1. Introduction
Carbon nano-objects are of great interest for different fields of medicine, pharmacology, and biotechnology due to their specific biological activity [
1,
2,
3]. It is known that high-dose exposures to bioactive compounds can inhibit physiological functions of multiple organisms and, hence, produce toxic effects, while low-dose exposures can activate the physiological functions due to optimization of complex metabolic processes [
4].
Fullerenols (F) are known to be rigid nanosized carbon particles, and are water-soluble polyhydroxylated derivatives of fullerenes.
Scheme 1 presents the hypothetical structure of F with 60 carbon atoms as an example.
Similar to fullerenes, fullerenols are electron-deficient structures and this property makes them efficient catalyzers in biochemical reactions, as well as in prospective medical drugs. Fullerenols are amphiphilic structures: “hydroxyl groups provide them with aqueous solubility, while the fragments of fullerene skeleton—with affinity to hydrophobic enzymatic fragments and lipid structures of cellular membranes” [
1,
2]. Amphiphilic properties and antiradical activity provide a wide range of fullerenols’ biological effects: from neutralization of free radicals [
5] to cell protection and drug transportation [
1,
5,
6,
7]. Fullerenols can be used in radiobiology, chemotherapy, and neurology [
5,
6], ensuring an important alternative to conventional pharmaceuticals. The antioxidant properties endow fullerenols with the ability to neutralize reactive oxygen and nitrogen species [
8,
9,
10,
11,
12], and to function as radioprotectors [
10], antitumor [
13], or neurological [
5,
10,
11,
12] drugs.
Structural properties influence the biological activity of fullerenols. Hydroxyl substituents distort the π-electron system conjugation of the fullerene skeleton, change the electron-acceptor ability of the nanoparticles, and, hence, can affect their catalytic activity. This can result in different toxicity and antioxidant activity of fullerenols with different number of hydroxyl substituents.
Biological activity of C
60-fullerenols with different number of hydroxyl groups have been studied over the last decades [
5,
6,
7,
8,
9]. Toxic and antioxidant effects of fullerenols were revealed. However, different biological test objects did not provide a comparability of the fullerenol biological activity. Comparable conditions were provided by Eropkin and co-authors [
14]. They studied the biological activity of a series of fullerenols, C
60(OH)
12–14, C
60(OH)
18–24, and C
60(OH)
30–38. It was found that C
60(OH)
12–14 was insoluble in water and did not show biological activity, while C
60(OH)
18–24 was soluble and showed maximum antiviral and protective properties. The nitric oxide-scavenging and protective activity of C
60(OH)
18–24 was demonstrated in [
5,
8]. However, in spite of extensive studies of fullerenol biological activity, dependencies of their toxic and antioxidant characteristics on concentrations were not revealed and compared.
Hydration of C
60-fullerenol with different number of hydroxyl groups (from 8 to 44) was theoretically studied in [
15]. It was proved that hydration of C
60(OH)
44 was less effective despite the large number of hydroxyl groups. The authors concluded that the involvement of >36 hydroxyl groups to the fullerenol structure resulted in effective intramolecular interactions of OH-groups, conflicting with the hydrogen bonds with the solvent.
Since an increase of a number of hydroxyl substituents reduces the available π-electron system conjugation, it can reduce fullerenols’ ability of reversible radical trapping [
14]. This might be a reason for the variation in content of reactive oxygen species (ROS) in aerated aqueous solutions, with effects on the following biological structures: cells, enzymes, low-molecular components, etc.
It is known that ROS groups include a number of free oxygen radicals or radical precursors, such as semiquinones, superoxide anion-radical (•O
2−), hydroxyl (•OH) and peroxide (HOO•) radicals, hydrogen peroxide (H
2O
2), peroxide anion (HOO
−), singlet oxygen (
1O
2) [
16], hypochloric acid (HOCl), peroxynitrite radical (ONOO-), and others. They are formed in cells as natural products of oxygen metabolism, their content is labile, and they can initiate formation of additional radicals. ROS play a role as mediators of important intracellular signaling pathways [
17], thereby regulating cellular processes (respiration, division, etc.), inducing the immune system, mobilizing ion transport systems, and triggering programmed cell death (apoptosis) [
18].
Fullerenols’ catalytic activity is due to electron donation/acceptance and oxygen radical scavenging. For example, the process of O
2•−- neutralization can be presented as follows:
where F plays the role of a catalyzer. A detailed description of possible mechanisms of oxygen radicals’ neutralization is presented in [
11].
