Multimodal evaluation of blood-brain barrier opening in mice in response to low-intensity ultrasound and a claudin-5 binder.

Background: The blood-brain barrier (BBB) is a major bottleneck in delivering therapeutics to the brain. Treatment strategies to transiently open this barrier include focused ultrasound combined with intravenously injected microbubbles (FUS+MB) and targeting of molecules that regulate BBB permeability. Methods: Here, we investigated BBB opening mediated by the claudin-5 binder cCPEm (a microorganismal toxin in a truncated form) and FUS+MB at a centre frequency of 1 MHz, assessing dextran uptake, broadband emission, and endogenous immunoglobulin G (IgG) extravasation. Results: FUS+MB-induced BBB opening was detectable at a pressure ≥0.35 MPa when assessed for leakage of 10 and 70 kDa dextran, and at ≥0.2 MPa for uptake of endogenous IgG. Treating mice with 20 mg/kg cCPEm failed to open the BBB, and pre-treatment with cCPEm followed by FUS+MB at 0.2 and 0.3 MPa did not overtly increase BBB opening compared to FUS+MB alone. Using passive cavitation detection (PCD), we found that broadband emission correlated with the peak negative pressure (PNP) and dextran leakage, indicating the possibility of using broadband emission for developing a feedback controller to monitor BBB opening. Conclusions: Together, our study highlights the challenges in developing combinatorial approaches to open the BBB and presents an additional IgG-based histological detection method for BBB opening.

Introduction

Purpose Drug delivery with BBB opening

Study Objective To evaluate whether the claudin-5 binder cCPEm, alone or combined with focused ultrasound plus microbubbles (FUS+MB), can open the blood-brain barrier in mice and to assess dextran/IgG leakage and passive cavitation signals as detection methods.

Animal model / Human subject Mouse C57BL/6J (mice), strain not specified, age not specified, sex not specified

Disease model Healthy

Cargo name and characteristics cCPEm; 10 kDa dextran; 70 kDa dextran

Route of administration Intravenous

Outcomes and Safety

Summary of Outcomes FUS+MB (1 MHz, 10 Hz PRF, 6 s sonications) produced BBB opening: 10 and 70 kDa dextran leakage occurred at ≥0.35 MPa (moderate at 0.35 MPa, substantial at 0.45 MPa; threshold between 0.3–0.35 MPa), whereas endogenous IgG extravasation was detectable at lower pressures (≥0.2–0.3 MPa). The claudin‑5 binder cCPEm (20 mg/kg) failed to open the BBB alone and did not augment FUS+MB at 0.2 or 0.3 MPa, and broadband cavitation emission correlated with peak negative pressure and dextran leakage (suggesting utility for feedback control).

Safety-related matter FUS+MB was generally safe at PNPs up to 0.35 MPa (safe BBB opening), with only one subtle petechial bleed observed at a single sonication spot at 0.45 MPa and intense bleeding consistently seen at 0.7 MPa (deemed unsafe). cCPEm at 20 mg/kg caused no detectable BBB opening or overt toxicity in this study, though the authors note potential antigenicity/toxicity with higher or repeated dosing from prior reports; broadband emissions in this study did not associate with tissue damage.

Brain Region

Visualization unavailable

Ultrasound Parameters

Ultrasound instrument Integrated focused ultrasound system (Therapy Imaging Probe System, TIPS, Philips Research, centre frequency of 1MHz

FUS Frequency 1 MHz

FUS Pressure 0.2 MPa, 0.3 MPa, 0.35 MPa

FUS Mode pulsed

Pulse duration 10 ms

Duration of a single FUS session 6 s

Focal Characteristics Focal depth: 80 mm; Focal length: None; Aperture size: 80 mm

Treatment frequency Single

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