Crossover between Anti- and Pro-oxidant Activities of Graphene

Sep 1, 2016 - Jing RuanYing WangFang LiRenbing JiaGuangming ZhouChunlin ShaoLiqi ... FanYu ChongRuibin LiCuicui GeChunying ChenJun-Jie Yin...
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Crossover between Anti- and Pro-oxidant Activities of Graphene Quantum Dots in the Absence or Presence of Light Yu Chong,†,‡ Cuicui Ge,*,†,‡ Ge Fang,† Xin Tian,† Xiaochuan Ma,† Tao Wen,‡ Wayne G. Wamer,‡,§ Chunying Chen,*,⊥ Zhifang Chai,†,⊥ and Jun-Jie Yin*,‡ †

School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China ‡ Division of Bioanalytical Chemistry and Division of Analytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740, United States ⊥ Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China and Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *

ABSTRACT: Graphene quantum dots (GQDs), zerodimensional carbon materials displaying excellent luminescence properties, show great promise for medical applications such as imaging, drug delivery, biosensors, and novel therapeutics. A deeper understanding of how the properties of GQDs interact with biological systems is essential for these applications. Our work demonstrates that GQDs can efficiently scavenge a number of free radicals and thereby protect cells against oxidative damage. However, upon exposure to blue light, GQDs exhibit significant phototoxicity through increasing intracellular reactive oxygen species (ROS) levels and reducing cell viability, attributable to the generation of free radicals under light excitation. We confirm that light-induced formation of ROS originates from the electron−hole pair and, more importantly, reveal that singlet oxygen is generated by photoexcited GQDs via both energy-transfer and electron-transfer pathways. Moreover, upon light excitation, GQDs accelerate the oxidation of non-enzymic anti-oxidants and promote lipid peroxidation, contributing to the phototoxicity of GQDs. Our results reveal that GQDs can display both anti- and pro-oxidant activities, depending upon light exposure, which will be useful in guiding the safe application and development of potential anticancer/antibacterial applications for GQDs. KEYWORDS: graphene quantum dots, free radicals, anti-oxidants, phototoxicity, lipid peroxidation

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anion, hydrogen peroxide, and hydroxyl radicals, are essential intermediates in physiological processes, and their levels within cells are tightly controlled by anti-oxidant systems.15−17 However, that intracellular redox homeostasis can be perturbed in many different ways: a burst of ROS can cause oxidative damage to essential biomolecules (e.g., proteins, lipids, and nucleic acids), resulting in growth arrest or apoptosis. Several studies have shown that GQDs can exhibit both ROS-quenching and -producing activities in biological systems.18,19 For example, Samuel et al.20,21 reported that carbon clusters having superoxide dismutase-like properties

ue to their superior optical properties, inorganic semiconductor quantum dots (e.g., CdSe, CdTe) have been used as bioimaging probes for nanomedical research.1,2 However, a major disadvantage of conventional inorganic quantum dots is the intrinsic toxicity associated with the release of heavy metal ions.3,4 Recently, ultrafine graphene quantum dots (GQDs), small fragments of graphene-based carbon materials, have attracted much interest because they possess both excellent luminescence properties and good biocompatibility.5−13 Interestingly, the photoluminescence of GQDs can be quenched in solution by either electron acceptor or donor molecules, which indicates GQDs have the potential to perform pro-oxidant and anti-oxidant activities.14 This behavior may have important implications for the formation or quenching of physiologically relevant reactive oxygen species (ROS). Those ROS, including singlet oxygen, superoxide © 2016 American Chemical Society

Received: June 19, 2016 Accepted: September 1, 2016 Published: September 1, 2016 8690

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Figure 1. Characterization of GQDs. (a) TEM image of as-prepared GQDs. (b) Top view AFM images of GQDs. (c) Statistics of GQD thicknesses measured by AFM. (d) UV−vis absorption spectrum of the GQDs dispersed in water. (e) Excitation and emission contour map of GQDs. (f) Observation of unpaired electron on GQD samples in solutions.

