Having conducting large-scale research, we already knew the following main biological properties of hydrated C60 fullerene (C60HyFn) by the end of 2005.

 

For example, experiments in vitro revealed that C60HyFn, in C60 concentrations up to 100 µM:
- Are not cytotoxic, including non-hepatotoxicity;
- Do not suppress, but rather promote the growth of isolated cell cultures; promote the survival rate and enhance the viability of spermatozoa in the process of “cryopreservation – thawing”;
- Stimulate seed germination and plant growth;
- Do not inhibit cell respiration;
- Do not inhibit the catalytic activity of membrane-bound ATPases;
- Have antioxidant properties that are more significant than ionol (BHT), vitamin E and B-carotene;
- Stabilise cell membranes and enhance their stability in unfavourable conditions;
- Do not affect blood coagulation systems;
- Enhance the temperature of biomolecules denaturation (DNA, albumin, collagen, etc.) by 5-10?C; 
- Increase the catalytic activity of isolated enzymes (e.g. serine proteinases).


Simultaneously with research at the molecular and cell level, experiments in vivo (with laboratory animals) have revealed that C60HyFn in wide scale of C60 total dose (from ultra small and up to 25 mg/kg of body weight):
- Is non-toxic, non-immunogenic, non-allergenic;
- Enhances the resistance of plasma membranes to disturbing factors;
- Has a positive influence on antioxidant systems;
- Has radioprotective properties due to the inhibition of the excessive level of free radicals;
- Has a strong and long-lasting antihistaminic and anti-allergic effect, i.e. can act as anti-inflammatory agents;
- Has a positive influence on the activity of adreno-, GABA-, histamine- and especially, serotonergic systems, and, as a result, enhances the organism’s adaptogenic functions;
- Has potent hepatoprotective activity;
- Has a remarkable neuroprotective (including Alzheimer's disease) and non-specific analgesic effect;
- Inhibits the development of tumour pathologies without killing cancer cells;
- Has antiatherosclerotic (antiatherogenic) properties;
- Is able to protect organisms against viral infections (e.g. influenza virus) (HyFn does not kill viruses, but prevents their effective penetration of the cell).

 

Nowadays, on the basis of a complex analysis of numerous experimental facts, official bio-medical trials, and our own observations, we have arrived at the conclusion that water solutions of hydrated fullerene C60 have a broad and universal spectrum of positive biological activity. In particular, they:
- Are “wise” and long-acting antioxidants, which not only normalise the processes of lipid peroxidation and lipid composition of cell membranes in the organism, but also protect and activate its own systems of antioxidant protection;
- Enhance cell membranes’ resistance to adverse influences and restore disturbed the energy supply inside the cells;
- Promote the processes of cell differentiation and tissue regeneration; 
- Are non-immunogenic, but have positive immune-modulating properties and protect the immune system against disturbing factors;
- Have adaptogenic and anti-stress properties, a fact that is related to the positive influence on the functions of the central nervous system and the ability to protect nervous tissue against the detrimental influence of free radicals;
- Have long-term antihistaminic and anti-inflammatory effects; 
- Protect the cardio-vascular system from the development of pathologies connected with atherosclerosis, and also effectively inhibit the causes of the development of atherosclerosis itself;
- Have remarkable neuroprotective properties, including the protection of brain tissues against disturbances caused by free radicals and other chemical agents. Thus, it can be used in therapy for different neurodegenerative diseases (e.g. Alzheimer's disease, Parkinson's disease, multiple sclerosis, etc.);
- Are highly effective hepatoprotectors in the therapy and prevention of liver pathologies of different etiology, including cirrhosis;
- Are effective in treating ulcer diseases of the stomach and duodenum;
- Are effective in treating diseases of gallbladder, urinary bladder and pancreas, including different forms of diabetes;
- Have remarkable anti-burn, regenerative, antiulcer and wound healing properties (especially in combination with mild antibacterial preparations);
- Shorten the period required for the organism to recover after surgeries;
- Offer natural protection from viral infections, acute respiratory diseases, and are rather effective in treating ENT-diseases;
- In the case of preventive intake, decrease the probability of developing oncological pathologies, and in the case if one has already developed and depending on its stage of development, help the organism to inhibit, restrain and control its spread. In addition to independent use, they are potent additional agents in combined, rational therapy of oncological conditions;
- Significantly prolong the period of remission in patients after surgically removing a tumour neoplasm and decrease the risk of relapse of an active tumour process;
- Promote a reduction in side effects caused by the use of active antitumor chemotherapy (improve its tolerance);
- Have a non-specific analgesic effect, and, in pain syndrome patients, improve their quality of life;
- Are radioprotectors and their administration is quite grounded for eliminating the side effects of radiation therapy;
- Alleviate alcohol withdrawal syndrome in the case of alcohol excess;
- Are effective in treating chronic fatigue syndrome;
- On the whole, have a generally positive effect in terms of supporting the normal functioning of the organism and inhibiting the development of negative manifestations connected with age-related changes (such as aging, menopause, etc.).