As the ROS impact on living organisms is evident [
19,
20,
21,
22,
23,
24,
25,
26,
27], a study of ROS content in fullerenol’ solutions can elucidate the mechanism of fullerenol’ biological effects.
Our paper used the physico-chemical approach to study the properties of bioactive compounds. This approach is based on the “structure-function” relations and allows for predicting the toxic and antioxidant properties of fullerenols. The relations can help in further applied studies, from synthesis to medical adaptation. Additionally, the relations can help to minimize further routine experiments with organs and whole organisms, which are usually time-consuming, expensive, and less reproducible.
The physico-chemical approach assumes an application of simple biological assays as models. We applied two types of bioassays of different levels of organization—cellular and enzymatic. Both bioassays used luminescence intensity as a test parameter. Luminescent registration provides high rates, low costs, and convenience of the bioassay procedures; it ensures simultaneous multiple analyses and, hence, proper statistical processing and reliability for the results of biomedical investigations.
Cellular bioassay is based on luminous marine bacterium. This bioassay is classic and has been widely used for more than five decades [
28,
29,
30,
31,
32,
33]. Enzymatic bioluminescence assay progressed from early 90s [
29,
34,
35,
36]. Solid immobilized bacterial and enzymatic preparations are developed now as a basis for bioluminescent biosensors [
34,
37,
38,
39,
40]. The bacterial and enzymatic bioassays are tools for investigation of the toxic mechanisms at the cellular and molecular levels, respectively. Classification of the toxic effects was suggested first in [
41] and developed later in [
42,
43,
44,
45]. It describes the (1) physicochemical, (2) chemical, and (3) biochemical basis for the toxic effects in the bioluminescence assay systems.
Antioxidant properties of bioactive compounds provided a new perspective for biosensor applications [
46,
47]. The bioassay systems, based on the luminous bacterium or its enzymatic reactions, are proper candidates for this application. Both assays can evaluate a general toxicity in the test samples under conditions of the oxidative stress. Additionally, the enzymatic bioassay is specific to oxidizers [
45] and can be applied to monitor the oxidative toxicity in the solutions. This type of toxicity is attributed to the redox properties of the toxic compounds only, while the other toxicity type, general toxicity, integrates all interactions of toxic compounds with the bioluminescent assay system: redox reactions, polar and non-polar interactions, etc. Previously [
48,
49], we evaluated the general and oxidative toxicities in solutions of inorganic and organic oxidizers (polyvalent metals and quinones) using the bioluminescent enzymatic assay system. Changes in general toxicity and oxidative toxicity under exposure to humic substances (bioactive compounds of natural origination, products of organics decomposition in soils) were studied in [
50,
51,
52]. The bioluminescence technique for evaluating the antioxidant activity of bioactive compounds was described in [
53,
54,
55]. Antioxidant properties of two fullerenols were studied for the first time and compared using bioluminescence enzymatic technique in [
53].
The current study develops a fundamental basis for fullerenol medical application. We review, analyze, and compare our new and previous experimental results, obtained under comparable conditions. Our paper analyzes antioxidant activity and toxicity of a series of fullerenols with different numbers of oxygen substituents. Chemical formulas of the fullerenols and their short abbreviations are presented in
Table 1. The fullerenols were attributed to two clusters involving either 24–28 or 40–42 oxygen substituents (F1, F2 or F3, F4, F5, respectively,
Table 1). Enzyme-based and cellular-based luminescent bioassays were applied to evaluate toxicity and antioxidant properties of the fullerenols. Ranges of toxic and antioxidant concentrations of fullerenols were of particular attention. Quantitative antioxidant characteristics (detoxification coefficients) are presented in a wide range of fullerenol concentrations. Possible antioxidant mechanisms and the role of ROS in the effects of the fullerenols are discussed. Toxic effect of a perspective endohedral metal-fullerenol with gadolinium atom involved (F5,
Table 1) is evaluated. A recommendation is provided for the synthesis of new low-toxic endohedral gadolinium-fullerenol preparation.
3. Materials and Methods
3.1. Preparations of Fullerenols
F1 and F2 were produced by fullerene hydroxylation in nitric acid followed by the hydrolysis of the polynitrofullerenes [
65,
66,
67,
68]. Preparation of F2 involved 60% of C
60O
y(OH)
x and 40% of C
70O
y(OH)
x. Fullerenes were preliminary synthesized by carbon helium high-frequency arc plasma at atmospheric pressure [
67,
69]. The carbon soot included 12.6% of fullerene. The fullerene mixture was extracted by toluene. Then, the individual fullerene C
60 was isolated by liquid chromatography based on turbostratic graphite with 3.42 Å interplanar distance (as a stationary phase) and a toluene/hexane mixture (as a mobile phase).
