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Biocompatible [60]/[70] Fullerenols: Potent Defense against Oxidative Injury Induced by Reduplicative Chemotherapy Yue Zhou, Jie Li, Haijun Ma, Mingming Zhen, Jun Guo, Liping Wang, Li Jiang, Chunying Shu, and Chunru Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08348 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 26, 2017

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ACS Applied Materials & Interfaces

Biocompatible [60]/[70] Fullerenols: Potent Defense against Oxidative Injury Induced by Reduplicative Chemotherapy Yue Zhou†,‡,§, Jie Li†,‡,§, Haijun Ma†,‡, Mingming Zhen*,†,‡, Jun Guo†, Liping Wang†,‡, Li Jiang†,‡, Chunying Shu†,‡ and Chunru Wang*,†,‡

†

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Molecular

Nanostructure and Nanotechnology, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡

University of Chinese Academy of Sciences, Beijing 100049, China

KEYWORDS: fullerenols, ROS scavenger, cytochrome P-450, oxidative injury, chemotherapy protection

ABSTRACT

Chemotherapy as a conventional cancer treatment suffers from critical systemic side effects, which is generally considered as the consequence of reactive oxygen species (ROS). Fullerenes have been widely studied for their excellent performance in radicals scavenging. In the present study, we report a solid-liquid reaction to synthesize fullerenols and their application as ROS scavengers in chemotherapy protection. The

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solid-liquid reaction is carried out without catalyst and suitable for mass production. The novel [60]/[70] fullerenols show a high stability in water and the [70] fullerenols (C70-OH) exhibit the superior radical scavenging capability to [60] fullerenols (C60-OH) in the chemotherapy protection. The mouse model for single and reduplicative chemotherapy-induced liver injury demonstrates their protective effects in the chemotherapeutic process, which is confirmed by histopathological examinations and haematological index. The increase of hepatic L-glutathione (GSH) level and down-regulated expression of the cytochrome P-450 2E1 (CYP2E1) give the possible mechanism associated with the impact of fullerenols on the metabolism of doxorubicin. The novel fullerenols may be promising protective agents to satisfy the demand for future clinical chemotherapy.

Introduction

Fullerene, a closed spherical molecule, has been widely applied in biomedical research since its discovery in 1985. Its unique physiochemical properties have received considerable attention and made it extremely versatile. However, its inherent hydrophobic character and poor dispersibility in water quite limit the application in physiological conditions. Various chemical derivatization methods are applied to achieve high water solubility by modifying hydrophilic functional groups or molecules onto the fullerenes.1-4 Among the numerous currently available fullerene derivatives, polyhydroxylated C60 synthesized by Chiang et al in 1992 is one of the earliest water-soluble fullerenes.5,

6

Nowadays polyhydroxylated fullerenes (known as


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fullerenols) have demonstrated a diversity of biomedical functions in diagnosis and therapy including imaging probes,7 antineoplastic agents,8, 9 drug carriers10, 11 and so on. The most prominent characteristic of fullerenols is the great capacity of scavenging free radicals, which attributes to their strong conjugated π-system to capture electrons. It has been widely reported that fullerenols (C60(OH)n n = 6-44) act as protective agents both in vitro and in vivo, and exert higher antioxidant activity than natural antioxidants like ascorbic acid12 and vitamin E.13 For instance, Chen et al. demonstrated C60(OH)22 can scavenge various kinds of reactive oxygen species (ROS), such as superoxide radical anion (O2•-), singlet oxygen, and hydroxyl radical (•OH). It exhibited a high cytoprotective ability against H2O2-induced oxidative damage.14 Besides, the protective effects of fullerenols against damage induced by gamma-rays15 or nephrotoxicity16, 17 and pulmotoxicity18 induced by chemotherapy agents were also achieved through their anti-oxidative and radical scavenging activities. Many studies are dedicated to the preparation of fullerenols under either acidic5, 19-21

or alkaline conditions.22, 23 Here we employ a novel solid-liquid reaction to prepare

fullerenols by directly introducing the hydroxyl moieties onto solid C60 in the presence of H2O2 and NaOH,3, 9 while the previously reported fullerenols are synthesized in the liquid state and the hydroxyl moieties from sodium hydroxide are connected to the C60/C70 molecules. This method is performed without the addition of catalyst and suitable for large-scale production. We further expand it to solid C70, and find the [70] fullerenol (C70-OH) exhibit better capability in scavenging ROS than that of [60]



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fullerenol (C60-OH), both in vitro or in vivo against hepatotoxicity induced by doxorubicin, a broad-spectrum chemotherapeutic drug. Results and Discussions

