Abstract 1070: Targeted Delivery of Macromolecules Using Ultrasound and Microbubbles is Regulated by Induction of Endocytosis and Pore Formation

Abstract

Ultrasound (US) contrast microbubbles 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. Primary bovine aortic endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 – 500 kDa) using 1-MHz US during 30s with 0.22 MPa peak-negative pressure. Duty cycle: 6.2%, pulse repetition frequency 20 Hz. Confocal laser microscopy showed the localization of 155 and 500 kDa dextrans in distinct vesicles after UMTD, indicating uptake via endocytosis. Independently inhibiting the main routes of active cellular uptake, i.e. clathrin- and caveolae-mediated endocytosis and macropinocytosis, caused significant decreases of 50 – 70% in intracellular delivery of 4.4 – 500 kDa dextrans (p<0.05). Furthermore, 3D immunofluorescence microscopy demonstrated dextran vesicles (500 kDa) to co-localize with both clathrin and caveolin-1. Finally, after energy depletion no uptake of 500 kDa dextran was observed after UMTD. In addition to triggering endocytosis, UMTD evoked transient pore formation, suggested by the homogeneous distribution through the cytosol of the 4.4 and 70 kDa dextrans. Formation of transient pores in the cell membrane was further supported by the influx of Ca2+ measured by live-cell fluorescence microscopy. This influx (peak mean fluorescence intensity (MIF) 186.4±3.4%) was not affected by the L-type Ca2+ channel blocker verapamil (MIF 172.5±3.2%), but significantly attenuated in a Ca2+ free buffer (MIF 111.1±2.0%, p<0.001). Finally, cells pre-loaded with fluorescent dextrans showed a significant decrease (p<0.05) of cytosolic fluorescence after US and microbubble exposure (4.4 kDa: 63.4±2.1%) and (155 kDa: 79.1±2.3%), compared to cells not exposed to US and microbubbles (100%). Endocytosis is a key mechanism of UMTD besides transient pore formation. The contribution of uptake via pores is dependent on molecular size. These findings are pivotal for optimizing US and microbubble intracellular delivery of therapeutic compounds.