Abstract 14619: Ultrasound-stimulated Microbubbles for Drug/gene Delivery: Microbubble-generated Shear Stress Induces Pores in Cellular Membranes

Abstract

Background: Ultrasound (US)-stimulated microbubbles (MBs) are emerging as novel target-specific non-viral gene/drug delivery vectors for treatment of vascular disease, e.g. to deliver VEGF plasmid DNA to promote arteriogenesis. The nature of the MB-target cell interactions that facilitate nucleic acid delivery across cell membranes, and hence strategies to optimize the efficiency of this platform, are poorly understood. The objective of this study was to gain mechanistic insight into MB-mediated increases in cell membrane permeability. Methods: Phospholipid coated MBs were allowed to lay adjacent to cultured human umbilical vein endothelial cells (HUVEC) at 37C. Propidium iodide (PI) was put into the media before US exposure to assess membrane permeability (sonoporation). Individual MB-cell pairs (n=351) were exposed to a single US pulse at 0.5, 1, or 2 MHz, with a duration of 4, 8 or 16 μs and pressures 0.1-0.8 MPa. Ultrafast microscopy recorded MB oscillations (Fig. 1a); a second camera simultaneously recorded fluorescence (PI uptake, Fig. 1b). US-induced MB radius change (excursion) was measured and used to calculate shear stress. HUVEC membranes were fluorescently labeled to visualize pore features. Results: Absolute MB excursion distance and associated shear stress were strong threshold indicators of sonoporation (PI uptake). For an 8 μs pulse, the oscillation (shear stress) threshold above which HUVECs porate was 3.9, 2.6, and 1.4 μm (7.8, 14.5, 22.7 kPa) at 0.5, 1 and 2 MHz respectively. At a given US frequency, increases in pulse duration decreased the required excursion and shear stress. Cell membrane imaging showed that MBs can induce apical and basal pores (sub-μm to μm sizes) that reseal (Fig. 1c). Conclusions: Sonoporation is related to absolute MB excursion distance and its associated shear stress and can occur with various US parameters and MB sizes. This data has implications on intelligent US-MB design for their use as payload delivery vehicles.