Contrast agents for ultrasound are now routinely used for diagnosis and imaging. In recent years, new promising possibilities for targeted drug delivery have been proposed that can be realized by using the microbubble composing ultrasound contrast agents (UCAs). The microbubbles can carry drugs and selectively adhere to specific sites in the human body. This capability, in combination with the effect known as sonoporation, provides great possibilities for localized drug delivery. Sonoporation is a process in which ultrasonically activated UCAs, pulsating nearby biological barriers (cell membrane or endothelial layer), increase their permeability and thereby enhance the extravasation of external substances. In this way drugs and genes can be delivered inside individual cells without serious consequences for the cell viability. Sonoporation has been validated both in-vitro using cell cultures and in-vivo in preclinical studies. However, today, the mechanisms by which molecules cross the biological barriers remain unrevealed despite a number of proposed theories. This chapter will provide a survey of the current studies on various hypotheses regarding the routes by which drugs are incorporated into cells or across the endothelial layer and possible associated microbubble acoustic phenomena.

Sonoporation is a physical method that has been successfully used to deliver drugs into living cells both in vitro and in vivo for experimental and therapeutic purposes. Despite numerous studies on this topic, often reporting successful outcomes, very little is known about the mechanisms involved in the hypothesized membrane permeabilization processes. In this study, electron microscopy was used to investigate the ultra-structural modifications of cell membranes, induced by sonoporation. Here, we demonstrate that sonoporation in the presence of microbubbles induces the formation of a significant number of transient and permeant structures at the membrane level. These structures were transient with a half-life of 10 min and had a heterogeneous size distribution ranging from a few nanometers to 150 nm. We demonstrated that the number and the size of these structures were positively correlated with the enhanced intracellular uptake of small molecules. In addition, we showed that these structures were associated with caveolae-dependent endocytosis for two thirds of the recorded events, with the remaining one third related to non-specific routes such as membrane disruptions as well as caveolae-independent endocytosis. In conclusion, our observations provide direct evidences of the involvement of caveolae-endocytosis in cell membrane permeabilization to small molecules after sonoporation.