Plasmonic photothermal photodynamic therapy in mice with colorectal cancer

• ¹SSMU named after. V.I. Razumovsky Ministry of Health of Russia, Saratov, Russia;

• ²SSU named after. N.G. Chernyshevsky, Saratov, Russia;

• ³FRC “Saratov Scientific Center of the Russian Academy of Sciences”, Saratov, Russia

The need for searching for new methods of antitumor therapy is due to the sharp increase in morbidity and mortality from cancer [1], as well as because of the fact that conventional chemo- and radiotherapy are often insufficiently effective owing to the development of tumor cells resistance [2]. Plasmonic photothermal therapy (PPT) is a method based on tumor hyperthermia by laser heating of metal nanoparticles with plasmon resonance properties [3]. Gold nanoparticles are most often used for PPT due to their low toxicity, colloidal stability and their ability to tune their plasmon resonance in accordance with the available laser wavelength [4]. The problems of PPT include insufficient accumulation of nanoparticles in the tumor that may lead to a low laser heating temperature and a continued tumor growth; there is also a need to develop optimal protocols for the introduction of nanoparticles and laser exposure modes for the maximum penetration into the tumor tissue, including an application of combined therapy technologies.

The aim of this study is to develop a combined technology of plasmonic photothermal therapy (PPT) and photodynamic therapy (PDT) in mice with transplanted colorectal cancer.

Materials and methods. The objects of the study were Balb mice, which had a tumor transplanted into the thigh muscle (colorectal cancer CT-26 ). Indocyanine green has been used as a photosensitizer, which was diluted in PEG (1:100). We have employed as the thermosensitizer the gold nanorods (GNRs) coated with polyethylene glycol, 41 ± 8 nm in length and 10 ± 2 nm in diameter, with a gold concentration of 400 μg/ml, corresponding to an optical density of 20 and a plasmon resonance of 808 nm. An IR laser was used for laser irradiation with a wavelength of 808 nm and a power density of 2.3 W/cm². To assess the vascularization of tumors, Dopplerographic examination of tumors in the power Doppler mode was applied with the VOCAL™ (Virtual

Organ Computer-aided AnaLysis) software. With the developed vascular network in the mouse tumor, the mice were randomly divided into 4 groups (n=4): 1) the animals who received PPT, when the polyethylene-gly-col coated GNR were introduced intratumorally into the mice as a thermosensitizer in a volume of 30% of that of the tumor; 2) the animals who underwent PDT, when indocyanine green was used as a photosensitizer, which was administered by intratumoral injection at a dose of 1 mg / kg; 3) with combined PDT and PPT, the mice were intratumorally introduced with gold nanorods and indocyanine green with the same doses at different location points; 4) the reference group to include the mice with transplanted tumors without PDT and PPT. Upon expiration of one hour after the injections, the tumor in the experimental groups of animals was irradiated transcu-taneously for 10 minutes with an 808 nm IR laser with a power density of 2.3 W/cm2.

Results. Upon completion of a single PDT session, the temperature of local heating of tumors increased slightly due to the effect of the IR laser (up to 37±2⁰). In case of application of PPT and combined PPT and PDT therapy, a pronounced increase in temperature was noted, up to 48-50⁰ C, and immediately after the completed therapy a burn surface was observed on the skin in the area of the laser exposure. 72 hours after the therapy, significant damage to tumor tissue was detected in mice in groups with PDT and combination therapy, where necrosis occupied up to 70-80% of the tumor section area; in the group with PPT therapy small areas of necrosis (up to 30%), hemorrhages and inflammatory infiltration were found. After 21 days, the tumor growth inhibition was observed in the group of mice with PDT, and the tumor growth inhibition index (TGII) by mass was 44%; the morphological examination revealed inflammatory infiltration and necrotic changes in tumors reaching up to 50% of the section area.

After PPT, a significant inhibition of the tumor growth was noted reaching up to 90%, and upon the morphological examination of tissues in the area of the laser exposure, intact tumor cells were noted only under the tumor capsule. When conducting combined therapy, a complete response was observed after 21 therapy days: the disappearance of all tumor foci in the mice; in the morphological examination no tumor cells were found in the area of the exposure.

Conclusion. The developed protocol of the combined PPT and PDT therapy leads to significant damage to the tumor tissue in mice with colorectal cancer, but however further studies of the safety of the proposed therapy are required.

The study was conducted within the framework of the advanced project No. SSMU-2022-002 , supported by the Samara State Medical University named after V.I. Razumovsky. Genin V.D., Genina E.A. and Tuchin V.V. were supported by the grant of the Russian Science Foundation No. 23-14-00287. The research work by N.G. Khlebtsov and B.N. Khlebtsov was completed within the framework of the state assignment by the Ministry of Education and Science of Russia for the Federal Research Center “Saratov Scientific Center at the Russian Academy of Sciences”.