Abstract 18652: Biophysics of Sonoporation for Gene Delivery: Shear Stress Initiates Endothelial Plasma Membrane Perforation and Calcium Signaling

• Published in: Circulation (New York, N.Y.) N.Y.), 2016-11, Vol.134 (Suppl_1 Suppl 1), p.A18652-A18652
• Main Authors: Helfield, Brandon, Chen, Xucai, Zeng, Jianxin, Watkins, Simon C, Villanueva, Flordeliza
• Format: Article
• Language: English
• ISSN: 0009-7322; 1524-4539

BackgroundUltrasound (US)-stimulated microbubbles (MBs) are emerging as a target-specific non-viral gene delivery platform for the treatment of vascular disease. The nature of MB-cell interactions that facilitate nucleic acid delivery across cell membranes (sonoporation), and hence strategies to optimize this platform, are poorly understood. The objective of this study was to investigate the biophysics of sonoporation.MethodsPhospholipid MBs were allowed to lay adjacent to cultured human umbilical vein endothelial cells (HUVEC) at 37C. To assess membrane permeability (sonoporation), propidium iodide (PI) was put into the media pre-US exposure. Individual MB-cell pairs (n=361) were exposed to a single US pulse at 0.5, 1, or 2 MHz, for 4, 8 or 16 μs at pressures of 0.1-0.8 MPa. Ultrafast microscopy recorded MB oscillations while a second camera simultaneously recorded fluorescence (PI uptake). US-induced MB oscillation was measured to calculate shear stress. For a subset of experiments, real-time confocal microscopy was employed to visualize HUVEC plasma membrane, cytoskeleton reorganization and intercellular calcium transients initiated by sonoporation.ResultsAbsolute MB radial excursion (Fig. 1a), i.e. shear stress, was a threshold indicator for sonoporation (Fig. 1b), requiring 7.8, 14.5, 22.7 kPa at 0.5, 1 and 2 MHz respectively to induce PI uptake (n=361). Sonoporation caused perforation through the apical and basal cell membranes, and triggered calcium signaling from the treated cell to adjacent, non-treated cells (Fig. 1b-e). A subset of studies reveals that sonoporation can rupture actin filaments and initiate cytoskeletal remodeling.ConclusionsSonoporation results in local shear stress magnitudes that perforate adjacent cell membranes, initiate calcium signaling, and re-organize actin networks. This data helps substantiate sonoporation as a viable payload delivery strategy and allows for further parametric optimization.