Chaofeng Qiao, Siyu Luo, Zhihui Liu, Yingxuan Bu, Yicong Cai, Zhuoyan Liu, Liying Wang, Claus Dieter Ohl, Fenfang Li
Ultrasound mediated microbubble cavitation holds great potential for non-invasive and targeted drug delivery. However, the interplay between acoustic parameters, bubble dynamics, and resulting cellular responses remains unclear, hindering the safety improvement and optimization of the technique. This study examined the effects of ultrasound pulse sequences on microbubble dynamics and bioeffects in endothelial monolayer using an acoustically coupled vessel-mimicking microchannels, where focused ultrasound exposure and concurrent recording of Ca 2+ signalling and membrane perforation were performed at flow conditions. A reduction of the total treatment time from 60 to 10 s avoided cell detachment. Microbubbles demonstrated brief oscillation and displacement under each of the 10 consecutive bursts of 40 µs short pulses with 1 ms interval while more intense bubble clustering, coalescence and displacement were observed under one continuous long pulse that lasted for around 9 ms. 10 s long pulse generated higher percentage and larger extent of cell membrane poration whereas short pulse induced wider spreading and larger Ca 2+ signalling across the cell population. Reactive oxygen species, extracellular Ca 2+ influx through mechanosensitive channels and internal Ca 2+ release were found critical in mediating Ca 2+ responses in short pulse condition. Further transwell experiments revealed that both pulse modes enhanced transport of 10 kDa FITC-dextran while a longer treatment of 60 s improved delivery efficiency for larger FITC-dextran of 40 kDa. These findings highlight the importance of pulse modes and total treatment time in tailoring Ca 2+ signalling mediated paracellular transport and sonoporation mediated transcellular transport, offering insights for optimizing ultrasound parameters for therapeutic drug delivery.