Evaluation of the C60 biodistribution in mice in a micellar ExtraOx form and in an oil solution

The article is devoted to the study of the pharmacokinetics of fullerene C60 in oil and micellar forms, analysis of its content in blood, liver, lungs, kidneys, heart, brain, adrenal glands, thymus, testicles, and spleen. The highest accumulation of C60 was found in the liver and adrenal glands. As a result of the studies carried out, it was shown that the bioavailability of C60 in the micellar form is higher than that in an oil solution.

www.nature.com/scientificreports/ indicating a relationship between the antioxidant effect of fullerene and lifespan of rats. Oral administration of a solution of fullerene in olive oil increased the lifespan of Wistar rats, and the authors attribute this to the antioxidant properties of fullerene. Due to these properties, fullerenes have found their application in cosmetology and dermatology. On the market for fullerene cosmetics, Japanese manufacturers are leading: the most successful company is Vitamin C 60 Research Corporation (a subsidiary of Mitsubishi Corporation), which produces the most common fullerene-containing products, RadicalSponge or Lipofullerene. According to Vitamin C 60 Research Corporation, more than 1,500 clinics in Japan use products in which fullerene serves as an active ingredient, and it is believed that there are more than 1000 such products. Compositions based on them act as nanotraps for radicals without causing toxic effects [32][33][34] . Due to the low solubility of fullerenes in water (< 10 −11 g·l −1 ) 35,36 , there is a problem of their study and application in medicine. An alternative way of injecting fullerenes into the body is to use solutions in such solvents as natural oils and animal fats. These systems have many advantages including: • fullerenes are decently soluble in these natural solvents from tenths to several grams of fullerenes per litre of a solution [37][38][39][40][41] ; • fullerenes form absolutely transparent true solutions stable in time with natural vegetable oils and animal fats 42 ; • such solutions are completely harmless and compatible with organisms of animals and humans, if they are prepared directly during the extraction of a fullerene mixture from fullerene soot by oils themselves; • fullerene solutions in oils and fats have pronounced bactericidal and antioxidant properties; they can also scavenge free radicals and radical ions from condensed phases, as well as photons in the UV region of the spectrum 43,44 .
Speaking about the specific toxicological effects of fullerene on a living organism, it is worth paying attention to ref. 45 , where the effect of an aqueous suspension of C 60 was studied when administered intraperitoneally into Sprague-Dawley rats. No acute toxicity was found; on the contrary, it turned out that fullerene exhibits hepatoprotective properties in the modelled CCl 4 -induced hepatitis. Also, no toxicological effects were found in the study of the C 60 complex with polyvinylpyrollidone 46,47 .
Ref. 48 was devoted to the toxic effects of fullerene. Fullerene C 60 was administered perorally to mice (female and male, age: 4 weeks) in corn oil, once a day at doses of 1, 10, 100 or 1000 mg·kg −1 per day for 29 days, followed by a 14-day recovery period. The study did not reveal any toxic effects of C 60 fullerene, but there was a slight increase in liver and spleen mass after a 14-day recovery period, which may be associated with the effect of oral intake of C 60 fullerene. Experiments on acute toxicity showed that administration of C 60 fullerene aqueous colloid solution in a concentration range of 75-150 mg·kg −1 to mice did not cause toxic effects. In an in vitro experiment, C 60 fullerene in the concentration range of 3.6-144 mg·ml −1 was found to have low toxicity towards human embryonic kidney line (HEK293), and the IC 50 value was 383.4 μg·ml −149 . Ref. 50 revealed that fullerene C 60 can stimulate the growth of cervical cancer cell line HeLa and human mesenchymal stem cells at low concentrations (6-12 μg·ml −1 ) and reduce cell viability at high concentrations (24 μg·ml −1 ).
This article is devoted to the study of the pharmacokinetics (PK) of C 60 . The relevance of the study is associated with the fact that it allows obtaining information on the biodistribution and bioavailability. The study of bioavailability reveals the organs and tissues in which fullerene penetrates most actively and accumulates, which can contribute to a more detailed understanding of the mechanisms of action of fullerene. For additional identification of fullerene, a number of physicochemical methods were used: NMR spectroscopy (NMR spectrometer Bruker Avance III 400 MHz). During the NMR study, the sample was placed in a rotor with an outer diameter of 4 mm, made of zirconium oxide and a rotation frequency of 12.5 kHz at a magic angle to the direction of a constant magnetic field. To record the spectra on 13 C nuclei, a cross-polarisation sequence of exciting pulses (CP/MAS technique) was used; the contact time was 2,000 ms. The resulting spectrum ( Fig. 1) contains one peak, which is related to the carbon atoms of the fullerene core. Figure 2 shows the mass spectrum of a C 60 fullerene sample obtained by negative ionisation using a Muldi mass spectrometer (Shimadzu Axima-Resonance). The presented spectrum shows a set of peaks corresponding to the C 60 isotopic distribution, which indicates the absence of impurities in the sample.

