A Multiparametric Study of Internalization of Fullerenol C60(OH)36 Nanoparticles into Peripheral Blood Mononuclear Cells: Cytotoxicity in Oxidative Stress Induced by Ionizing Radiation.

The aim of this study was to investigate the uptake and accumulation of fullerenol C60(OH)36 into peripheral blood mononuclear cells (PBMCs). Some additional studies were also performed: measurement of fullerenol nanoparticle size, zeta potential, and the influence of fullerenol on the ionizing radiation-induced damage to PMBCs. Fullerenol C60(OH)36 demonstrated an ability to accumulate in PBMCs. The accumulation of fullerenol in those cells did not have a significant effect on cell survival, nor on the distribution of phosphatidylserine in the plasma membrane. However, fullerenol-induced depolarization of the mitochondrial membrane proportional to the compound level in the medium was observed. Results also indicated that increased fullerenol level in the medium was associated with its enhanced transport into cells, corresponding to its influence on the mitochondrial membrane. The obtained results clearly showed the ability of C60(OH)36 to enter cells and its effect on PBMC mitochondrial membrane potential. However, we did not observe radioprotective properties of fullerenol under the conditions used in our study.


Introduction
Nanomaterials (NMs) are structure components with at least one dimension less than 100 nm. NMs should possess superior properties with enhanced performance that typical materials do not have [1]. Nanoparticles (NPs) are created due to van der Waals forces, electrostatic interactions, hydrogen bonds, as well as capillary bridges. The ability of NPs to assemble and interconnect strongly depends on their physical and chemical characteristics and their surface properties, including size, shape, ratio of electric charges and hydrophobicity, and last, but not least, on the concentration and the presence of functional groups [2,3]. These factors, as well as ability to interact with superficial cellular receptors also determine the rate and mechanism of entry of NMs into a cell [4].
Fullerene C 60 is a nanomaterial consisting of 60 carbon atoms connected by sp 2.5 -bonds. Due to those bonds, it has a pseudo-aromatic structure resulting from delocalization of π-electrons over the carbon core. This structure allows easy reaction with oxygen free radicals [4][5][6]. It was shown that pristine C 60 fullerene was non-toxic at low concentrations, and it was able to penetrate cellular plasma membrane. It has also been shown to exhibit antioxidant properties [7]. Fullerene increases the activity of antioxidant enzymes and prevents deleterious effects of oxidative stress by direct reactive oxygen species (ROS) scavenging [8]. The C 60 fullerene nanoparticle does not exert any genotoxic effect on human lymphocytes. C 60 reduces the genotoxic effect of doxorubicin (DOX) in normal human lymphocytes [9]. New fullerene derivatives can also be used for upconversion luminescence (UCL) and magnetic resonance imaging (MRI). That may be of great importance in "image-guided therapy" [10]. Fullerenes may be introduced anywhere in the body and they accumulate in specific organs, such as the liver, kidneys, and the spleen. Scientists suggested that fullerenes were able to pass into the blood from the gut and could be transported with blood [11,12]. Studies have shown that fullerenes affected the order of membrane lipids and induced membrane damage by lipid peroxidation. It was demonstrated that they accumulated in lysosomes of oyster hepatopancreas cells. The disposition of carbon NPs in biological systems is independent on the site of contact between NPs and an organism. It is related to the passage across cell membrane. Fluorescence quenching showed that C 60 (OH) [18][19][20][21][22] accumulated in outer regions of the membrane bilayer, whereas C 60 was found in deeper regions of the bilayer [13,14]. Other water-soluble fullerene derivatives, such as the dicarboxyfullerenes, concentrate in mitochondria or in endosome-or lysosome-like vesicles [15].
Fullerenols are bioactive compounds, polyhydroxylated water-soluble derivatives of fullerenes, a third natural allotropic variation of carbon [5,16,17]. The chemical structure of fullerenol C 60 (OH) x is presented in Figure 1. The solubility of fullerenol in water increases with the increasing number of hydroxyl groups [18]. Fullerenols and malonic acid fullerene species are two major groups of functional fullerene derivatives possessing effective protective antioxidative properties both in vitro and in vivo, and having also antitumor potential and antimetastatic activity [19][20][21]. The results of the study carried out by Yin et al. (2009) [22] indicated that C 60 (C(COOH) 2 ) 2 , C 60 (OH) 22 , and Gd@C 82 (OH) 22 could protect cells against H 2 O 2 -induced oxidative damage, stabilized the mitochondrial membrane potential, and reduced intracellular ROS production. Due to their hydrophilic properties and the ability to scavenge free radicals, fullerenol could provide a valuable alternative to conventional pharmacological agents [17]. The effect of polyhydroxylated fullerenol NPs on adult male Wistar rats treated with DOX was investigated by Jacevic et al. (2017) [23]. The authors reported that fullerenol increased the survival rate, body and liver weight, as well as decreased the level of thiobarbituric acid reactive substances (TBARS), antioxidative enzyme activity, and hepatic damage score in DOX-treated rats. Fullerenol exhibits excellent biocompatibility and has other advantages, including high aqueous solubility and neutral pH. Its susceptibility to further modifications makes fullerenol a promising agent for medical applications [24,25]. Previous experimental results suggested that fullerenol C 60 (OH) 24 induced ion permeability of a model membrane via the formation of ion pores or conductive defects. A preference for cations over anions was also observed [26]. carbon core. This structure allows easy reaction with oxygen free radicals [4][5][6]. It was shown that pristine C60 fullerene was non-toxic at low concentrations, and it was able to penetrate cellular plasma membrane. It has also been shown to exhibit antioxidant properties [7]. Fullerene increases the activity of antioxidant enzymes and prevents deleterious effects of oxidative stress by direct reactive oxygen species (ROS) scavenging [8]. The C60 fullerene nanoparticle does not exert any genotoxic effect on human lymphocytes. C60 reduces the genotoxic effect of doxorubicin (DOX) in normal human lymphocytes [9]. New fullerene derivatives can also be used for upconversion luminescence (UCL) and magnetic resonance imaging (MRI). That may be of great importance in "image-guided therapy" [10]. Fullerenes may be introduced anywhere in the body and they accumulate in specific organs, such as the liver, kidneys, and the spleen. Scientists suggested that fullerenes were able to pass into the blood from the gut and could be transported with blood [11,12]. Studies have shown that fullerenes affected the order of membrane lipids and induced membrane damage by lipid peroxidation. It was demonstrated that they accumulated in lysosomes of oyster hepatopancreas cells. The disposition of carbon NPs in biological systems is independent on the site of contact between NPs and an organism. It is related to the passage across cell membrane. Fluorescence quenching showed that C60(OH)18-22 accumulated in outer regions of the membrane bilayer, whereas C60 was found in deeper regions of the bilayer [13,14]. Other water-soluble fullerene derivatives, such as the dicarboxyfullerenes, concentrate in mitochondria or in endosome-or lysosome-like vesicles [15]. Fullerenols are bioactive compounds, polyhydroxylated water-soluble derivatives of fullerenes, a third natural allotropic variation of carbon [5,16,17]. The chemical structure of fullerenol C60(OH)x is presented in Figure 1. The solubility of fullerenol in water increases with the increasing number of hydroxyl groups [18]. Fullerenols and malonic acid fullerene species are two major groups of functional fullerene derivatives possessing effective protective antioxidative properties both in vitro and in vivo, and having also antitumor potential and antimetastatic activity [19][20][21]. The results of the study carried out by Yin et al. (2009) [22] indicated that C60(C(COOH)2)2, C60(OH)22, and Gd@C82(OH)22 could protect cells against H2O2-induced oxidative damage, stabilized the mitochondrial membrane potential, and reduced intracellular ROS production. Due to their hydrophilic properties and the ability to scavenge free radicals, fullerenol could provide a valuable alternative to conventional pharmacological agents [17]. The effect of polyhydroxylated fullerenol NPs on adult male Wistar rats treated with DOX was investigated by Jacevic et al. (2017) [23]. The authors reported that fullerenol increased the survival rate, body and liver weight, as well as decreased the level of thiobarbituric acid reactive substances (TBARS), antioxidative enzyme activity, and hepatic damage score in DOX-treated rats. Fullerenol exhibits excellent biocompatibility and has other advantages, including high aqueous solubility and neutral pH. Its susceptibility to further modifications makes fullerenol a promising agent for medical applications [24,25]. Previous experimental results suggested that fullerenol C60(OH)24 induced ion permeability of a model membrane via the formation of ion pores or conductive defects. A preference for cations over anions was also observed [26].  The action of ionizing radiation on biological systems is mostly associated with the formation of ROS by the radiolysis of water. ROS action results in oxidative damage of proteins, lipids, deoxyribonucleic acid (DNA) and, as a consequence, leads to loss of cell function and cell death [20,27,28]. Cell death is related to damage to nuclear DNA and formation of DNA double-strand breaks (DSBs). Damage to the DNA can be caused directly by radiation or indirectly, through mediation of radicals, peroxides, and superoxides [29]. In order to reduce adverse radiation effects on biological systems, radioprotective compounds are researched [27]. Fullerens and their hydroxyl derivatives are a group of compounds studied for their possible application as antioxidants and radioprotective compounds [30,31].
The aim of the present study was to investigate pathways and processes involved in the uptake, translocation, and accumulation of fullerenol C 60 (OH) 36 in peripheral blood mononuclear cells (PBMCs). We chose PBMCs as they are commonly used in toxicological studies. Moreover, we decided to perform some additional studies: measurement of fullerenol size and zeta potential, and the influence of fullerenol on the ionizing radiation-induced damage to PMBCs.

