Monitoring of hydrogen peroxide production under photodynamic treatment using protein sensor HyPer

An interest to H₂O₂ accumulation under photodynamic treatment can be explained by its participation in intracellular signal cascades. It is important not only to detect H₂O₂ generation, but also to trace the dynamics of its intracellular content. In the present study the dynamics of cellular H₂O₂ content under photodynamic treatment was analyzed using genetically encoded reversible H₂O₂-sensitive sensor HyPer. Real-time detecting of H₂O₂ production after photodynamic treatment was performed using the protein sensor and individual features of action of different photosensitizers were revealed. Photodynamic treatment with a number of chlorin and phthalocyanine photosensitizers was found to induce secondary production of H₂O₂ in the cells. Three types of dynamic responses were registered: monotonous increase of H₂O₂ level during the entire observation time in the presence of Fotoditazin and Holosens; transient short-term accumulation in the presence of Radachlorin and Phthalosens; and relatively low-level stable increase in the presence of Photosens. The listed photosensitizers differ significantly in intracellular localization and physicochemical properties, which can determine the differences in the response of H₂O₂ after the photodynamic treatment. In general, it has been shown that the rapid transient H₂O₂ response is typical for hydrophobic compounds localized in membrane cell structures, whereas in the presence of more hydrophilic dyes a prolonged monotonous H₂O₂ accumulation occurs.