Ultrasound-cavitation-enhanced drug delivery via microbubble clustering induced by acoustic vortex tweezers.

The application of acoustic vortex tweezers (AVT) in conjunction with ultrasound (US) cavitation pulses presents a promising noninvasive approach for the delivery of high concentrations of therapeutic agents. This methodology facilitates the aggregation of drug-loaded microbubbles (MBs) into clusters, which are subsequently destroyed to release their contents. Nevertheless, prior investigations have not thoroughly examined the resonance frequency and cavitation activity of MB clusters, critical factors that could enhance the efficiency of payload release. Theoretically, the resonance frequency of an MB cluster is expected to approximate that of a single large bubble of comparable size, thus being significantly lower than that of the individual MBs constituting the cluster. Accordingly, this study aims to optimize the release of payloads from AVT-trapped MB clusters, which measure 15 to 40 μm (mean radius: 24.7 μm) in size, by employing US at their resonance frequency of 100 kHz, henceforth referred to as "on-resonance US." In this investigation, MBs were loaded with the model drug DiI, resulting in the formation of DiI-MBs, which were then clustered utilizing AVT. On-resonance US excitation was subsequently applied to enhance the release of the drug payload. The dimensional characteristics of the DiI-MB clusters formed via 3-MHz AVT were measured to determine the range of resonance frequencies. Concurrent optical and acoustic analyses were conducted to evaluate the size, oscillation dynamics, and cavitation activity of the DiI-MB clusters in response to on-resonance US excitation. Additionally, the payload release from these clusters was quantitatively assessed. Our results indicate that significant oscillations of individual DiI-MB clusters commenced at a pressure of 44 kPa during 100 kHz US excitation. Further quantitative experiments demonstrated that the synergistic combination of AVT and 100-kHz US at 65 kPa significantly enhanced the payload release efficiency to 93 %. This efficiency surpassed that achieved with either method independently, with increases of 1.8-fold relative to AVT alone and 2.3-fold compared to 100-kHz US alone. The acoustic analyses revealed the onset of inertial cavitation at 44 kPa, which strongly correlated with payload release efficiency (R