Abstract-Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4-and 70-kDa dextrans through the cytosol, and localization of 155-and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin-and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4-to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size. Key Words: ultrasound microbubble targeted delivery Ⅲ cell membrane pore Ⅲ endocytosis Ⅲ dextran Ⅲ endothelial cells C onventional delivery methods for drugs or genes, such as systemic administration via intravenous injection or oral administration, often do not suffice for therapeutic compounds such as peptides, silencing RNAs and genes. 1 A recent development in delivery systems for therapeutic compounds is the microbubble-ultrasound (US) interaction. 2,3 Before its use as a clinical modality, it is of utmost importance to obtain new physiological insights into the mechanisms of uptake by US and microbubble-exposed cells.Microbubbles were originally developed as US contrast agents and are administered intravenously to the systemic circulation to enhance scattering of blood in echocardiography. They consist of a gas core stabilized with an encapsulation, ranging from 1 to 10 m in diameter. 4 Nowadays, research focuses on the use of US and microbubbles for therapeutic applications. It has been demonstrated that USexposed microbubbles can achieve safe and efficient local delivery of a variety of drugs 5,6 and genes.

Ultrasound and microbubbles targeted gene delivery (UMTGD) is a promising technique for local gene delivery. As the endothelium is a primary target for systemic UMTGD, this study aimed at establishing the optimal parameters of UMTGD to primary endothelial cells. For this, an in vitro ultrasound (US) setup was employed in which individual UMTGD parameters were systematically optimized. The criteria for the final optimized protocol were: (1) relative high reporter gene expression levels, restricted to the US exposed area and (2) induction of not more than 5% cell death. US frequency and timing of medium replacement had a strong effect on UMTGD efficiency. Furthermore, US intensity, DNA concentration and total duration of US all affected UMTGD efficiency. Optimal targeted gene delivery to primary endothelial cells can be accomplished with Sonovue microbubbles, using 20 microg/ml plasmid DNA, a 1 MHz US exposure of Ispta 0.10 W/cm(2) for 30 s with immediate medium change after UMTGD. This optimized protocol resulted in both an increase in the number of transfected cells (more than three fold) and increased levels of transgene expression per cell (170%).