Standardized acoustic characterization and temperature monitoring reveal platform-dependent outcomes in liposomal PpIX-mediated sonodynamic therapy

To compare sonodynamic therapy (SDT) efficacy mediated by protoporphyrin IX-loaded liposomes (PLP) and heating effects across three non-focused ultrasound (US) platforms under approximately matched geometric-area-normalized intensity ( I MGA ). Three platforms were evaluated using a standardized direct-contact 96-well 4T1-cell model. Acoustic power was measured using a radiation force balance, with I MGA derived from measured power and visible transducer-face area. Cell viability was assessed by CCK-8, intracellular reactive oxygen species (ROS) generation by DCFH-DA-based imaging and flow cytometry, and the heating effect by infrared thermography supplemented with thermocouple measurements. Despite matched I MGA , platforms differed in cytotoxicity and heating. At low I MGA , US-alone cytotoxicity was limited, whereas PLP-dependent viability loss was observed for Platforms A and B. At high I MGA , Platforms A and B showed US-alone cytotoxicity and spatial heterogeneity, whereas Platform C retained PLP-dependent cytotoxicity. PLP + US enhanced intracellular ROS generation across all three platforms. Endpoint temperature was inversely associated with US-alone cell viability at high I MGA for Platforms A and B. At a fixed displayed intensity, measured time-averaged acoustic power scaled approximately with duty cycle. PLP-mediated SDT efficacy remained platform-dependent at approximately matched I MGA . Independent verification of acoustic output and temperature monitoring are essential for reproducible cross-platform interpretation of SDT studies.