Review of Synthesis and Antioxidant Potential of Fullerenol Nanoparticles
This review describes the chemical synthesis of polar polyhydroxylated fullerene C60 derivatives, fullerenols C60(OH)n, , C60HzOx(OH)y, and polyanion fullerenols C60(OH)15(ONa)9, ranging from the very first synthetic methods up to some contemporary approaches to synthesis and separation. It also provides some basic information about physical characteristics of fullerenols. With the increasing number of hydroxyl groups, water solubility of fullerenols increases as well. Fullerenols both in water and biological media build nanoparticles of different dimensions and stability. In different chemical and biological model systems a large number of various polyhydroxylated fullerene derivatives were tested and they showed both their antioxidative and prooxidative characteristics. Several mechanisms have been proposed for the antioxidant activity of fullerenol. In addition, this paper also provides insight into patents referring to the antioxidant properties of fullerenol.
Since its discovery by Kroto et al. in 1985, fullerene C60 molecule has had a significant impact on many scientific directions with a very interesting history . Starting from fundamental research of cluster carbon structures all the way to industrial production, fullerenes and their derivatives have now found a place in commercial products. Fullerene C60, unlike graphite and diamond, is chemically very reactive. So far, a large number of different chemical reactions and derivatives of fullerene C60 have been published in scientific papers [2, 3]. Spherical fullerene C60 behaves as an electron-deficient alkene and readily reacts with electron-rich species. Attachment of various polar functional groups or molecules on the fullerene core overcomes the almost complete insolubility of C60, while retaining the unique inherent fullerene properties, and achieves reasonable biological availability [3–5]. Several synthetic paths of fullerenols with various degrees of fullerenes hydroxylation , , polyanion fullerenols C60(OH)15(ONa)9, metallofullerenes Gd@C82(OH)22, and other fullerene derivatives have been published [6–20]. In aqueous solutions, depending on the pH value, fullerenols are more or less deprotonated and exist in the form of fullerenol nanoparticles (FNP). FNP are mostly important in the biological application of fullerenes, especially due to their antioxidant properties. Several mechanisms of FNP antioxidant activity are proposed here: the radical-addition reaction of radicals to the remaining olefinic double bonds of the fullerenol core, the ability of the hydroxyl radical to abstract hydrogen or an electron from fullerenol, and the formation of coordinative bonds with prooxidant metal ions. It has been shown in different model systems that FNP prevent the process of lipid peroxidation and possess superoxide, hydroxyl radical, and nitric oxide scavenging activity. The unique electronic -system of fullerene C60 and its derivatives make them potential photosensitizers upon the absorption of UV or visible light.
2. Fullerene C60
The fullerene C60 form of carbon was named after the American architect Buckminster Fuller, who was famous for designing a large geodesic dome which slightly resembles the molecular structure of C60. Fullerene is a compound composed solely of an even number of carbon atoms which form a three-dimensional cage-like fused ring polycyclic system with 12 five-membered rings and the rest are six-membered rings. All fullerenes have an even number of carbons. Spherical fullerene C60, known as buckyball, is the most representative member of the fullerene family with the shape of an icosahedron, containing 12 pentagons and 20 hexagons. Fullerene carbon atoms are considered to be equivalent, since C60 shows a single line at ppm in its 13C NMR spectrum. C60 behaves as a three-dimensional electron-deficient polyolefin. The pentagonal structures in C60 molecule contain single bonds, and the bridging bonds between pentagonal and hexagonal structures contain double bonds. All fullerenes which obey the so-called isolated pentagon rule are considered to be stable. Fullerene C60 is practically insoluble in water and other polar solvents and slightly soluble in toluene and benzene; however, it is soluble in 1,2-dichlorobenzene, dimethylnaphthalenes, and 1-chloronaphthalene. The chemical properties of fullerene C60 are based on the fact that the bonding has delocalized molecular orbitals extending throughout the structure, and the carbon atoms are a mixture of sp2 and sp3 hybridized systems. Fullerene C60 is not “superaromatic” as it tends to avoid double bonds in the pentagonal rings, resulting in poor electron delocalization. As a result, C60 behave as an electron deficient alkenes and reacts readily with electron-rich species. The main types of chemical reactions of C60 are nucleophilic addition, pericyclic reactions, radical additions, oxidation, electrophilic addition, halogenations, and the formation of endohedral complexes M@C60, where M usually refers to an atom of metal . Figure 1 presents the main chemical reactions on fullerene C60.
The principal reactions are electrophilic addition reactions and are therefore exothermic in most cases (these reactions are accompanied by a charge of hybridization of the carbon atoms from sp2 to sp3, which reduces angular strain in the cage). The number of addends decreases the exothermic heat of the reaction. Therefore, adducts with a high degree of addition become unstable. As a result, a great number of isomers are formed that is one of the biggest problems in the synthesis of only one derivative. For example, two addends C60X2 can have eight regioisomers (23 stereoisomers). The chemical properties of C60 (nucleophilic and electrophilic additions, pericyclic reactions, and radical additions) enable the covalent bonding of many different organic compounds and functional groups on its cage. Water-soluble fullerene-based derivatives are the most important for the biological application of fullerenes.
