Comparison of Sonication Patterns and Microbubble Administration Strategies for Focused Ultrasound-Mediated Large-Volume Drug Delivery

Neurology Medical Laboratory Science Medicine Bioengineering Oncology Life Sciences Immunology
Focused Ultrasound Microbubbles Large-Volume Drug Delivery Blood-Brain Barrier Immune Checkpoint Inhibitor

Comparison of Acoustic Patterns and Microbubble Delivery Strategies in Large-Volume Drug Delivery Mediated by Focused Ultrasound for Blood-Brain Barrier Opening

Background

Diffuse Intrinsic Pontine Glioma (DIPG) is the most common and lethal brainstem tumor in children. Due to the usually intact blood-brain barrier (BBB) in DIPG, drug penetration is effectively hindered, posing a significant challenge for treatment. Recently, the Focused Ultrasound-mediated BBB opening (FUS-BBBO) technique combined with microbubbles has shown great potential in overcoming this barrier. Considering the high diffuse nature of DIPG, there is a need for a large-volume FUS-BBBO treatment strategy that can cover the entire tumor region effectively. The aim of this study is to identify an optimal treatment strategy for achieving efficient and uniform large-volume BBBO in the brainstem, facilitating the effective delivery of immune checkpoint inhibitors (such as anti-PD-L1 antibodies) to the brainstem region.

Research Origin

The study was authored by Yan Gong, Dezhuang Ye, Chih-Yen Chien, Yimei Yue, and Hong Chen. They are all from the Department of Biomedical Engineering at Washington University in St. Louis. The article was published in the November 2022 issue of the IEEE Transactions on Biomedical Engineering. The original research work was supported by the National Institutes of Health and a fellowship from Washington University in St. Louis Taiwan.

Research Methods

Research Process

The study designed four combinations of two key parameters to evaluate the effect of FUS-BBBO: acoustic pattern (interleaved vs sequential) and microbubble injection method (bolus vs continuous). The study first compared the delivery effect of Evans blue under different strategies and then tested the delivery of anti-PD-L1 antibody (APD-L1) under different acoustic pressures with the selected optimal strategy.

  1. Animal Experiment Design

    • A total of 39 adult mice were used in the experiments. In the first experiment, 24 mice were divided into four groups, with six mice in each group, for testing the following combinations: interleaved-bolus injection, interleaved-continuous injection, sequential-bolus injection, and sequential-continuous injection. The acoustic pressure was maintained at 0.45 MPa. After selecting the optimal strategy, the remaining mice were divided into three groups in the second experiment to compare the delivery effect of APD-L1 under different acoustic pressures.
    • Microbubbles (Definity) were activated before the experiment and administered via tail vein injection or infusion.

  2. Focused Ultrasound Treatment

    • Ultrasound treatment was performed using an image-guided focused ultrasound system. The sound waves were applied using a 3x3 grid of multi-point sonication, covering the entire brainstem region.
    • 2D passive cavitation imaging (PCI) was used to monitor cavitation events in real-time during large-volume sonication.

  3. In Vivo Fluorescence Imaging and Quantification

    • After FUS treatment, 4% Evans blue or fluorescently labeled APD-L1 was injected. The mice were sacrificed post-treatment, and brain samples were collected, fixed, sectioned, and imaged for fluorescence. Fluorescence intensity was quantified using built-in software.

  4. Uniformity Analysis

    • Spatial uniformity of delivery was assessed by calculating the coefficient of variation (CV) of the pixel fluorescence intensity of Evans blue in the brainstem. A lower CV indicates higher uniformity.

  5. PCI Monitoring

    • Signals were passively received by the ultrasound probe at a frequency of 40 FPS. The total dose of cavitation was integrated over the entire sonication duration by recording the cavitation intensity at each time point.

  6. Physiological Monitoring

    • During anesthesia, the heart and respiration rates of the mice were recorded to assess any physiological changes induced by FUS treatment.

  7. Histological Analysis

    • Hematoxylin and eosin (HE) staining was used to evaluate tissue damage under different acoustic pressures, and microhemorrhage density was quantified using ImageJ software.

Research Results

  1. Efficiency and Uniformity of Evans Blue Delivery

    • The interleaved-bolus injection strategy achieved the highest delivery efficiency and uniformity. Its fluorescence intensity was 1.29 to 2.06 times higher than other strategies. The CV was 0.66, compared to 0.68 to 0.88 for other strategies.

  2. APD-L1 Delivery Effect

    • APD-L1 delivery was successfully achieved at 0.30 MPa and 0.45 MPa. Quantified fluorescence intensity indicated higher delivery efficiency at 0.45 MPa compared to 0.30 MPa, although both had similar uniformity. No significant delivery effect was observed at 0.15 MPa.

  3. Safety of Large-Volume FUS-BBBO

    • Monitoring showed no significant changes in heart and respiration rates, indicating safety. Histological analysis revealed microhemorrhages in four mice at 0.45 MPa, but no tissue damage was observed at 0.15 MPa and 0.30 MPa.

Conclusion

This study demonstrates that the interleaved acoustic pattern combined with bolus injection of microbubbles is the optimal strategy for achieving efficient and uniform large-volume FUS-BBBO. This strategy also enables safe drug delivery at relatively low acoustic pressures (0.30 MPa). PCI monitoring proved highly effective in evaluating delivery efficiency and spatial uniformity, correlating strongly with the final drug delivery results. The study provides critical guidance for future clinical application of FUS-BBBO in treating DIPG.

Academic Significance and Application Value

This study not only reveals the best strategy for achieving efficient and uniform large-volume drug delivery but also offers a real-time assessment and control technique through PCI monitoring. Additionally, good drug delivery effects were achieved while ensuring treatment safety, exploring new possibilities for treating DIPG and other similar brainstem tumors. In the future, the clinical application of FUS-BBBO technology could significantly improve the treatment outcomes for this deadly disease.