Self-dependent antitumor effects produced by metal-containing nanoparticles in in vivo experiments

• 1    - National Medical Research Centre for Oncology

Russia, Rostov-on-Don, 2 - RosNOU, Moscow, Russia

At present, the scope of research of the medical and biological effects produced by metal-containing nanoparticles (NPs) is very impressive. At the same time, the vast majority of scientific research therein is carried out to cover three main areas: visualization of neoplasms (improvement of image contrast in MRI), targeted delivery of various antitumor factors (cytostatics, vaccines, etc.) and 30

magnetic fluid hyperthermia [1–3]. In the last decade, the fourth area of exploration is forming, associated with the study of the self-dependent effects of metal-containing NPs, which can be considered as a new option in cancer immunotherapy. At the same time, domestic researches carried out at the National Medical Research Center for Oncology in the period 2007-2016 [1, 3] allowed to obtain fundamental results earlier than foreign works of a similar essence came out [2].

The aim of our research was the study of self-dependent (without the use of any other, specific, antitumor agents) effects produced by various metal-containing NPs on transplanted tumors and the body of laboratory tumor-bearing animals.

Material and methods . The experiments were carried out in outbred rats of both sexes (500) and outbred male mice (40), as well as in C57BL/6 mice of both sexes (150). We used transplanted sarcoma 45, Pliss lymphosarcoma, Guerin's carcinoma, murine sarcomas 37 and 180, melanoma B16. The effects produced by NPs of magnetite (10 ± 2 nm), Cu (70–80 nm), Fe (30–50 nm), and ZnO (18–20 nm) NPs were studied. We began injecting of NPs after the formation of the tumors, when they reached the sizes, at which their spontaneous regression was considered to be unlikely, in some cases, even with the larger tumor sizes (with a volume exceeding 3 cm³). Various single doses of the metal-containing NPs (0.25–35.5 mg/kg) and various methods of their injection - peritumoral, intratumoral, intraperitoneal - were used, with a total number of the NP introductions of 4-10. In separate experiments on white outbred rats and mice of the C57BL/6 line, weak (up to 3.2 mT) infra-low-frequency (up to 9 Hz) electromagnetic radiation (ILF EMR, Gradient-4M device) was applied to the brain (in rats) or the organism as a whole (in mice) in the regimes of activation therapy. Changes in the blood count and tissues in the animals were analyzed using cytology, histology, histochemistry, immunohistochemistry, electron microscopy, biochemistry, and flow cytometry. The adaptational status of the animals was assessed by their hematological parameters and the morphofunctional state of their immune system organs (the thymus and the spleen). The Statistica 6–10 software was used for statistical processing of the results. The Wilcoxon-Mann-Whitney and Pearson (χ2) tests were used.

Main results . The tested NPs, used as monofactors, had their antitumor effect in 33-100% of the animals in different sets of the experiments. The antitumor effect produced by NPs was manifested in an increase in the survival (at least by a factor of 1.5), inhibition of the tumor growth (by 33–78%), partial (by 30–60%) and complete regression of tumors, tumor growth arrest, and various combinations of the above effects. The form and the intensity of the effects depended on the type of NP, the type of the tumor, the dose, the method of introduction, the biological kind and the sex of the animals, the use of weak ILF EMR, and the season of the year. In the white outbred rats, in general, more pronounced antitumor effects were obtained as compared with those found in the mice. The high efficiency of NPs at minimal doses was demonstrated. The histochemical and 31 Cardiometry, Issue 24, November 2022

electron microscopic examinations showed signs of increased lymphoproliferative processes in the organs of the immune system, activation of intercellular interactions involving macrophages in the tumor zone, as well as death of malignant cells by apoptosis, autophagy, and necrosis. ILF EMR, which had a systemic antistress effect, enhanced the antitumor effect of magnetite ILF in rats with Pliss lymphosarcoma, which increased regression rates by 20-30%.

The results of flow cytometry indicated the dominance of the subpopulation of the CD3+ T-lymphocytes within the tumor zone with the detected growth inhibition and regression. The delayed onset of the regression of Pliss lymphosarcoma, the dynamics of such regression, the recorded large and very large sizes of the completely regressed tumors (10-30 cm3) in the absence of toxic reactions indicated the development of the antigen-presentation processes in those cases and the death of the tumor cells by apoptosis as a result of effective tumor-specific T- killing.

Based on the obtained evidence data and the available literature data on the relationship between the macrophage phenotype and the ratio of the activity of iron transport proteins ferritin and ferroportin, a hypothesis was formulated that the antitumor effect of iron-containing NPs is due to a change in the polarization of tumor-associated macrophages M2 → M1 [3, June 2016]. The hypothesis was confirmed by the results of the studies of the tumor-growth-preventing effect made by ferrumoxitol, which were conducted by the group of H.E. Daldrup-Link [2, e, September 2016]. Similar, but somewhat less pronounced, effects were obtained in the rats with Pliss lymphosarcoma after the use of copper NPs.

The novelty of our obtained evidence data is supported by 6 patents for inventions.

The study results and their interpretation differ from foreign ones in the following:

• 1.    The injection using metal-containing NPs was applied to the fully developed tumors, while foreign researchers started the injection of NPs in parallel with the introduction of tumor cells [2].

• 2.    Foreign researchers associate the antitumor effects produced by the iron-containing NPs with the free radical processes only, due to the Fenton reaction initiated in the tumor-associated macrophages, which have undergone M2 → M1 polarization [2]. Based on the data on the dynamics of regression of the large Pliss lymphosarcoma tumors, we have made an assumption that, upon the polarization of the tumor-associated macrophages under the influence made by magnetite NPs, there is a possibility of the restoration of their ability to recognize tumor cells and effectively destroy them with the T-killers by apoptosis

• 3.    Studies of the antitumor effects of Cu and ZnO NPs were unique for a long time and did not have foreign analogues.

Conclusion . The results suggest using biogenic metal NPs as factors in antitumor immunotherapy aimed at overcoming the mechanisms of malignant cell evasion from immune surveillance.