Characterization of microbubble cavitation in theranostic ultrasound-mediated blood-brain barrier opening for gene delivery

Rationale: The characterization of microbubble (MB) activity in ultrasound-mediated blood-brain barrier (BBB) opening has proven critical in assessing the method's safety and efficacy in drug delivery. In this study, we build upon our previous work on theranostic ultrasound (ThUS)-mediated BBB opening (ThUS-BBBO) and conduct for the first time a comprehensive characterization of the role of MB cavitation in ThUS-BBBO and gene delivery with adeno-associated viruses (AAV). Methods: A repurposed diagnostic ultrasound imaging phased array was used to generate ultra-short focused transmits at 1.5 MHz center frequency and simultaneously record acoustic emissions. MB activity was first characterized using a separate passive cavitation detector (PCD) in a flow phantom using pulse lengths ranging from 1.5 to 20 cycles and varying MB flow rates. A comprehensive in vivo study in mice was then conducted to characterize and correlate the resulting cavitation with AAV transgene expression. The transcranial MB activity was first detected using a PCD to confirm the findings of the flow phantom study. Next, three mouse studies were conducted to evaluate the relationship between cavitation and AAV delivery; one with different MB size distributions including polydisperse and size-isolated MB, one with variable burst length and burst repetition frequency, and one with different AAV serotypes and injection doses. Electronic beam steering enabled bilateral BBB opening with 1.5 cycle on the left and 10 cycles on the right hemisphere. Cavitation dose, BBBO volume, transgene expression, and histological safety were assessed following each experimental condition. Results: Frequency domain analysis in the phantoms revealed increasing harmonic cavitation with longer pulses. However, analysis in the time domain showed that longer pulses induce higher MB collapse. In the transcranial in vivo experiments, the PCD detected increased harmonic cavitation for 10-cycle pulses. The MB study showed that 3-5 mu m MB resulted in the largest cavitation doses, BBBO volumes and transgene expression compared to the smaller MB groups. However, non-significant differences in BBBO volume and transgene expression were found when the MB dose was normalized by gas volume. The burst sequence study revealed that shorter bursts and faster burst repetition frequencies induce larger BBBO volumes and AAV transduction due to MB replenishment within the focal volume. Transgene expression was also increased with injection dose, and AAV9 showed the highest brain transduction efficiency compared to AAV2 and AAV5. Conclusions: The phantom and in vivo studies showed that the mechanism of ThUS-BBBO is transient cavitation dominant and MB collapse increases with pulse length. Increased cavitation dose resulted in larger BBBO volumes and transgene expression in vivo. Overall, these results offer insights into the mechanism of AAV delivery across the BBB with ThUS and indicate that despite modulation of ThUS parameters and MB sizes, particular AAV serotypes are better-suited for maximal ThUS-mediated transgene expression in the brain after systemic injection.