Simultaneous Application of Methylene Blue and Chlorin e6 Photosensitizers: Investigation on a Cell Culture

The application of photosensitizers for inhibition of oxidative phosphorylation in order to temporally decrease oxygen uptake by tumor cells in the course of photodynamic therapy (PDT) evokes growing interest. The aim of the study is to overcome tumor hypoxia for further photodynamic therapy with simultaneous use of type I photosensitizer methylene blue (MB) and type II photosensitizer chlorin e6.

A photodynamic activity of MB and its combined use with chlorin e6 has been studied on the HeLa cell culture, their effect on cell metabolism in their co-accumulation and subsequent irradiation has also been assessed.

MB generates reactive oxygen species in the cells in contrast to chlorin e6, which produces singlet oxygen. Besides, MB is converted to a colorless leucoform at low concentrations in the process of de-oxygenation. Incubation of cells with MB concurrently with chlorin e6 results in its greater fluorescence as compared to the incubation with MB only. MB concentration in the range of 1-10 mg/kg and the laser radiation dose of 60 J/cm² do not cause cell death, probably, due to the MB transition to the photodynamically inactive leucoform. Cell death is observed after PDT in all samples with chlorin e6 and with MB at the 0-20 mg/kg concentration ranges and at 60 J/cm² radiation dose. The phototoxicity of MB together with chlorin e6 is higher than that of chlorin e6 alone. The analysis of metabolic NADH cofactor lifetime after the incubation of the cells with MB and chlorin e6, and after PDT with them has revealed the presence of stress seen as an extension of NADH fluorescence cloud along the metabolic axis. After PDT with low concentrations of MB, the NADH fluorescent cloud on the phasor diagram shifts to the right towards short lifetimes (closer to anaerobic glycolysis along the NADH metabolic trajectory). The PDT with MB and chlorin e6 leads to the shift of the NADH fluorescence cloud on the phasor diagram to the left towards long lifetimes (closer to oxidative phosphorylation along the NADH metabolic trajectory). In this case, the cells die due to necrosis.

The co-accumulation of MB with chlorin e6 prevents MB reduction to a colorless leucoform, decreasing the oxygen uptake by the cells and making it possible to use simultaneously type I and II photodynamic reactions.