Development of optimized synthesis techniques of metal-containing nanoparticles having a pronounced effect on tumor tissue, and integrated technologies for enhancing antitumor resistance mechanisms using nanomaterials and personalized low-intensity systemic effects.
Activity End Date: 31.12.2016
Supervisor: Alexander Soldatov
Co-workers: Oleg Polozhentsev / Tatiana Lastovina
The project in 2015 aimed to develop optimized synthesis techniques of iron-based nanoparticles having a pronounced effect on tumor tissue, and highly effective pathogenetically justified methods of complex cancer treatment using nanotechnology and personalized low-intensity systemic exposure using factors of electromagnetic and chemical nature.
New synthesis techniques for obtaining pure and doped with metal atoms magnetic iron-based nanoparticles were developed. These materials were synthesized with different composition, size and structure. Varying of synthesis techniques allowed obtaining particles with different size and shape. The smallest nanoparticles (4,5 nm) were obtained using modified co-precipitation in microwave oven with adding of gold nanoparticles as nucleation sites. The biggest particles (30 nm) were obtained using the varying of conditions in classical co-precipitation technique. Particles with size 6-17 nm were obtained using microwave synthesis. Using of polyol synthesis resulted in formation of spherical particles in most cases. Solvothermal synthesis with 2,2’-bipirydine as stabilizer allowed obtaining needle-like nanoparticles, and modified co-precipitation method with ultrasound treatment reaction mixture during the synthesis leads to plate-like nanoparticles. Obtained nanoparticles could form agglomerates. The most separate particles were obtained in system oleylamine/oleic acid using microwave treatment. The influence of solvent nature, composition of the organic solvent, and microwave synthesis conditions on the average size, size distribution and agglomeration degree of the particles was determined.
Laboratory characterization of size distribution, morphology, atomic and electronic structures of bare and doped iron-based magnetic nanoparticles of different size, composition and structure was performed. The synthesized materials were investigated by means of several experimental techniques. The variations of the synthesis methods allow obtaining magnetite, maghemite, or a mixture of these iron oxides. Microwave synthesis in glycerol at temperatures up to 275 ºС was the only method of synthesis resulted in formation of α-Fe2O3. The fraction of the surface layer, consisted of maghemite (γ-Fe2O3), is smaller comparing with magnetite phase. For bare nanoparticles (~30 nm) this layer is about 0.9 nm, and for nanoparticles doped with samarium (~10 nm) it is about ~0.3 nm.
Magnetic properties, such as magnetic saturation and magnetic moment, were determined by means of vibrational magnetometer and X-ray magnetic circular dichroism (XMCD). The synthesized bare and doped with rare-earth elements magnetic nanoparticles exhibit mainly superparamagnetic behavior. The doping of the nanoparticles using small amounts of the rare-earth elements allowed to improve their magnetic properties. For example, magnetization for bare microwave synthesized magnetite nanoparticles with sized up to 30 nm covered by PEG was about 75 emu/g, while for the nanoparticles prepared following the similar method but doped with samarium, saturation was about 122 emu/g at room temperature.
Measurements of XANES and EXAFS spectra of bare and doped with rare-earth elements of magnetic iron-based nanoparticles of different composition, size and structure were performed. Analysis of the absorption spectra allowed us to determine the structure parameters of a local atomic structure, the degree of oxidation of the iron and rare earth elements in doped magnetic nanoparticles, the doping sites of the doping elements in the structure of magnetic nanoparticles. The process of oxidation of nanoparticles in the form of nanosized powders and colloids was examined. The doping with rare earth elements in a small amount allowed to reduce the average particle size, to obtain a narrow size distribution, and to increase resistance to oxidation.
The synthesis techniques of the nanoparticles for the X-ray photodynamic therapy with solvothermal method and microwave synthesis were dveloped.
