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

TitleMicrobubble-mediated drug delivery revealed at nanosecond and nanometer resolution
Image caption

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.

First nameKlazina
Last nameKooiman

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

Additional authors
Session7. Biomedical applications
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