Microbubble gas volume: A unifying dose parameter in blood-brain barrier opening by focused ultrasound.

Authors: Song KH, Fan AC, Hinkle JJ, Newman J, Borden MA, Harvey BK

Focused ultrasound with microbubbles is being developed to transiently, locally and noninvasively open the blood-brain barrier (BBB) for improved pharmaceutical delivery. Prior work has demonstrated that, for a given concentration dose, microbubble size affects both the intravascular circulation persistence and extent of BBB opening. When matched to gas volume dose, however, the circulation half-life was found to be independent of microbubble size. In order to determine whether this holds true for BBB opening as well, we independently measured the effects of microbubble size (2 vs. 6 µm diameter) and concentration, covering a range of overlapping gas volume doses (1-40 µL/kg). We first demonstrated precise targeting and a linear dose-response of Evans Blue dye extravasation to the rat striatum for a set of constant microbubble and ultrasound parameters. We found that dye extravasation increased linearly with gas volume dose, with data points from both microbubble sizes collapsing to a single line. A linear trend was observed for both the initial sonication (R<sup>2</sup>=0.90) and a second sonication on the contralateral side (R<sup>2</sup>=0.68). Based on these results, we conclude that microbubble gas volume dose, not size, determines the extent of BBB opening by focused ultrasound (1 MHz, ~0.5 MPa at the focus). This result may simplify planning for focused ultrasound treatments by constraining the protocol to a single microbubble parameter - gas volume dose - which gives equivalent results for varying size distributions. Finally, using optimal parameters determined for Evan Blue, we demonstrated gene delivery and expression using a viral vector, dsAAV1-CMV-EGFP, one week after BBB disruption, which allowed us to qualitatively evaluate neuronal health.

Introduction

Purpose Drug delivery with BBB opening

Study Objective To determine whether microbubble gas volume dose, rather than microbubble size, governs the extent of focused-ultrasound-induced blood–brain barrier opening and to demonstrate gene delivery using the optimized parameters in the rat striatum.

Animal model / Human subject Rat (Rattus norvegicus), strain: None, age: None, sex: None

Targeted brain region(s) Striatum

Cargo name and characteristics Evans Blue (small-molecule dye used to assess BBB extravasation); dsAAV1-CMV-EGFP (double-stranded AAV serotype 1 viral vector encoding EGFP under CMV promoter for gene delivery/expression)

Outcomes and Safety

Summary of Outcomes Focused ultrasound (1 MHz, ~0.5 MPa at focus, 5 min sonications) with microbubbles produced linear, gas-volume-dose-dependent BBB opening (1–40 µL/kg gas volume) such that both 2 µm and 6 µm microbubbles gave equivalent effects when matched by gas volume, and this enabled localized AAV1-CMV-eGFP gene delivery (successful demonstration using 20 µL gas volume with 6‑µm MBs at ~2×10^8 MB/kg) with minimal neuronal loss.

Duration of biological effect 5 min

Safety-related matter Limited hemorrhaging was observed immediately after BBB disruption at higher microbubble gas volume doses—suggesting possible inertial cavitation—but this hemorrhage was not present one week later in AAV experiments. The selected microbubble volume balanced efficient permeabilization with minimal hemorrhage and yielded robust neuronal transduction with no noticeable neuronal loss by NeuN staining, though the authors caution these findings may not generalize to other ultrasound parameters.

Brain Region

Ultrasound Parameters

FUS Frequency 1 MHz

FUS Pressure 0.5 MPa

Focal Characteristics Focal depth: None; Focal length: None; Aperture size: None

Treatment frequency Multiple sessions

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