Assessing photodynamic therapy under in vitro hypoxia: photochemical pathways and methodological challenges

Abstract

Photodynamic therapy (PDT) is a light-activated modality that kills cells through photosensitizer-triggered reactive oxygen species, but its efficacy is strongly shaped by oxygen availability and by whether photochemistry proceeds through Type I or Type II pathways. In oxygen-rich settings, Type II PDT mainly generates singlet oxygen, whereas hypoxia suppresses this route and shifts interest toward Type I mechanisms that produce radical species such as superoxide, hydroxyl radicals, and hydrogen peroxide. This review argues that hypoxia-driven resistance in vitro is not explained by oxygen depletion alone, but by interactions among photochemical pathway choice, cellular adaptation, photosensitizer uptake and localization, and assay limitations. It highlights that hypoxia can reshape membrane composition, vesicular trafficking, and organelle targeting, which in turn alters reactive oxygen species patterns and photodamage outcomes. Methodologically, the work recommends Controlled hypoxia models, including 2D low oxygen cultures and 3D spheroids, together with orthogonal readouts of hypoxia and oxidative damage. For ROS profiling, it supports comparing normoxic and hypoxic conditions using probes for total ROS, superoxide, hydroxyl radicals, and singlet oxygen, because no single assay captures the full mechanistic picture. Finally, it emphasizes that lipid and protein oxidation should be interpreted with caution in hypoxic PDT, since FOX, TBARS, iodometric methods, and BODIPY-C11 mainly report secondary or localized products and may miss Type I driven damage unless combined with imaging, membrane integrity assays, and time-resolved measurements.