Bubbles, membranes and molecules: sequence of events from sonication to intracellular delivery

To elucidate mechanisms and control bioeffects for ultrasound-mediated drug and gene delivery, we carried out an experimental study to quantitatively measure the effects of ultrasound and other physical parameters on the sequence of events leading from sonication to drug and gene delivery. Using a Coulter counter, the number and size distribution of bubbles was measured as a function of ultrasound pressure and time, and found to decrease as a function of acoustic energy exposure. The effects of these bubbles on cells were measured by electron and confocal microscopy, which indicated that cavitation created cell membrane defects that could be actively repaired and permitted the intracellular transport of molecules. Using flow cytometry, levels of molecular uptake and cell viability were quantified over a broad range of conditions and correlated with acoustic energy, the ratio of cells-to-cavitation nuclei and the size distribution of bubbles. Finally, levels of gene expression were quantified as a function of acoustic and other parameters and related to cavitation bubble dynamics. By examining each of the steps, rather than only measuring endpoints, we can understand and ultimately control the process by which ultrasound delivers drugs and genes into cells.