CC BY
0Spectroscopic study of methylene blue to the leucomethylene blue
transition in vitro and in vivo
D.V. Pominova1'2*, A.V. Ryabova1'2, I.V. Markova2, A.S. Skobeltsin1'2, I.D. Romanishkin1
1-Prokhorov General Physics Institute of Russian Academy of Sciences, Moscow, Russia 2- National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Moscow, Russia
Methylene blue (MB) is second-generation FDA-approved photosensitizer [1]. Photodynamic therapy with MB is reported to be effective [2], but the results are promising not for all tumor types. This may be related to the different bioavailability of MB in target tissues due to the transition of MB to the colorless leucoform, which reduces its photodynamic activity. In addition to this, the reduction of MB into the leucomethylene blue (LMB) can play a role in various redox processes occurring in the body and affect metabolism. For example, MB can interact with the mitochondrial electronic circuit, donating electrons to complexes I and III and/or providing partial restoration of the Krebs cycle [3], which not only determines its neuroprotective properties [4], but can also be used to increase the effectiveness of antitumor therapy. The interaction of MB with coenzymes, such as NADH is important in this aspect. A high ratio of NADH/NAD+ has been reported to be a key feature of malignant cells [5]. When interacting with NADH, MB is reduced to the leucoform, and NADH is oxidized to NAD+, providing also metabolic changes: increase of pyruvate:lactate ratio and reactivation of electron transport chain [3]. Under the influence of molecular oxygen and reactive oxygen species, LMB can be oxidized back to MB.
The purpose of this work was to study the MB to LMB transition under the influence of various factors, such as coenzymes, oxygen and reactive oxygen species, as well as under the influence of laser radiation using spectroscopic methods.
Absorption and fluorescence spectra, as well as fluorescence lifetimes of MB in solutions during interaction with coenzymes and products of biochemical reactions (NADH, FADH, lactate, pyruvate, glutathione) under normal oxygen conditions and hypoxia were studied. Then studies were carried out on cell cultures with different metabolism (glycolysis and oxidative phosphorylation) and finally verified in vivo on small laboratory animals. A model was proposed that describes the transition of MB-LMB in various tissues depending on their metabolism, redox state, of the microenvironment, oxygen supply and MB concentration. The proposed model will allow optimizing MB concentrations for effective photodynamic therapy or for its use as an antioxidant and neuroprotector.
The study was funded by a grant from the Russian Science Foundation (project N 22-72-10117).
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[4] E. Poteet, et al, Neuroprotective Actions of Methylene Blue and Its Derivatives, PLoS ONE, 7, e48279 (2012).
[5] A. Chiarugi, C. Dolle, R. Felici, M. Ziegler, The NAD metabolome — a key determinant of cancer cell biology, Nat Rev Cancer, 12, 741752 (2012).
