Low frequency nanobubble-enhanced ultrasound mechanotherapy for noninvasive cancer surgery

Scaling down the size of microbubble contrast agents to the nanometer level holds the promise for noninvasive cancer therapy. However, the small size of nanobubbles limits the obtained bioeffects as a result of ultrasound cavitation, when operating near the nanobubble resonance frequency. Here we show that coupled with low energy insonation at a frequency of 80 kHz, well below the resonance frequency of these agents, nanobubbles serve as noninvasive therapeutic warheads that trigger potent mechanical effects in tumors following a systemic injection. We demonstrate these capabilities in tissue mimicking phantoms, where a comparison of the acoustic response of micro- and nano-bubbles after insonation at a frequency of 250 or 80 kHz revealed that higher pressures were needed to implode the nanobubbles compared to microbubbles. Complete nanobubble destruction was achieved at a mechanical index of 2.6 for the 250 kHz insonation vs. 1.2 for the 80 kHz frequency. Thus, the 80 kHz insonation complies with safety regulations that recommend operation below a mechanical index of 1.9. In vitro in breast cancer tumor cells, the cell viability was reduced to 17.3 ± 1.7% of live cells. In vivo, in a breast cancer tumor mouse model, nanobubble tumor distribution and accumulation were evaluated by high frequency ultrasound imaging. Finally, nanobubble-mediated low frequency insonation of breast cancer tumors resulted in effective mechanical tumor ablation and tumor tissue fractionation. This approach provides a unique theranostic platform for safe, noninvasive and low energy tumor mechanotherapy.