Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles

Ultrasound application in the presence of microbubbles is a promising strategy for intracellular drug and gene delivery, but it may also trigger other cellular responses. This study investigates the relationship between the change of cell membrane permeability generated by ultrasound-driven microbubbles and the changes in intracellular calcium concentration ([Ca(2+)](i)). Cultured rat cardiomyoblast (H9c2) cells were exposed to a single ultrasound pulse (1MHz, 10-15cycles, 0.27MPa) in the presence of a Definity(TM) microbubble. Intracellular transport via sonoporation was assessed in real time using propidium iodide (PI), while [Ca(2+)](i) and dye loss from the cells were measured with preloaded fura-2. The ultrasound exposure generated fragmentation or shrinking of the microbubble. Only cells adjacent to the ultrasound-driven microbubble exhibited propidium iodide uptake with simultaneous [Ca(2+)](i) increase and fura-2 dye loss. The amount of PI uptake was correlated with the amount of fura-2 dye loss. Cells with delayed [Ca(2+)](i) transients from the time of ultrasound application had no uptake of PI. These results indicate the formation of non-specific pores in the cell membrane by ultrasound-stimulated microbubbles and the generation of calcium waves in surrounding cells without pores.