Characterization of Fullerenol Nanoparticles
Fullerenol NPs were characterized using atomic force microscopy (AFM) and dynamic light scattering (DLS) techniques. Figure 2A presents morphology images of fullerenol deposited by droplet precipitation on cleaved mica substrate, from aqueous solution, acquired using AFM. Measurements were performed after complete evaporation of the solvent. The AFM study revealed some randomly distributed agglomerates visible as white, round-shaped objects with the approximate local height of 1.4 nm. The size of analyzed fullerenol in solution was determined with the use of dynamic light scattering, while the particle surface potential (zeta potential) was determined by electrophoretic light scattering. Particle size distribution of fullerenol in aqueous solution and in 0.02 M phosphate buffer (pH 7.4) is presented in Figure 2B,C, respectively. DLS data indicated that NPs with an average diameter of 40 nm and 110 nm predominated in C 60 OH 36 aqueous suspensions, suggesting the presence of hydrated clusters formed by several fullerene molecules. Zeta potential measurements, evaluated by electrophoretic mobility of NPs (Table 1; Figure 3), indicated that hydrated NPs of fullerene acquired a negative surface charge (−27.5 mV for water and −37.4 mV for buffer). Negative values of zeta potential suggest that fullerene NPs were surrounded by a well-organized layer of hydrogen-bonded water molecules (a stable hydrophilic shell), which promoted a negative surface charge and prevented interaction with similar neighboring molecules.

Direct Evidence of Fullerenol Internalization by Confocal Microscopy and Flow Cytofluorometry
Confocal microscopy techniques ( Figure 4) were used to analyze the internalization of NPs into PBMCs after 24-h and 48-h incubation with fullerenol at the concentrations of 75 mg/L and 150 mg/L. We used the fullerenol property for autofluorescence at 345 nm excitation wavelength and at 470 nm emission wavelength in aqueous solution (own studies). Figure 5 shows that uptake of nanoparticles was concentration-dependent. More efficient internalization of nanoparticles was observed after incubation of PBMCs with NPs at the higher concentration. Internalization of fullerenol NPs was still observed when fullerenol was removed from the cell suspensions by centrifugation and fresh medium was added. Intensity-weighted average values were used to determine the hydrodynamic size, while volume distribution data was used to determine relative amounts of nanoparticles (NPs). Graphs in Figure  2B,C were produced by the Zetasizer Nano-ZS software.
DLS data indicated that NPs with an average diameter of 40 nm and 110 nm predominated in C60OH36 aqueous suspensions, suggesting the presence of hydrated clusters formed by several fullerene molecules. Zeta potential measurements, evaluated by electrophoretic mobility of NPs (Table 1; Figure 3), indicated that hydrated NPs of fullerene acquired a negative surface charge (−27.5 mV for water and −37.4 mV for buffer). Negative values of zeta potential suggest that fullerene NPs were surrounded by a well-organized layer of hydrogen-bonded water molecules (a stable hydrophilic shell), which promoted a negative surface charge and prevented interaction with similar neighboring molecules. Intensity-weighted average values were used to determine the hydrodynamic size, while volume distribution data was used to determine relative amounts of nanoparticles (NPs). Graphs in Figure 2B

Direct Evidence of Fullerenol Internalization by Confocal Microscopy and Flow Cytofluorometry
Confocal microscopy techniques ( Figure 4) were used to analyze the internalization of NPs into PBMCs after 24-h and 48-h incubation with fullerenol at the concentrations of 75 mg/L and 150 mg/L. We used the fullerenol property for autofluorescence at 345 nm excitation wavelength and at 470 nm emission wavelength in aqueous solution (own studies). Figure 5 shows that uptake of nanoparticles was concentration-dependent. More efficient internalization of nanoparticles was observed after       Flow cytofluorometry was used to determine the extent of adsorption of fullerenol (50-150 mg/L) on the cell surface and/or the compound internalization after 1 h, 24 h, and 48 h of incubation. Figure 6 shows that, as in the case of confocal microscopy, the uptake of nanoparticles by the cells depended on the concentration and incubation time. The highest cellular uptake of NPs was observed for their concentration of 150 mg/L.