3. Water-Soluble Fullerene C60 Based Derivatives, Fullerenol C60(OH)n
The attachment of various polar functional groups or molecules to the fullerene core overcomes the almost complete insolubility of fullerene C60, while it retains its unique inherent fullerene properties and achieves reasonable biological availability [3–5, 53, 54]. Fullerene derivatives have been widely investigated in various chemical and biological experimental models. Special attention has been paid to the investigation of carboxyfullerenes , where the tris(dicarboxymethyl)-fullerene C3 isomer has been most extensively studied, as well as bisphosphonate fullerene derivatives and amino derivatives of fullerene [3–5].
Several synthesis paths of fullerenols with various degrees of hydroxylation and a general formula of , or have been published since 1992. The solubility of a fullerene molecule is dependent on the number of introduced hydroxyl groups. The low-degree hydroxylated fullerenols can dissolve in some polar solvents, for example, THF, dimethylformamide (DMF), and dimethyl sulfoxide (DMSO), and the medium-degree fullerenols C60(OH)16 and are reported to dissolve even in water. The specific behavior of fullerenols is a consequence of their structural flexibility, the rotation of the OH groups around the axes going through the C–O bonds, and the distribution of these groups across different carbon sites of the fullerene surface . Fullerenol in a molecular state can be obtained at concentrations below 20 mg/dm3. The sonication of fullerenol solutions increased their agglomeration and caused the formation of nanofullerenol clusters predominately with diameters of 10.7 or 102 nm, suggesting that clusters of these sizes were more stable and, hence, energetically more favored, which was supported by zeta potential measurements . The relationship between fullerenol concentration and zeta potential warrants a more in-depth sensitivity analysis in order to assess how higher concentrations impact biological response . Fullerenols simultaneously have both attractive (C–OH) and repulsive (C–O−) sites. The acidic protons could be involved in attractive hydrogen bonding interactions with other fullerenol molecules, driving nanocluster formation which would decrease the hydrophobic portion of the molecular surface area . Depending on the number of hydroxyl groups per C60 molecule, the pH values and concentration of fullerene stable nanoclusters range from 10 to 250 nm. Since the protonation state of polyhydroxylated C60 is pH dependent, in aqueous solutions, depending on the pH value, they are more or less deprotonated and exist in the form of stable polyanion nanoparticles. Most of the investigations of fullerene derivatives on biological model systems (especially investigations of antioxidant potential) were conducted with the polyhydroxylated derivatives and endohedral fullerenes listed below: , , C60(OH)16, , , C60(OH)24, C60(OH)36, C60(OH)44, , C60O5(OH)18, C60(OH)15(ONa)9, Gd@C82(OH)22, (, , and ).
3.1. Synthesis and Characterization of Hydroxylated Fullerene
Many methods for synthetizing polyhydroxylated fullerene C60 have been reported in scientific publications. Commercially available fullerenols (http://buckyusa.com/Polyhydroxy.htm) as well those synthetized in laboratory conditions have a certain variability in the chemical composition of the overall oxygen and monooxygenated surface groups. These products do not have good reproducibility in structural characterization which creates difficulties for experimental studies.
3.2. Polyhydroxylated Fullerene Derivatives C60(OH)n
In the early 1990s Li et al. synthesized C60 fullerenol with 24–26 hydroxyl groups directly by the reaction of fullerene C60 with aqueous NaOH in the presence of tetrabutylammonium hydroxide (TBAH), the most effective catalyst in aerobic conditions and at room temperature . Methanol was used for the separation of the reaction mixture. IR spectra showed characteristic absorption bands at 3430 cm−1 (–OH), 1400, 1070 cm−1 (C–O), and 1600 cm−1 (C=C). In the 1H NMR spectrum in DMSO-6 the mean peak was found at ppm, while in D2O 6 the mean peak was found at ppm. The 13C NMR spectrum had one broad peak at ppm. Using elemental microanalysis Li et al. determined that synthesized fullerenol had 26,5 hydroxyl groups. The same procedure of fullerenol synthesis was done in an argon flow. The reaction was slower and a maximum 10 hydroxyl groups were attached to the fullerene core.
3.3. Fullerenol Synthetized Using Hemiketal Groups
In brief, a fullerene mixture of C60 (84%) and C70 (16%) was treated with oleum (H2SO4–SO3), and the solution was stirred to give a green solution with suspension. An excess of potassium nitrate (KNO3) was then added to this acid suspension at 5°C. The resulting aqueous acid solution was filtered through Celite under vacuum to remove insoluble particles. The filtrate was basified until the pH reached 9.0 or higher. During base neutralization, the color of the solution slowly turned dark with fine, brown suspensions. The precipitate was separated from the solution by a centrifuge technique and washed several times with a NaOH solution (1 mol/L) and methanol to provide brown solids of polyhydroxylated fullerene derivatives . The spectral characteristics of the obtained fullerenol were as follows: IR = 3424 (–OH), 1595, 1392, 1084, and 593 cm−1; 13C NMR (D2O) , 140.3, 100.0, and 79.0 ppm; and solid-state 13C NMR , 141.1, 103.1, and 78.3 ppm. The basic analysis of the obtained fullerenol resulted in the following: C-43.5, H-3.1, O-46.9, N-0.52, Na-2.3, and S-1.6%. In the second method, the fullerenol was prepared as follows: a fullerene mixture of C60 (84%) and C70 was treated with concentrated sulfuric acid and concentrated nitric acid. The mixture was slowly heated to 115°C and stirred at that temperature for 4–6 h. It was cooled to room temperature and basified until the pH of the product solution reached 9.0 or higher. To provide brown solids of polyhydroxylated fullerene, the above explained separation procedure was carried out. The X-ray photoelectron spectroscopy analysis (XPS) indicated that obtained fullerenol molecule had monooxygenated carbons (287.9 eV, 23%) such as ethereal or hydroxylated carbons, dioxygenated carbons (289.7 eV, 9%) such as carbonyl (C=O), ketal (RO–C–OR), or hemiketal (RO–C–OH) carbons, and nonoxygenated carbons (286.1 eV, 68%). The estimation is that the average number of hydroxyl additions is 14–16 with approximately 6-7 hemiketal moieties per fullerene molecule. The solid-state 13C NMR showed peaks at ppm (hydroxylated carbons), ppm (hemiketal carbons), ppm (unreacted olefinic carbons), and ppm (vinyl ether carbons). These spectra provided consistent evidence to support the structural assignment of fullerenols containing hemiketals with vinyl ether linkages. A TGA-mass spectroscopy analysis of fullerenol detected the thermal elimination of H2O, CO from monooxygenated carbons and CO2 from dioxygenated carbons.