Experimental part
The transmission spectrum was recorded on a Shimadzu apparatus, after mixing the investigated powdered fullerene C 60 with microcrystalline potassium bromide (KBr) and subsequent pressing of the microtablet from this mixture. The spectrum (Fig. 3) shows bands at 1427.4, 1180.5, 574.8, 525.6 cm −1 , caused by vibrations of free bonds of fullerene C 60 . The data obtained are in good agreement with the literature ones 51,52 .
When studying the pharmacokinetics, the oil and micellar forms of fullerene C 60 were used. A micellar solution of fullerene C 60 contained an oil solution of C 60 fullerene, as well as a surfactant TWEEN-80 purchased from Sigma-Aldrich (molecular weight 1310 g·mol −1 ) approved for the use in cosmetic, pharmaceutical, and food industry and applied to form micelles. Moreover, it was determined that this surfactant does not possess cancerogenic and mutagenic activity. TWEEN-80 is a non-ionic surfactant derivative of polyethoxylated sorbitan and oleic acid (Fig. 4). Its hydrophilic-lipophilic balance value is 16.7 which determines the hydrophilicity of that kind of a surfactant, and consequently an ability to stabilise an oil-in-water emulsion. In addition, micellar www.nature.com/scientificreports/ form is more bioavailable in comparison to oil one. The micellar form of fullerene in oil was obtained using the original ExtraOx technology developed by "AQUANOVA RUS" JSC 53 . This technology makes it possible to obtain micelles up to 30 nm in size, in which various biologically active substances are encapsulated in a shell consisting of surfactants. The structure of such a micelle mimics the micelle that is formed in the human body when lipids are absorbed during digestion. Due to the creation of micellar forms of various biologically active substances, the bioavailability of the active ingredient is significantly increased, as well as the stability during storage is improved. The content of fullerene C 60 in the oil form is 0.1 wt.%, in the micellar form is 0.01 wt.%.
The distribution of C 60 micellar particles was studied by dynamic light scattering on a Malvern Zetasizer 3000 device (Malvern Instruments, Malvern, Worcestershire, United Kingdom). The obtained results are presented in Fig. 5. It can be seen that the average micelle size is 10 ± 5 nm.

Equipment and research methods. Study of the solubility of C 60 in olive oil.
To determine the saturation time of fullerene solutions, the kinetics of fullerene dissolution was studied. The determination of the polythermal solubility of C 60 in olive oil was carried out using the isothermal saturation method. According to this method, olive oil was added to the flask with a notorious excess of C 60 . Then the flask was thermostated    Concentrations were determined by means of SOLAR CM 2203 spectrofluorometer (Solar CJSC, Minsk, Belarus). As is known, fullerene C 60 has a characteristic transmission spectrum with well-pronounced absorption maxima in the near UV and visible regions (300-600 nm) [54][55][56][57][58] . The obtained spectrum of fullerene C 60 ( Fig. 6) shows an almost complete absence of solvatochromic effects, which makes it possible to use reliably the following formula 42 : where D 335 and D 472 are optical densities of the solution reduced to an absorbing layer of 1 cm, C(C 60 ) is a fullerene concentration (mg·l −1 ).
Modelling of the structural and dynamic properties of a fullerene solution in olive oil was carried out by the molecular dynamics (MD) method. To analyse the interaction of a fullerene molecule and olive oil components in solution, a model system was chosen containing one fullerene molecule and 20 triglyceride molecules (Fig. 7), composed of two residues of oleic acid and one residue of palmitic acid. At the first stage, the electronic structure and equilibrium geometry of the triglyceride molecule were calculated by the DFT method in the DMol 3 program from the Materials Studio package (PBE functional, basis DNP 4.4); then the charges on the atoms were determined according to the Mulliken scheme. For calculations by the method of MD, a cell was formed (Fig. 8), consisting of a fullerene molecule and triglyceride molecules, with the calculated charges. The calculations were carried out in the Forcite program (UFF force field) from the Materials Studio package. At the first stage, an integration step of 1 fs, time 1000 ps, NPT ensemble was used. A system was obtained with good agreement with the experimental density value of olive oil. At the second stage, an integration step of 0.1 fs, a time of 100 ps, an NVT ensemble was used. Thus, at each stage, 1 million steps were simulated.