Effects of Fullerenol on the Viability of PBMCs
Annexin V is a protein demonstrating a high affinity for phosphatydylserine (PS). Externalization of PS occurs at early stages of apoptosis, which can be detected by Annexin V staining. Annexin V-FITC/propidum iodide (PI) double staining is useful for distinguishing between viable cells (unstained) and apoptotic cells (stained with Annexin V-FITC), and necrotic cells (stained with PI). Figure 7 shows quantitative changes in PBMCs after 24 h incubation with fullerenol (50-150 mg/L). According to those results, C60(OH)36 does not affect cell viability compared to the control (viable cells ~85%).

Effects of Fullerenol on the Viability of PBMCs
Annexin V is a protein demonstrating a high affinity for phosphatydylserine (PS). Externalization of PS occurs at early stages of apoptosis, which can be detected by Annexin V staining. Annexin V-FITC/propidum iodide (PI) double staining is useful for distinguishing between viable cells (unstained) and apoptotic cells (stained with Annexin V-FITC), and necrotic cells (stained with PI). Figure 7 shows quantitative changes in PBMCs after 24 h incubation with fullerenol (50-150 mg/L). According to those results, C 60 (OH) 36 does not affect cell viability compared to the control (viable cells~85%).

The Impact of Fullerenol on Human Peripheral Blood Mononuclear Cells under Radiation-Generated Oxidative Stress
Radioprotective properties of fullerenol were also tested. PBMCs were irradiated (5, 10, and 15 Gy) in the absence and presence of fullerenol C 60 (OH) 36 and labeled with PI/ calcein-AM (c-AM) after 24 h from the end of irradiation. No significant apoptotic changes were detected in the control, after incubation with fullerenol alone, and at 5-Gy and 10-Gy irradiation. In response to radiation, statistically significant changes were observed for the combination of 10 Gy radiation with the highest concentration of fullerenol. The results obtained by means of this method were similar to those for Annexin V-FITC/PI staining. The number of apoptotic cells increased with increasing fullerenol concentration and a dose of radiation ( Figure 8).
It is possible that structural disorders of plasma membrane (demonstrated in previous studies) [32] caused by fullerenol C 60 (OH) 36 were responsible for the effect of this substance observed during irradiation of PBMCs. Previous studies [33] proved that fullerenol lowered the post-radiation hemolysis, potassium efflux, and oxidation of thiol groups in human anucleate erythrocytes. However, fullerenol enhanced toxic effects of radiation toward membrane acetylcholinesterase.
Annexin V is a protein demonstrating a high affinity for phosphatydylserine (PS). Externalization of PS occurs at early stages of apoptosis, which can be detected by Annexin V staining. Annexin V-FITC/propidum iodide (PI) double staining is useful for distinguishing between viable cells (unstained) and apoptotic cells (stained with Annexin V-FITC), and necrotic cells (stained with PI). Figure 7 shows quantitative changes in PBMCs after 24 h incubation with fullerenol (50-150 mg/L). According to those results, C60(OH)36 does not affect cell viability compared to the control (viable cells ~85%).

The Impact of Fullerenol on Human Peripheral Blood Mononuclear Cells under Radiation-Generated Oxidative Stress
Radioprotective properties of fullerenol were also tested. PBMCs were irradiated (5, 10, and 15 Gy) in the absence and presence of fullerenol C60(OH)36 and labeled with PI/ calcein-AM (c-AM) after 24 h from the end of irradiation. No significant apoptotic changes were detected in the control, after incubation with fullerenol alone, and at 5-Gy and 10-Gy irradiation. In response to radiation, statistically significant changes were observed for the combination of 10 Gy radiation with the highest concentration of fullerenol. The results obtained by means of this method were similar to those for Annexin V-FITC/PI staining. The number of apoptotic cells increased with increasing fullerenol concentration and a dose of radiation (Figure 8).
It is possible that structural disorders of plasma membrane (demonstrated in previous studies) [32] caused by fullerenol C60(OH)36 were responsible for the effect of this substance observed during irradiation of PBMCs. Previous studies [33] proved that fullerenol lowered the post-radiation hemolysis, potassium efflux, and oxidation of thiol groups in human anucleate erythrocytes. However, fullerenol enhanced toxic effects of radiation toward membrane acetylcholinesterase.

Assessment of Mitochondrial Membrane Potential after Incubation with Fullerenol
The fluorescent probe JC-1 was used to determine the effect of fullerenol on PBMCs with or without radiation-generated oxidative stress. Figure 10 shows changes in mitochondrial membrane potential of PBMCs depending on the concentration of fullerenol (50-150 mg/L) and irradiation (5, 10, and 15 Gy) after 24-h incubation (37 • C, 5% CO 2 atmosphere, 100% relative humidity). Fullerenol reduced mitochondrial membrane potential in PBMCs in a concentration-dependent manner. At its highest concentration (150 mg/L) fullerenol showed a similar effect to the 10-Gy ionizing radiation, reducing mitochondrial potential down to approximately 80% of the control value (measured in cells that were not treated with fullerenol nor irradiated). Fullerenol at none of the applied concentrations (50-150 mg/L) showed protective activity against ionizing radiation, nor intensified the effect of irradiation.