3.4. Fullerenol Synthesis Using Hydroborate
An excess of the BH3-THF complex was added to a solution of fullerene dissolved in dry toluene. The reaction mixture became increasingly brown due to precipitation of a solid intermediate, , leaving the supernatant toluene colorless . The intermediate was then treated with a solution of H2O2 followed by NaOH. The resulting mixture was stirred for 3 h and allowed to settle overnight. The obtained brown precipitate was soluble in dimethyl sulfoxide and pyridine, sparingly soluble in diluted HCl and slightly soluble in water. The IR spectrum of the obtained precipitate was 3430, 1631, 1385, 1090, and 450–550 cm−1 (from unreacted fullerene). The above described procedure of fullerene derivatization in water-soluble form produces fullerenol with a variable number of hydroxyl and other functional groups. In the second procedure, a slight variation of reaction conditions was used for the synthesis of . The intermediate was than treated with glacial acetic acid and washed with NaHCO3 solution. The IR spectrum of the residual solid in toluene gave characteristic IR stretching bands of C–H and O–H groups; 1H NMR spectrum in C6D6 was found with a peak of ppm (C–H group) and two unidentified peaks at and 6.03 ppm.
3.5. Fullerenol Synthesis from Polybrominated Derivative
The procedure for catalytical bromination of C60 with elementary bromine with FeBr3 as a catalyst is described in the paper published by Djordjević et al. In this procedure only one reaction product—C60Br24—was obtained without any occluded bromine molecules . The excess of unreacted bromine was evaporated and the catalyst was separated from the reaction mixture by washing it with an acidic aqueous solution pH 2. A thermogravimetric analysis showed that in the process of thermal transformation all bromine atoms are lost, which is a characteristic of the completely symmetrical distribution of bromine over the C60 molecule. FTIR and ray analysis were in accordance with published data.
The polyhydroxylated polyanion C60 derivative, fullerenol , was obtained by complete substitution (SN2 mechanism) of bromine atoms from C60Br24 with hydroxyl groups in alkaline aqueous solution pH 12. The aqueous solution of fullerenol with residual amounts of NaOH and NaBr was applied on the top of the combined ion-exchange resin and eluted with demineralized water until discoloration. The solution of fullerenol (pH = 7) was evaporated under low pressure; a dark brown powder substance of fullerenol C60(OH)24 remained (see the following) .
Synthesis of fullerenol C60(OH)24 from polybrominated derivative C60Br24  is as follows:
Analysis: FTIR C60(OH)24: 3426 cm−1, 1596 cm−1, 1359 cm−1 1062 cm−1; 1H NMR singlet peak at ppm; 13C NMR peaks at ppm and ppm, UPLC retention time at 4.49 min MS/MS 1128 ; UV/VIS maximum at 211 nm; °C (moisture), 252°C and 455°C. Water and DMSO as a cosolvent, physiological saline solution, cell culture media (DMEM, RPMI 1640), and human blood serum provide conditions for the good stability of fullerenol nanoparticles as is the case with water (or water/DMSO) and physiological saline.
The solubility of fullerenol in water was 11 mg/mL, while in the DMSO/water mixture (9 : 1 v/v) it was more than 37 mg/mL. The size distribution of particles by number analysis revealed the presence of particles of dimensions ranging from 10 to 50 nm, with a maximum of 15.7 nm. Fullerenol nanoparticles dissolved in water pH 6.5 had a negative charge mV. A change in the pH of the aqueous solution (from 2 to 11) affected the negative charge of the nanoparticles. Fullerenol nanoparticles are formed from the more organized molecules that can aggregate, and they form stable agglomerates ranging in dimension within 20–60 nm. AFM images of fullerenol nanoparticles in aqueous solution pH 6.5 are presented in Figure 2. AFM measurements of fullerenol nanoparticles are made by using the standard AFM tapping mode with a tip radius lower than 10 nm. Highly orientated pyrolytic graphite (HOPG) was used as a surface.
Structures of fullerenol molecule C60(OH)24 and fullerenol polyanion nanoparticles are presented in Figure 3. The space between the polyanion molecules in nanoparticles is filled with water molecules connected with hydrogen bonds. The ability of to self-assemble opens the possibility of the application of nanoparticles as a nanodelivery system of active principles in biological models.
