Bi-modal confirmation of liposome delivery to the brain after focused ultrasound-induced blood-brain barrier opening.

Focused ultrasound-mediated opening of the blood-brain barrier offers a great opportunity to deliver therapeutics into hard-to-treat brain tumors such as glioblastoma multiforme or diffuse midline glioma. However, the potential of the technique to offer a time window for efficient nanomedicine delivery has not been thoroughly studied. Non-invasive and targeted delivery of large drug-loaded nanocarriers, such as liposomes, could offer a safe and scalable method of personalized therapy for the treatment of brain pathologies. Additionally, it is essential to monitor the safety and efficacy of such treatments, tracking drug delivery in real-time through quantitative medical imaging. In this study, liposomes were modified to have an MRI contrast agent (i.e., Gd) in both lipid membrane and core, while an infrared dye (i.e., CW800) was coupled to lipids introduced in the lipid bilayer for bimodal detection and treatment verification. Targeted delivery of 110 nm-in-diameter liposomes to the brain was quantified using 9.4-T MRI and near infrared fluorescence imaging. The spatiotemporal distribution of liposomes in vivo was assessed up to 4 h post treatment using T1 weighted MRI. In vivo MRI signal co-localized with NIRF signal from excised brains ex vivo. Passive acoustic detection during treatments revealed a correlation between acoustic signal and MRI contrast, providing a scalable metric for assessing clinical treatment efficacy in real-time. In conclusion, therapeutic ultrasound exposure can enhance delivery of large trackable nanoparticles into the brain, while enabling real-time treatment monitoring and verification.

Introduction

Purpose Drug delivery with BBB opening

Study Objective To design and optimize imageable 110 nm liposomes and quantify their focused ultrasound-mediated delivery across the blood–brain barrier in mice using MRI and near-infrared fluorescence, and to evaluate correlation with passive acoustic detection.

Animal model / Human subject Mouse (Mus musculus), CD-1 strain, 4-6 weeks old, female

Disease model Healthy

MRI or image guidance method Stereotactic targeting: mice fixed in a stereotactic frame and targeted using a metallic grid referenced to the lamboid (lamboidal) suture (anatomical coordinates provided: sonication location +2 mm AP, −2 mm ML; caudate targeted at −2 mm lateral, +2 mm ventral from lamboid suture)

Targeted brain region(s) Caudate

Target coordinates AP +2 mm, ML −2 mm, DV +2 mm (ventral) relative to the lamboid/lamboidal suture (caudate target). Figure also notes +2 mm A/P, −2 mm M/L from the lamboidal structure.

Cargo name and characteristics Thermosensitive liposomal nanoparticles (liposomes, ~110 nm diameter) — PEGylated lipid formulation (DPPC:DSPC:MSPC:DSPE‑PEG2000) dual‑labelled for imaging: Gd.DOTA.DSA incorporated into the lipid membrane and Gadovist® (Gd) encapsulated in the core, plus CW800 near‑infrared dye conjugated to a lipid (CW800.DSA) in the bilayer. Formulations denoted iTSL30[Gd] (30 mg/mL) and iTSL50[Gd] (50 mg/mL).

Route of administration Intravenous (tail vein catheter)

Outcomes and Safety

Summary of Outcomes Focused ultrasound BBB opening (0.5 MHz) enabled delivery and MRI/NIRF-detectable accumulation of 110 nm Gd/CW800 liposomes in mouse hippocampus at 2–4 h post-treatment, with delivery volume and contrast correlating with passive acoustic emissions; mild, generally reversible T2 changes (edema/occasional micro-hemorrhage) were observed in some animals. Successful FUS parameters included a 0.5 MHz transducer at ~320 ± 30 kPa peak-negative pressure (tested ~300–350 kPa) with BR1 microbubbles (2 mL/kg) and a single sonication (liposomes 50 mg/mL, 8 mL/kg).

Duration of biological effect 4 h

Safety-related matter T2 changes were observed in all mice; two mice had small hyperintense regions consistent with mild cerebral edema, and histology revealed micro-hemorrhage or red blood cell extravasation in hypointense areas. These findings diminished between 2 and 4 h indicating acute recovery, and the contralateral (non-sonicated) hemisphere was unaffected.

Brain Region

Ultrasound Parameters

Ultrasound instrument 0.5-MHz single-element FUS transducer (Part No. H-204; Sonic Concepts, Bothell, WA, USA); passive cavitation detector (Part No. U8423539, V320; Olympus Industrial, Waltham, MA, USA), diameter: 12.7 mm, focal depth: 76.2 mm

FUS Frequency 0.5 MHz; Passive cavitation detector: 7.5 MHz; High-pass filter cutoff: 1.2 MHz

FUS Pressure 0.32 ± 0.03 MPa

FUS Mode pulsed

Pulse duration 1 ms

Duration of a single FUS session 120 seconds (2 minutes)

Focal Characteristics Focal depth: 76.2 mm; Focal length: None; Aperture size: 12.7 mm

Treatment frequency Single session

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