F3 was produced from a powder mixture of fullerene soot and acetylacetonate FeIII (Fe(acac)
3). The mixture was heated up to spontaneous ignition at 180 °C. Then, the combustion process proceeded without additional heating. The product of the combustion reaction was exposed to boiling hydrochloric acid and the dissolved part of the product was removed. Retreatment with acid was provided to remove the metal salt. The solid residue of fullerenes was washed with water and used as a precursor in the synthesis of polyhydroxylated fullerenes (fullerenols). F3 was produced by precursor hydroxylation in nitric acid followed by the hydrolysis of the polynitrofullerenes [
65,
66,
67,
68].
F4 (two molecules of C
60-fullerenol are combined by an iron atom) was produced from the powder which involved Fe-containing C
60 fullerene soot and acetylacetonate of FeIII (Fe(acac)
3). This mixture was heated up to spontaneous ignition (180 °C); then the temperature increased up to 250 °C in the smoldering regime. The product was treated with concentrated nitric acid (90 °C). The red cinnamonic solution was evaporated and treated by distilled water. The procedure provided the hydrolysis of poly-nitro-fullerene to poly-hydroxylated fullerene [
65,
66,
68].
F5 (gadolinium atom inside C
82-fullerenol) was produced by Gd@C
82-fullerene hydroxylation in nitric acid followed by hydrolysis of the polynitrofullerenes [
65,
66,
68,
70]. Mixture of fullerenes, involving Gd@C
82-fullerene, was preliminary synthesized by carbon helium high-frequency arc plasma at 98 kPa [
67,
70]. The Gd@C
82-fullerene content in carbon soot was about 4.8%. The reaction of complexation with Lewis acids (TiCl
4) was used for enrichment of the extract of fullerene mixture by endohedral metallofullerenes (Gd@C
82) [
71]. Then, Gd@C
82 was extracted with carbon disulfide from carbon soot.
The fullerenol preparations were characterized with IR and photoelectron spectroscopies [
72,
73].
3.2. Bioluminescence Assay Systems and Experimental Data Processing
Antioxidant activity and toxicity of fullerenols were evaluated using bioluminescence assay systems, both cellular and enzymatic: (1) bacterial assay, i.e., Microbiosensor 677F, was based on the lyophilized luminous bacteria
Photobacterium phosphoreum from the collection of the Institute of Biophysics SB RAS (CCIBSO 863), strain 667F IBSO, and (2) enzyme preparation was based on the coupled enzyme system NADH:FMN-oxidoreductase from
Vibrio fischeri (0.15 a.u.) and luciferase from
Photobacterium leiognathi, 0.5 mg/mL [
74]. All the biological preparations were produced at the Institute of Biophysics SB RAS (Krasnoyarsk, Russia).
Chemicals used were: NADH from ICN, USA; FMN and tetradecanal from SERVA, Germany; 1,4-benzoquinone from Aldrich, USA; and sodium chloride (NaCl) from Khimreactiv, Russia.
Antioxidant activity of fullerenols was assessed in water solutions of model oxidizer, 1,4-benzoquinone.
To construct the enzymatic assay system, we used 0.1 mg ml−1 enzyme preparation, 4 × 10−4 M NADH, 5 × 10−4 M FMN, and 0.002% tetradecanal solutions. The enzymatic assay was performed in 0.05 M phosphate buffer, pH 6.8, at 20 °C.
The enzymatic assay system is based on two coupled enzymatic reactions:
Measurements of bioluminescence intensity were carried out with bioluminometers BLM-3606 (Nauka Special Design Bureau, Russia) and TriStar LB 941 (Berthold Technologies, Germany).
Toxic effects of fullerenols on bioluminescence of bacterial and enzymatic assay systems were characterized by relative bioluminescence intensity,
:
where,
and
are maximal bioluminescence intensities in the absence and presence of fullerenols, respectively.
To compare toxic effects of fullerenols, their effective concentrations that inhibited bioluminescence intensity by 50% ( = 0.5), EC50, were determined.
General toxicity (
GT) of the model oxidizer solutions (1,4-benzoquinone) was evaluated with relative bioluminescence intensity,
:
where,
and
are maximal bioluminescence intensities in the absence and presence of the oxidizer, respectively, as shown in
Figure 7. The effective concentration of model organic oxidizer (1,4-benzoquinone) that inhibited bioluminescence intensity by 50% (
= 0.5),
EC50, were determined using bacterial and enzymatic bioluminescence assays. The
EC50 values of 1,4-benzoquinone were 2.5 × 10
−7 M and 10
−4 M for bacterial and enzymatic assays, respectively. The values were close to those determined in previous studies [
49,
52].