Biological study of pure and doped with metal atoms of magnetic i of iron-based magnetic nanoparticles of different size, composition and structure was performed. Incubation of human blood with doped metallic nanoparticles produced no injuring effect on membrane receptors characterizing lymphocytes` immunophenotype and did not enhance their apoptotic or necrotic death. Non-doped magnetic nanoparticles are likely to have some influence on early stages of T- lymphocytes` main subsets activation, causing marked decrease of CD38 expression on CD3+CD4+ cells and slight stimulation of CD69 predominantly on CD3+CD8+ cells. Doping of magnetic nanoparticles with Eu (europium) but not with Sm (samarium) is able to prevent their proapoptogenic effect on blood monocytes, possibly due to modulation of their phagocytosis. In spite of 100-fold difference of the applied nanoparticles` concentrations no dose-dependent discrepancy of all the studied parameters was noted. In summary the results suggest that doping of magnetic nanoparticles especially with Eu (europium) makes them more inert to the donors` immunocytes. Microscopic investigation of human lung carcinoma samples implanted to rats in plastic chambers showed that magnetic Fe nanoparticles injected intraperitoneally caused the increase of damaged tumor cells` amount by 20-30% in comparison with the control.
Our results demonstrated the absence of toxic effect of the studied nanoparticles after their administration in high dose (single dose 17.7 mg/kg, total dose 106 mg/kg) in tumor-bearing and tumor-free rats. Histological and histochemical study of liver, lungs, kidneys, adrenal glands, organs of immune system (thymus, spleen) as well as of the area of injection revealed no structural or functional injures of cells or tissues. After peritumoral administration of magnetic nanoparticles only local structural injure of muscle tissue was noted. In the neighbouring muscle fibers no structural damage was observed. In tumor-bearing animals visceral changes correlated with the extent of antitumor effect and reflected antistress influence of the studied nanofactors which was especially distinct in the microscopic picture of the organs of immune system. In animals without antitumor effect no significant discrepancy from the control group was noted.
Injection of Sm-doped magnetic nanoparticles to rats with lymphosarcoma induced normalization of the endogen intoxication indices: increase of total, effective concentration and binding capacity of albumin (by 35% and 23% respectively). Intoxication ratio in animals receiving all the kinds of doped nanoparticles had no significant changes from normal data. No dose-dependent difference in indices characterizing endotoxicosis was seen after application of doped nanoparticles in various doses.
High antioxidative activity of ceruloplasmin and decrease of POL intensity in plasma were observed. In cases of supplementary magnetic impact in animals with tumor regression effective concentration and binding capacity of albumin were significantly higher (by 1.7-8 times) than in animals with tumor growth; this resulted in triple decrease of toxicity index and almost double decrease of intoxication coefficient. Effects of magnetic nanoparticles in high dose differed in rats with lymphosarcoma, with sarcoma 45 and in С57 Black mice with melanoma B16. Complete or partial regression of lymphosarcoma was noted in 20-50% of tumor-bearing rats. In sarcoma 45- bearing rats effect resulted in inhibition of tumor growth (up to 3 times) and complete or partial tumor regression in more than a half of animals (up to 80%).
Antitumor effect was demonstrated in 40-80% of melanoma-bearing mice. It resulted in inhibition of tumor growth by 75-78% and/or prolongation of life span from the time of tumor visualization by 1.5 times. No cases of marked regression of melanoma B16 were observed. Effect varied significantly in the number of experiments that needs explanation during the following study.
In rats application of magnetic nanoparticles in a low dose (single dose 1.25 mg/kg) induced more significant effect than higher dose (17.7 mg/kg). It was expressed in 1/3-increasing of rats with tumor regression and in double increase of complete regression cases. After application of Cu nanoparticles in a low dose maximal antitumor effect was demonstrated in animals receiving intratumoral injection.
Unlike Cu nanoparticles application of magnetic ones doped with Sm in the same (low) dose induced maximal effect after intraperitoneal administration. The next most effective way of injection of doped magnetic nanoparticles was intratumoral which was more effective than peritumoral one.
The effect of peritumoral challenge of non-doped magnetic nanoparticles was similar to the effect of doped ones after intraperitoneal administration.
Peritumoral administration of magnetic nanoparticles both Sm-doped and non-doped ones accompanied by activation magnetic therapy caused increase of the incidence of tumor regression by 20-33% in animals including double increase of the marked regression. The result was comparative to the effect of cAMP-modified chemotherapy with cyclophpsphamide. In melanoma-bearing C57Black mice extra application of magnetic therapy caused twice prolongation of life span in 40% of animals while the index for the whole group was 1.5-fold.
Distinct correlation of antitumor effect of the studied nanoparticles with systemic antistress effect estimated according to hematologic and morphologic characteristics of thymus and spleen was observed in all the cases.