Non-invasive molecularly-specific millimeter-resolution manipulation of brain circuits by ultrasound-mediated aggregation and uncaging of drug carriers.

Non-invasive, molecularly-specific, focal modulation of brain circuits with low off-target effects can lead to breakthroughs in treatments of brain disorders. We systemically inject engineered ultrasound-controllable drug carriers and subsequently apply a novel two-component Aggregation and Uncaging Focused Ultrasound Sequence (AU-FUS) at the desired targets inside the brain. The first sequence aggregates drug carriers with millimeter-precision by orders of magnitude. The second sequence uncages the carrier's cargo locally to achieve high target specificity without compromising the blood-brain barrier (BBB). Upon release from the carriers, drugs locally cross the intact BBB. We show circuit-specific manipulation of sensory signaling in motor cortex in rats by locally concentrating and releasing a GABA<sub>A</sub> receptor agonist from ultrasound-controlled carriers. Our approach uses orders of magnitude (1300x) less drug than is otherwise required by systemic injection and requires very low ultrasound pressures (20-fold below FDA safety limits for diagnostic imaging). We show that the BBB remains intact using passive cavitation detection (PCD), MRI-contrast agents and, importantly, also by sensitive fluorescent dye extravasation and immunohistochemistry.

Introduction

Purpose Drug delivery WITHOUT BBB opening

Study Objective To develop and validate a noninvasive focused-ultrasound approach that aggregates and uncages drug-loaded carriers to deliver small molecules across an intact blood–brain barrier for focal, circuit-specific modulation of brain activity.

Animal model / Human subject Rattus norvegicus (Long Evans), age not specified (200–300 g body weight), female

MRI or image guidance method Stereotactic (stereotaxic frame using bregma-referenced coordinates and angled positioning); focal alignment validated with needle hydrophone and 3D skull scanning

Targeted brain region(s) Vs1 (Vibrissal Primary Somatosensory Cortex)

Target coordinates craniotomy above vM1: (AP 0–2.5 mm, ML 0–2 mm, DV not specified); vM1 probe: (AP 1–2 mm, ML 0.5–1 mm, DV 1.5–2 mm from pia); vS1 FUS focal: (AP −2.3 mm, ML 6 mm, DV 3.3 mm from skull surface); vS1 (vS1-V1) FUS: (AP −2.3 mm, ML 6.5 mm, DV 3.3 mm from skull surface); V1 electrode: (AP −5.5 to 6.0 mm, ML 3.2–3.5 mm, DV 1.6–2.0 mm); vS1 (simultaneous) probe: (AP −2.3 mm, ML 6 mm, DV 1.1 mm from pia); vM1 (simultaneous) probe: (AP 1.5 mm, ML 1 mm, DV 0.8 mm from pia); skull-scan/focus alignment: (AP +4 mm rostral to bregma, ML not specified, DV 7 mm ventral from skull surface).

Cargo name and characteristics Muscimol — small-molecule GABA_A receptor agonist (therapeutic cargo, loaded into liposomal UC-carriers and also used systemically); Sodium fluorescein — small-molecule fluorescent tracer (experimental cargo loaded into UC-carriers).

Route of administration intravenous

Outcomes and Safety

Summary of Outcomes Aggregating-and-uncaging FUS (AU-FUS) enabled noninvasive, focal, reversible inhibition of targeted cortical circuits in rats by locally uncaging muscimol, reducing evoked responses by around 56% with around 1300 fold enhancement over systemic dosing, while preserving BBB integrity

Duration of biological effect 50 min

Safety-related matter The authors report their AU-FUS method uses very low ultrasound pressures (≈20-fold below FDA diagnostic limits) and preserves BBB integrity with no detectable off-target effects or signatures of BBB opening by multiple assays, avoiding tissue heating and damage. They note that higher-intensity or BBB-opening FUS approaches can cause BBB opening, sterile inflammation, nonspecific neuronal and behavioral effects, systemic off-target drug exposure, and cite typical drug side effects (e.g., ketamine's psychotomimetic, perceptual, and cardiovascular complications) as risks that focal delivery could mitigate.

Brain Region

Ultrasound Parameters

Ultrasound instrument Custom-made transducer (Sonic Concepts); 2.5 MHz center frequency; 40 mm diameter (aperture); 30 mm working distance; 20.65 mm focal depth; theoretical focal volume 0.5 × 0.5 × 2.5 mm (−6 dB); used with custom impedance matching network (Sonic Concepts)

FUS Frequency 2.5 MHz

FUS Mode pulsed

Focal Characteristics Focal depth: 20.65 mm; Focal length: 30 mm; Aperture size: 40 mm

Treatment frequency multiple AU-FUS cycles within a single experimental session

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