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PDT with a genetically-encoded photosensitizer miniSOG by continuous wave or pulsed laser irradiation on tumor spheroid model

D.V. Yuzhakova1; M.V. Shirmanova 1; M.M. Lukina1; A.I. Gavrina 1; A.I. Izosimova 1, 2; V.A. Kamensky3

1 Privolzhsky Research Medical University, Nizhny Novgorod, Russia;
2 Lobachevsky State University of Nizhny Novgorod, 23 Gagarina Avenue, 603950, Nizhny Novgorod, Russia;
3 Institute of Applied Physics RAS, Nizhny Novgorod, Russia


Despite the significant progress in PDT of cancer, there is still a wild field for research, including the development of new photosensitizers and improvement of the treatment regimens. Fluorescent flavoprotein miniSOG (mini Singlet Oxygen Generator) from a novel class of genetically encoded photosensitizers generates singlet oxygen with a high yield and demonstrates the strong phototoxic properties in vitro in cancer cells. However, the effective approaches for PDT with miniSOG have not been developed so far. Therefore, future investigation of miniSOG requires the optimization of PDT regimen. The advantages of pulse light irradiation over classical continuous wave (CW) regimens in PDT have been demonstrated in the resent studies.
The purpose of this study was to investigate the phototoxicity of miniSOG in HeLa Kyoto 3D tumor spheroids upon CW and pulsed periodic laser irradiation and develop an effective regimen of PDT.
HeLa Kyoto spheroids stably expressing miniSOG were generated by cultivation of 5x105 cells per well in the 96-well ultra-low attachment round bottom plates during 5 days. Fluorescence microscopy of tumor spheroids was performed using Axio Zoom.V16 (Zeiss, Germany). A diode laser Epistar LED (Taiwan), 470 nm, 65, 35 or 120 mW/cm2, was used for continuous wave and pulsed periodic modes with a repetition rate of 50 Hz and pulse duration of 10, 5, 2.5, or 1.25 ms. Cell viability assay was performed 4 h after irradiation using Apoptosis/Necrosis detection kit (Abcam, UK). Tumor spheroid growth was estimated.
Pulse periodic mode with pulse duration 2.5 ms provide higher photobleaching of miniSOG (75% vs 60%) on tumor spheroids compared to CW mode. PDT in CW mode demonstrated a moderate expansion of central necrotic core of tumor spheroids, while pulse periodic mode provided an extreme increase in number of apoptotic cells, including late stages of apoptosis, in all zone of the spheroid. PDT with miniSOG in CW mode slightly inhibited spheroids growth, while pulse periodic mode led to complete suppression of spheroid growth.
In summary, we report for the first time on an effective regimen for PDT with miniSOG in a tumor spheroid model upon pulsed periodic laser irradiation.
This work was supported by the Russian Foundation for Basic Research (grant # 18-42-520027).

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Diana Yuzhakova
Privolzhsky Research Medical University


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