Photobleaching kinetics of Photofrin in vivo and in multicell tumour spheroids indicate two simultaneous bleaching mechanisms

We present a detailed investigation of Photofrin photobleaching and photoproduct accumulation. Fisher rats were sensitized with 10 mg kg(-1) Photofrin and irradiated 24 h later with 514 nm light at 5 or 100 mW cm(-2). Fluorescence spectra were collected from the skin throughout treatment, and sensitizer bleaching and fluorescent photoproduct formation were quantified using spectral analysis. Photofrin bleaching was slightly more rapid at the higher irradiance under these conditions. However, accumulation of photoproduct was significantly enhanced at lower irradiance. To interpret these unexpected findings, we developed a new mathematical model in which reactions between singlet oxygen (1O2) and the photosensitizer and reactions between the sensitizer triplet and biological targets are both allowed to contribute to bleaching. Predictions of this model were tested in experiments performed on EMT6 spheroids sensitized with concentrations of 2.5, 10 and 30 microg mL(-1) Photofrin and subjected to PDT. Photofrin bleaching and photoproduct formation in these spheroids were measured using confocal fluorescence spectroscopy. In qualitative agreement with the mixed-mechanism model predictions, at the highest drug concentration Photofrin bleaching was more efficient via 1O2 reactions, while at the lowest concentration triplet reactions were more efficient. At all concentrations, photoproduct accumulation was greater under conditions of abundant oxygen.