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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access   Full Text Article                                                                                                                                                                                                   Mini Review 

Nanoparticles of Cerium Dioxide and Pristine (Unmodified) Fullerene C₆₀ Protect Living Cells against Adverse Environmental Exposure: Does it Work?

Oksana V. Falko¹*, Viktor V. Chizhevskiy¹, Vladimir K. Klochkov², Viktoria V. Yevlash³

1 – Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv, Ukraine

2 – Institute for Scintillation Materials of the National Academy of Sciences of Ukraine, Kharkiv, Ukraine

3 – Kharkiv State University of Food Technology and Trade, Ukraine

Recently, the rapid development of nanotechnology has made it possible to create innovative nanomaterials with fundamentally new properties and great potential to be used in biology, medicine and pharmacy. Unique in structure and chemical qualities the nanoparticles demonstrate a wide range of biological action and capabilities as a tool for influencing the functional systems of cells and the body as a whole ^1,2,3,4,5.

However, the toxicity of these compounds remains a serious problem ^6,7,8,9. Undoubtedly, the disputes and misunderstandings in this regard are due to the fact that the studying of biological properties of nanoparticles does  not always comprehensively  assume the conditions of synthesis, methods of stabilizing the particles in colloidal systems, their size, transport to biological targets etc. ^5,10,11 i.e. the parameters depending on which the biological effects of nanoparticles can vary from cytoprotective to cytotoxic ones.

For example, there are quite a number of water-soluble derivatives of fullerene C₆₀, used in various medical and biological experiments. However, it should be borne in mind that there is a significant difference between the physicochemical and, consequently, biological properties of the solutions of unmodified fullerene and those of its water-soluble derivatives. When converting a hydrophobic fullerene molecule into an aqueous solution, the final products may strongly differ by properties; demonstrate new aspects of activity due to changes in the acquisition of biological activity by them. In our opinion, the use of pure unmodified fullerene is promising for the detection of a direct biological action of fullerene C₆₀.

Due to the physicochemical structure of fullerenes C₆₀ and nanocrystalline cerium dioxide (NCD) have pronounced antioxidant properties, as they are able to participate in biological processes as the ROS regulators and free radical acceptors ^12,13,14.

In particular, the protective effect of cerium dioxide nanoparticles has been shown in a model of oxidative stress induced by tobacco smoke toxicants ¹⁵. 

NCD and water-soluble unmodified C₆₀ fullerene exhibit radioprotective properties and can strongly reduce the harmful effects of ionizing radiation ^16,17. Cerium dioxide nanoparticles also have a photoprotective effect to the cells against ultraviolet light ^18,19. There are some reported data that nanoparticles of unmodified C₆₀ fullerene protect skeletal muscles from ischemic lesions ^20,21, have a stabilizing effect on enzymes, protecting them from thermal and oxidative inactivation ²².

That is, the protective properties of the presented nanoparticles can be promising when searching the effective means of artificial protection of living cells from the negative effects of environmental factors.

The aim of this research was to study the biological effect of aqueous solutions of nanocrystalline cerium dioxide and unmodified C₆₀ fullerene on the culture of Spirulina platensis when stored in distilled water at moderately low temperatures (4-5°C).

2. MATERIALS AND METHODS

(a) Subjects

The research was performed in the culture of Spirulina platensis cyanobacteria and aqueous solutions of nanocrystalline cerium dioxide (NCD) as well as unmodified hydrated fullerene C₆₀ (C₆₀FWS).

In our studies we used a water-soluble hydrated pristine (unmodified) fullerene C₆₀ (C₆₀FWS - C₆₀HyFn), (manufactured by LLC «Institute of Physiologically Active Compounds (IPAC)», Ukraine, Kharkiv).

Also in these investigations we applied the nanocrystalline cerium dioxide (NCD) with a particle size of 2 nm obtained by wet synthesis (produced by the Institute for Scintillation Materials of the National Academy of Sciences of Ukraine, Kharkiv).

(b) Protocol

Before the experiments, the culture of Spirulina platensis was washed from the growth medium with distilled water using a nylon filter and placed in aqueous solutions of fullerene C₆₀ with a final concentration of 14.4 mg / l (2x10ˉ⁵ M) and NDC – 20 mg / l. Each experimental sample contained 300 μl of Spirulina platensis biomass in 1.0 ml of the studied solution of nanoparticles (number of trichomes made ~ 3.75 x10⁹ / l). The selected concentration of fullerene solutions C₆₀ and NCD was the maximum allowable, taking into account the possible toxic effects of nanoparticles ^{6, 23}.

Experimental samples of cyanobacteria were stored in distilled water at a temperature of 4–5°С in a household refrigerator. To assess the safety of cells we used the method of quantitative counting of Spirulina platensis intact trichomes. 

Biomass growth of Spirulina platensis was evaluated by the suspension optical density using a Solar PV1251C spectrophotometer (Belarus) at a wavelength of 750 nm. Measurements were performed in cuvettes with a working side length of 1 cm.

Washed from the Zarrouk’s nutrient medium the culture of Spirulina platensis was divided into the following experimental groups, which were stored: 1 - in distilled water; 2 - with adding the C₆₀FWS; 3 - with the NCD adding; 4 - in the Zarrouk’s cultural medium. Observations were performed for 28 days. These results were statistically processed according to Student's criterion with 0.95 reliability.

