Evaluation of blood-tumor barrier permeability and doxorubicin delivery in rat brain tumor models using additional focused ultrasound stimulation.

Focused ultrasound (FUS) has emerged as a promising technique for temporarily disrupting the blood-brain barrier (BBB) and blood-tumor barrier (BTB) to enhance the delivery of therapeutic agents. Despite its potential, optimizing FUS to maximize drug delivery while minimizing adverse effects remains a significant challenge. In this study, we evaluated a novel FUS protocol that incorporates additional FUS stimulation without microbubbles (MBs) ("FUS protocol") prior to conventional BBB disruption with MBs ("BBBD protocol") in a rat brain tumor model (n = 35). This approach aimed to validate its effectiveness in enhancing BBB/BTB disruption and facilitating doxorubicin delivery. T1-weighted contrast-enhanced and dynamic contrast-enhanced (DCE) MRI demonstrated significant increases in signal intensity and permeability (K<sub>trans</sub>) in the tumor region under the "FUS + BBBD protocol", with 2.65-fold and 2.08-fold increases, respectively, compared to the non-sonicated contralateral region. These values were also elevated compared to the conventional "BBBD protocol" by 1.45-fold and 1.25-fold, respectively. Furthermore, doxorubicin delivery in the targeted region increased by 1.91-fold under the "FUS + BBBD protocol", compared to a 1.44-fold increase using the conventional "BBBD protocol". This novel FUS approach offers a promising, cost-effective strategy for enhancing drug delivery to brain tumors. While further studies are required to assess its applicability with different chemotherapeutics and tumor types, it holds significant potential for improving brain tumor treatment in both preclinical and clinical settings.

Introduction

Purpose Drug delivery with BBB opening

Study Objective To evaluate whether an additional focused ultrasound stimulation without microbubbles before conventional microbubble-mediated BBB disruption enhances BBB/BTB permeabilization and doxorubicin delivery in a rat brain tumor model.

Animal model / Human subject Rat (Rattus norvegicus), Sprague-Dawley strain, 5 weeks old, male

Disease model Glioma (9L gliosarcoma brain tumor model in rats)

MRI or image guidance method MRI-guided focused ultrasound (MRgFUS) using a 9.4T MRI; T2-weighted images were scanned, registered and synchronized with the MRgFUS system for targeting

Targeted brain region(s) Tumor Region (9L Gliosarcoma Implantation Site: 3 Mm Lateral, 1 Mm Anterior To Bregma, 5 Mm Depth)

Target coordinates AP +1 mm (anterior to bregma), ML 3 mm lateral to bregma, DV 5 mm below dura

Cargo name and characteristics Doxorubicin hydrochloride — small-molecule anthracycline chemotherapeutic (DOX); administered IV at 5.67 mg/kg to assess tumor delivery and quantified by tissue fluorescence (480 nm excitation / 590 nm emission). Also used as experimental agents: Evans blue — small-molecule dye (4 mg/kg IV) to mark BBB opening; Gd-DOTA (gadoterate meglumine) — gadolinium-based MRI contrast agent (0.2 mM/kg) for BBB/BTB permeability imaging.

Route of administration Intravenous

Outcomes and Safety

Summary of Outcomes Adding a preliminary focused ultrasound (FUS) stimulation without microbubbles before conventional microbubble-mediated BBB/BTB disruption ('FUS + BBBD') increased tumor permeability (T1 contrast and Ktrans) and enhanced doxorubicin delivery (up to ~1.9×) without histologically detectable damage. Effective tested parameters were the 'FB' conditions (0.5FB, 1.0FB — the most effective, — and 2.0FB), while FUS alone (1.0F) was ineffective and the conventional BBBD ('B') produced intermediate effects.

Duration of biological effect 24 h

Safety-related matter Histopathological analysis (H&E) showed no significant tissue damage or differences between brain regions sonicated with '1.0FB', 'B', and '1.0F'; the authors report no observed adverse effects from the additional FUS stimulation, consistent with prior studies showing no tissue damage or heating.

Brain Region

Ultrasound Parameters

Ultrasound instrument RK-100 (FUS Instruments, Toronto, Canada); single-element spherical piezoelectric transducer, diameter 75 mm, radius of curvature 60 mm, resonant frequency 1 MHz

FUS Frequency 1 MHz

FUS Pressure 0.5 MPa, 1.0 MPa, 2.0 MPa, 0.72 MPa

FUS Mode pulsed

Pulse duration 10 ms

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

Focal Characteristics Focal depth: Not reported; Focal length: 60 mm; Aperture size: 75 mm

Treatment frequency single

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