Study of methylene blue photodynamic activity on erythrocyte suspensions in vitro
https://doi.org/10.24931/2413-9432-2025-14-3-4-13
Abstract
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 (φΔ ) 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) (φΔ = 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.
Keywords
Supporting agencies: The study was funded by a grant from the Russian Science Foundation (project N 22-72-10117).
About the Authors
I. V. Markova
Prokhorov General Physics Institute of Russian Academy of Sciences; National Research Nuclear University MEPhI (Moscow Engineering Physics Institute)
Russian Federation
Russian Federation
Moscow
A. V. Ryabova
Prokhorov General Physics Institute of Russian Academy of Sciences; National Research Nuclear University MEPhI (Moscow Engineering Physics Institute)
Russian Federation
Russian Federation
Moscow
I. D. Romanishkin
Prokhorov General Physics Institute of Russian Academy of Sciences
Russian Federation
Russian Federation
Moscow
D. V. Pominova
Prokhorov General Physics Institute of Russian Academy of Sciences; National Research Nuclear University MEPhI (Moscow Engineering Physics Institute)
Russian Federation
Russian Federation
Moscow
References
1. Foote C. S. Definition of type I and type II photosensitized oxidation, Photochemistry and Photobiology, 1991, vol. 54(5), pp. 659–659. doi: 10.1111/j.1751-1097.1991.tb02071.x.
2. Baptista M. S., Cadet J., Di Mascio P. et al. Type I and Type II Photosensitized Oxidation Reactions: Guidelines and Mechanistic Pathways, Photochemistry and Photobiology, 2017, vol. 93(4), pp. 912–919. doi: 10.1111/php.12716.
3. Li X., Kwon N., Guo T. et al. Innovative Strategies for Hypoxic Tumor Photodynamic Therapy, Angewandte Chemie International Edition, 2018, vol. 57(36), pp. 11522–11531. doi: 10.1002/anie.201805138.
4. Zhao X., Liu J., Fan J. et al. Recent progress in photosensitizers for overcoming the challenges of photodynamic therapy: from molecular design to application, Chemical Society Reviews, 2021, vol. 50(6), pp. 4185–4219. doi: 10.1039/D0CS00173B.
5. Xu J., Bonneviot L., Guari Y. et al. Matrix Effect on Singlet Oxygen Generation Using Methylene Blue as Photosensitizer, Inorganics, 2024, vol. 12(6), pp. 155. doi: 10.3390/inorganics12060155.
6. Lucky S. S., Soo K. C., Zhang Y. Nanoparticles in Photodynamic Therapy, Chemical Reviews, 2015, vol. 115(4), pp. 1990–2042. doi: 10.1021/cr5004198.
7. Filonenko E. V. Clinical implementation and scientific development of photodynamic therapy in Russia in 2010-2020, Biomedical Photonics, 2022, vol. 10(4), pp. 4–22. doi: 10.24931/2413-9432-2021-9-4-4-22.
8. Semyonov D. Yu., Vasil’ev Yu. L., Dydykin S. S. et al. Antimicrobial and antimycotic photodynamic therapy (review of literature), Biomedical Photonics, 2021, vol. 10(1), pp. 25–31. doi: 10.24931/2413-9432-2021-10-1-25-31.
9. Trushina O. I., Filonenko E. V., Novikova E. G. et al. Photodynamic therapy in the prevention of HPV-induced recurrences of precancer and initial cancer of the cervix, Biomedical Photonics, 2024, vol. 13(3), pp. 42–46. doi: 10.24931/241-9432-2024-13-3-42-46.
10. Tseimakh A. E., Mitshenko A. N., Kurtukov V. A. et al. Effectiveness of palliative photodynamic therapy for unresectable biliary cancer. Systematic review and meta-analysis, Biomedical Photonics, 2024, vol. 13(2), pp. 34–42. doi: 10.24931/2413-9432-2024-13-2-34-42.
11. Shanazarov N. А., Zinchenko S. V., Kisikova S. D. et al. Photodynamic therapy in the treatment of HPV-associated cervical cancer: mechanisms, challenges and future prospects, Biomedical Photonics, 2024, vol. 13(1), pp. 47–55. doi: 10.24931/2413-9432-2023-13-1-47-55.
12. Panaseykin Y. A., Kapinus V. N., Filonenko E. V. et al. Photodynamic therapy in treatment of squamous cell carcinoma of oral cavity with chlorine e6 photosensitizer with long-term follow up, Biomedical Photonics, 2024, vol. 13(1), pp. 28–38. doi: 10.24931/2413-9432-2023-13-1-28-38.
13. Taldaev A., Terekhov R., Nikitin I. et al. Methylene blue in anticancer photodynamic therapy: systematic review of preclinical studies, Frontiers in Pharmacology, 2023, vol. 14, pp. 1264961. doi: 10.3389/fphar.2023.1264961.
14. DeRosa M. Photosensitized singlet oxygen and its applications, Coordination Chemistry Reviews, 2002, vol. 233–234, pp. 351–371. doi: 10.1016/S0010-8545(02)00034-6.
15. Redmond R. W., Gamlin J. N. A compilation of singlet oxygen yields from biologically relevant molecules, Photochemistry and Photobiology, 1999, vol. 70(4), pp. 391–475. doi: 10.1111/j.1751-1097.1999.tb08240.x.
16. Tardivo J. P., Del Giglio A., De Oliveira C. S. et al. Methylene blue in photodynamic therapy: From basic mechanisms to clinical applications, Photodiagnosis and Photodynamic Therapy, 2005, vol. 2(3), pp. 175–191. doi: 10.1016/S1572-1000(05)00097-9.
17. Medhi D., Hazarika S. Formation of dimer and higher aggregates of methylene blue in alcohol, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2025, vol. 329, pp. 125490. doi: 10.1016/j.saa.2024.125490.