3.6. Fullerenol Synthesis Using PEG 400 as a Catalyst
Zhang et al. synthesized fullerenols via the direct reaction of fullerene with aqueous NaOH comprising polyethylene glycol (PEG) 400 as a catalyst . The substitution of TBAH with PEG 400 as a catalyst represents a modification of the method described by Li et al. . Depending on the reaction conditions, either water-soluble C60 fullerenol (fullerenol 1) or water-insoluble C60 fullerenol (fullerenol 2) could be obtained selectively. The elemental analyses of fullerenols 1 and 2 showed an average composition of ( and 27 for 1 and 2, resp.). Both fullerenols showed similar IR spectra: 3432 cm−1, 1063 cm−1, and 1600 cm−1; 1H NMR spectra were also similar: a single strong peak centered at ppm, corresponding to hydroxyl protons. With the increase of the concentration of PEG and NaOH, the conversion of fullerene to water insoluble fullerenol (fullerenol 2) was significantly accelerated. Longer reaction time was needed when the reaction was carried out in N2 than in air, which proved that the PEG 400 was a more effective catalyst than some other catalysts such as TBAH. Addition of the aqueous NaOH to the benzene solution of C60 obtained a high percentage of water-soluble fullerenol 2.
3.7. Synthesis of Fullerenol Covered by More Than 18 Hydroxyl Groups
The starting material for the synthesis of fullerenol with more than 18 hydroxyl groups  was fullerenol 1 C60(OH)12, sodium free, synthesized by the method reported by Chiang et al. . The starting material C60(OH)12 (fullerenol 1) was added to a 30% hydrogen peroxide solution, and the mixture was vigorously stirred for 4 days under air at 60°C until the suspension turned into a clear yellow solution. After the solution cooled down, the addition of a mixed solvent of 2-propanol, diethyl ether, and hexane gradually yielded a milky white precipitate. Drying of the residue gave 67% of pale yellow-brown powder of C60(OH)36·8H2O (fullerenol 2). Similar treatment of C60(OH)12 (fullerenol 1) for a prolonged reaction time at 60°C for up to 2 weeks, within the same workup as given above, provided 68% of C60(OH)40·9H2O (fullerenol 3) as a milky white powder. The IR spectra of fullerenols 2 and 3 were 3400, 1080, 1370, and 1620 cm−1. The elemental analysis of fullerenol 2 resulted in C60(OH)36·8H2O and fullerenol 3 resulted in C60(OH)40·9H2O. The solubility (25°C, pH 7) of fullerenol 2 was 17.5 mg/mL and fullerenol 3 58.9 mg/mL, while the solubility of polyanion fullerenol was more than 200 mg/mL despite the moderate number of hydroxyl groups . Such a type of water-soluble fullerenol might include a few sodium ions because of the synthetic process using NaOH as hydroxylation or neutralization reagent and the difficulty in complete removal of the sodium ion from the weakly acidic or chelation-natured fullerenol [7, 16]. Presumed mechanisms of fullerenol formation in an alkaline medium and by oxidation with molecular oxygen are shown in Figure 4 .
Because the simple acidification of fullerenol must induce the acid-catalyzed pinacol rearrangement, it is difficult to remove the sodium ion completely without using a column chromatography process. It is noteworthy that the water solubility of fullerenol 3 was much higher than that of 2 because of the greater number of hydroxyl groups of the former. The weight loss of fullerenol 2 (C60(OH)36·8H2O) was observed in three temperature ranges, that is, room temperature to 130°C, 130–350°C, and 350°C. The first weight loss is assigned to the secondary bound water; the second reduction might be attributed to dehydration of the introduced hydroxyl groups and, for example, by possible thermal pinacol rearrangement, whereas the third reduction might be attributed to the decomposition of the fullerene nucleus. The particle size of fullerenol 2 measured using dynamic light scattering (DLS) analysis was 1 nm. The addition of NaOH to the solution of fullerenol 2 up to pH 12 revealed a high extent of aggregation (50–100 nm) of the fullerenol, although the addition of HCl (pH 2.6) essentially did not affect the particle size. The observed phenomenon was rationalized on the basis of a strong interaction between the metal cation (Na+) and the fullerenol, leading to aggregation or finally precipitation. Precipitation phenomena have not been noticed with alkali metals, while complete precipitation of fullerenol occurred with alkaline earth metals and transition metals . Addition of a mixture of 2-propanol, diethyl ether, and hexane (5 : 5 : 5) into the reasonably concentrated aqueous solution of the fullerenol 2 or 3 led to the formation of fullerenol aggregation. The addition of the poor solvent probably reduced the solvation of the fullerenol by water molecules and increased the intermolecular hydrophobic interaction. The synthesis of C60(OH)36·8H2O and C60(OH)40·9H2O is presented in Figure 5.
A possible reaction mechanism for the formation of the fullerenol with a high number of hydroxyl groups is that the basic hydroxide ion –OH induces hydroperoxide ion –OOH formation as a result of the slightly higher acidity of H2O2 than that of H2O (Figure 6) [15, 18]. The formed –OOH attacks C60 to give fullerene epoxide C60O, followed by the attack of –OH and protonation. The obtained fullerene epoxide was susceptible to subsequent nucleophilic attacks of –OH and –OOH because of the higher strain.