The data on the solvent content in crystal solvates were obtained as follows: the solid phase freshly precipitated from the corresponding solution was washed twice with ethyl alcohol, then dried at 20 °C for 30 min, after which the resulting solid phase was weighed. After that, the solid phase was repeatedly washed in a Soxhlet apparatus with ethyl alcohol (at 78 °C, 1 atm.) and dried in a vacuum (0.1 mm Hg) at a temperature of 200 °C for 1 h, and then weighed again. The change in the mass of the solid phase was used to determine the solvent content in the initial crystal solvate.  www.nature.com/scientificreports/ Study of pharmacokinetics. The study was approved by the Local Ethics Committee of the Pavlov First Saint Petersburg State Medical University, and the followed procedures were in accordance with University guidelines and ethical standards. All experiments were complied with ARRIVE guidelines. In the experiment, mice from the Rappolovo nursery were used: males weighing 30 ± 3 g, a total of 54 pcs. 12 h before the experiment, the animals were starved, without restricting access to water. The method of administration of the test solutions was the following: once 15 mg·kg −1 for the oil form of C 60 and 1.5 mg·kg −1 for the micellar one. Both forms were administered intragastrically via gavage. The dose of C 60 in oil was chosen according to ref. 59 . Preliminary experiments on the accumulation of C 60 in oil and in the micellar form in HEK293 cells showed that the micellar form has almost an order of magnitude more accumulation in cells.
Mice were sacrificed 24 h after administration of the final dose of fullerene. The tissues of the liver, lungs, kidneys, heart, brain, adrenal glands, thymus, testicles, and spleen were collected, weighed, and stored at − 80 °C. Blood samples were collected in tubes containing 5 IU·ml −1 heparin sodium salt.
A portion of the biological material was frozen and homogenised. Fullerene was extracted with 1 ml of toluene in an ultrasonic bath for 30 min. Proteins precipitated in the extracts obtained, thereafter, the extracts were centrifuged at 12,000 g for 15 min at 4 °C. The centrifugate was diluted with the mobile phase in a 1:2 ratio, re-centrifuged, and the resulting solution was analysed by electrospray ionisation gas chromatography-mass spectrometry (AgilentVarian 500-MSLC), chromatographic column Microsorb-MV 100-5 (250 mm × 4.6 mm, 5 μm, 100 A), the voltage across the capillary was − 4787 V. Figure 9 shows an example of the obtained chromatogram.

Results and discussion
Solubility study. Analysis of the temperature dependence of solubility showed that fullerene C 60 is quite well compatible with olive oil: solubility is from tenths to 1.2 g of fullerenes per litre of a solution in the temperature range from 0 to 80 °C (Fig. 10).
Analysis of the composition of the crystal solvate formed at relatively low temperatures (t ≤ 40 °C) showed that the weight loss of the crystal solvate sample after repeated washing with ethanol followed by drying in vacuum is Δm ≈ 18 ± 5 rel. wt.%. The average molecular weight of the mixed triglycerides that make up the basis of olive oil is M ≈ 885 ± 15 u. In the calculation, it was assumed that one averaged triglyceride molecule in olive oil contained 2.10 oleic acid residues CH 3 (CH 2 ) 7 CH=CH(CH 2 ) 7 COOH, 0.45 acid residue of palmitic acid CH 3 (CH 2 ) 14 COOH and 0.45 acid residue of linoleic acid CH 3 (CH 2 ) 3 (CH 2 CH = CH) 2 (CH 2 ) 7 COOH. Then the calculation makes it possible to obtain the values of the average composition of the crystal solvate C 60 (0.17 ± 0.05) TG (TG-a triglyceride of olive oil). Thus, one acidic residue in the triglyceride holds two C 60 fullerene molecules. It can be concluded that C 60 forms crystal solvates with a very low solvent content with natural vegetable oils.
The structural peculiarities of investigated system were focused on the most probable distances between the fullerene molecule and the atoms of the triglyceride molecule. Table 1 shows the determined maxima of the radial distribution functions (RDF) between the fullerene molecule and triglyceride atoms. The fullerene molecule www.nature.com/scientificreports/ interacts to a greater extent with the hydrophobic hydrocarbon chains of carboxylic acid residues as compared with the atoms of the glycerol backbone and oxygen atoms of carbonyl groups. This is due to the hydrophobicity of hydrocarbon groups and fullerene. The residues of oleic and palmitic acids create a connected network for a sufficient movement of a fullerene molecule in olive oil. Density field of oleic residues is presented in Fig. 11.
