Extracellular adenosine-5′-triphosphate release kinetics following microbubble cavitation in cultured human endothelial cells

Abstract

Ultrasound Targeted Microbubble Cavitation (UTMC) causes vasodilation, which can improve radiotherapy efficacy, but the mechanisms remain unknown. Herein, we characterize extracellular Adenosine Triphosphate (eATP) release kinetics in vitro following UTMC using live microscopy. eATP was measured in real-time with a bioluminescent Luciferin-Luciferase (LL) assay. Human endothelial cells were grown in microfluidic chips. A microbubble solution (Definity: 107 MB/ml) containing propidium iodide (25 μg/ml) and LL was added to the chips before sending a single ultrasound pulse (A303S-SU, 1MHz, 0.5 in., Olympus, pressure: 300 kPa; 10, 100, or 1000 cycles). The bioluminescence signal of eATP reaction with the LL was captured with an EMCCD camera and converted into eATP released quantity, as in the study by Tan et al. (2019). After the acquisition, a viability assay was done using calcein-AM (4 μg/ml). We found a significantly higher total eATP released by dead cells than live sonoporated cells. The eATP release rate was also significantly higher in dead cells than in sonoporated cells at 4s after the pulse. At that time, we could estimate the eATP released by individual cells within a cell cluster before the individual bioluminescent signals merged due to diffusion. This will able us to understand better the eATP release mechanism in vitro and in vivo.