Ryabova A V, Romanishkin I D, Markova I V, Pominova D V
Senior Researcher, Laser Biospectroscopy Laboratory, Light-Induced Surface Phenomena Department, Natural Sciences Center; Prokhorov General Physics Institute of the Russian Academy of Sciences, 38 Vavilov St., Moscow, 119991, Russia; Associate Professor, Department 87 "Laser Micro-, Nano-, and Biotechnologies, Engineering Physics Institute for Biomedicine"; National Research Nuclear University MEPhI, 31 Kashirskoye Highway, Moscow, 115409, Russia.
Junior Researcher, Laser Biospectroscopy Laboratory, Light-Induced Surface Phenomena Department, Natural Sciences Center; Prokhorov General Physics Institute of the Russian Academy of Sciences, 38 Vavilov St., Moscow, 119991, Russia.
Sovrem Tekhnologii Med. 2025;17(1):58-68. doi: 10.17691/stm2025.17.1.06. Epub 2025 Feb 28.
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. 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/cm 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/cm 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.
为了在光动力疗法（PDT）过程中暂时减少肿瘤细胞的氧摄取，应用光敏剂抑制氧化磷酸化引起了越来越多的关注。目的是通过同时使用I型光敏剂亚甲蓝（MB）和II型光敏剂氯e6来克服肿瘤缺氧，以进行进一步的光动力治疗。
研究了MB的光动力活性及其与氯e6的联合使用对HeLa细胞培养物的影响，还评估了它们在共积累和随后照射时对细胞代谢的影响。
与产生单线态氧的氯e6不同，MB在细胞中产生活性氧。此外，在脱氧过程中，低浓度的MB会转化为无色的隐色体形式。与仅用MB孵育相比，将细胞与氯e6同时与MB孵育会导致其荧光更强。1-10mg/kg范围内的MB浓度和60J/cm的激光辐射剂量不会导致细胞死亡，可能是由于MB转变为光动力学无活性的隐色体形式。在所有使用氯e6以及浓度在0-20mg/kg范围内的MB且辐射剂量为60J/cm的样品中，光动力疗法后均观察到细胞死亡。MB与氯e6共同的光毒性高于单独的氯e6。在用MB和氯e6孵育细胞后以及用它们进行光动力疗法后，对代谢NADH辅因子寿命的分析揭示了存在应激，表现为NADH荧光云沿代谢轴的延长。在用低浓度MB进行光动力疗法后，相量图上的NADH荧光云向右移动至短寿命（沿NADH代谢轨迹更接近无氧糖酵解）。用MB和氯e6进行光动力疗法会导致相量图上的NADH荧光云向左移动至长寿命（沿NADH代谢轨迹更接近氧化磷酸化）。在这种情况下，细胞因坏死而死亡。
MB与氯e6的共积累可防止MB还原为无色的隐色体形式，减少细胞的氧摄取，并使得同时使用I型和II型光动力反应成为可能。