3. RESULTS

(a) Results of morphology analysis

An important point in favor of choosing the Spirulina platensis cyanobacteria as a biological model in our studies was its increased sensitivity to any environmental changes. The response of Spirulina platensis to any changes is an instant death of the trichomes. That is why this object could be used as a biological indicator of the first link in the food chain when studying an environmental pollution ²⁴.

In our previous studies, we demonstrated that the addition of fullerene C₆₀ nanoparticles at a final concentration of 14.4 mg / l or NCD of 20 mg / l did not show toxic effects on Spirulina platensis, stored in Zarrouk's medium at a temperature of 18 ÷ 20ºС (Table 1).

Table 1: Preservation of Spirulina platensis culture in the studied media at 18 ÷ 20ºC

The morphology of Spirulina platensis trichomes after storage for 7 days at 18 ÷ 20ºC in the studied media is shown in Figure 1.

                 а)                                             б)                                              в)

Figure 1: Spirulina platensis culture, stored: a) in the Zarrouk’s cultural medium; b) with the C₆₀ adding; c) with NCD adding, magnification х40.

Thus, the morphology of Spirulina platensis trichomes and their number after storage at a temperature of 18 ÷ 20°C for 7 days in a nutrient medium with the addition of nanoparticles of cerium dioxide and hydrated fullerene C₆₀ did not differ from the samples stored just in Zarrouk's medium. Therefore, the obtained experimental results indicated the absence of toxic effects of NCD and hydrated fullerene C₆₀ on Spirulina platensis culture.

Based on the main idea of our study, to determine the possible biological protective effect of nanoparticles against the negative influence of the environment, we intended to create unfavorable living conditions for the Spirulina platensis culture, namely: lack of nutrients, reduced pH of the storage medium, lack of light and lower temperatures. Therefore, later the culture of Spirulina platensis of the four studied groups was stored in a household refrigerator; the condition of the samples was examined at intervals of 7 days. Morphology of Spirulina platensis trichomes and their number after storage at 4-5ºC for 21 days in groups 2 and 3 did not differ from the samples stored in Zarrouk’s  medium (p> 0.05), while in group 1 all the trichomes were damaged. After 3-4 weeks of storage there was a decrease in the number of Spirulina platensis trichomes in the samples of group 2, compared with those of groups 3 and 4 (p <0.05) (Table 2).

Table 2: Preservation of Spirulina platensis culture trichomes in NCD and hydrated fullerene C₆₀ presence

(b) Results preservation the Spirulina platensis

To determine the preservation rate of the experimental samples, the Spirulina platensis culture proliferative activity was examined. Cells of each experimental group after each storage step (first day; 1 – 4 weeks) were subsequently cultured for 5 weeks in Zarrouk’s medium. Optical density Indices of Spirulina platensis culture suspension, significantly changed depending on the period of previous hypothermic storage of cyanobacteria and the composition of the storage is shown in Figure 2.

Figure 2: Optical density of Spirulina platensis suspension at different times of its cultivation in Zarrouk’s medium after hypothermic storage.

In general, we can conclude that the culture of Spirulina platensis is able to survive in "deadly" conditions of existence in the presence of nanoparticles studied by us.

We noted above that the biological action of nanoparticles depended on many physicochemical factors, synthesis conditions, etc., the issue of the behavior of C₆₀ fullerene and NCD in the presence of electrolytes in the dissolution medium deserves special attention. According to the published reports, the presence of, for example, Na + ions, K +, promotes the agglomeration of nanoparticles of unmodified fullerene C₆₀ and NCD, which ultimately affects their bioavailability ^{10, 11, 25}. Therefore, our study of the effect of nanoparticles on the culture of Spirulina platensis, stored in distilled water, suggests that the nanoparticles of unmodified fullerene C₆₀ and NCD"were able" to maximize their own biological action.

It is also important that the aqueous solution of unmodified fullerene C60FWS used in the research  is registered and certified for application  in Ukraine as a dietary supplements, namely as a "Fullerene C₆₀ hydrated" concentrate (C60FWS). That is, it enables the application of research results for the needs of food industry, cosmetology and others.

We believe, that the biological effect of the solutions of nanosized compounds studied by us may be related to their antioxidant activity, the ability to prevent the negative effects of free radical compounds that occur during storage of cells. The same opinion is expressed by some authors, arguing that hydrated fullerene C₆₀ is able to simulate free radical life processes at different levels of biological organization - to stabilize the state of enzymes under adverse conditions, blood parameters, etc. ²⁶. In turn, the protective effect of CeO₂ nanoparticles may also be due to their antioxidant activity, the ability to stimulate the expression of key genes involved in the cell response to oxidative stress ²⁷. However, our findings necessitate further study of the mechanisms of protective action of nanoparticles and detailed development of techniques for the use of unmodified C₆₀ fullerene and NCD in practical biology.

CONCLUSIONS

1. The presence of nanoparticles of cerium dioxide of 2 nm size in 0.02 g / l concentration and hydrated fullerene C₆₀ with a concentration of 14.4 mg / l allows the storage of Spirulina platensis cells at f 4-5°C for 4 weeks without the use of Zarrouk’s nutrient medium. 

2. The use of nanosized particles of fullerene C₆₀ and NCD stimulates the proliferation of Spirulina platensis after hypothermic storage.

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