Study of methylene blue photodynamic activity on erythrocyte suspensions in vitro

In this paper we studied the photodynamic activity (the rate of molecular oxygen utilization during irradiation) of methylene blue (MB) in erythrocyte suspensions in vitro. Using spectroscopy and confocal microscopy with fluorescent sensors for singlet oxygen and other active oxygen species, it was shown that with an increase in the MB concentration (10–100 mg/kg), the molar photodynamic activity decreases. It was found that 5–10% of the MB added to erythrocytes tightly binds to the erythrocyte membranes, and the generation of singlet oxygen (¹O₂) is suppressed in favor of type I reactions (formation of H₂O₂, O₂•⁻, •OH). Another 40% of the MB added to erythrocytes is converted into a colorless leuco form, but is reoxidized back to MB under photodynamic exposure. The maximum relative quantum yield of ¹O₂ generation (φ Δ ) among those measured in erythrocyte suspensions was 0.014 for a 10 mg/kg MB concentration, which is an order of magnitude lower than the values for MB in organic solvents and for the aluminum sulfonated phthalocyanine comparison photosensitizer (PS) (φ Δ = 0.38). Interaction with erythrocytes (aggregation, reduction to the leuco form, competition for oxygen) explains the decrease in the MB efficiency under physiological conditions compared to organic solvents. The obtained results are important from the point of view of optimizing the systemic use of MB in photodynamic therapy.