Mosiyenko V.S.1, Kushchevska N.F.2, Burlaka A.P.1, Shlyakhovenko V.A.1, Lukin S.M.1, Iakovliev P.A.3, Karnaushenko A.V.1, Verbynenko A.V.1
1R.E. Kavetsky Institute of Experimental Pathology, Oncology, and Radiobiology of NAS of Ukraine
2University “Ukraine”, Chair of modern nanotechnology and nanomaterials
3Health Recover Center “Kalhivi”, Kyiv, Ukraine
Nowadays, due to the unique chemical, physical, biological, and pharmacological properties, many scientists in the world have been widely investigated nanomaterials of various origins and developed new nanotechnologies [1-4]. Scientific works are aimed at study of the effect of nanoparticles on biological objects at all levels from the molecular one to the whole living organism. The use of nanotechnology is enabling us to manufacture qualitatively new drugs and to improve treatment of many diseases including cancer [5-7]. Nanomedicine was included by the US National Institute of Health to the five high-priority areas of medicine of the XXI century. The Institute’s scientists believe that nanoparticles and nanotechnologies will help to treat successfully patients with various forms of cancer [8-10].
Rapid development of nanotechnologies in the world led to the emergence of convergent technologies in relation to synergistic combinations of the main branches of science and technology (N-nano-, B-bio-, I-info, and C-cogno-technologies) [11-14].
The most medically studied are metal nanoparticles and nanocomposites formed by them. In paricular, these are nanoparticles of iron, gold, copper, platinum, and other metals. A new class of organic cluster compounds containing ferromagnetic nanoparticles has unique pharmacological properties. They are similar to hemoglobin and effective in the treatment of viral and neoplastic diseases, of anemia and of immunodeficiencies.
Today, there is an urgent need to study the pharmacological and toxicological properties of iron nanoparticles, their anticancer activity and side effects. Ferromagnetic iron nanoparticles penetrate cell membranes and blood-brain barrier and accumulate in the cells of various organs and tissues . R. Elliott at all  believe that the main problem in oncology research of the XXI century is the study of iron metabolism in tissues, of mitochondrial respiration disorders, and of immunosuppressive state in patients with tumor disease.
Ferromagnetic nanoparticles cause oxidative stress and inflammation leading to epigenomic and genomic changes in cells involved in the cell division process. Iron nanoparticles can block DNA repair and cause its methylation which leads to genes malfunction [17,18]. Oxidative stress can activate intracellular signal transmission paths, including factor NF-kb. DNA damage by iron nanoparticles can activate tumor suppression gene p-53 and run mechanism of apoptosis or, vice versa, can stop these processes . Ferromagnetic nanoparticles form hydrogen peroxide which is the engine for generation of highly active hydroxyl radicals, for lipid peroxidation, and for damage of proteins and nucleic acids. Increase of concentrations of iron and metabolites of lipid peroxidation activates polyamineoxidase deamining (oxidizing) polyamines and their precursors which leads to the formation of highly toxic aldehydes which in turn can inhibit the growth of malignant cells [20,21].
Thus, researches and practical introduction of ferromagnetic nanoparticles and their preparation technologies cause rapid convergence of other sciences and technologies in solving such highly complex tasks as diagnostics, prevention, treatment, and rehabilitation in cases of malignant disease.
This paper aims at study of the effect of different doses of ferromagnetic nanoparticles (FN) and their composites in combination with inhibitors of the polyamines synthesis on the development of experimental mice lymphocytic leukemia L1210 and of Ehrlich ascite carcinoma.
Objects and methods.
Experiments were conducted with 300 non-linear and linear BDF1 and CDF1 mice of both sexes, weighing from 17 up to 23 g, taken from vivarium of R.E. Kavetsky Institute of Experimental Pathology, Oncology, and Radiobiology of National Academy of Sciences of Ukraine (IEPOR NANU), which, after two weeks of quarantine, were grafted with lymphocytic leukemia L1210 or Ehrlich ascite carcinoma using standard procedures. Leukemic cells or cells of Ehrlich carcinoma (300-500 thousands) in 0.2 ml sterile solution of sodium chloride were Injected into the abdominal cavity. Tumor strains were obtained from the bank of cell lines of human and animal tissues of the IEPOR NANU.
