A spectroscopic study of the photophysical properties of methylene blue (MB) in aqueous solutions was carried out. Absorption and fluorescence spectra as well as fluorescence lifetime were recorded. The concentration dependence of the intensity and shape of the spectra allowed establishing the ranges of MB concentrations for in vitro and in vivo studies at which aggregation is not observed (up to 0.01 mM, which corresponds to 3.2 mg/kg). Studies of photodegradation in biological media showed that photobleaching of more than 80% in plasma and culture media is observed already at a dose of 5 J/cm2 , while in water at this concentration and dose photobleaching is not yet observed, and at a dose of 50 J/cm2 photobleaching of MB is about 30%. It was found that in media containing proteins and having an alkaline pH, photobleaching occurs significantly faster than in neutral aqueous media. The ionic strength of the solution has no effect on the photobleaching rate. Such photobleaching is caused by the photodegradation of MB rather than the transition to the leucoform.The efficiency of singlet oxygen generation and photodynamic activity were evaluated in vitro. In the investigated range of MB concentrations, the efficiency of singlet oxygen generation is rather low, because positively charged MB binds to negatively charged cell membranes, which leads to a change in the type of photodynamic reaction. The emergence of other reactive oxygen species (ROS), different from singlet oxygen, in cells has been demonstrated. The generation of ROS and the low quantum yield of singlet oxygen generation indicate the tendency of MB to provide the type I photosensitization mechanism (electron transfer with the formation of semi-reduced and semi-oxidized MB+ radicals) rather than to the type II mechanism (energy transfer to oxygen with the formation of singlet oxygen) in biological media and in vivo.