Antioxidant activity of fullerenols was evaluated in model solutions of oxidizer (1,4-benzoquinone). EC50 of the oxidizer was used in these experiments. To exclude the peculiar toxic effects of the fullerenols, concentration ranges (CR) of the fullerenols inhibiting the bioluminescence intensity less than 10% ( > 0.9) were preliminary determined and used in the experiments.
Both bioluminescent assays (bacterial and enzymatic) were applied to study changes in general toxicity (
GT) under the addition of fullerenols. Detoxification coefficients
DGT were determined as follows:
where
,
are relative bioluminescence intensities in oxidizer solutions at
EC50, in the absence and presence of fullerenols, respectively, calculated according to Equation (1a). Values of
DGT were determined at different fullerenol concentrations.
To characterize oxidative toxicity (
OxT) in the oxidizer solutions, the bioluminescence enzyme assay was used. Changes of
OxT under fullerenol exposure were characterized with detoxification coefficients,
DOxT:
where
and
are bioluminescence induction periods in oxidizer solutions in the absence and presence of fullerenols, respectively (
Figure 7b). The
DOxT values were determined and plotted vs. fullerenol concentrations.
Values of DGT > 1 or DOxT > 1 revealed a decrease in GT or OxT under the exposure to fullerenols, i.e., detoxification of solutions of oxidizers. Values of DGT ≈ 1 or DOxT ≈ 1 revealed an absence of the fullerenol effect.
The SD values for DGT or DOxT did not exceed 0.1. The data for the DGT or DOxT processing were obtained in three experiments with five samplings from all control and fullerenol solutions.
It should be noted that all experiments with ’colored’ solutions of fullerenols excluded the effect of the “optic filter” [
29]; this effect did not skew the results the toxicological measurements.
3.3. Luminol Chemiluminescence Assay
Luminol was obtained from Sigma-Aldrich, potassium hydroxide from Khimreactiv (Russia), and 3% solution of hydrogen peroxide from Tula Pharmaceutical Factory (Russia). The 10−4 M aqueous alkaline luminol solution was used.
The chemiluminescence luminol reaction was initiated by a solution of K3[Fe(CN)6] and maximal value of chemiluminescence intensity was determined. All measurements were carried out in 25–40 replicates using TriStar LB 941 bioluminometer with injector system. Average and SD values did not exceed 0.05.
The dependence of chemiluminescence intensity on H2O2 concentration was initially determined and it was used as a calibration dependent in the following experiments to evaluate concentrations of peroxide compounds in the solutions of fullerenols. Peroxides were considered to be components of the ROS group. The ROS content was plotted vs. concentrations of fullerenols.
To compare the effects of fullerenols on ROS content, their effective concentrations that decreased chemiluminescence intensity by 50%, EC50, were determined.
4. Conclusions
The current paper analyzed toxicity and antioxidant activity of a series of fullerenols studied under comparable conditions. In summary, all the fullerenols inhibited bacterial and enzymatic bioluminescence at high concentrations (>0.01 g L
−1,
Table 2), producing a toxic effect, while the antioxidant activity of all the fullerenols was evident at low and ultralow concentrations (<0.001 g L
−1,
Table 2). We found that the toxic and antioxidant characteristics of the fullerenols depended, additionally, on the number of oxygen substituents. Quantitative characteristics of the fullerenols (effective concentrations, concentration ranges, and detoxification coefficients) were determined and compared.
Lower toxicity and higher antioxidant activity were demonstrated for the fullerenols with fewer substituents: C60Oy(OH)x and C60,70Oy(OH)x, where x+y = 24–28. The differences were attributed to fullerenol’ ability to disturb ROS balance in aqueous solutions. Further investigations, including theoretical studies, should be carried out to understand the physical and chemical basis of these differences. The investigations should be aimed at such structural fullerenol peculiarities as an interrelation between the number of oxygen-containing groups and hydrophobic π-conjugated surface fragments, with the latter responsible for the reversible electron acceptance and, hence, nonspecific catalytic activity in chemical and biochemical processes.
As an outlook, a recommendation can be made for the selection and synthesis of fullerene’ water-soluble derivatives: a high number of oxygen substituents (up to 40 and more) provided higher toxicity and lower antioxidant activity.
Hence, the study demonstrated a suitability and high potential for the bioluminescence-based biosensing procedure for the complex study of the carbon nanoparticles, which are promising pharmaceutical agents.