18. Junqueira H. C., Severino D., Dias L. G. et al. Modulation of methylene blue photochemical properties based on adsorption at aqueous micelle interfaces, Physical Chemistry Chemical Physics, 2002, vol. 4(11), pp. 2320–2328. doi: 10.1039/b109753a.
19. Severino D., Junqueira H. C., Gugliotti M. et al. Influence of negatively charged interfaces on the ground and excited state properties of methylene blue, Photochemistry and Photobiology, 2003, vol. 77(5), pp. 459–468. doi: 10.1562/0031-8655(2003)0772.0.co;2.
20. Ball D. J., Luo Y., Kessel D. et al. The induction of apoptosis by a positively charged methylene blue derivative, Journal of Photochemistry and Photobiology B: Biology, 1998, vol. 42(2), pp. 159–163. doi: 10.1016/S1011-1344(98)00061-X.
21. Curry S. Methemoglobinemia, Annals of Emergency Medicine, 1982, vol. 11(4), pp. 214–221. doi: 10.1016/S0196-0644(82)80502-7.
22. Sass M. D., Caruso C. J., Axelrod D. R. Accumulation of methylene blue by metabolizing erythrocytes, The Journal of Laboratory and Clinical Medicine, 1967, vol. 69(3), pp. 447–455.
23. Sakai H., Leong C. Prolonged functional life span of artificial red cells in blood circulation by repeated methylene blue injections, Artificial Cells, Nanomedicine, and Biotechnology, 2019, vol. 47(1), pp. 3123-3128. doi: 10.1080/21691401.2019.1645157.
24. Sakai H., Li B., Lim W. L. et al. Red Blood Cells Donate Electrons to Methylene Blue Mediated Chemical Reduction of Methemoglobin Compartmentalized in Liposomes in Blood, Bioconjugate Chemistry, 2014, vol. 25(7), pp. 1301–1310. doi: 10.1021/bc500153x.
25. May J. M., Qu Z., Cobb C. E. Reduction and uptake of methylene blue by human erythrocytes, American Journal of Physiology-Cell Physiology, 2004, vol. 286(6), pp. C1390–C1398. doi: 10.1152/ajpcell.00512.2003.
26. McDonagh E. M., Bautista J. M., Youngster I. et al. PharmGKB summary: methylene blue pathway, Pharmacogenetics and Genomics, 2013, vol. 23(9), pp. 498–508. doi: 10.1097/FPC.0b013e32836498f4.
27. Harrop Geo. A., Barron E. S. G. STUDIES ON BLOOD CELL METABOLISM, Journal of Experimental Medicine, 1928, vol. 48(2), pp. 207–223. doi: 10.1084/jem.48.2.207.
28. Pominova D., Ryabova A., Skobeltsin A. et al. The use of methylene blue to control the tumor oxygenation level, Photodiagnosis and Photodynamic Therapy, 2024, vol. 46, pp. 104047. doi: 10.1016/j.pd-pdt.2024.104047.
29. Pominova D. V., Ryabova A. V., Skobeltsin A. S. et al. Spectroscopic Study of Methylene Blue Interaction with Coenzymes and Its Effect on Tumor Metabolism, Sovremennye tehnologii v medicine, 2025, vol. 17(1), pp. 18. doi: 10.17691/stm2025.17.1.02.
30. Ryabova A. V., Stratonnikov A. A., Loshchenov V. B. Laser spectroscopy technique for estimating the efficiency of photosensitisers in biological media, Quantum Electronics, 2006, vol. 36(6), pp. 562–568. doi: 10.1070/QE2006v036n06ABEH013291.
31. Stratonnikov A. A., Loschenov V. B. Evaluation of blood oxygen saturation in vivo from diffuse reflectance spectra, Journal of Biomedical Optics, 2001, vol. 6(4), pp. 457. doi: 10.1117/1.1411979.
32. Pominova D. V., Ryabova A. V., Romanishkin I. D. et al. Spectroscopic study of methylene blue photophysical properties in biological media, Biomedical Photonics, 2023, vol. 12(2), pp. 34–47. doi: 10.24931/2413-9432-2023-12-2-34-47.
33. Kalyanaraman B., Darley-Usmar V., Davies K. J. A. et al. Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations, Free Radical Biology and Medicine, 2012, vol. 52(1), pp. 1–6. doi: 10.1016/j.freeradbiomed.2011.09.030.
34. Marques S., Magalhães L., Tóth I. et al. Insights on Antioxidant Assays for Biological Samples Based on the Reduction of Copper Complexes—The Importance of Analytical Conditions, International Journal of Molecular Sciences, 2014, vol. 15(7), pp. 11387–11402. doi: 10.3390/ijms150711387.
35. George A., Pushkaran S., Li L. et al. Altered phosphorylation of cytoskeleton proteins in sickle red blood cells: The role of protein kinase C, Rac GTPases, and reactive oxygen species, Blood Cells, Molecules, and Diseases, 2010, vol. 45(1), pp. 41–45. doi: 10.1016/j.bcmd.2010.02.006.
36. Gomes A., Fernandes E., Lima J. L. F. C. Fluorescence probes used for detection of reactive oxygen species, Journal of Biochemical and Biophysical Methods, 2005, vol. 65(2–3), pp. 45–80. doi: 10.1016/j.jbbm.2005.10.003.
Review
For citations:
Markova I.V., Ryabova A.V., Romanishkin I.D., Pominova D.V. Study of methylene blue photodynamic activity on erythrocyte suspensions in vitro. Biomedical Photonics. 2025;14(3):4-13. https://doi.org/10.24931/2413-9432-2025-14-3-4-13
Views:
22
Email this article 