Real-time investigation of irreversible cell membrane damage induced by acoustic droplet vaporization

Acoustic droplet vaporization (ADV) has shown a great potential in extravascular tumor-targeting theranostics. The mechanical perturbations produced during ADV process (e.g. initial volume expansion, cavitation, and movement of the bubble) on the nearby cells could cause cell dysfunction with irreversible cell membrane damage or facilitate drug delivery due to sonoporation. A confocal acousto-optical microscopic system was used to investigate the bioeffects of ADV on nearby cells in real time with high-speed microscopic imaging and fluorescence imaging. A 5 MHz single-element transducer driven by a high power pulser was focused confocally with the microscopy to induce ADV nearby the cells. Calcein-AM and propidium iodide (PI) were added for real-time evaluating cells viability and tracking cell membrane integrity after ADV, respectively. HeLa cervical cancer cells were cultured in the Opticell chamber and perfluoropentane (PFP) nanodroplets were added prior to experiments. Upon ultrasound exposure, the rapid vaporization of PFP nanodroplets into gaseous bubbles and subsequent ADV bubble-cell interactions were observed by high-speed imaging, including the bubble growth, aggregation, coalescence and translational motion above the apical cell membrane layer or near the cells. A pronounced and continuous increase in the intracellular PI fluorescence intensity in the cells near ADV was observed in the fluorescence images post-ADV. It indicated the loss of cell membrane integrity, which was also confirmed by a complete leakage of Calcein. It revealed that the rapid ADV event and formed ADV bubbles forced by ultrasound pulses can cause the irreversible pores on the cell membrane, which results in significant cell damage and eventually cell death. Understanding the real-time characteristics of ADV-mediated bioeffects on cells is significant to develop this approach in vivo and then in clinic.