1. Preparation and Characterization of [60]/[70] fullerenols

Both of C70-OH and C60-OH fullerenols were simply prepared by the solid-liquid method in the presence of H2O2 and NaOH under heating condition (scheme in Figure 1a), suitable for mass production (Figure S1). Then the obtained solutions were precipitated by ethanol and further purified by dialysis. To determine the average number of hydroxyl groups introduced for the C70-OH and C60-OH, we employed a previously reported method combining elemental analysis (Table S1) with a water content measured by thermogravimetric analysis (Figure S2).20,

24

The first loss is

assigned to the secondary bound water (C70-OH: 3.7%; C60-OH: 4.5%) and the second loss might belong to the dehydration of hydroxyl groups on the surface of carbon cage. Along with the hydrogen contents from elemental analysis and the ratio of carbon to hydrogen (C/H), we could assess the average number of hydroxyl groups per fullerene molecule, twenty-four for C70-OH and twenty-six for C60-OH, respectively. Therefore, the average structures of the two fullerenols were deduced as C70(OH)24•5H2O and C60(OH)26•5H2O. However, due to the specificity of solid-liquid reaction, the average molecular formula are not entirely representative of the fullerenol structures. The surface of the nanoparticle is inevitably modified with more hydroxyl moieties. The chemical state of the two fullerenols’ surfaces was further confirmed by FT-IR


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and XPS spectra. As shown in Figure 1b, the FT-IR spectra showed there were three main peaks located at 3400, 1600 and 1350-1400 cm-1 corresponding to the vibrational bands of -OH, C=C and stretching bands of C-O or C-C , respectively. The vibrational bands of -OH (ca. 3400 cm−1) combined with the asymmetric (ca. 1350 cm−1) and symmetric stretching bands (ca. 1080 cm−1) demonstrated the presence of hydroxyl group. The weak peak (ca. 1750 cm−1) revealed a small quantity of hemiketal groups, which were probably attributed to the pinacol rearrangement and subsequent keto-enol isomerization.3, 25, 26 According to the surface functional groups and different valence states of carbon in XPS (Figure S3), we judged that the two fullerenols have similar chemical compositions on the surfaces due to the same synthesis condition. The aggregation behavior is also a reflection of the chemical state, which is almost with the same hydrodynamic diameters in aqueous solution (140 nm for C70-OH and 145 nm for C60-OH) (Figure 1d), as well as the zeta potential (-45 mV for C70-OH and -44 mV for C60-OH) (Figure S4). The single peak in dynamic light scattering and the negative value of zeta potential indicate the samples are stable enough in water. Additionally, it also revealed their high stability in physiological mediums, such as in the phosphate buffer saline (PBS), the fetal bovine serum (FBS) and the dulbecco's modified eagle medium (DMEM), without forming any aggregation even after centrifugation at 8000 rpm for 10 min (Figure 1d). 2. Scavenging capability of hydroxyl radical Hydroxyl radical (•OH) is the most common free radical produced from oxygen. Its high reactivity and extremely short half-life time (approximately 10-9 seconds) are



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doomed to the danger to the organism.27,

28

Thus hydroxyl radical is chose as a

representative for evaluating the radical scavenging capability of fullerenols. The EPR techniques showed in Figure 1e revealed the different effects of the two samples on eliminating hydroxyl radicals with various concentrations (Figure S5). Both C70-OH and C60-OH reduced the intensity of the DMPO-OH adduct, but C70-OH notably cut down the quantity of generated hydroxyl radicals (70.6%), much higher than that of C60-OH (48.6%) at the same concentration. The radical-scavenging ability of fullerenes is closely associated with the molecular structure. First, The average structures of the two fullerenols were determined as C70(OH)24•5H2O and C60(OH)26•5H2O according to the combination of the elemental analysis and TGA. In average, comparatively more hydroxyls were bonded to C60 rather than C70. XPS revealed the similar percentage of C=C in the two samples (Figure S3). Therefore, with a larger quantity of intrinsic conjugated double bonds in C70, there are more residual conjugated double bonds in C70-OH compared with C60-OH, indicating that C70-OH got larger capability to capture ROS than C60-OH. This might be the key cause that leads to the apparent different scavenging behavior between C70-OH and C60-OH. Second, other factors like the electron affinity may also affect their radical scavenging property. C60 has been reported to be 2.65 eV while the larger fullerene, C70, has an electron affinity of 2.72 eV.29-31 Last but not least, the large polarizability of fullerenes would facilitates the attachment of electrons and radicals to the nanosurface of fullerenes.32-34


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Figure 1. Characterizations of C70-OH and C60-OH nanoparticles. (a) The schematic diagrams of the synthesis of the two fullerenols. (b) FT-IR spectra for C70-OH and C60-OH nanoparticles. (c) Hydrodynamic size distributions of C70-OH and C60-OH nanoparticles in water. (d) Optical imagines of C70-OH and C60-OH nanoparticles in water, PBS, FBS and DMEM, before (left) and after (right) centrifugation at 8000 rpm for 10 min. (e) Scavenging capability of hydroxyl radical measured by X-band EPR spectra.

