Real-time detection of intracellular reactive oxygen species and mitochondrial membrane potential in THP-1 macrophages during ultrasonic irradiation for optimal sonodynamic therapy

• Introducing a real-time fluorescence detection system during ultrasonic sonication.
• Quantifying intracellular ROS generation and MMP loss in real-time during SDT.
• This system can help facilitate a better understanding of the mechanisms involved in SDT.
• This system can be applied as a dosimetric tool for achieving optimal SDT.

Reactive oxygen species (ROS) elevation and mitochondrial membrane potential (MMP) loss have been proven recently to be involved in sonodynamic therapy (SDT)-induced macrophage apoptosis and necrosis. This study aims to develop an experimental system to monitor intracellular ROS and MMP in real-time during ultrasonic irradiation in order to achieve optimal effect in SDT. Cultured THP-1 derived macrophages were incubated with 5-aminolevulinic acid (ALA), and then sonicated at different intensities. Intracellular ROS elevation and MMP loss were detected in real-time by fluorospectrophotometer using fluorescence probe DCFH-DA and jc-1, respectively. Ultrasound at low intensities (less than 0.48 W/cm2) had no influence on ROS and MMP in macrophages, whereas at an intensity of 0.48 W/cm2, ROS elevation and MMP loss were observed during ultrasonic irradiation. These effects were strongly enhanced in the presence of ALA. Quantitative analysis showed that ROS elevation and MMP loss monotonically increased with the rise of ultrasonic intensity between 0.48 and 1.16 W/cm2. SDT at 0.48 and 0.84 W/cm2 induced mainly apoptosis in THP-1 macrophages while SDT at 1.16 W/cm2 mainly cell necrosis. This study supports the validity and potential utility of real-time ROS and MMP detection as a dosimetric tool for the determination of optimal SDT.