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Graphene Oxide Quantum Dots as Novel Nanozymes for Alcohol Intoxication Anqi Sun, Li Mu, and Xiangang Hu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00306 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 23, 2017

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ACS Applied Materials & Interfaces

Graphene Oxide Quantum Dots as Novel Nanozymes for Alcohol Intoxication

Anqi Sun†, Li Mu‡, Xiangang Hu†,*

†

Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of

Education), Tianjin Key Laboratory of Environmental Remediation and Pollution Control, College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China. ‡

Institute of Agro-environmental Protection, Ministry of Agriculture, Tianjin 300191,

China

Correspondence should be addressed to: College of Environmental Science and Engineering, Nankai University, Tianjin. E-mail address: [email protected] (X.G. Hu). Tel: 0086-022-23507800; Fax: 0086-022-23507800.

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ACS Paragon Plus Environment


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ABSTRACT Alcohol overconsumption as a worldwide issue results in alcoholic liver disease (ALD), such as steatosis, alcoholic hepatitis and cirrhosis. The treatment of ALD has been widely investigated but remains challenging. In this work, the protective effects of graphene oxide quantum dots (GOQDs) as novel nanozymes against alcohol overconsumption are discovered, and the specific mechanisms underlying these effects are elucidated via omics analysis. GOQDs dramatically alleviate the reduction of cell viability induced by ethanol and can act as nanozymes to accelerate ethanol metabolism and avoid the accumulation of toxic intermediates in cells. Mitochondrial damage and the excessive generation of free radicals were mitigated by GOQDs. The mechanisms underlying the cellular protective effects were also related to alterations in metabolic and protein signals, especially those involved in lipid metabolism. The moderately increased autophagy induced by GOQDs explained the removal of accumulated lipids and the subsequent elimination of excessive GOQDs. These findings suggest that GOQDs have an antagonistic capacity against the adverse effects caused by ethanol and provide new insights into the direct applications of GOQDs. In additional to traditional antioxidation, this work also establishes metabolomics and proteomics techniques as effective tools to discover the multiple functions of nanozymes.

Keywords: Graphene; Nanozyme; Ethanol; Hepatocyte; Oxidative stress; Artificial enzyme 2


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1. INTRODUCTION Alcohol overconsumption is a worldwide health issue that leads to alcoholic liver disease (ALD), including early-mild steatosis, advanced fibrosis, superimposed cirrhosis and, finally, hepatic carcinoma,1 and liver cirrhosis accounts for 16.6% of mortality worldwide.2 The development of detoxification methods has become a global topic and the alcohol detoxification work is going worldwide. In order to accelerate ethanol excretion, the utilization of antioxidant enzymes and hepatic transferase is considered to be an effective detoxification method.3 Unfortunately, the use of natural enzymes has many drawbacks, such as low stability, weak recovery, ultrahigh selectivity and high cost.4 Compared with metal-based nanomaterials, graphene-family nanomaterials (GFNs) exhibit high biocompatibility and represent a promising substitute for natural enzymes because of their distinctive electronic properties and high catalytic activities.5-10 Currently, GFNs are used as nanozymes in bioimaging and biosensing, but limited information about their activities for applications against toxins and for the prevention of diseases, such as ALD, is available. Liver is the main organ that metabolizes alcohol. Following consumption, alcohol is oxidized to acetaldehyde, which generates oxidative stress that causes structural and functional abnormalities in the liver.11 Oxidative stress is the first issue that must be addressed using nanozymes. However, the cytotoxicity of nanozymes has drawn substantial attention because their peroxidase-like activity can decompose hydrogen peroxide to produce highly toxic hydroxyl radicals under acidic conditions.12-14 In 3