 

Undoubtedly, this list of positive biological activity will grow. Based on already known facts, one can assume that, in general, the effect of hydrated fullerene C60 and its water solutions directed at restoring disturbed functions at the level of cells, organs and systems, and the normalisation of numerous biochemical indexes of the whole macro-organism. All of these are being realised, thanks to improvements in adaptive, immune, metabolic and reparative processes.

 

Hydrated fullerenes (CnHyFn) can be received only in the form of their water solutions (CnFWS - “Fullerene Water Solutions”, where n is the quantity of carbon atoms in the fullerene molecule).

 

The general principle of obtaining CnFWS (i.e. hydrated Cn fullerene solution) is as follows.

The solution of fullerene (e.g., C60) in organic solvent, where it can exist as single molecules, is added to water and sonicated under special conditions. Under the chosen processing conditions, what occurs is the instantaneous substitution of the solvate shell, comprised of molecules of the organic solvent and initially surrounding the С60 molecule with a spherical hydrated (aqueous) one. In such a shell, 6 water molecules take part in the formation of donor-acceptor contacts with symmetrically-located electron-acceptor centres on the fullerene surface and 18 more water molecules via joint hydrogen bonds and, together with 6 molecules on the surface, form the uniform and spherical net around C60 (= hydrated C60 fullerene, C60HyFn). In other words, there is “collapse” of bulk water around the spherical molecule of C60 with the formation of a strong water shell, which fullerene cannot get out of. Now water itself (6+18=24 molecules) has different properties from those of bulk ones. Fullerene cannot be practically removed from this net. For example, it is not extracted from FWS with nonpolar organic solvents and its water shell does not disintegrate upon heating (at least, up to a temperature of 125?C) or under the effect of inorganic and organic coagulants.

 

It is interesting that in heavy water, in D2O, a solution similar to C60FWS cannot be obtained, as heavy water is not capable of forming stable and strong hydrated shells around the C60 molecule.

 

As was pointed out above, for C60HyFn production, a method of fullerene molecule transfer from the organic phase into the water phase is used. Under conditions of ultrasonic cavitation and the heating of the reaction mixture above the boiling point of the azeotropic mixture of the organic solvent with water, what occurs is the substitution of organic molecule, the surrounding fullerene molecules, and water molecules.

 

The organic phase is removed from water solution as an azeotropic mixture with water. The resultant solution undergoes multi-stage additional purification from the residue of organic solvent and other technological impurities. After completion of all the necessary technological procedures, we obtain an aqueous concentrate of C60HyFn, containing only fullerene C60, water and small quantity of inorganic cations.

 

In the process of hydrated fullerenes’ production, much attention is paid to the purity of the resultant solutions. As raw materials, we use fullerene C60 with a purity of at least 99.9%. Water and other components taking part in the technological process preliminarily undergo additional deep purification. The C60HyFn concentration is determined on the basis of the absorption spectrum of its water solution. The coefficient of molar extinction in the maximum absorption band at 343 nm is 68 000 LxM-1xcm-1.

At present, it has been possible to achieve concentrations of hydrated C60 in water solution of about 4 mg/ml, which exceeds C60 solubility in organic solvent, toluene, which is considered to be a good solvent for fullerenes.

 

For biological purposes, solutions of hydrated C60 undergo sterilisation at 100-120?C.

 

There is no doubt that the rapid development of nanotechnologies requires a clear understanding about the safety of the developed nanomaterials, both for humans and for the environment. And the biological properties of nanoparticles depend not only on their composition, but mainly on their size, form, physical and chemical properties of their surface, determined by the peculiarities of their synthesis method. All of this also holds true for fullerenes – most simple representatives of nano-objects – for which we already knew dozens of ways to obtain their nanoparticles, especially as water dispersions. Nevertheless, one can often face a rather simplified and superficial approach to understanding the definite physical and chemical properties of fullerene nanoparticles synthesised in different ways. In turn, it is well known from scientific literature that this approach has produced many contradicting results during toxicological trials of fullerene nanoparticles.