3.8. Synthesis of Fullerenol Prepared by the Direct Oxidation Route
Semenov et al.  started their synthesis of fullerenol by using fullerenol (fullerenol-d, i.e., fullerene-direct) synthesized by the method reported by Li et al. . Briefly, a near-saturated solution of С60 in benzene was prepared and NaOH solution and solution of tetrabutylammonium hydroxide were added. Benzene was distilled and the resulting mixture was stirred for 12–15 h, during which time the resulting fullerenol-d was extracted to the aqueous phase. Adding methanol to the resulting solution caused the salting out of fullerenol-d from the aqueous solution as a brown flaky precipitate. The precipitate was separated from the liquid phase and additionally washed repeatedly with methanol until neutral pH was obtained, after which it was dried. The yield of red-brown crystals of fullerenol-d was 72%. FTIR spectra were 3420 cm−1, 1590 cm−1, 1450 cm−1, and 1040 cm−1; HPLC analysis determined the following: a broad peak maximum near 6.1 min. This indicates that the column that Semenov et al. employed did not allow the separation of the main product, fullerenol-d, since fullerenol-d is a mixture of polyalcohols С60(OH)n, oxypolyalcohols C60(OH)n1On2, or their salts C60(OH)n1On2(ONa)n3. Qualitative mass spectra of fullerenol-d have distinctive peaks corresponding to ≈ 970–1317. The mean expectancies formula for fullerenol ≈ 1094–1128 was .
3.9. Synthesis and Separation of Fullerenol Based on Dialysis
Fullerenol, prepared according to a two-phase reaction by using NaOH, contains Na ions . A dialysis-based method was developed by Yao et al. to remove Na ions in fullerenol preparation . The used dialysis membrane had a molecular weight cut-off (MWCO) of 8–15 kDa. A dialysis route for fullerenol prepared by the reaction of fullerene with aqueous NaOH and tetrabutylammonium hydroxide (TBAH) is shown in Figure 7.
FTIR spectrum for purified fullerenol resulted in 1080 cm−1, 1380 cm−1, 1600 cm−1, and 3400 cm−1; 1H NMR spectrum ppm. More Na elements are eliminated by the prolonged dialysis time.
3.10. Synthesis of Fullerenol as a Single Nanoparticle
Kokubo et al. synthesized fullerenol C60(OH)44 in a facile one-step reaction from the toluene solution of C60 by hydroxylation with hydrogen peroxide in the presence of a phase-transfer catalyst, tetra-n-butylammonium hydroxide (TBAH) . The mixture was stirred under air at 60°C until the purple toluene layer turned into a colorless transparent solution. An aqueous solution was separated and a mixed solvent of 2-propanol, diethyl ether, and hexane (7 : 5 : 5) was added to yield a milky white precipitate. The residual solid was washed with diethyl ether and dried. A pale yellow powder of fullerenol was obtained. To remove residual TBAH, fullerenol was dissolved in deionized water and the resulting yellow solution was passed through an active magnesium silicate. Addition of a mixed solvent afforded a brownish-yellow precipitate. The solid was washed with diethyl ether. Drying of the solid gave purified fullerenol 2 (67%) as a milky white to yellow powder. The IR spectrum of fullerenol 2 was 3400, 1080, 1370, and 1620 cm−1. Weight losses for fullerenol 2 were observed in three temperature ranges on the TGA trace recorded: room temperature to 120°C, 120–250°C, and >250°C. The first weight loss can be assigned to secondary bound water; the second reduction can be attributed to the dehydration of the introduced hydroxyl groups, while the weight loss at the highest temperature (>250°C) can be attributed to the decomposition of the fullerene nucleus. The average structure of fullerenol 2 was deduced to be C60(OH)44·8H2O from elemental analysis. Fullerenol 2 exhibited high water solubility, up to 64.9 mg/mL, under neutral (pH = 7) conditions. The narrow distribution of the particle sizes by number ( nm) indicates that fullerenol 2 is highly dispersed at a molecular level and that the usual aggregation of fullerenols is not prevalent. This could be because fullerenol 2 is surrounded by solvent-water molecules as a result of the strong hydrogen bonding with the introduced hydroxyl groups. The particle size distribution obtained from the induced grating method (IG method) was consistent with the previously mentioned DLS results. The average particle size was determined to be 0.806 nm ± 0.022 nm. To compare and verify the data obtained by the DLS and IG methods, Kokubo et al. conducted the particle size measurement again by means of scanning probe microscopy (SPM). The average particle size of fullerenol 2 was determined to be 1.03 nm ± 0.28 nm. The results of the particle size measurement by three different methods confirm that the highly hydroxylated fullerenol nanoparticles have a highly dispersed nature in water. The surface nanostructure of fullerenol 2 in powder form was also observed by SPM. It revealed nanoscale spherical structures of about 30–50 nm in diameter which combine with a second particle to form a larger third particle on a microscale. The solid state of fullerenol therefore exists in an aggregated form but disperses at a molecular level once it is dissolved in water. Table 1 shows the methods of synthesis of hydroxylated derivatives of C60, fullerenols.
