Objectives Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) loss have recently been shown to be involved in sonodynamic therapy (SDT)-induced macrophage death. This study aims to develop an experimental system of monitoring intracellular ROS and MMP in real-time during ultrasonic irradiation for the optimal SDT.

Methods Cultured THP-1 derived macrophages were incubated with either sonosensitizer 5-aminolevulinic acid (ALA) or phosphate-buffered saline, and then irradiated by ultrasound at different intensities. During ultrasonic irradiation, intracellular ROS generation and MMP loss were determined by fluorospectrophotometer using fluorescence probe DCFH-DA and jc-1, respectively. Cell apoptosis and necrosis after SDT were determined using Annexin V-FITC and propidium iodide assay.

Results Ultrasound at low intensities (100-600 mV) has no influence on ROS and MMP in macrophages during irradiation, whereas at a high intensity (600 mV) it stimulated ROS generation and MMP loss. The effects were enhanced in the presence of ALA, while ALA alone had little effect. Quantitative analysis showed that the levels of ROS generation and MMP loss increased with ultrasonic intensity. Consequently, SDT with 600 and 800 mV ultrasonic intensities tended to induce apoptotic change, whereas a higher intensity (1000 mV) tended to induce necrotic change.

Conclusions This study supports the validity and potential utility of real-time ROS and MMP detection as a dosimetric tool for the optimal SDT, as well as confirming the pivotal role of ROS in SDT.