 

As a consequence of the above, if you enter the key words “toxicity, fullerene” in an Internet search, you can see a long list of fearful denominations and phrases connected with two works carried out in the USA in 2004: an article by V. Colvin with his co-workers from Rice University, and one by E. Oberd?rster from Dallas University [1]. As a “toxic” fullerene, water dispersions of nano-C60 (nC60, THF/nC60) were used for the purposes of this research, prepared with one and the same method, where rather toxic tetrahydrofuran (THF) was used as an intermediate solvent. We reproduced this method and thoroughly analysed the resultant water dispersion of C60. We obtained evidence that standard manipulations with such a dispersion had no desired result in terms of getting rid of THF and that gas chromatography analysis always detects THF and products of its degradation that are integral and prevailing components of nano-C60 particles. It is THF and products of its oxidising modification and their further polymerisation that stipulate the negative biological effects of nano-C60 particles. Our critical remarks as for the pseudo-sensation about fullerenes’ toxicity can be found in the English-language article [2] or its Russian variant, written as an open letter to the scientific community.

 

Considering our criticism, E. Oberd?rster repeated her experiments with aquarian fish and water flea (Daphnias) in 2005. But this time she used fullerene water solution, obtained via 2-month mixing of fullerene powder in water. In this case, any influence of harmful impurities of organic solvents was excluded, but she received water dispersions of fullerene particles of 10–200 nm, whose surfaces consisted of modified hydroxylated (oxidised) fullerenes. However, there was no significant toxicity of fullerenes to any extent, as there had been in previous research work. It was only at very high concentrations (~35 mg/l) of oxidised fullerene particles that any adverse and unreliable effects were observed, which were mainly connected with the very high level of turbidity of the medium, where the water organisms lived and were tested.

 

At the same time, scientists from France [3] demonstrated that the use of water dispersions of fullerene C60 during experiments on rats had no toxic effects, even in very high doses of C60 (? 2.5 g/kg). Moreover, pure fullerene C60 perfectly protected the animal’s organism from the influence of strong toxicant and proved to have powerful antioxidant and hepatoprotective properties.

 

In spite of this, the idea that fullerenes can be toxic gave occasion for theorists from Vanderbilt University to “play” with computer modelling with fullerene C60 molecules and DNA. However, the real physical and chemical properties of fullerene and DNA molecules in water systems were not considered; speculative conclusions were received as to the possible genotoxicity of fullerenes. You can see our critical remarks (in Russian) on this issue on the Internet-site “Elements”.

 

Then, in spite of the fact that, back in 1997, Russian scientists clearly demonstrated the absence of any mutagenic, DNA-disturbing action on the part of pure fullerenes (Genetica, 1997, vol. 33, pp. 405-409), two works from American groups appeared in 2006-07 (V. Tarabara et al. from Michigan State University, and S. Pacheco, K. Arcaro et al. from the University of Massachusetts), which included high-sounding statements to the effect that they had established the genotoxicity of fullerene C60 dispersions in water. In addition, in testing done by the second group of researchers, they apparently discovered that colloidal particles of silica (the main component of usual sand!) had DNA-disturbing effects and that they were similar in their extent to those of fullerenes!

 

A detailed analysis of the results provided by American research works have revealed the scientific incorrectness and fallibility of further conclusions, which were connected with the fundamental non-applicability of the chosen method – the Single-Cell Gel Electrophoresis Assay or Comet assay with Olive Tail Moments (OTM) observation – for an analysis of the genotoxicity of nanoparticles and nanomaterials. The conclusion of our critical analysis (in Russian) can be expressed by the following way: “Pure fullerenes are no more dangerous than regular sand”!

 

Before these publications appeared, for the previous 20 years, nobody from many laboratories of the world had noticed any toxicity of fullerenes; and fullerenes in their toxicological characteristics have always been equated and continue to be equated to amorphous technical carbon, to carbon black (carbon black, U.S. Department of Labour, OSHA's Hazard Communication Standard, 29 CFR 1910.1000 and 29 CFR 1910.1200).

 

Numerous biological experiments with our molecular colloidal solutions of hydrated fullerenes (FWS) virtually always start with the testing of their probable toxic effects. And none of the in vitro and in vivo tests have revealed any signs of their toxicity.

