Quantification of optison bubble size and lifetime during sonication dominant role of secondary cavitation bubbles causing acoustic bioeffects

Acoustic cavitation has been shown to deliver molecules into viable cells, which is of interest for drug and gene delivery applications. To address mechanisms of these acoustic bioeffects, this work measured the lifetime of albumin-stabilized cavitation bubbles (Optison) and correlated it with desirable (intracellular uptake of molecules) and undesirable (loss of cell viability) bioeffects. Optison was exposed to 500 kHz ultrasound (acoustic pressures of 0.6-3.0 MPa and energy exposures of 0.2-200 J/cm2) either with or without the presence of DU145 prostate cancer cells (10(6) cells/ml) bathed in calcein, a cell-impermeant tracer molecule. Bubble lifetime was determined using a Coulter counter and flow cytometer, while bioeffects were evaluated by flow cytometry. The lifetime of Optison cavitation nuclei was found to decrease and bioeffects (molecular uptake and loss of cell viability) were found to increase with increasing acoustic energy exposure. These bioeffects correlated well with the disappearance of bubbles, suggesting that contrast agent destruction either directly or indirectly affected cells, probably involving unstabilized cavitation nuclei created upon the destruction of Optison. Because Optison solutions presonicated to destroy all detectable bubbles also caused significant bioeffects, the indirect mechanism involving secondary cavitation bubbles is more likely.