Fabrication and characterization of echogenic microbubbles for in vitro modeling of blood-brain barrier (BBB) disruption via focused ultrasound

The blood-brain barrier (BBB) presents a major obstacle to the delivery of many promising pharmacological agents to the central nervous system. The BBB is composed of tightly connected endothelial cells that regulate molecular exchange to maintain brain homeostasis. While this is beneficial for protecting the brain, over 98% of small-molecule and almost 100% of large-molecule drugs are blocked by the BBB, limiting treatment options for neurological diseases. Recently, bubble-assisted focused ultrasound (BaFUS) has emerged as a non-invasive strategy to transiently disrupt the BBB and facilitate targeted drug delivery by injected gas bubbles in the bloodstream. We previously developed a custom ultrasound-transparent organ-on-chip (UST-OoC) platform for modeling BaFUS-mediated BBB disruption in vitro . In this study, we report on the optimization and characterization of a microbubble formulation for use in BaFUS. Microbubbles were synthesized using a phospholipid-based protocol and characterized by dynamic light scattering (DLS) and zeta potential measurements. The formulation produced ~1 µm microbubbles with a neutral zeta potential and a concentration of 2.4 × 10 10 bubbles/mL. Acoustic testing showed strong, sustained bubble cavitation across flow rates of 150-300 µL/min. An acoustic dose-response sweep revealed non-linear stable cavitation and inertial cavitation thresholds to be 0.08 and 0.32 MPa. These lab-fabricated microbubbles have the advantages of low cost and easy adjustment of size and concentration. The UST-OoC platform enables human-based in vitro modeling for real-time visualization of barrier disruption, quantitative permeability assessment, and systematic optimization of acoustic parameters, including pressure, frequency, and duty cycle under controlled flow conditions. This integrated system provides a scalable, reproducible preclinical tool for evaluating microbubble performance and BaFUS protocols prior to in vivo translation. By establishing human-based BBB models, standardized methods for microbubble fabrication, acoustic characterization, and barrier disruption modeling, this platform addresses key translational gaps in developing BaFUS-enhanced drug delivery for brain disorders.