Effects of shear stress cultivation on cell membrane disruption and intracellular calcium concentration in sonoporation of endothelial cells.

摘要

Microbubble facilitated ultrasound (US) application can enhance intracellular delivery of drugs and genes in endothelial cells cultured in static condition by transiently disrupting the cell membrane, or sonoporation. However, endothelial cells in vivo that are constantly exposed to blood flow may exhibit different sonoporation characteristics. This study investigates the effects of shear stress cultivation on sonoporation of endothelial cells in terms of membrane disruption and changes in the intracellular calcium concentration ([Ca(2+)](i)). Sonoporation experiments were conducted using murine brain microvascular endothelial (bEnd.3) cells and human umbilical vein endothelial cells (HUVECs) cultured under static or shear stress (5 dyne/cm(2) for 5 days) condition in a microchannel environment. The cells were exposed to a short US tone burst (1.25 MHz, 8 mus duration, 0.24 MPa) in the presence of Definity microbubbles to facilitate sonoporation. Membrane disruption was assessed by propidium iodide (PI) and changes in [Ca(2+)](i) measured by fura-2AM. Results from this study show that shear stress cultivation significantly reduced the impact of ultrasound-driven microbubbles activities on endothelial cells. Cells cultured under shear stress condition exhibited much lower percentage with membrane disruption and changes in [Ca(2+)](i) compared to statically cultured cells. The maximum increases of PI uptake and [Ca(2+)](i) were also significantly lower in the shear stress cultured cells. In addition, the extent of [Ca(2+)](i) waves in shear cultured HUVECs was reduced compared to the statically cultured cells.