4. Antioxidative and Prooxidative Potential of Fullerenols
4.1. Scavenging Potential of Various Free Radical Types of Polyhydroxylated Derivatives of Fullerene
Many of the water-soluble fullerene derivatives have been recognized for their antioxidant properties: amphiphilic monoadducts of fullerene , C3 and D3—trismalonyl derivative , endohedral fullerenol Gd@C82(OH)22, and fullerenol C60(OH)22 [60–62]. Several mechanisms for the antioxidant activity of fullerenol nanoparticles (FNP) have been proposed. In aqueous solution, nanomolecules of fullerenol form hydrogen bonds with H2O and other molecules of fullerenol, creating stable negatively charged nanoparticles. Electron spin resonance (ESR) spectroscopy revealed that fullerenol has the ability of the dose-dependent inhibition of the ESR signal intensity of DPPH (2,2-diphenyl-1-picrylhydrazyl) radical. The possible mechanism of the antioxidative activity of fullerenol C60(OH)24 is the radical-addition reaction of radicals to the remaining olefinic double bonds of the fullerenol core to yield (), in a dose-dependentmanner. The other proposed mechanism is the possibility of a hydroxyl radical to abstract a hydrogen from fullerenol, including the formation of a relatively stable fullerenol radical . In addition, a hydroxyl radical may abstract one electron from fullerenol yielding the radical cation . One more proposed mechanism is that the polyanion nanoparticles have numerous free electron pairs from oxygen, distributed around the FNP, and have a great capacity to form coordinative bonds with prooxidant metal ions . In a liposome model system of cell membranes, Mirkov et al. showed that FNP prevents the process of lipid peroxidation. Treatment of liposomes with FeSO4 and ascorbic acid led to the oxidation of polyunsaturated fatty acid in liposomes and formation of TBARS. The results showed that fullerenol-induced dose-dependent inhibition of FeSO4/ascorbic acid-stimulated formation of TBARS. In parallel, the authors examined the effect of butylated hydroxytoluene (BHT) on lipid peroxidation and the obtained results demonstrated that fullerenol possesses similar efficiency in the prevention of lipid peroxidation as BHT. For the determination of the superoxide radical scavenging activity of FNP, the authors applied fullerenol into the xanthine/xanthine oxidase system which caused a decrease in the reduction rate of cytochrome c compared to the control. The obtained result demonstrated that fullerenol in the range of nanomolar and micromolar concentrations decreased the reduction of cytochrome c between 5 and 20%, while concentration of 1 mM decreased reduction of cytochrome c for 40% . The hypothetical mechanism of action of the polyanion fullerenol C60(OH)24 with the superoxide anion radical is presented in Figure 8 .
The antioxidant ability of the water-soluble derivative of fullerene C60(OH)32·8H2O was assessed by DMPO-spin trap/ESR method. This C60 derivative had an ability to diminish the ESR spectrum attributed to hydroxyl radicals. Meanwhile, a singlet radical-signal different from OH-attributed signals increased in a manner dependent on the concentrations of C60(OH)32·8H2O. These results suggest that C60(OH)32·8H2O scavenges owing to the dehydrogenation of C60(OH)32·8H2O and is simultaneously oxidized to a stable fullerenol radical . The antioxidant ability of C60(OH)32·8H2O was also confirmed in beta-carotene bleaching assay .
The first proof of the nitric oxide scavenging activity of FNP in different model systems was in the solution of SNP which is a spontaneous liberator of NO in the presence of light irradiation. The obtained results showed that the presence of fullerenol in a SNP solution decreased the levels of nitrite, in comparison to the nitrite levels obtained when SNP was dissolved alone. To test the possible in vivo NO-scavenging activity of FNP, the antioxidant defense in adult rat testis was used as a model system. The effects of the NO-scavenging activity of FNP on the activities of testicular antioxidant enzymes were investigated after intratesticular (i.t.) injection of SNP and fullerenol into each testis. Pretreatment of the rats with an i.t. injection of fullerenol completely prevented an SNP-induced reduction in the activities of catalase, glutathione S-transferase, and glutathione peroxidase. FNP, applied alone, did not induce any changes in the activity of the studied antioxidant enzymes, with the exception of decreased glutathione transferase activity. These results suggest that FNP possess NO-scavenging activity in vivo . The scavenger activity of fullerenol with a smaller or moderate number of hydroxyl groups with OH radicals can be explained by addition to sp2 carbon atoms [63, 66]. Table 2 presents the results of fullerenol scavenger activities in different biological systems.
4.2. Phototoxic Properties of Water-Soluble Fullerene Derivatives
The unique electronic -system of fullerenes and its derivatives makes them potential photosensitizers upon the absorption of UV or visible light. Fullerenol C60(OH)24 produces a mixture of reactive oxygen species (ROS) under both visible and ultraviolet irradiation through two types of photochemical mechanisms , with the greatest rates of oxygen consumption at acidic pH (pH = 5) (see the following).
Potential reaction mechanisms of ROS generation via photosensitization of fullerenol C60(OH)24  are as follows:
Evidence of both singlet oxygen (1O2) and superoxide production () was obtained and when compared to other known sensitizers of reactive oxygen, fullerenol C60(OH)24 produced more ROS at a rate at least two times that of other sensitizers. Because of all these features, fullerenol and other water-soluble derivatives could exhibit high toxicity toward epithelial cells and promote photocatalytic degradation of environmental hazards.
The formation of superoxide anion radical was observed when a solution of fullerenol C60(OH)24 was irradiated (>400 nm). Comparing phototoxicity toward HaCaT of (γ-CyD)2/C60 (c-cyclodextrin bicapped C60) and fullerenol, Zhao et al. concluded that fullerenol was less phototoxic . The aggregation of fullerenol in aqueous solution results in a loss of its intrinsic photochemical reactivity with respect to the production of superoxide and singlet oxygen [69, 70]. The free radical (type I) mechanisms are considered to be involved in fullerenol phototoxicity.