Ferromagnetic nanoparticles (FN) was first obtained by D.Sc., Prof. N.F. Kushchevskaya using the original method, particularly with the ratio 40% Fe2O3 + 60% Fe3O4. Anti-leukemic effect of FN was assessed by dynamic of growth of lymphocytic leukemia cells in ascite suspension, by its volume, by dynamics of animals weight growth and by average lifespan of mice. The main research results were analyzed using the Student t-test.
Results and discussion.
It is known from the literature [17,18] that, during the researches, attention is not driven to action of ferromagnetic nanoparticles by themselves while studying them mostly as carriers for designing new drugs on the basis of already known drugs, including platinum, to achieve their rapid targeted transportation and deep penetration into the pathological focus or to increase prolongation of their action in order to improve the antitumor and anti-inflammatory effects and to reduce side effects.
In the study of antitumor activity of FN, we applied doses comprising one half, one tenth, and one hundredth of LD50 (8,41 g) per kg of body weight of mice and introduced orally 3 times per day in 0.3 ml of distilled water every day (for preventive option) three days before grafting with Ehrlich ascite carcinoma.
As a result of experiment using prevention option, it was found that the growth rate of ascite tumor cells during the first two weeks was the lowest in the group of mice treated with a dose of 16 mg FN per kg of body weight of the animals. For the first week, this growth rate averaged 0,1 ± 0,04 g, and for the second one – 1,2 ± 0,8 g. In the control group of mice during this period, weight of animals was increased by an average of 1,9 ± 0.6 g and of 2,1 ± 0.6 g, respectively. Other groups of mice showed tendence to increase weight gain, but this gain was lower than in the control group.
As to average life expectancy (ALE), it turned out to be the longest in mice treated with the lowest dose of nanoparticles (27,8 ± 2,4 days) in comparison with the control group of mice where ALE was equal to 24,1 ± 2,7 days. In the other two groups of mice, ALE only tended to prolongation. It should be noted that none of these large doses introduced into mice in preventive mode did lead to growth stimulation of Ehrlich ascite carcinoma.
Because iron particles are almost insoluble in water and alcohol and forming clusters, to obtain uniform suspension, silica nanoparticles (Silard medicinal preparation) were used. In other experiments, to obtain a uniform suspension, sucrose, bentonite, and fullerene C60 containing medicinal preparation “Water of Life” was added in certain proportions, as well as polyamine biosynthesis inhibitors DFMO (Difluoromethylornithine) and MGBG (methylglyoxal-bis-guanylhydrazone). FN and their composites were administered orally in 0.3 ml volume daily (10-12 injections per course), starting the next day after transplantation of L1210 lymphocytic leukemia cells.
For the experiment, 50 CDF1 female mice were taken; L1210 lymphocytic leukemia cells were transplanted to them; and then animals were divided into 5 groups of 10 mice in each: I – control group, II – group which mice were administered orally with FN composite (5 µg + 250 µg SiO2) per day per mouse in 0.2 ml of distilled water (10 injections); III – group which mice were injected with FN composite (50 micrograms per mouse for the above described scheme); IV – group which mice were injected with FN composite (500 micrograms per mouse for the above described scheme); V – group which mice were injected with FN composite (50 micrograms per mouse for the above described scheme without silicon oxide).
The most effective was the composite of FN (50 micrograms per mouse). The average mice life span was 18,5 ± 0,3 and 18,9 ± 0,5 days, respectively. In the second and the fourth mice groups, an average mice lifespan was only slightly different from the control group, in which this value was 17,3 ± 0,7 days (Fig. 1).
Fig. 1 Average life span of mice grafted with lymphocytic leukemia L1210 and treated with FN alone and with their composites in different doses.
When studying the effect of FN in a dose of 50 µg + 250 µg of bentonite and of FN at the same dose plus 500 µg of sucrose per mouse, it appears that FN composite with bentonite has different impact on life expectancy compared with a group of mice received FN alone which was 18,8 ± 0,6 and 18,3 ± 0,5 days, respectively, and FN composite with sucrose has even reduced life expectancy of mice compared to the control group which was 15,6 ± 0,4 and 17,0 ± 0,7 days, respectively (Fig. 2).
Fig. 2. The average life span of mice grafted with lymphocytic leukemia L1210 and treated with FN and their composites in a dose of 50 micrograms per mouse.
Analyzing the dynamics of body weight changes, it has been shown that it changed wave-like with the least mice weight change in the group received FN with bentonite.
