Sonophotodynamic therapy with Protoporphyrin IX in single cells: a bubble as a new experimental model

Single cell experiments offer a powerful approach for characterizing therapeutic effects at the single-cell level, free from the confounding influences of cell density and intercellular signaling, which is especially interesting when combined techniques are used. In this work, we present a novel experimental model, the bubble model, consisting of a liquid latex membrane containing cells in suspension, enabling simultaneous exposure to light and ultrasound under controlled, interface-free conditions. Using B16-F10 murine melanoma cells and Protoporphyrin IX (PpIX) as a photosensitizer, we investigated the isolated and combined effects of Photodynamic Therapy (PDT) and Sonodynamic Therapy (SDT). Results demonstrate that PDT produces a dose-dependent reduction in cell viability in single cells, whereas SDT alone yields negligible cytotoxicity under these conditions, consistent with the volumetric nature of sonodynamic mechanisms, which require a critical cell density to manifest. Notably, the combined application of PDT and SDT (Sonophotodynamic Therapy, SPDT) produces a strongly amplified response exceeding the sum of individual effects. These findings provide mechanistic evidence supporting the use of SPDT and establish the bubble model as a reproducible platform for investigating fundamental aspects of light- and ultrasound-based therapies at the single-cell level.