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5An innovative approach to phototherapy of model cancer in rats: skin optical clearing and combined PDT/PTT
E.A. Genina1,2*, V.D. Genin1'2, A.B. Bucharskaya3, N.A. Navolokin3, D.A. Mudrak3, G.N. Maslyakova3, B.N. Khlebtsov4, N.G. Khlebtsov4, V. Tuchin12 5
1-Institution of Physics, Saratov State University, 83, Astrakhanskaya St., Saratov, 410012 Russia 2- Laboratory of Laser Molecular Imaging and Machine Learning, National Research Tomsk State University,
36, Lenin Ave., Tomsk, 634050 Russia
3- Department of Faculty Surgery and Oncology, Saratov State Medical University, 112, Bolshaya Kazachya St.,
Saratov, 410012 Russia
4- Institute of Biochemistry and Physiology of Plants and Microorganisms of the Russian Academy of Sciences,
FRC "Saratov Science Center of the RAS", 13, Entuziastov Ave., Saratov, 410049 Russia
5- Institute of Precision Mechanics and Control, FRC "Saratov Scientific Centre of the RAS", 24, Rabochaya St.,
Saratov, 410028 Russia
In this study Wistar rats with transplanted cholangiocarcinoma PC-1 were used. For photodynamic therapy (PDT), photosensityzers indocyanine green (ICG-PDT) or Photosence® (PS-PDT) were chosen. A solution of ICG in polyethylene glycol (MW=300 Da) had a maximum absorption wavelength of about 790 nm; and PS dissolved in saline had a maximum absorption wavelength of about 670 nm. Gold nanorods (GNRs) with an aspect ratio of 4:1 were used for photothermal therapy (PTT). A plasmon resonance of GNRs was observed in the spectral range of 800-820 nm. All components were injected intratumorally. For ICG-PDT/PTT and PS-PDT, laser irradiation was used for 15 min with a wavelength of 808 nm at a power density of 2.3 W/cm2 or with a wavelength of 660 nm at a power density of ~0.5 W/cm2, respectively. Optical clearing agent (aqueous solution of 70% glycerol and 5% DMSO) was applied topically to the skin above the tumor before the therapy. Skin heating was monitored using an IR visualizer. Diffuse reflectance spectra were registered before and after the procedures in the spectral range of 400-900 nm.
The withdrawal of animals and sampling of tumor tissues for histological examination was performed 72 hours after combination therapy. Morphological studies of tumor tissue were performed on tumor sections stained by standard methods and with immunohistochemical staining for the proliferation marker Ki-67 and the apoptosis marker Bax.
a b c d
Fig. 1. Diffuse reflectance spectra of the surface of rat skin above the tumor before and after laser exposure and the morphological picture of cholangiocarcinoma 72 hours after combined exposure to PS-PDT/PTT a), b) and ICG-PDT/PTT c), d), respectively. Magnification of
histological images is 246.4.
A significant rise in tumor heating temperature, up to 60±4.10 C, was observed during combined PDT/PTT. Heating caused significant changes in the spectral range of hemoglobin absorption (Fig. 1). After 72 hours, pronounced damage to tumor tissue was noted in rats. In a morphological study after PDT therapy, necrosis fields occupied from 30 to 50% of the section area; with single PTT and combined PDT/PTT, necrosis occupied up to 80% of the tumor section area. Intact tumor cells were observed only at the periphery of the tumor. An immunohistochemical study revealed a significant decrease in the expression of the proliferation marker Ki-67 and an increase in the expression of the apoptosis marker Bax in intact tumor cells.
The work was supported by the RSF grant no. 23-14-00287.