 

Thus, we can fully confidently answer the question about fullerenes’ toxicity: fullerene molecules are not toxic and cannot be more toxic than coal, graphite, diamond or usual sand. What can be toxic are both the chemical derivatives of fullerenes (which is determined by the properties of the chemical groups attached to the fullerene core) and nanoparticles in the form of crystalline solvates (or clathrates) of fullerene molecules with other toxic molecules. In contrast with that, FWS contain only pure fullerenes and water; therefore, one cannot expect any toxic effects upon applying hydrated fullerenes and their aqueous solutions.

 

Incidentally, at the end of 2005, we gave E. Oberd?rster a sample of our FWS and suggested that he carry out comparative biological testing, but there has been no answer as of yet. Moreover, this is probably due to the fact that the myth of fullerenes’ toxicity, which appeared with much fuss in 2004, is being constantly shattered under pressure from reliable and unquestionable scientific findings. Water dispersions, known as nano-C60 or nC60, or THF/nC60, or nC60/THF, which scientists in the West like to work with, are absolutely unsuitable objects for the objective assessment of pure fullerenes’ toxicity. That is exactly why conclusions on the toxicity of fullerene C60, obtained on the basis of such objects, should be considered false ones and they should not be taken into consideration in future.

[1] Oberdorster E. Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in brain of Juvenile Largemouth bass. Environ Health Perspect., 112(10), (2004) 1058–1062.

[2]. G.V. Andrievsky, V.K. Klochkov, L.I. Derevyanchenko. IS C60 FULLERENE MOLECULE TOXIC?! Fullerenes, Nanotubes and Carbon Nanostructures, 13 (4), (2005) 363-376.

[3] Najla Gharbi, Monique Pressac, Michelle Hadchouel, Henri Szwarc, Stephen R. Wilson and Fathi Moussa. [60]Fullerene is an in vivo Powerful Antioxidant With no Acute or Sub-acute Toxicity. Nano Letters, 5 (12) (2005) 2578-2585.

 

Fullerenes exist everywhere in nature, and especially where carbon and high energy exist. They exist near carbon stars, in interstellar space, in places where lighting has previously struck or close to volcano craters. They even form when gas burns in the household gas cooker or in the flame of a regular lighter.


Fullerenes have also been found in places of ancient carbon rock accumulation. The Karelian minerals – schungite have a special place in this regard. These rocks, comprises of up to 80% of pure carbon, are about 2 billion years old. The nature of their origin is still not clear. One suggestion is that it was due to the impact of a big carbon meteorite. In 1992, in a scientific journal, it was announced that natural fullerenes were found in schungite. In 1999, using the ideology of obtaining hydrated fullerene water solutions, we also succeeded in extracting and identifying fullerene, but only C60, in high-carbon type I schungite.


Since the time of Peter I (Peter the Great), there has been a medicinal spring, known as “Marcial waters” in Karelia. For many years, no one could ultimately explain the reason for the multiple therapeutic properties of this spring. It was assumed that increased iron content is the cause of its healing effect. But there are many springs containing iron and, as a rule, the medicinal effects after their use are rather limited. It was only when fullerenes were found in schungite rocks, which the spring flows through, that the assumption came about that fullerene is the quintessence of the medicinal effect of Marcial water. But the medicinal properties of this water, similar to thawed water, are preserved for a rather short time. The following day, it tends to lose its properties. Marcial water, passing over rocks containing fullerene and fullerene-like structures, only “saturates” with the structures, imposed by the rock. When stored, these life-giving clusters disintegrate. Fullerene does not dissolve in water spontaneously. That is why there is no structure-forming element that is able to preserve ordered water clusters for a long time. Such water soon takes on the properties of regular water. In addition, the ions present therein rebuild native water structure themselves, forming their own hydrated clusters.


Having obtained molecular-colloidal solutions of hydrated fullerenes in water once, we tried to reproduce the natural and medicinal properties of Marcial water under laboratory conditions. With this purpose, we took highly purified water and added hydrated fullerene C60 in “homeopathic” dosage. Biological trials were conducted on different models. The results were striking. On virtually any pathology model, we observed a positive biological effect. The experiments lasted for more than 15 years. Many pathological changes in a living organism are eliminated and it returns to its normal, healthy condition. This is not a medicinal preparation of purposeful action, nor a foreign chemical substance, but just a carbon ball, dissolved in water. It seems that hydrated C60 fullerene assists an organism to return to its “a normal condition” in the case of any negative changes and it does so due to the restoration and maintenance of those structures it has generated as a matrix in the process of the origin of life.