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contrast, the free radical-scavenging ability of GFNs is their most exploited property in biomedical studies because of their sp2 structures.15-16 In long-term studies, graphene oxide quantum dots (GOQDs) have been shown to exhibit lower cytotoxicity than graphene oxide (GO) and pristine graphene because of their more efficient degradation and excretion.17-19 In general, at the same concentration, the catalytic activity of nanozymes is inversely proportional to the size distribution; thus, quantum dots with large edge effects, high charge density and quantum confinement may be a desirable option.20-21 Therefore, we investigated GOQDs as potential nanozymes to protect hepatocytes against ALD. Currently, most work on nanozymes is limited to the antioxidant defense system, although oxidative stress is critical to cellular physiological homeostasis, as described above. The mechanisms of nanozymes against the adverse effects of toxins are expected to be more effective than the antioxidant defense system alone; however, limited information about other detoxification pathways is available.22 In this context, beyond antioxidation, the possible pathogenesis responsible for ALD may include toxic metabolites, unbalanced lipid metabolism and biomacromolecule disorders.23-24 As non-specific assessment techniques for metabolite and protein profiles, metabolomics and proteomics can be employed as effective tools to elucidate the potential mechanisms of biological responses.25-26 To gain sensitive and detailed insight into the detoxification mechanisms of nanozymes, metabolomics and proteomics studies were performed. To test the above hypothesis that GOQDs are potential antidotes for ALD, suitable 4


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doses of ethanol and GOQDs were screened. Then, the effects of GOQDs on cell viability, oxidative stress, mitochondria and hepatic alcohol-metabolizing enzymes were analyzed in the context of alcohol overconsumption. Cell metabolism, especially lipid metabolism, and mediation mechanisms related to the protein profile were explored. This paper describes an effective method for preventing ALD using GOQDs and reports the discovery of the global functions of nanozymes based on an omics analysis. 2. EXPERIMENTAL SECTION 2.1. Characterization of nanomaterials GOQDs were obtained from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). The details of the nanomaterial characterization were described in our previous study.27 Briefly, the morphology of the GOQDs was examined with field-emission transmission electron microscopy (TEM, JEM-2010 FEF, JEOL, Japan) and atomic force microscopy (AFM, Nanoscope IV, VEECO). The size distribution was measured using a ZETAPALS/BI-200SM instrument equipped with a 30-mW, 635-nm laser (Brookhaven Instruments Corporation, USA). The surface groups of the GOQDs were investigated with Fourier transform infrared spectroscopy (FTIR, Bruker Tensor 27, Germany). 2.2. Cell culture and viability assay Buffalo rat liver (BRL) cells were cultured in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) at 37°C in an incubator with 5% CO2. The viability of 5



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BRL cells in the presence of ethanol or GOQDs was assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and the fluorescein diacetate (FDA) method. Cells were plated at a density of 5×104 cells/mL in 96- and 6-well microplates for the MTT and FDA assays, respectively. To determine the suitable concentrations of ethanol and nanomaterials, cells were cultured in a complete medium with the addition of ethanol (200, 300, 400, or 500 mM) or GOQDs (1, 10, or 100 mg/L) for 12 h, followed by the MTT assay using an automatic microplate reader (Bio Tek, USA) at 490 nm. Subsequently, the cells in the complete medium were exposed to 10-mg/L GOQDs for 12 h and then washed with phosphate-buffered saline (PBS). Next, these cells were exposed to 300-mM ethanol for 12 h. The cells in the 6-well microplate were stained with FDA (2.5 µg/mL) for 15 min in the dark. After staining, the cells were trypsinized, washed and re-suspended in PBS, and then, the FDA assay was performed using a fluorescence spectrophotometer (LS55, PerkinElmer, USA) with excitation at 480 nm and emission at 530 nm. 2.3. Cellular ultrastructure The cellular ultrastructure was observed by TEM. After treatment with GOQDs and ethanol, the cells seeded in the 6-well microplates were collected and fixed in 2.5% glutaraldehyde at 4°C and then subjected to post-fixation in 1% osmium tetroxide and dehydration in a graded ethanol series. Finally, the samples were embedded in an epoxy resin, cut into ultrathin sections (70 nm) and stained for TEM observation (HT7700, Hitachi, Japan). 2.4. Mitochondrial membrane potential loss 6


