Intracellular delivery of nanoparticles using ultrasound

The effects of ultrasound on cellular uptake of FITC-dextrans governed by sonoporation or endocytosis were studied. Hela cells in suspension with FITC-dextran (MW 4-2000 kDa) were exposed to ultrasound using different acoustic parameters (0.0 to 0.58 MPa peak negative pressures, 33 μs pulse length, 3 kHz pulse repetition frequency and 120s insonication time) in the presence or absence of microbubbles. After ultrasound exposure, the cellular uptake of FITC-dextran and cell viability were measured using flow cytometry. Confocal microscopy was used to localize the uptake of nanoparticles in cells. The role of endocyctosis was investigated using endocytic inhibitors; genistein, wortmannin and chlorpromazine inhibiting respectively caveolae-mediated endocytosis, macropinocytosis and clathrin-mediated endocytosis. Ultrasound in the presence of microbubbles enhanced the cellular uptake of dextran more than ultrasound alone. At a constant duty cycle (10%) and insonication time (120s), the percentage of cells internalizing dextran increased to 65% with increase in acoustic peak negative pressure (145 to 576 kPa, i.e., MI of 0.26 to 1.05). There was no statistical difference (p ≥ 0.3) between the uptakes of different sizes of dextran (MW 4-2000 kDa) in the presence of microbubbles. Inhibition of the endocytic pathways resulted in significant decrease in the cellular uptake (29% for genistein, 37% for wortmannin and 45% for chlorpromazine). The result indicates that ultrasound in the presence of microbubbles enhances cellular uptake of nanoparticles whereas ultrasound alone has hardly any effect. The improved uptake might be due to both poration and endocytosis. The mechanism of uptake of dextran is size independent (up to 2 MDa), thus either the size of the pores is larger than the largest dextran molecule used, or endocytosis is size independent. The results show that ultrasound enhances cellular uptake of therapeutic molecules and has the potential to impro- e cancer therapy.