Comparative analysis of the photobleaching process and formation of fluorescent photoproducts of chlorin e6 and methylene blue in solutions and in HeLa cells

Chlorin e6 and methylene blue were shown to undergo significant photodegradation under photodynamic treatment. The photosensitizers exhibited substantially different photostability, while both were generating fluorescent photoproducts with spectral characteristics distinct from the initial compounds. Photobleaching of chlorin e6 resulted in 2-fold decrease of the mean weighted fluorescence lifetime, whereas methylene blue exhibited photodegradation-induced 50% increase in this parameter due to potential contribution of the fluorescent photoproducts. The photoproducts also altered the absorption and emission spectra of the solutions of both drugs. The nonexponential kinetics of fluorescence intensity decay observed for both substances can be attributed to a combination of photobleaching of the initial compound followed by subsequent photobleaching of generated fluorescent photoproducts. Similar patterns of changes in fluorescence parameters were also observed in HeLa cells during photodynamic treatment, with the exception of an initial increase in methylene blue fluorescence intensity. This difference was explained by oxidation of the reduced leuco-form present in the reducing cellular microenvironment. The observed pattern of the photobleaching process of these two photosensitizers accompanied with formation of photoproducts can be used for real-time monitoring in photodynamic treatment and adjustment of the irradiation dose.