁵Kooiman et al, J Contr Rel 2011, 154:35; ⁶Chin et al., Rev Sci Instru 2003, 74:5026

Confocal microscopy images of cellular response before and after ultrasound (US) and the corresponding excursion amplitude of the microbubble recorded with the Brandaris 128 ultra-high speed camera.

Department of Biomedical Engineering, Thoraxcenter, Erasmus MC, Rotterdam, the Netherlands