CC BY
6DOI 10.24412/cl-37136-2023-1-189-192
DEVELOPMENT OF COMBINED PHOTOTHERMAL/PHOTODYNAMIC THERAPY
USING A MODEL TUMOR: PILOT STUDY
VADIM D. GENIN1,2*, ALLA B. BUCHARSKAYA1,2,3, NIKITA A. NAVOLOKIN1,3, GEORGY S. TERENTYUK3, BORIS N. KHLEBTSOV4, NIKOLAI G. KHLEBTSOV1,4, VALERY V. TUCHIN1,2,5, ELINA A. GENINA1,2
1 - Saratov State University, st. Astrakhanskaya, 83, 410012, Saratov, Russia 2 - Tomsk State University, 36 Lenin Ave., 634050, Tomsk, Russia 3 - Saratov State Medical University, st. Bolshaya Kazachya, 112, 410012, Saratov, Russia 4 - Institute of Biochemistry and Physiology of Plants and Microorganisms, Federal Research Center "Saratov Scientific Center of the RAS", Entuziastov Ave., 13, Saratov, 410049 Russia
5 - Institute ofPrecision Mechanics and Control Problems of the Russian Academy of Sciences, Federal Research Center "Saratov Scientific Center of the RAS", st. Rabochaya, 24, Saratov, 410028 Russia
*e-mail: [email protected]
The growth of a number of oncological diseases stimulates extensive development of both early tumor diagnostics and therapy methods. A promising area of research is using a combination of several methods, for example, photodynamic therapy (PDT) and plasmonic photothermal therapy (PPT) [1, 2]. Many research groups have proposed new treatment models that are based on the combination of PDT and PPT, using various types of nanoparticles and photosensitizers [3-6].
The purpose of the study was to develop a combined technology of PPT and PDT in rats with model tumors.
As a material of the study 4 outbreed albino male rats with a subcutaneously grafted experimental model tumors of liver bile ducts cancer (cholangiocarcinoma of the PC-1 line) were used. All rats were injected with a photosensitizer solution and a suspension of gold nanorods (GNRs) intratumorally at three points each. The points were equidistant from the center of the tumor to ensure a more uniform distribution of both nanoparticles and dyes inside the tumor. GNRs were functionalized with PEG-300. For PDT two rats were injected by Photosens (Niopik, Russia) solution (PS) and two ones were injected by indocyanine green (Sigma-Aldrich Cheme Gmbh, Germany) solution (ICG). The maximum absorption of the PS solution was at a wavelength of 680 nm, and the maximum absorption of the ICG solution was at a wavelength of 795 nm. The details of intratumoral injections design are presented in Table 1.
An hour after the injections, the tumors of rats 1 and 2 were irradiated percutaneously with a 660 nm laser Latus-T (Atkus, Ltd, Russia) at a power density of 0.5 W/cm2 for 15 min (for PDT) and then with an 808 nm infrared laser (LS-2-N-808-10000, Russia) at a power density of 2.3 W/cm2 for 15 min (for PPT). The tumors of rats 3 and 4 were irradiated percutaneously only with an 808 nm infrared laser at a power density of 2.3 W/cm2 for 15 min (for PDT and PPT simultaneously). Figure 1 is shows the design of the experiment. The diffuse reflectance spectra before laser irradiation, after PDT and after PPT in range 450-2100 nm were taken from all tumors using commercial available spectrometers USB4000-Vis-NIR (Ocean Optics, USA) and NIRQuest (Ocean Optics, USA).
The withdrawal of animals and sampling of tumor tissues for histological examination was performed 72 hours and 21 days after the 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.
Table 1. Design of intratumoral injections of photosensitizer solutions and GNRs suspension.
Rat io. Tumor me, cm3 Photosensitizer Solvent Volume of photosensitizer solution Photosensitizer centration in the solution Volume of R suspension GNR :ntration in the uspension
Rat 1 ~3.8 Photosens Saline solution 0.30 ml 2 mg/ml 1.25 ml 400 mg/ml
Rat 2 ~8.2 0.45 ml 2 mg/ml 2 ml 400 mg/ml
Rat 3 ~2.5 Indocyanine green PEG-300 0.18 ml 10 mg/ml 0.8 ml 400 mg/ml
Rat 4 ~4.7 0.20 ml 10 mg/ml 1.57 ml 400 mg/ml
Figure 1. The design of the experiment
The local heating temperature of the skin above the tumor was measured with an IRI4010 thermal imager (IRYSYS, UK). The kinetics of skin surface heating over tumors is presented in Figure 2.
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Figure 2. Kinetics of skin surface over the tumor heating averaged by groups of rats
During the combined therapy in PS and ICG groups, there was a pronounced rise in the temperature of local heating of the tumor up to 55.2±2.8 and 62.4±5.6 °C, respectively. Necrosis of up to 95% of the tumor was observed in the PS group after 72 hours. In rat 4 (ICG group) after 72 hours, pronounced necrotic changes were observed in the tumor tissue, necrosis fields occupied up to 80% of the area. Preserved tumor cells were found only on the periphery of the tumor; they showed a decrease in the expression of the proliferation marker and an increase in the expression of the apoptosis marker. In rat 3 (ICG group) after 21 days, there was a complete destruction of the tumor.
Diffuse reflectance spectra of rat tumors at different stages of the experiment are presented in the Figure
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Figure 3. Diffuse reflectance spectra of rat skin surface over the tumor at different stages of the experiment
averaged by groups
The diffuse reflectance spectra of all tumors demonstrate the presence of hemoglobin in the oxygenated form in the tumor (Q-bands at 546-548 and 576 nm). It can be seen that in all rats after the PPT the bands corresponded to the hemoglobin absorption peaks is smoothed out, which may be due to the transition of hemoglobin to a deoxygenated form. Also visible are dips due to the presence of PS (680 nm in rats 1 and 2) or ICG (800 nm in rats 3 and 4) in the tumors. In rats 3 and 4, the absorption band at 800 nm disappears after laser irradiation, which may be due to the photobleaching effect. Absorption bands of water are observed at 14321434 and 1941-1958 nm. The change in the NIR absorption bands of water is associated with dehydration due to high temperature during PPT.
The proposed combination PPT/PDT therapy technology caused pronounced damage to tumor tissue in rats with transplanted cholangiocarcinoma. Our results are in a good agreement with the result of other authors [1-6]. For example, Yan et al, which used ICG liposomes for PTT/PDT treatment of mice breast tumors [3]. The tumors were irradiated by the 808 nm laser at a power density of 1.0 W/cm2 for 10 min. Almost complete regression of the tumor was achieved.
The work was supported by the RSF grant no. 23-14-00287. REFERENCES
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