Ultrasound-microbubble-mediated drug delivery efficacy and cell viability depend on microbubble radius and ultrasound frequency

We have been investigating ultrasound-mediated drug delivery from microbubbles (MBs) as an alternative therapy to reduce hyper-proliferation of smooth muscle cells. We sought to determine if MB size affects drug delivery, cell viability, or cell adherence. Control, Dil, and drug-incorporated-rapamycin microbubbles (R-MBs) were size sorted into "small" and "large" sub-populations with number-average mean diameters of 1.7μm and 3.7μm respectively. Drug dose was maintained by keeping the surface area between MB sub-populations constant. Vascular smooth muscle cells (SMCs) were exposed to size-sorted R-MBs and insonated at 1MHz, 300 kPa, with 50 cycle sinusoids at a PRF of 100Hz for 8s. Insonation with large MBs reduced the proliferation rate of cells by 66.0% vs. 56.1% (n=8, p=0.46) with small MBs. The LIVE/DEAD® Viability/Cytotoxicity assay revealed a significant reduction in live cells (61.% live vs. 77.6% live), n=4, p=0.41), and a greater, but not statistically significant, increase in the permeabilized cells after insonation (1MHz) with large MBs (15.6%) versus small MBs (11.1%). We also investigated the effect of ultrasound frequency (1, 2.25, 5, and 10MHz) on MB on rupture and viability following 20 ultrasound pulses (20 cycle-sinusoids) at a peak negative pressure 600 kPa. 1MHz insonation ruptured the most microbubbles of either size, permeabilized the most cells (50% & 43% for small and large MBs respectively) and killed (33% & 31%) the most cells. More small (d = 1.8 μm) MBs were ruptured at every frequency (p<;0.001, see Fig. 4). Permeabilization of cells was approximately inversely related to frequency of insonation. 1MHz ultrasound permeabilized the most cells, but also killed the most cells. The permeable/dead cell ratio was 64% for large MBs and 98% for small MBs indicating a better therapeutic ratio when using small MBs.