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After treatment with the GOQDs and ethanol, the cells attached to the 6-well microplates were stained with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1, 10 µg/mL) for 15 min in the dark. The fluorescence assay was conducted using a fluorescence spectrophotometer (LS55, PerkinElmer, USA). For the J-aggregate (red fluorescence) assay, the excitation and emission wavelengths were 585 and 590 nm, respectively. For the monomer (green fluorescence) assay, the excitation and emission wavelengths were 514 and 529 nm, respectively. The mitochondrial membrane potential loss was recorded as the ratio of the intensity of the red fluorescence to that of the green fluorescence. 2.5. Oxidative stress The generation of intracellular reactive oxygen species (ROS) was probed using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The cells were incubated with DCFH-DA (10 µM) for 30 min. The fluorescence intensity was measured using an automatic microplate reader (Bio Tek, USA) with excitation at 485 nm and emission at 530 nm. The malondialdehyde (MDA) content was determined with an assay kit (Nanjing Jiancheng Bioengineering Institute, China) using an ultraviolet-visual (UV-vis) spectrophotometer (TU-1900, Persee, China). Since glutathione (GSH) is widely considered as one of the major antioxidants in cells,28 GSH (10 mM) instead of GOQDs was used as a positive control for ROS and MDA measurements. According to the kit’s instructions (Nanjing Jiancheng Bioengineering Institute, China), the activities of superoxide dismutase (SOD) and catalase (CAT) were 7



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determined with an automatic microplate reader (Bio Tek, USA). The SOD/CAT-like activity of GOQDs was also determined. To further verify the CAT-like activity, hydrogen peroxide (30%) and GOQDs (10 mg/mL) were mixed to determine whether oxygen was produced. Moreover, the effects of GOQDs on hydroxyl radicals and nitroxide radicals were elucidated using an electron paramagnetic resonance (EPR) spectrometer (Magnettech MiniScope 400 EPR, Germany) with MiniScope Control software. Hydroxyl radicals were obtained through the Fenton reaction with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap agent. 2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) was employed as the source of nitroxide radicals. For the EPR measurements, the tested concentrations of GOQDs were 1, 10 and 100 mg/L. 2.6. Hepatic alcohol-metabolizing enzymes The activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) of the cells and GOQDs were determined. Briefly, cells were seeded in 75-cm3 flasks and collected and then subjected to pre-lysis with 2 ml of Triton X-100 (2%) and complete lysis by sonication. For the ADH assay, the lysates were mixed with reaction buffer (v/v=1/4) containing 3-mM nicotinamide adenine dinucleotide (NAD+), 0.5-M Tris-HCl and 300-mM ethanol. For the ALDH assay, the lysates were mixed with reaction buffer (v/v=1:4) containing 3-mM NAD+, 100-mM NaPO4, 10-mM pyrazole and 1-mM propionaldehyde. To determine the ADH/ALDH-like activity of the GOQDs, the lysates were replaced with GOQDs, and the final concentration of the GOQDs in the reaction buffer was 10 mg/L. The catalytic activity was measured by 8


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an automatic microplate reader (Bio Tek, USA) at 340 nm. The activities of both ADH and ALDH were expressed in units of nmol of nicotinamide adenine dinucleotide (NADH) generated/min/mg protein. The catalytic activity of the GOQDs was expressed in units of nmol NADH generated/min/mg. 2.7. Metabolic analysis Precooled methanol/chloroform (900 µL, v/v=2/1) was mixed with approximately 106 cells and then sonicated in an ice bath at 300 W for 3 min. Subsequently, 300 µL of precooled chloroform and 540 µL of ultrapure water were added. The suspension was centrifuged for 20 min at 9447 g and 4°C, and the metabolites were collected from the upper and lower phases. The organic solvents and water were removed by nitrogen blow-off and lyophilization, respectively. Finally, derivatization was conducted with 50 µL of methoxamine hydrochloride (20 mg/mL) and 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide. The metabolites were analyzed by gas chromatography coupled to quadruple mass spectrometry (6890N/5973, Agilent, USA). 2.8. Proteomics analysis Approximately 106 cells were processed via filter-aided sample preparation (FASP) using the standard two step digestion29 followed by mass spectrometry-based proteome analysis. Specifically, FASP includes the filtration of low-molecular weight components using a urea buffer, thiol carboamidomethylation, protein digestion and peptide elution. Proteome analyses were conducted using liquid chromatography (Thermo EASY-nLC, USA) coupled with mass spectrometry (Thermo Obitrap Fusion, 9



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USA). Proteins were identified and quantified with Thermo Proteome Discoverer 1.4 and MaxQuant database, respectively. The quantification results were corrected by the total proteins of each group. 2.9. Statistical analysis At least three replicate experiments were performed to ensure credible data detection. The collected data exhibited a normal distribution and were analyzed using one-way analysis of variance (ANOVA). A value of p

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