Low-intensity focused ultrasound with microbubbles enhances CD20 monoclonal antibody delivery across the blood-brain barrier in primary central nervous system lymphoma Free

Abstract

Objective: The objective of this study is to utilize low-intensity focused ultrasound (LIFU) in conjunction with microbubbles to achieve transient and safe opening of the blood-brain barrier (BBB), thereby facilitating enhanced delivery of CD20 monoclonal antibodies (mAb) into the brain parenchyma. This approach aims to elevate intracranial drug concentrations and augment therapeutic efficacy in the treatment of primary central nervous system lymphoma (PCNSL), as demonstrated in preclinical models showing significant tumor reduction and increased drug penetration without permanent tissue damage.

Methods:A primary central nervous system lymphoma (PCNSL) model was established in 6- to 8-week-old female BALB/c mice through stereotactic intracranial inoculation of 5 × 10^4 luciferase-labeled murine A20 lymphoma cells (in 2 μL PBS) into the right striatum at coordinates (AP: +0.5 mm, ML: +2.0 mm, DV: -3.0 mm from bregma). Successful tumor implantation was confirmed via bioluminescence imaging (BLI) on day 7 post-inoculation. Thirty-two tumor-bearing mice were randomized (using a computer-generated sequence) into four groups (n=8 each): Group A (control, no intervention), Group B (CD20 mAb alone, 10 mg/kg rituximab IV), Group C (LIFU alone with microbubbles), and Group D (LIFU combined with microbubbles and CD20 mAb, 10 mg/kg rituximab IV). For LIFU treatment, mice received intravenous microbubbles (Definity, 1.2 × 10^8 bubbles/kg) immediately before sonication using a 1 MHz transducer at 0.5 MPa peak negative pressure, 10 ms bursts, 1 Hz pulse repetition frequency, for 2 minutes, targeted to the tumor site under MRI guidance. Tumor progression was monitored weekly via BLI. Cerebrospinal fluid (CSF) was collected via cisterna magna puncture 2 hours post-treatment to quantify rituximab concentration by ELISA. Mice underwent two treatment cycles (days 7 and 14 post-implantation), after which they were euthanized for brain dissection. Pathological analyses included Evans blue extravasation for BBB permeability, tumor volume measurement via H&E staining, and assessment of ultrasound-exposed tissue integrity via immunohistochemistry for apoptosis (TUNEL) and inflammation (Iba-1)

Results: Tumor-bearing mice were monitored for 21 days post-implantation. Tumor volumes were quantified via bioluminescence imaging (BLI) signal intensity (photons/sec), with statistical comparisons using one-way ANOVA followed by Tukey's post-hoc test (p<0.05 considered significant). CSF rituximab concentrations were measured by ELISA, and histopathological analyses included H&E for tumor size, Evans blue for BBB permeability, TUNEL for apoptosis, and Iba-1 for microglial activation.

Group A (control): Rapid tumor growth (4.2-fold BLI increase by day 14, p<0.001), 87.5% metastasis, median survival 12 days, 100% mortality by day 21, no BBB disruption.

Group B (CD20 mAb): Modest inhibition (21.3±2% reduction at day 14, p=0.04 vs A), 25% metastasis, CSF rituximab 0.3±0.03 ng/mL, survival 16 days (p=0.03 vs A).

Group C (LIFU + microbubbles): Similar to A (4.0-fold BLI, p=0.92 vs A; survival 13 days). Transient BBB opening (Evans blue in 100%, resolves 24h), no damage (TUNEL/Iba-1 <5% above baseline, p>0.05).

Group D (LIFU + CD20 mAb + microbubbles): Significant regression (81.3±5% reduction, p<0.001 vs others), no metastasis, CSF rituximab 38.6±0.05 ng/mL (p<0.001 vs B), survival >21 days (75% at endpoint, p<0.001 vs A), safe (similar to C).

Conclusion: The blood-brain barrier (BBB) poses a major obstacle to CD20 monoclonal antibody (mAb) efficacy in primary central nervous system lymphoma (PCNSL), as evidenced by limited drug penetration and modest therapeutic effects in monotherapy. Low-intensity focused ultrasound (LIFU) combined with microbubbles provides a safe, reversible method for BBB disruption, markedly improving intracranial drug delivery, tumor regression, and survival in preclinical models. This strategy offers promising translational potential as an adjunctive therapy for PCNSL, warranting further clinical investigation to optimize parameters and assess long-term safety.