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Methylene Blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for the topical treatment in photodynamic therapy (PDT). MB+ can potentially be used as a redox indicator to detect the important redox reactions that are induced during PDT. MB+ induced PDT was successful in the intraluminal treatment of inoperable esophageal tumors and in the topical treatment of psoriasis. In order to improve the therapy, the reaction mechanism of MB+ was investigated in vivo by local injection of MB+ in a xenotransplanted subcutaneous tumor (adeno-carcinoma, G-3) in female nude mice. The MB+ preparation 'MB+1%' was applied both undiluted and diluted to 0.1% and 0.01% with isotonic sodium chloride. After an incubation period of 1 h, the tumors were irradiated at 662 nm. Treatment with 1% MB+ and subsequent irradiation with 100 J/cm2 led to complete tumor destruction in 79% of the treated animals. A decrease of the fluence rate from 100 mW/cm2 to 50 mW/cm2 significantly increased the phototoxic response, which was attributed to oxygen depletion but also to nonlinear redox reactions. In addition, fractionated light application with 15 s interruption intervals enhanced the effect. When 0.1% MB+ was used, complete tumor destruction was observed only in 10% of the treated animals. Below a relatively high threshold dose the therapeutic response was not significant. The efficiency of the therapy was correlated with nonlinear dynamics of MB+ on a subcellular level, using laser scanning microscopy. During MB+-PDT nonlinear redox- reactions were induced. This could be deduced from local fast changes of the MB+-fluorescence as well as the pH-value during irradiation of single cells. The light induced reaction of MB+ seems to be correlated with the nonlinear production of reactive oxygen species (ROS). As a consequence below a threshold dose the reducing ability of MB+ prevents tissue from oxidative damage. However, above this dose, as a point of no return, MB+ acts as an extremely potent oxidant.
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