Characterization of Photofrin photobleaching for singlet oxygen dose estimation during photodynamic therapy of MLL cells in vitro

A singlet oxygen dose model is developed for PDT with Photofrin. The model is based on photosensitizer photobleaching kinetics, and incorporates both singlet oxygen and non-singlet oxygen mediated bleaching mechanisms. To test our model, in vitro experiments were performed in which MatLyLu (MLL) cells were incubated in Photofrin and then irradiated with 532 nm light. Photofrin fluorescence was monitored during treatment and, at selected fluence levels, cell viability was determined using a colony formation assay. Cell survival correlated well to calculated singlet oxygen dose, independent of initial Photofrin concentration or oxygenation. About 2 x 10(8) molecules of singlet oxygen per cell were required to reduce the surviving fraction by 1/e. Analysis of the photobleaching kinetics suggests that the lifetime of singlet oxygen in cells is 0.048 +/- 0.005 micros. The generation of fluorescent photoproducts was not a result of singlet oxygen reactions exclusively, and therefore did not yield additional information to aid in quantifying singlet oxygen dose.