Assessment of Mitochondrial Membrane Potential after Incubation with Fullerenol
The fluorescent probe JC-1 was used to determine the effect of fullerenol on PBMCs with or without radiation-generated oxidative stress. Figure 10 shows changes in mitochondrial membrane potential of PBMCs depending on the concentration of fullerenol (50-150 mg/L) and irradiation (5, 10, and 15 Gy) after 24-h incubation (37 °C, 5% CO2 atmosphere, 100% relative humidity). Fullerenol reduced mitochondrial membrane potential in PBMCs in a concentration-dependent manner. At its highest concentration (150 mg/L) fullerenol showed a similar effect to the 10-Gy ionizing radiation, reducing mitochondrial potential down to approximately 80% of the control value (measured in cells that were not treated with fullerenol nor irradiated). Fullerenol at none of the applied concentrations (50-150 mg/L) showed protective activity against ionizing radiation, nor intensified the effect of irradiation.

Discussion
Numerous hydroxyl groups present on the surface of fullerenol enable the formation of multiple hydrogen bonds to various biomolecules, such as protein domains of the cell membrane and hydrophilic lipid heads. Fullerenol can be adsorbed onto cytoskeletal proteins and this interaction may be used as a drug transport mechanism [32][33][34][35]. Furthermore, fullerenol exhibits antioxidative properties [36,37].
More and more carbon NPs enter the environment and living organisms [38][39][40]. Therefore, it is important to identify the mechanisms involved in NPs' transport, and assess their impact on cells, including blood cells. Blood cells are a valuable research model, as they are commonly exposed to xenobiotics and are easy to separate. This paper is the first to present results of the study clearly showing penetration of fullerenol

