Microbubbles manufactured using a tissue homogeniser: DoE-guided optimisation for a narrower size distribution and...

Microbubbles are clinically approved as ultrasound contrast agents because they oscillate in response to ultrasound (cavitation) and are increasingly explored for therapeutic applications. Microbubble diameter governs dynamic behaviour under ultrasound; therefore, a narrow size distribution is essential for predictable performance and optimal responsiveness. However, producing phospholipid-coated microbubbles with narrow size distributions using simple methods remains challenging. This study evaluated a bead-type tissue homogeniser as an alternative to probe sonication for generating DSPC-PEG40S (9:1), air-filled microbubbles, using design of experiments (DoE) to identify influential parameters. A three-level full-factorial design assessed effects on mean microbubble diameter, concentration, and polydispersity index (PDI). Optical microscopy with an ImageJ analysis pipeline quantified size and concentration, while passive cavitation detection characterised acoustic response. Liquid volume significantly affected mean diameter, concentration, and PDI, whereas homogenisation speed significantly influenced PDI only. Response optimisation identified 5 m·s⁻¹, 45 s, and 500 µl as optimal speed, time, and volume settings. Compared with sonication, homogenisation generated less heat and achieved higher production rates. Resulting microbubbles were smaller, more uniform in size, and higher in concentration. At 37 °C, homogenised suspensions remained above 10⁸ MB/ml at 6 h, indicating improved short-term stability. Acoustic emissions increased with pressure and were comparable between methods when normalised to gas volume. Overall, bead homogenisation offers a simple, less thermogenic, high-throughput method for reproducible phospholipid microbubble production.