Use of transcranial low-intensity focused ultrasound for targeted delivery of stem cell-derived exosomes to the brain.

The blood-brain barrier (BBB) presents a significant challenge for targeted drug delivery. A proposed method to improve drug delivery across the BBB is focused ultrasound (fUS), which delivers ultrasound waves to a targeted location in the brain and is hypothesized to open the BBB. Furthermore, stem cell-derived exosomes have been suggested as a possible anti-inflammatory molecule that may have neural benefits, if able to pass the BBB. In the present study, transcranial low-intensity focused ultrasound (LIFU), without the use of intravenous microbubbles, was assessed for both (1) its ability to influence the BBB, as well as (2) its ability to increase the localization of intravenously administered small molecules to a specific region in the brain. In vivo rat studies were conducted with a rodent-customized 2 MHz LIFU probe (peak pressure = 1.5 MPa), and injection of labeled stem cell-derived exosomes. The results suggested that LIFU (without microbubbles) did not appear to open the BBB after exposure times of 20, 40, or 60 min; instead, there appeared to be an increase in transcytosis of the dextran tracer. Furthermore, the imaging results of the exosome study showed an increase in exosome localization in the right hippocampus following 60 min of targeted LIFU.

Introduction

Purpose Drug delivery WITHOUT BBB opening

Study Objective To evaluate whether transcranial low-intensity focused ultrasound (LIFU) without microbubbles can alter blood–brain barrier permeability and enhance localization of intravenously administered small molecules and stem cell-derived exosomes to a targeted brain region in rats.

Animal model / Human subject Rattus norvegicus (Wistar), 90 days old, female; Rattus norvegicus (Lewis), 6 months old, male

Disease model Healthy

MRI or image guidance method Stereotactic — animal head mounted on a stereotaxic apparatus (ear and nose bars); FUS transducer positioned using anatomical coordinates (6.2 mm rostral from interaural, 2.00 mm lateral right) with a water-filled standoff to set focal depth; no MRI/image guidance reported.

Targeted brain region(s) Hippocampus

Target coordinates AP: 6.2 mm rostral from interaural; ML: 2.00 mm right; DV: None

Cargo name and characteristics Stem cell–derived exosomes (XoGlo Pro Extravesicles), extracellular vesicles labeled via biotinylated CD9/CD63 and streptavidin–Renilla luciferase (Rluc); and fluorescent dextran tracers: FITC–dextran (10 kDa, small MW tracer) and Rhodamine–dextran (70 kDa, large MW tracer) used to assess BBB permeability.

Route of administration Intravenous (tail vein)

Outcomes and Safety

Summary of Outcomes Transcranial LIFU (2 MHz, peak pressure ~1.5 MPa) delivered without microbubbles did not overtly open or damage the BBB but increased endothelial transcytosis of a dextran tracer and produced significantly greater accumulation of IV stem cell‑derived exosomes in the targeted right hippocampus; these effects were time‑dependent and were observed after 60 min of LIFU (minimal effects at 20–40 min). Tested LIFU parameters: 2 MHz, 1.5 MPa peak pressure, exposure durations of 20, 40, and 60 min — 60 min exposure was effective.

Duration of biological effect 60 min

Safety-related matter No adverse effects were observed: LIFU (20–60 min) did not damage the BBB and there was no edema, bleeding, tissue damage, micro‑bleeding, or other untoward effects. Exposures were below the FDA derated maximum and designed to avoid cavitational/thermal effects, though the authors state further studies are needed to confirm safety.

Brain Region

Ultrasound Parameters

Ultrasound instrument Rodent-customized 2 MHz LIFU probe (peak pressure 1.5 MPa); commercial single-element focused ultrasound transducers (Olympus NDT and Technisonic), 2 MHz, 25 mm diameter, 48 mm nominal focal depth

FUS Frequency 2 MHz

FUS Intensity 0.668 W/cm2 (I_SPTA.0), 0.632 W/cm2 (I_SPTA.3)

FUS Pressure 1.5 MPa

FUS Mode pulsed

Pulse duration 0.06 ms

Duration of a single FUS session 60 min

Focal Characteristics Focal depth: 48 mm; Focal length: 48 mm; Aperture size: 25 mm

Treatment frequency Single session

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