The energy parameters of a simulated structure are presented in Table 2.    Figure 11. A density field of oleic residues in a system under study. www.nature.com/scientificreports/ biomacromolecules of blood and tissues. Figure 12 shows the dependence of the content of fullerene C 60 in whole blood on time, which was interpolated using ninth-order polynomial in PKSolver software 60 : where a i (i = 0-9) are polynomial coefficients. The values of polynomial coefficients are summarised in Table 3. Based on experimental data (Fig. 12), PK parameters were calculated (Table 4). Area under the concentration time curve (AUC(0-t)) was calculated by ninth-order polynomial interpolation (see Eq. 2) and integration from zero time up to the last measured concentration: where C p (t) is concentration time function.
Area under the concentration time curve extrapolated to infinite time (AUC (0-∞)) was calculated using Eq. 4:   where C p (t = 0) is the value of concentration after extrapolation to zero time.
Biological half-life (t 1/2 ) is the time required for C 60 to be reduced to half of its maximum concentration: where k e is elimination rate constant. Oral clearance (CL/F) describes how efficiently C 60 is eliminated when administered orally: where D is dose (mg kg −1 ). Finally, apparent volume of distribution during terminal phase (V/F) was calculated using Eq. 8 61 : Analysis of Table 4 reveals that, in both cases, C 60 fullerene was found in the bloodstream achieving its highest concentration after 4 h after administration. In almost 5-6 h, C 60 concentration decreases twice. It can be noted that biological half-life for C 60 oil solution is less and it is eliminated faster than the micellar one. The comparison of all PK parameters reveals that the micellar form of C 60 is more suitable as a basis for further development of prolongated nanocompositions.
Based on the results of Table 4, the bioavailability of the micellar form of fullerene C 60 relative to the oil form was determined by the following equation: Taking into account that the administered dose is 15 mg·kg −1 for C 60 in oil and 1.5 mg·kg −1 for the micellar form, it follows that the micellar form is 12.2 times more bioavailable.
Figs. S1-S7 of the Supplementary Information and Table 5 demonstrate the dependence of the content of fullerene C 60 on time in the lungs, thymus, heart, liver, spleen, kidneys and adrenal glands. It was found that fullerene C 60 , administered to mice in oil and micellar forms, appears in the blood of experimental mice for 15 min after its oral administration, reaching a maximum at 60 min and persists for 6 h. After 8 h, a decrease in the concentration of C 60 and a further gradual a decrease up to a time of 24 h. Fullerene C 60 , administered to mice in oil and micellar forms, accumulates in the tissues of the liver, lungs, kidneys, heart, adrenal glands, thymus, and spleen. The detectable amounts of C 60 fullerene are also determined 24 h after the administration of the oil solution and the micellar dispersion of C 60 fullerene. The highest content of fullerene C 60 was found in the liver and adrenal glands. It is important to note that fullerene C 60 , administered to mice in oil and micellar forms, was not detected in the brain tissue and testes. This is probably due to the presence of tissue barriers in these organs, namely blood-brain and blood-testis, respectively.
In conclusion, we can say that since fullerenes have a number of unique properties, a relevant direction is the creation of systems for the delivery of these molecules and ways to increase their bioavailability. One of these Table 4. PK parameters of micellar and oil dispersions of C 60 in conventional mice (m = 25 g) after oral administration of C 60 in micellar and in oil forms (n = 10). *t max is time of maximum plasma concentration. **C max is maximum plasma concentration. www.nature.com/scientificreports/ forms can be the micellar form of C 60 in olive oil, obtained using TWEEN-80. The prospect of studying C 60 in olive oil was confirmed by the authors of ref. 62 , in which they carried out molecular and cytogenetic studies and showed that C 60 in olive oil reduces CdCl 2 -induced genotoxicity in liver, kidney and bone marrow tissues of rats. Moreover, in ref. 63 in experiments on erythrocyte integrity, platelet aggregation, and blood factors involved in coagulation, haemocompatibility of aqueous dispersions of C 60 was revealed. On the other hand, analysis of the literature shows that fullerenes can be used as platforms for targeted drug delivery. For example, a conjugate based on C 60 modified with glycine and docetaxel increases cellular uptake, efficacy of docetaxel and has an improved pharmacokinetic profile 64 .

Conclusion
In this work, for the first time, studies on the biodistribution of fullerene C 60 in two forms were carried out: a solution in olive oil and a micellar form under the ExtraOx trademark, created according to the original technology of "AQUANOVA RUS" JSC. As a result of the studies, it was found that the bioavailability of the micellar form of fullerene C 60 increases 12 times compared to the oil form.  Table 5. Fullerene content in various organs after administration of an oil dispersion of fullerene to mice after 1 h (C 1 ) and 24 h (C 24 ) (data are given as mean ± standard deviation of the sample).