4.3. Structures and Stabilities of Fullerenols
Antioxidative characteristics of the polyhydroxylated fullerene derivatives depend both on the number of hydroxyl groups and their arrangement on the C60 sphere [55, 67, 71]. Semiempirical calculations suggest that, in terms of thermodynamics, fullerenols are the most stable with 6 and 12 hydroxyl groups which are symmetrically arranged on the sphere of the C60 and with the smallest number of double bonds, 5, 6 [14, 72, 73]. Another method, such as density functional theory, suggests that the structures with 7 hydroxyl groups arranged on the one side of the C60 sphere are the most stable. The next stable structure is the one with 14 hydroxyl groups symmetrically arranged on both sides of the C60 [74, 75]. Theoretically speaking, the fullerenol forms with 24 hydroxyl groups which are arranged on the equator of the C60 sphere are the most stable . Fullerenols with more than 24 hydroxyl groups have a tendency to open and destabilize cages. Characteristic functional groups that may appear in an open cage include hydroxyls, epoxies, carbonyls, and hemiacetals . Pitek et al. used theoretical models to show that a small cluster of fullerenol C60(OH)24 with 7 molecules is the most stable . Fullerenols with about 20 hydroxyl groups form negatively charged nanoagglomerates in a wide pH range in water media and in the presence of cosolvents such as DMSO [7, 51].
4.4. Patents Related to the Antioxidant Properties of Fullerenol
The patents related to the antioxidant properties of fullerenol are listed in Table 3.
The paper presents the syntheses, stability, and main antioxidant characteristics of the fullerenol molecule on biological models. The largest number of fullerenol synthesis procedures was performed in acidic and alkaline conditions. The process of synthesis over a polybrominated precursor results in a reaction product with 24 hydroxyl groups on C60. With an increase in the number of hydroxyl groups, the water solubility of fullerenols increases as well. Fullerenols with a larger number of hydroxyl groups were derived by alkaline procedure synthesis. With the increasing number of hydroxyl groups per C60 sphere, the number of other potential functional groups, such as carbonyls and epoxies, increases likewise. Defining the fullerenol structure in such cases is more complex. Thermodynamically, the most stable fullerenol structure is the one with 24 hydroxyl groups, which is theoretically described with the OH groups arranged on the C60 sphere. The experimentally proven structure with 24 hydroxyl groups is characterized by the symmetrically arranged distribution of the OH groups on the C60 cage. Fullerenols with up to 26 hydroxyl groups tend to form agglomerates of nanometric sizes in aqueous solutions. Fullerenols have shown excellent antioxidant characteristics in many biological models. In certain photoinduction cases fullerenols show prooxidative characteristics. The scavenging activity of the polyanion fullerenols with 24 hydroxyl groups with O2 is explained through the formation of the peroxyradicals on fullerenol. The greatest number of biological studies has been conducted with fullerenols . The characteristic of these fullerenols (with the mean number of hydroxyl groups) to form stable polyanion nanoagglomerates both in water and other biological media indicates a possible basic path of antioxidative characteristics in biological models.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by a grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant no. III 45005.
A. Hirsch and M. Brettreich, “Cluster modified fullerenes,” in Fullerenes, pp. 345–358, Wiley, 2005.View at: Google Scholar
F. Cataldo and T. Da Ros, Medicinal Chemistry and Pharmacological Potential of Fullerenes and Carbon Nanotubes, Springer, 2008.
A. Djordjevic, G. Bogdanovic, and S. Dobric, “Fullerenes in biomedicine,” Journal of the Balkan Union of Oncology, vol. 11, no. 4, pp. 391–404, 2006.View at: Google Scholar
J.-M. Zhang, W. Yang, P. He, and S.-Z. Zhu, “Efficient and convenient preparation of water-soluble fullerenol,” Chinese Journal of Chemistry, vol. 22, no. 9, pp. 1008–1011, 2004.View at: Google Scholar
D. N. Johnson-Lyles, K. Peifley, S. Lockett et al., “Fullerenol cytotoxicity in kidney cells is associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction,” Toxicology and Applied Pharmacology, vol. 248, no. 3, pp. 249–258, 2010.View at: Publisher Site | Google Scholar
M. P. Gelderman, O. Simakova, J. D. Clogston et al., “Adverse effects of fullerenes on endothelial cells: fullerenol C60(OH)24 induced tissue factor and ICAM-I membrane expression and apoptosis in vitro,” International Journal of Nanomedicine, vol. 3, no. 1, pp. 59–68, 2008.View at: Google Scholar
K. Stankov, I. Borisev, V. Kojic, L. Rutonjski, G. Bogdanovic, and A. Djordjevic, “Modification of antioxidative and antiapoptotic genes expression in irradiated K562 cells upon fullerenol C60(OH)24 nanoparticle treatment,” Journal of Nanoscience and Nanotechnology, vol. 13, no. 1, pp. 105–113, 2013.View at: Publisher Site | Google Scholar