3.

Protective

effects

of

[60]/[70]

fullerenols

on

human

epidermal

keratinocytes-adult (HEK-a) cells against oxidative stress First, we investigated the cytotoxicity of the samples by different cells (HEK-a and



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HUVECs). After incubation with C70-OH and C60-OH nanoparticles at various concentrations (50-450 µM), the cells exhibited higher viability than the control (Figure S6), indicating both of them have negligible cytotoxicity and facilitate the cell growth. We selected the HEK-a cells to evaluate the protective effects of the two fullerenols against oxidative stress. By referring to the concentrations of EPR measurement, the HEK-a cells were treated with the C70-OH and C60-OH nanoparticles at different concentrations from 50 to 450 µM for 3 h and then incubated with 800 µM H2O2 for 1 h. From the cell viability and confocal laser scanning microscopy (CLSM) examinations, we could conclude that the two fullerenols both exhibited the protection in vitro and the group treated with C70-OH showed a higher cell viability than C60-OH (Figure 2a). As shown in Figure 2b-e, fluorescence costaining and imaging gave an intuitive confirmation of the cytoprotective effects. The two dyes, Calcein AM and PI, were applied to distinguish the live (green) and dead (red) cells. The untreated cells displayed a uniformly green fluorescence (Figure 2b), while the H2O2-damaged HEK-a cells suffered extensive apoptosis with the red fluorescence (Figure 2c). The C70-OH and C60-OH nanoparticles protected the cells from oxidative stress to different degrees, which was represented by the reduction of red fluorescence (Figure 2d and e).


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Figure 2. (a) Cell viability of HEK-a cells that were treated with C70-OH and C60-OH nanoparticles with various concentrations (50-450 µM) separately for 3 h and then cultured in 800 µM H2O2 for 1 h. Cells treated with only PBS were used as a control. (b-e) Confocal images of HEK-a cells costained with Calcein AM (green fluorescence) and PI (red fluorescence) after incubation with (c) PBS, (d) C70-OH and (e) C60-OH nanoparticles at 350 µM separately for 3 h and then treatment with 800 µM of H2O2 for 1 h. Untreated cells were used as a control (b). 4. Protective effects of [60]/[70] fullerenols against DOX-induced hepatotoxicity in vivo 4.1 The single damage model To investigate the protective effects against oxidative injury in chemotherapy, we



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took DOX, a broad-spectrum chemotherapeutic agent, as a representative to trigger toxicity towards liver. First, we also investigated the protective effects of different dosages on the tested mice by detecting a short-term change of haematological parameters (Table. S2). With the low dosage (0.25 mM), C70-OH showed a limited alleviation compared with the control. As the increase of fullerenols’ concentration, the protective efficacy was strengthened stepwise. Both the middle (0.50 mM) and high (1.00 mM) dosages held relatively effective condition. Thus we chose the concentration of 0.5 mM for system studies in vivo. For comparison, the dosage of C60-OH kept consistent with C70-OH. Twenty-four mice were randomly distributed into four groups (n = 6) as follows: control healthy group, DOX + saline group, DOX + C70-OH group and DOX + C60-OH group. The DOX-treated groups (the latter three groups) were pre-treated with saline, C70-OH and C60-OH for seven days, respectively. On the 8th day they received a single injection of DOX to induce hepatotoxicity (scheme in Figure 3a). The control healthy group was only i.p. injected with saline in the whole course of treatment. Considering the high toxicity usually results in abnormity of the organ coefficient, we checked the body and liver weights on sacrificing the mice and calculated their coefficients of liver. The hepatomegaly appeared to varying degrees in the DOX-treated groups, which is reflected in the increased value of liver coefficient (Figure 3b). Both C70-OH and C60-OH exhibited the inhibiting role in liver swelling, especially C70-OH. Moreover, the administrations of C70-OH and C60-OH prolonged the survival rate of the severely liver-injured mice (Figure 3c). Notably, 70% of the mice treated with C70-OH


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survived in fifteen days while the saline group died within nine days.

Figure 3. (a) The schedule of single damage model treated with C70-OH and C60-OH nanoparticles in DOX-induced hepatotoxicity of mice. (b) The liver coefficient of different groups after sacrificing. *P

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