Insights into Photodynamic Therapy Dosimetry: Simultaneous Singlet Oxygen Luminescence and Photosensitizer Photobleaching Measurements

Photodynamic therapy (PDT) is generally based on the generation of highly reactive singlet oxygen (O-1(2)) through interactions of photosensitizer, light, and oxygen (O-3(2)). These three components are highly interdependent and dynamic, resulting in variable temporal and spatial O-1(2) dose deposition. Robust dosimetry that accounts for this complexity could improve treatment outcomes. Although the 1270 nm luminescence emission from O-1(2) provides a direct and predictive PDT dose metric, it may not be clinically practical. We used O-1(2) luminescence (or singlet oxygen luminescence (SOL)) as a gold-standard metric to evaluate potentially more clinically feasible dosimetry based on photosensitizer bleaching. We performed in vitro dose-response studies with simultaneous SOL and photosensitizer fluorescence measurements under various conditions, including variable O-3(2), using the photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC). The results show that SOL was always predictive of cytotoxicity and immune to PDT's complex dynamics, whereas photobleaching-based dosimetry failed under hypoxic conditions. However, we identified a previously unreported 613 nm emission from mTHPC that indicates critically low O-3(2) levels and can be used to salvage photobleaching-based dosimetry. These studies improve our understanding of PDT processes, demonstrate that SOL is a valuable gold-standard dose metric, and show that when used judiciously, photobleaching can serve as a surrogate for O-1(2) dose.