Discussion
Numerous hydroxyl groups present on the surface of fullerenol enable the formation of multiple hydrogen bonds to various biomolecules, such as protein domains of the cell membrane and hydrophilic lipid heads. Fullerenol can be adsorbed onto cytoskeletal proteins and this interaction may be used as a drug transport mechanism [32][33][34][35]. Furthermore, fullerenol exhibits antioxidative properties [36,37].
More and more carbon NPs enter the environment and living organisms [38][39][40]. Therefore, it is important to identify the mechanisms involved in NPs' transport, and assess their impact on cells, including blood cells. Blood cells are a valuable research model, as they are commonly exposed to xenobiotics and are easy to separate. This paper is the first to present results of the study clearly showing penetration of fullerenol C 60 (OH) 36 NPs into cells and their adsorption on plasmatic membrane. Earlier reports [41,42] indirectly indicated adsorption and penetration of those polar NPs into cells. Molecular dynamics studies [43] also demonstrated a possible transport of fullerenol into cells.
Methods of confocal microscopy and flow cytometry were applied. Fluorescent properties of fullerenol were also used to directly demonstrate the penetration of highly hydroxylated fullerenol into cells. The obtained results indicated that C 60 (OH) 36 entered PBMCs in the amount proportional to its concentration used and cell incubation time. This study also showed that at longer incubation time, intracellular accumulation of this NP prevailed over its adsorption on the external, hydrophilic surface of the cellular membrane ( Figure 5).
Our previous study demonstrated that fullerenol was able to adsorb on membrane surface, particularly in the vicinity of integral membrane proteins, such as transmembrane ionic pumps: ATPases K/Na, Mg, and Ca, irreversibly inhibiting the activity of those enzymes [32].
NPs can easily enter living cells by phagocytosis, macropinocytosis, caveolae-mediated endocytosis, or clathrin-mediated endocytosis [44,45]. Physicochemical properties of NPs (size, shape, surface charge, and surface chemistry) influence their uptake efficiency by cells [46]. Aggregation of NPs may be changed depending on the composition of media and functional coatings [47].
In this study, zeta potential measurements evaluated by electrophoretic mobility of NPs (Table 1; Figure 3) indicated that hydrated NPs of fullerene acquired a negative surface charge (−27.5 mV for water and −37.4 mV for phosphate buffer, pH 7.4). Negative values of zeta potential suggest that fullerenol NPs were surrounded by a well-organized layer of hydrogen-bonded water molecules (a stable hydrophilic shell), which promoted a negative surface charge and prevented interaction with similar neighboring molecules.
Existence of a layer of water molecules was confirmed by the results of DLS and AFM measurements. The mean diameter of fullerenol NPs in water solution was 40-110 nm. However, AFM results revealed that fullerenol particles, which stripped off the aqueous shell, were approximately 2 nm in diameter.
Electrostatic interactions between fullerenol molecules and between those molecules and the cell membrane may be largely responsible for the biological effect of C 60 (OH) 36 in cells. That was confirmed by observations made by other researchers, who reported opposed effects of fullerenol, even with the same number of OH groups bound to the C 60 fullerene core [48,49]. Similarly, this study indicated the absence of a direct linear correlation between the cellular fullerene content and its concentration in the medium ( Figure 5).
Fullerenols C 60 (OH) x -water-soluble derivatives of fullerenes-are currently being intensively studied in the context of their possible application in biomedicine [50]. Due to its hydrophilic properties and the ability to eliminate free radicals, fullerenol may in the future provide a solid alternative to currently used pharmacological methods in chemotherapy, treatment of neurodegenerative diseases, and radiobiology [51,52].
However, depending on the research protocol applied, fullerenol may also act as prooxidant [53]. This dualistic nature of fullerenols may contribute to the future development of some new biomedical and environmental applications of these agents [54][55][56].
This study demonstrated that fullerenol C 60 (OH) 36 at the concentrations up to 150 mg/L showed no toxic effect on PBMCs. No statistically significant differences in the induction of apoptosis or necrosis were observed between fullerene untreated and treated PBMCs (Figure 7, Figure 8, Figure 9). It is worth noting that the toxic effect of fullerenol was assessed using two methods for apoptosis detection. The first method monitored externalization of phosphatidylserine on the outer leaflet of cell membrane (Annexin V-FITC), and the second determined changes in the intracellular calcium level (calcein-AM staining). Consistent results were obtained from both methods, indicating an absence of toxic effect of fullerenol C 60 (OH) 36 over the analyzed concentration range on PBMCs.
Bacchetta et al. [57] showed that shape and size were important factors influencing carbon NPs uptake by cells and tissues. Investigations imply that cytotoxicity is strongly correlated with the size and shape of NPs, physical parameters of cell culture media, and biochemical interactions with the cell occurring following internalization [58]. The results of this study indicate that fullerenol particles of 40-110 nm in diameter possessing a negative surface charge are absorbed by PBMCs and do not initiate cell death, even at the concentration of 150 mg/L.
It was observed, however, that fullerenol caused depolarization of the mitochondrial membrane potential in PBMCs in a concentration-dependent manner ( Figure 10). Yang et al. [59] studied isolated mitochondria and demonstrated the same effect of fullerenol C 60 (OH) 44 . Moreover, C 60 (OH) 44 increased the permeability of the mitochondrial inner membrane to H + and K + and induced mitochondrial permeability. The effect was concentration-dependent and was inhibited by cyclosporin A and EDTA.
As ionizing radiation is known to cause depolarization of the mitochondrial membrane potential [60], and numerous reports indicated that fullerenols were effective scavengers of free radicals and exhibited antioxidative activity [33,61], this study analyzed the effect of fullerenol C 60 (OH) 36 on the mitochondrial potential in irradiated PBMCs. Unfortunately, it was found that the presence of fullerenol failed to protect from depolarization of the mitochondrial membrane potential caused by ionizing radiation at doses of up to 15 Gy. Similarly, no protective effect of fullerenol on radiation-induced death of PBMCs was observed. PBMCs irradiated with 15 Gy demonstrated a reduced viability following a 24-h incubation started directly after irradiation. Simultaneous exposure of cells to fullerenol and irradiation showed that this NP failed to protect PBMCs from death. Similar results were reported by Nowak et al. [62], who did not observe a protective action of fullerenol following a 24-h incubation, for the very high dose of 50 Gy. On the other hand, the electron spin resonance spectroscopy (ESR) studies confirmed that C 60 (OH) 24 was a powerful radical scavenger for superoxide, hydroxyl, and lipid radicals. Those data suggested that C 60 (OH) 24 was a mitochondrial protective antioxidant with direct radical scavenging activity and indirect antioxidant activity [61].
The free radical mechanism of ionizing radiation suggests that scavengers of free radicals should protect cellular structures against damage. It is possible, however, that depolarization of the mitochondrial membrane potential, as well as structural disorders of plasma membrane (demonstrated in previous studies) [32] caused by fullerenol C 60 (OH) 36 were responsible for the effect of this substance observed during the irradiation of PBMCs. It is worth noting that the effects of fullerenol and ionizing radiation were neither synergistic, nor additive.
The mitochondrial membrane potential (∆Ψm) is a result of the difference in the concentration of ions, including H + , on both sides of the inner mitochondrial membrane. Therefore, it depends on the electrochemical potential gradient and the pH gradient. Calcium ions play an important role in the maintenance of homeostasis of ∆Ψm because they control the opening of the mitochondrial permeability transition (MPT) and adenosine triphosphate (ATP) production. Depolarization of the inner ∆Ψm enhances outflow of cations from and inflow of anions to mitochondria, wherein the outflow is not proportional, but exponentially dependent on ∆Ψm [63]. Excessively high potential of the mitochondrial membrane favors the production of ATP, but also of ROS. Over-production of ROS leads to damage to the mitochondrial membrane, and further generation of ROS. A cell is able to tolerate some short-term changes of the mitochondrial membrane potential, but long-term ∆Ψm disorders lead to cell death.
Ionizing radiation induces both cellular and mitochondrial oxidative stress, leading to the generation of ROS, as reviewed thoroughly in [64]. Despite the fullerenol-induced mitochondrial membrane depolarization observed in this study, this effect did not trigger apoptosis or necrosis of PBMCs (Figure 7, Figure 8). This finding may be explained by the ability of fullerenol to bind to membrane proteins, and by-at least partial-inhibition of outflow of cations from mitochondria, as a result of mitochondrial membrane depolarization. Irreversible binding of fullerenol to ion-dependent ATPases was confirmed in previous studies [32]. Fullerenol binding and inhibition of ionic transport through the outer mitochondrial membrane resulting from block of the voltage-dependent anion channel 1 (VDAC1) cannot be excluded either. It is known that reduced mitochondrial membrane potential predispose channels to transport some large ions, even up to 5 kDa [65].
Using density functional theory calculations, Wang et al. [66]  and H 2 O 2 ) production, no protective effect of fullerenol was observed in analyzed systems, which confirms the previous statement of direct interference between fullerenol and cellular structures.
Considering the size of fullerenol molecules determined in this study, and their tendency to form aggregates/conglomerates, the actual concentration of NPs may be insufficient to compete with ROS, despite a nominally high concentration.