The effect of FN in a dose of 50 micrograms per mouse, together with fullerene C60 (“Water of Life”), at lymphocytic leukemia L1210 was studied . Fullerene C60 solution normalizes water structure which becomes similar to the structure of water of normal human tissues. It also normalizes metabolism in the cells, especially in various pathological conditions, and shows antioxidant, anti-inflammatory, and anticancer properties. Hydrated fullerene C60 were administered to mice orally in the morning and at the end of the working day daily in volume 0,7 ml 10 times 30 minutes after administration of FN. Another group of mice grafted with lymphocytic leukemia was administered with DFMO every other day by injection into the abdominal cavity at a dose of 16 mg per mouse 6 times or with MGBG at a dose of 0.2 ml per mouse using exactly the same scheme. At 9th day after lymphocytic leukemia grafting, in 3 of 10 mice of each group, all the ascites liquid from the abdominal cavity was taken, together with liver, kidneys, blood, lymphocytic leukemia cells for EPR studies, and also with thin and large intestine to determine the mitotic index of epithelial cells of these organs.
During the analysis of the experimental data, it was found that the volume of ascites liquid of lymphocytic leukemia cells was greatest in the group of mice treated with fullerene C60 and averaged 9,3 ml compared with 8,3 ml in the control group of mice. In other groups of mice, ascites liquid volume was lower than in the control group. Numbers of lymphocytic leukemia cells were higher in the control group of mice and in the mice group treated with fullerene C60, averaging 1385×106 and 1431×106, respectively, and were lower in groups of mice treated with DFMO, FN, and MGBG, averaging 550×106, 977×106, and 977×106 cells, respectively. In the group of mice treated with FN and C60 fullerene, number of leukemia cells did not differ from such number in the control group of mice.
Mice body weight dynamics was different. The gain in difference between the initial and final body weight of mice treated by FN alone was 0.38 g; the largest increase in body weight of mice (0.62 g) was in the group received fullerene C60 alone; the smallest gain was in the group of mice treated with MGBG and in the control group, being negative and equal to -0.88 and -0,2 g, respectively (Fig. 3).
Fig. 3. The difference between the initial and final body weight of mice with lymphocytic leukemia L1210 treated with FN, with fullerene C60, with their composite, and with modulators of metabolism of polyamines.
Differences between the initial and final mice body weight data correlated with the volume of ascites liquid of lymphocytic leukemia L1210 in different mice groups. As for the difference in body weights of mice treated with MGBG and of the control mice group, it appeared negative, although the volume of ascites liquid in these groups of mice was much higher than in other experimental groups. This indicates that the toxic effects of lymphocytic leukemia cells and of polyamines modulator MGBG onto the mice were significantly higher than in groups of animals treated with FN and fullerene C60 and that these nanoparticles reduce or neutralize this negative impact and thus increase antitumor resistance.
The average life span of mice with lymphocytic leukemia L1210 was the longest (p <0.05) in the group of animals received FN alone (19,9 ± 1,3 days) and in the group of mice treated with FN + DFMO (18,6 ± 1.6 days), compared with an average life expectancy of control mice group (17,0 ± 1,4 days); the lowest life expectancy of mice appeared in the group of mice treated with fullerene C60 (average 13,6 ± 1,2 days) (Fig. 4).
Fig. 4. The average life span of mice grafted with lymphocytic leukemia L1210 ascites after the treatment by FN in a dose of 50 micrograms per mouse and by composite of fullerene C60, DFMO, and MGBG. 1. FN; 2. Fullerene C60; 3. FN + fullerene C60; 4. Control; 5. DFMO + FN; 6. MGBG.
Effects of different doses of FN on the activity of mitochondrial FeS-proteins – N-2, on the activity of cytochrome P-450, on the generation of superoxide and NO-radicals in the kidneys, liver, peripheral blood neutrophils, and in L1210 lymphocytic leukemia cells in vivo were studied by EPR spectrometry with spin trap technology using computerized radiospectrometer EPR RE 1307. It is known  that significant increase in the generation of oxygen radicals in leukemic cells leads to increase in their death rate.
In the course of treatment by FN, it was found that these nanoparticles, serving as hemoglobin, normalize electron transport in mitochondria, activate mNOS, improve oxidative phosphorylation, and reduce the level of superoxide radicals. It was shown that FN reduce the NO level in leukemic cells, leading to a reduction in their growth rate. FN normalize the activity of hepatic cytochrome P-450.
