Tuning endothelial barrier permeability with ultrasound: a pulse-length-dependent interplay between bubble dynamics...

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 Ca²⁺ 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 Ca²⁺ signalling across the cell population. Reactive oxygen species, extracellular Ca²⁺ influx through mechanosensitive channels and internal Ca²⁺ release were found critical in mediating Ca²⁺ 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 Ca²⁺ signalling mediated paracellular transport and sonoporation mediated transcellular transport, offering insights for optimizing ultrasound parameters for therapeutic drug delivery.