Low-concentration photodynamic therapy induces oxidative stress-driven cytoskeletal remodeling and formulation-dependent cell responses

Photodynamic therapy (PDT) induces localized oxidative stress through the light-activated generation of reactive oxygen species (ROS), leading to targeted cell death. While conventional PDT protocols inducing marked photocytotoxicity have been extensively characterized, the cellular responses to low-concentration PDT, here defined as a reduced photosensitizer concentration under standard light fluence and irradiance, remain poorly understood. In this study, the effects of low-concentration PDT (0.33 µM) using pheophorbide a (Pheo), either in free form or encapsulated within poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-PCL) micelles, were examined in three cell models: primary human dermal fibroblasts, human HCT-116 carcinoma cells, and canine MDCK-1 epithelial cells. At the reduced Pheo concentration, PDT induced consistent morphological alterations across all models, including cytoskeletal remodeling, cell rounding, and early apoptotic features. These effects appeared earlier and were more pronounced with encapsulated Pheo, underscoring the influence of formulation on PDT efficiency. Antioxidant treatments (Sodium-L-ascorbate or N-Acetyl-L-cysteine) mitigated the morphological changes and cell death, supporting the involvement of oxidative stress in these responses. Compared with conventionally cytotoxic PDT, the low-concentration approach triggered slower cell-death dynamics, with caspase activation preceding plasma membrane permeabilization. Together, these findings show that low-concentration PDT induces oxidative stress responses across diverse cell types, while their magnitude and kinetics remain formulation-dependent. These findings provide a framework for further investigating the temporal and mechanistic dimensions of PDT-induced oxidative stress and their potential relevance to photodynamic priming, immune modulation, and combination therapies.