Y. Saitoh, A. Miyanishi, H. Mizuno et al., “Super-highly hydroxylated fullerene derivative protects human keratinocytes from UV-induced cell injuries together with the decreases in intracellular ROS generation and DNA damages,” Journal of Photochemistry and Photobiology B: Biology, vol. 102, no. 1, pp. 69–76, 2011.View at: Publisher Site | Google Scholar
X. Cai, H. Jia, Z. Liu et al., “Polyhydroxylated fullerene derivative C60(OH)24 prevents mitochondrial dysfunction and oxidative damage in an MPP+-induced cellular model of Parkinson's disease,” Journal of Neuroscience Research, vol. 86, no. 16, pp. 3622–3634, 2008.View at: Publisher Site | Google Scholar
A. M. da Rocha, J. R. Ferreira, D. M. Barros et al., “Gene expression and biochemical responses in brain of zebrafish Danio rerio exposed to organic nanomaterials: carbon nanotubes (SWCNT) and fullerenol (C60(OH)18−22(OK4)),” Comparative Biochemistry and Physiology A: Molecular and Integrative Physiology, vol. 165, no. 4, pp. 460–467, 2013.View at: Publisher Site | Google Scholar
M. Roursgaard, S. S. Poulsen, C. L. Kepley, M. Hammer, G. D. Nielsen, and S. T. Larsen, “Polyhydroxylated C60 fullerene (fullerenol) attenuates neutrophilic lung inflammation in mice,” Basic and Clinical Pharmacology and Toxicology, vol. 103, no. 4, pp. 386–388, 2008.View at: Publisher Site | Google Scholar
T.-H. Ueng, J.-J. Kang, H.-W. Wang, Y.-W. Cheng, and L. Y. Chiang, “Suppression of microsomal cytochrome P450-dependent monooxygenases and mitochondrial oxidative phosphorylation by fullerenol, a polyhydroxylated fullerene C60,” Toxicology Letters, vol. 93, no. 1, pp. 29–37, 1997.View at: Publisher Site | Google Scholar
R. Injac, M. Boskovic, M. Perse et al., “Acute doxorubicin nephrotoxicity in rats with malignant neoplasm can be successfully treated with fullerenol C60(OH)24via suppression of oxidative stress,” Pharmacological Reports, vol. 60, no. 5, pp. 742–746, 2008.View at: Google Scholar
B. Srdjenovic, V. Milic-Torres, N. Grujic, K. Stankov, A. Djordjevic, and V. Vasovic, “Antioxidant properties of fullerenol C60(OH)24 in rat kidneys, testes, and lungs treated with doxorubicin,” Toxicology Mechanisms and Methods, vol. 20, no. 6, pp. 298–305, 2010.View at: Publisher Site | Google Scholar
A. Djordjevic, R. Injac, D. Jovic, J. Mrdjanovic, and M. Seke, “Bioimpact of carbon nanomaterials,” in Advanced Carbon Materials and Technology, pp. 193–271, John Wiley & Sons, 2014.View at: Google Scholar
I. Rade, R. Natasa, G. Biljana, D. Aleksandar, and S. Borut, “Bioapplication and activity of fullerenol C60(OH)24,” African Journal of Biotechnology, vol. 7, no. 25, pp. 4940–4950, 2008.View at: Google Scholar
A. Djordjevic, J. M. Canadanovic-Brunet, M. Vojinovic-Miloradov, and G. Bogdanovic, “Antioxidant properties and hypothetic radical mechanism of fullerenol C60(OH)24,” Oxidation Communications, vol. 27, no. 4, pp. 806–812, 2004.View at: Google Scholar
K. Kokubo, Water-Soluble Single-Nano Carbon Particles: Fullerenol and Its Derivatives, InTech, 2012.
S. Kato, H. Aoshima, Y. Saitoh, and N. Miwa, “Highly hydroxylated or γ-cyclodextrin-bicapped water-soluble derivative of fullerene: the antioxidant ability assessed by electron spin resonance method and β-carotene bleaching assay,” Bioorganic and Medicinal Chemistry Letters, vol. 19, no. 18, pp. 5293–5296, 2009.View at: Publisher Site | Google Scholar
H. Ueno, S. Yamakura, R. S. Arastoo, T. Oshima, and K. Kokubo, “Systematic evaluation and mechanistic investigation of antioxidant activity of fullerenols using carotene bleaching assay-carotene bleaching assay,” Journal of Nanomaterials, vol. 2014, Article ID 802596, 7 pages, 2014.View at: Publisher Site | Google Scholar
B. Zhao, Y.-Y. He, C. F. Chignell, J.-J. Yin, U. Andley, and J. E. Roberts, “Difference in phototoxicity of cyclodextrin complexed fullerene [(γ-CyD)2/C60] and its aggregated derivatives toward human lens epithelial cells,” Chemical Research in Toxicology, vol. 22, no. 4, pp. 660–667, 2009.View at: Publisher Site | Google Scholar
M. A. Orlova, T. P. Trofimova, A. P. Orlov, and O. A. Shatalov, “Perspectives of fullerene derivatives in PDT and radiotherapy of cancers,” British Journal of Medicine and Medical Research, vol. 3, no. 4, pp. 1731–1756, 2013.View at: Google Scholar
B. Zhao, P. J. Bilski, Y.-Y. He, L. Feng, and C. F. Chignell, “Photo-induced reactive oxygen species generation by different water-soluble fullerenes (C60) and their cytotoxicity in human keratinocytes,” Photochemistry and Photobiology, vol. 84, no. 5, pp. 1215–1223, 2008.View at: Publisher Site | Google Scholar
Z. Slanina, X. Zhao, L. Y. Chiang, and E. Osawa, “Biologically active fullerene derivatives: computations of structures, energetics, and vibrations of C60 and C60,” International Journal of Quantum Chemistry, vol. 74, no. 3, pp. 343–349, 1999.View at: Google Scholar
J. G. Rodríguez-Zavala and R. A. Guirado-López, “Stability of highly OH-covered C60 fullerenes: role of coadsorbed O impurities and of the charge state of the cage in the formation of carbon-opened structures,” The Journal of Physical Chemistry A, vol. 110, no. 30, pp. 9459–9468, 2006.View at: Publisher Site | Google Scholar