Materials and Methods
The following components were used for the synthesis of fullerenol C 60 OH 36 : fullerene C 60 99.5% (SES Research, Houston, TX, USA), sodium hydroxide, methanol and hydrogen peroxide 30% (Avantor Performance Materials Poland SA, Gliwice, Poland) and Amberlit MB20 (Sigma-Aldrich, St. Louis, MO, USA). Peripheral blood mononuclear cells were obtained from leukocyte platelet buffy coat from healthy donors purchased from the Regional Centre of Blood Donation and Blood Treatment, Lodz, Poland.

Synthesis of Fullerenol Nanoparticles
Fullerenol C 60 (OH) 36 was synthesized from fullerene C 60 (99.5%). Sodium hydroxide, perhydrol (H 2 O 2 , 30%) and Amberlit MB20 were used for the synthesis of fullerene. Polyhydroxylated fullerene was obtained using the method described previously by Krokosz et al. [67]. First, fullerene C 60 was reacted with solid NaOH and perhydrol (H 2 O 2 , 30%) at room temperature. Hydrolysis was carried out for 24 h. Fullerenol was precipitated with methanol. Subsequently, NaOH residues were removed by ion-exchange chromatography on AmberlitMB-20. The structure of the obtained C 60

AFM Characterization of Fullerenol
A drop of fullerenol C 60 (OH) 36 water solution was deposited on freshly cleaved muscovite (mica) surface without any further pretreatment. Surface topography was analyzed with the use of atomic force microscopy (AFM)-Bruker Dimension Icon operating in ambient conditions in air. NSC35/Si 3 N 4 /AlBS (NT-MDT) cantilevers were used for the AFM tapping mode measurements. Typical scan sizes of the acquired images were: 0.5 µm × 0.5 µm, 1 µm × 1 µm, and 2 µm × 2 µm. The size of C 60 (OH) 36

Preparation of Peripheral Blood Mononuclear Cell Suspensions
Peripheral blood mononuclear cells (PBMCs) were separated from erythrocytes and granulocytes by density gradient centrifugation with Histopaque 1077 (at 800 RCF for 25 min). After rinsing with a separation medium, PBMCs were cultured in RPMI 1640 medium with 10% FBS, 2 mM L-glutamine, 10 mg/mL streptomycin and 1000 U/mL penicillin. For the experiments, PBMCs demonstrating viability above 95% were only used (the analysis with Trypan Blue 0.4%). The cells were incubated in sterile conditions in a Smart Cell incubator (37 • C, 5% CO 2 atmosphere, 100% relative humidity).

Fullerenol Treatment Conditions
Fullerenol C 60 (OH) 36 from the stock solution (3 mg/mL) was added to cell suspensions to final concentrations of 50, 75, 100, or 150 mg/L. The solution of fullerenol was sonicated for 1 h before being added to the cell suspension.

