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The evaluation of novel photosensitizers (PSs) for photodynamic therapy (PDT) is difficult due to the limitations of two-dimensional cell culture and multiple parameters (dose, light intensity, uptake time), which complicate progression to in vivo experiments and clinical translation. Three-dimensional (3D) cell culture models like multicellular cancer tumor spheroids (MCTS) show great similarities to in vivo avascular tumor conditions, improving the speed and accuracy of screening novel compounds with various treatment combinations. In this study, we utilize C8161 human melanoma spheroids to screen PDT treatment combinations using protoporphyrin IX (PpIX) and drug-loaded carbon dot (CD) conjugates PpIX-CD and PpIX@CD at ultralow fluence values (&lt;10 J/cm 2 ). Conjugates show equivalent light-induced damage to PpIX from 1 μg/mL with significantly less dark cytotoxicity up to 72 h after exposure, shown by LDH release and dsDNA content. Fractionated treatments, carried out by dividing light exposure with 24 h intervals, demonstrate an enhanced PDT effect compared to single exposure at equal concentrations. Light sheet fluorescence microscopy combined with live/dead staining demonstrates that spheroids sustain extensive damage after PDT, with PpIX and PpIX-CD showing improved uptake compared to PpIX@CD. We show that PDT parameter screening can be carried out using a low-cost and convenient combination of assays to improve the efficiency of evaluating novel compounds.

acs biomaterials science and engineering

<i>In Vitro</i> Low-Fluence Photodynamic Therapy Parameter Screening Using 3D Tumor Spheroids Shows that Fractionated Light Treatments Enhance Phototoxicity

In Vitro Low-Fluence Photodynamic Therapy Parameter Screening Using 3D Tumor Spheroids Shows that Fractionated Light Treatments Enhance Phototoxicity