Using a novel rapid alternating steering angles pulse sequence to evaluate the impact of theranostic ultrasound-mediated ultra-short pulse length on blood-brain barrier opening volume and closure, cavitation mapping, drug delivery feasibility, and safety

Abstract

Background: Focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening is a noninvasive, safe and reversible technique for targeted drug delivery to the brain. Most preclinical systems developed to perform and monitor BBB opening are comprised of a separate geometrically focused transducer and passive cavitation detector (PCD) or imaging array. This study builds upon previous work from our group developing a single imaging phased array configuration for simultaneous BBB opening and monitoring called theranostic ultrasound (ThUS), leveraging ultra-short pulse lengths (USPLs) and a novel rapid alternating steering angles (RASTA) pulse sequence design for simultaneous bilateral sonications with target-specific USPL. The RASTA sequence was further employed to evaluate the impact of USPL on BBB opening volume, power cavitation imaging (PCI) pixel intensity, BBB closing timeline, drug delivery efficiency, and safety. Methods: A P4-1 phased array transducer driven by a Verasonics Vantage ultrasound system was operated using a custom script to run the RASTA sequence which consisted of interleaved steered, focused transmits and passive imaging. Contrast-enhanced magnetic resonance imaging (MRI) confirmed initial opening volume and closure of the BBB by longitudinal imaging through 72 hours post-BBB opening. For drug delivery experiments, mice were systemically administered a 70 kDa fluorescent dextran or adeno-associated virus serotype 9 (AAV9) for fluorescence microscopy or enzyme-linked immunosorbent assay (ELISA) to evaluate ThUS-mediated molecular therapeutic delivery. Additional brain sections were also H&E-stained to evaluate histological damage, and IBA1- and GFAP-stained to elucidate the effects of ThUS-mediated BBB opening on stimulation of key cell types involved in the neuro-immune response, microglia and astrocytes. Results: The ThUS RASTA sequence induced distinct BBB openings simultaneously in the same mouse where volume, PCI pixel intensity, level of dextran delivery, and AAV reporter transgene expression were correlated with brain hemisphere-specific USPL, consistent with statistically significant differences between 1.5, 5, and 10-cycle USPL groups. BBB closure after ThUS required 2-48 hours depending on USPL. The potential for acute damage and neuro-immune activation increased with USPL, but such observable damage was nearly reversed 96 hours post-ThUS. Conclusion: ThUS is a versatile single-array technique which exhibits the potential for investigating a variety of non-invasive therapeutic delivery applications in the brain.

Rapid Short-Pulses of Focused Ultrasound and Microbubbles Deliver a Range of Agent Sizes to the Brain

“…This reduces, compared with long-pulse sequences [26,31,54] , the residual time for which the BBB remains disrupted, enabling rapid restoration of normal BBB functioning and, thus, neuroprotection 55 . Reduced BBB closing times following ultrasound exposure have also been reported in other mouse studies using short pulses of ultrasound [25,56] .…”

“…Despite our ndings of a size threshold of approximately 8 to 11 nm using RaSP at 0.35 MPa, we stress that short-pulse sequences can be used to deliver larger agents using higher acoustic pressures [42] . Recently, Batts et al reported the delivery of a 4.0 MDa adeno-associated virus serotype 9 (hydrodynamic diameter: ~ 25 nm) using a different short-pulse sequence at 1.0 MPa [25] . We have also previously demonstrated the delivery of 98 nm liposomes at 0.53 MPa using the same RaSP sequence used in this study [42] .…”

“…We targeted the delivery of model agents to the hippocampus due to its importance in learning and memory, and its pathological signi cance in numerous neurological disorders such as Alzheimer's disease, schizophrenia and temporal lobe epilepsy [36][37][38] . Fluorescent dextran was selected as a model agent due to its commercial availability in a range of sizes, biological and chemical inertness, and its precedent as a tracer in BBB disruption studies, offering good spatial resolution and contrast in brain tissue [19,21,25,30,[32][33][34][39][40][41] .…”

“…We also aimed to combine these ndings with previous RaSP-mediated deliveries and the subsequent detection of endogenous protein extravasation in mouse brain tissue to indicate size thresholds for delivery by RaSP at a speci ed pressure. By correlating the delivery characteristics of the model agents with their measured hydrodynamic diameters [25,[30][31][32][33][34] , we demonstrate the potential for RaSP-mediated deliveries of pharmacologically-relevant agents of comparable dimensions.…”

“…Efforts have been made to tune the activity of microbubbles within the vasculature to optimize the e ciency of treatments and improve their biosafety [16][17][18] . One strategy towards this is the use of short-pulse ultrasound sequences [19][20][21][22][23][24][25] .…”