Measurement of Fullerenol Internalization by Confocal Microscopy
After the isolation and above-mentioned treatment of PBMCs with NPs, drops of samples were placed on a slide and covered with a cover glass. The Leica TCS SP8 confocal microscope (Leica-Microsystems, Wetzlar, Germany) featuring 20× and 63×/1.20 lenses (HC PL APO CS2 63×/1.40 OIL) was used for microscopic imaging. To analyze the uptake of NPs, the 405-nm LED was used, with excitation and emission parameters of 420-530 nm. The LAS X Software (Leica-Microsystems, Wetzlar, Germany) was used to examine the fluorescence intensity of NPs in 100 cells.

Measurement of Fullerenol Internalization by Flow Cytofluorometry
Fullerenol exhibits fluorescence at 345-nm excitation wavelength and at 470-nm emission wavelength. In order to determine the adsorption of fullerenol on cell surface and/or internalization, flow cytofluorometry was used (flow cytometer FACS, LSR®II from Becton-Dickinson, San Jose, CA, USA; 355 nm laser). In each experiment, 10,000 events were counted after gating of viable cells. Median of fluorescence intensity was calculated for each cell population. Untreated cells were used as a control.

Conditions of Irradiation
PBMCs were irradiated using the Stabilipan X-ray machine (Siemens, Germany) equipped with a 2-mm aluminum filter. Dosimetry was performed before irradiation of PBMCs on a 6-well plate. The radiation dose delivered to the plate was 1.75 Gy/min, 185 kV, and 10 mA. Cells were irradiated with doses of 5, 10, and 15 Gy (irradiation on a 6-well plate) with or without fullerenol. The final concentration of fullerenol C 60 (OH) 36 in tested samples was 50-150 mg/L. Before irradiation, the cells were incubated with or without the analyzed compound for 1 h at 37 • C, in 5% CO 2 atmosphere with 100% relative humidity. After irradiation, the samples were incubated for 24 h and 48 h at 37 • C in 5% CO 2 atmosphere and 100% relative humidity.

Cell Viability
Cell viability was determined using calcein-AM (green, viable cells) and propidium iodide (PI) (red, non-viable cells). Calcein-AM, an acetoxymethyl ester of calcein, is highly lipophilic and easily penetrates cell membrane. In viable cells, calcein, formed from calcein-AM by esterase, emits green fluorescence (excitation: 490 nm, emission: 515 nm). PI cannot penetrate a viable cell membrane. It is able to penetrate a damaged membrane of a dead (necrotic) cell. Following entering a non-viable cell, the compound interacts with DNA and emits red fluorescence (excitation: 535 nm, emission: 617 nm).

Detection of Apoptosis
During apoptosis, the cell is characterized by loss of structural asymmetry of cell membrane, and unrestricted permeability. Phospholipid phosphatidylserine (PS) is translocated from the inner to the outer surface of the membrane. In order to assess the type of cell death induced by fullerenol C 60 (OH) 36 (50-150 mg/L for 24 h), double staining with Annexin V-FITC and PI was used. The double staining was carried out using the FITC-Annexin V Apoptosis Detection Kit II (BD Biosciences-Pharmingen, San Jose, CA, USA). PBMCs were suspended in binding buffer and stained with Annexin V-FITC and PI, respectively, and incubated for 15 min at room temperature. The study was carried out using the FACS flow cytometer (LSR®II from Becton-Dickinson, San Jose, CA, USA).

Detection of Mitochondrial Membrane Potential
Mitochondrial membrane potential (∆Ψm) is an important parameter of mitochondrial function.

Statistical Analysis
All data are presented as mean ± standard deviation and asterisks are used to mark statistical significance of differences. Statistical analysis was performed using one-way and two-way ANOVA followed by the Tukey post hoc test for multiple comparisons. All statistics were calculated using the Statistica software (StatSoft, Tulsa, OK, USA). Values of p < 0.05 were considered statistically significant.

Conclusions
We have demonstrated that fullerenol C 60 (OH) 36 could accumulate in peripheral blood mononuclear cells. The cellular accumulation of fullerenol had no significant effect on the survival of the cells, nor on the distribution of phosphatidylserine in the plasma membrane. However, fullerenol-induced depolarization of the mitochondrial membrane potential proportional to the concentration of NPs studied in the medium was observed. The obtained results have indicated that increased fullerenol concentration in the medium resulted in increased transport of this substance into the cells, reflected by its effect on the mitochondrial membrane. The obtained results have clearly shown the ability of C 60 (OH) 36 to enter cells and the influence the mitochondrial membrane potential. However, we did not observe radioprotective properties of fullerenol under the conditions used in this study. These results may have important implications for the use of highly hydroxylated fullerenols in biomedical applications.

Conflicts of Interest:
The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.