Thus, summarizing the research results with ESR spectrograms, it was concluded that FN act on the energy potential of mitochondria and on oxygen exchange of leukemic and normal cells and peripheral blood neutrophils, increase detoxic activity of parenchymal organs (especially liver), change NO level, and thus, reducing immunosupressiveness, enhance antitumor resistance.
Due to the fact that the body bearing the tumor is in immunosuppressed state [24, 25], we have investigated the effect of FN on immune mechanism in mice with ascites lymphocytic leukemia L1210.
It is known that almost 70% of immune cells located in the small and large intestine, and FN was introduced through the gastrointestinal tract.
We have studied the mitotic activity of the epithelium of the small intestine and liver of mice under course dose of FN and their composites with fullerene C60. Change in mitotic index (MI ‰) was calculated by the following formula:
The results indicate that (MI ‰) in the small intestine of mice with lymphocytic leukemia L1210 was increased in study groups to the different extent: the largest value was in the group of mice treated with FN + C60 fullerene, and the lowest level of this index, compared with control animals treated with distilled water, was in the group of mice administered with fullerene C60 or FN alone (Fig. 5, Table 1).
Fig. 5. Increased mitotic activity in the crypts of the small intestine with the application of FN + Fullerene C60. Hematoxylin-eosin. Zoom x200.
Table 1. Mitotic index of the epithelium of the small intestine of mice after a course of treatment by FN, their composite, and fullerene C60.
The above data indicate the presence of effects of the studied compounds on mitotic activity of the epithelium of the small intestine, especially when using FN + Fullerene C60 which means that the immune mechanism is activated under action of FN and their composites. It is true especially because histological studies conducted in liver of mice of the aforementioned groups found activation of cells of the reticuloendothelial system and extramedullary hematopoiesis, indicating the presence of immune adjustment in the organs and systems of test animals. The greatest severity of this index was in the liver of mice injected with FN.
In the study of influence of FN dose, for 4 and 20 mg introduced orally per mouse with ascites lymphocytic leukemia L1210 on the next day after grafting (11 times), the best therapeutic results were obtained. The longest average life span appeared in the group of mice treated with 4 mg FN per mouse (19,0 ± 2,1 days; 28.5%) compared with the control group of mice. For ascites lymphocytic leukemia L1210 strain, small doses appeared to be the most effective and showed statistically significant (P <0.05) increase of the life expectancy of mice.
Thus, for the first time, it was found that ferromagnetic nanoparticles, after oral administration at doses of 2 to 50 mg per mouse, delay growth of ascites leukemia L1210 cells, reduce the volume of ascites liquid, and prolong the life expectancy of mice by 7-28,5% depending on the dose received. To create a uniform FN suspension, the following composites were used: SiO2 (Syllard preparation), bentonite, sucrose, fullerene C60, which are differently influenced the anti-leukemic action of FN. It was shown in therapeutic experiments that fullerene C60 and sucrose stimulated the growth of leukemia cells, SiO2 nanoparticles and bentonite were not affect significantly the growth of leukemia cells and almost did not lengthen the life of mice compared to control animals. The conclusion was drawn that the use of iron nanoparticles as targeted transport media apparently can not always just enhance antitumor activity, but also can cause reverse negative effects. Treatment by FN in combination with inhibitors of polyamines MGBG and DFMO of mice with lymphocytic leukemia L1210 were not significantly increased their life expectancy compared to the FN alone.
During the study of the mechanism of action by ESR, it was found that FN normalize the process of electron transport in mitochondria electric transport chain of leukemic and normal cells, and this FN effect was more pronounced in malignant cells, which contributed to the reduction of superoxide radicals level. Thus, in the mitochondria of cells, levels of NO and of complex NO – FeS-proteins decrease. Nanoparticles of iron increase almost twice the activity of cytochrome P-450 in liver increasing antitumor resistance of the body.
As the significant number of immune cells located in the small intestine, we have studied histologically mitotic index of the epithelium of the small intestine and shown that the impact of FN and their composites statistically significantly increased mitotic index. This indicates that the FN act not only on oxygen exchange, but also increase the body protective immune mechanisms of tumor.
The positive data obtained in therapeutic experiments on grafting of ascites lymphocytic leukemia L1210 confirm perspectiveness of studies related to cell regulation mechanisms of influence on the malignant cells of ferromagnetic nanoparticles and their composites and improve body antitumor resistance.
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