A Remote Electro-Optical Technique for Monitoring Singlet Oxygen Generation During Photodynamic Therapy

Abstract

Photodynamic therapy (PDT) is a form of local cancer treatment in which cell death is caused by photochemical reactions involving an exogenous photosensitizer. The photosensitizer, which is preferentially retained in malignant tissues, is photoactivated and cell death results from the generation of reactive products -most likely excited molecular (singlet) oxygen. The development of in vivo PDT dosimetry would be greatly aided by the ability to directly measure the local concentration of this product by non-invasive means. In condensed media singlet oxygen will, with some small probability, undergo a radiative transition to the ground state with emission at 1270 nm. This infrared phosphorescence may provide a means for monitoring the production of singlet oxygen in vivo. Unfortunately the background infrared fluorescence observed from tissue may be many times the expected magnitude of the 1270 nm phosphorescence, even within the bandwidth encompassing the peak. The principal aim of this project was the design of a system optimized for the in vivo detection of the singlet oxygen emission. The system makes use of the most sensitive commercially available detector and uses phase sensitive detection to discriminate against infrared fluorescence. The system's performance matched theoretical expectations for the photosensitizer Photofrin II in aqueous and methanol solutions. However, a discrepancy in the observed and theoretical values was noted for aluminum chlorosulphonated phthalocyanine suggesting a deviation from simple first order kinetics. Singlet oxygen phosphorescence was not observed during PDT of cell suspensions or mouse tumours even though considerable cell death and tumour necrosis were observed. The most likely explanation of this failure is that, due to quenching by biomolecules, the lifetime of singlet oxygen in cells or tissue is much lower than in solution so that the probability of emission is reduced accordingly. Quantitative calibration of the system yielded a lower limit of approximately 0.1 us on the singlet oxygen lifetime in tissue. This suggests that singlet oxygen is generated in a protein environment.