method.26,27 As previously reported, these GQDs are highly soluble in water and have good biocompatibility.13 Figure 1a presents a TEM image of as-synthesized GQDs, which range in size from 3 to 6 nm. Figure 1b illustrates the topographic morphology of GQDs, as characterized by AFM; the heights of that topography are mainly distributed between 0.4 and 2 nm, corresponding to a single layer of graphene. The GQDs show two UV−vis absorption peaks at around 230 and 280 nm, as shown in Figure 1c, representing a typical absorption of a π system from benzene structures and functional groups. As our data show, broad absorption with a gradual reduction up to the long wavelength indicates the existence of a band tail, attributable to defect states.28 The 2-D fluorescence topographical maps showed the excitation wavelength maximized at 375−505 nm (Figure 1d). The ESR spectrum, shown in Figure 1e, revealed an intrinsic GQD unpaired electron signal of Lorentzian shape centered at g = 2.0025 with a line width of 2 G, which was very stable at room temperature. Free Radical-Scavenging Activity of GQDs. The presence of the defects and unpaired electrons at the surface of GQDs indicate their potential for quenching reactive ROS. Moreover, the π-conjugated nature of GQDs is likely to facilitate charge transfer and electron storage.29 Therefore, to analyze the free radical-scavenging capability of GQDs, we developed a set of cell free radical-producing systems for measuring the most common ROS and nitrogen-centered free radicals using ESR, which is the most reliable and direct method for identifying and quantifying short-lived free radicals. It is well known that the high reactivity of hydroxyl radicals (•OH) can cause oxidative damage to lipids, proteins and DNA. The •OH for our experiments were generated using the classical Fenton reaction between Fe2+ and H2O2.30 As shown in Figure 2a,b, the strong ESR signals indicated the formation of DMPO/•OH adduct. The addition of GQDs significantly decreased the ESR signal intensity in a dose-dependent manner. A 10 μg/mL of GQDs decreased the ESR signal by approximately 56%, demonstrating the strong •OH-scavenging

could improve cerebrovascular dysfunction in a rat model of traumatic brain injury. Volarevic et al.22 studied the immunomodulatory and cytoprotective effects of GQDs in a mouse model of immune-mediated liver damage. These effects may be related to the modulation of ROS. In contrast to their moderating effects on ROS, when GQDs in suspension are exposed to photoexitation, they can also generate singlet oxygen, making them potential candidates for use in photodynamic therapies.23−25 However, there have been neither systematic studies of how GQDs generate or quench ROS nor explorations of subsequent biological effects. Indeed, the mechanism underlying the effects GQDs exert on ROS has not yet been clearly explained. Achieving a better understanding of these anti-oxidative and pro-oxidative properties of GQDs would greatly facilitate development of safe and practical applications. In our initial experiments using the electron spin resonance (ESR) technique, we discovered that GQDs can efficiently quench a series of free radicals and inhibit lipid peroxidation, a process involved in many disease states, including inflammation, neurodegenerative diseases, and cancers. To explore further, we evaluated the cytoprotective effects of GQDs against the oxidative damage induced by H2O2 or X-rays on human umbilical vein endothelial cells (HUVECs). However, we found that upon irradiation with blue-violet light, the presence of GQDs was associated with bursts of ROS, resulting in remarkable phototoxicity, as determined in human A549 lung cancer cells. We then used ESR spin trapping methods in a cellfree system to identify and quantify the species of ROS generated following photoexcitation of GQDs. The mechanism by which GQDs initiated free radical generation was further investigated. In addition, we measured the effects of GQDs on anti-oxidant system and peroxidation of lipids in liposomes.

RESULTS AND DISCUSSION Preparation and Characterization of GQDs. Our GQDs were synthesized using a chemical oxidation and cutting 8691

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Figure 2. Scavenging of different types of free radicals by GQDs. (a) ESR spectra of DMPO/•OH adducts were collected after incubation 1 min of samples containing 20 μM Fe2+, 50 mM DMPO, 20 μM H2O2, 10 mM PBS buffer (pH 7.27), and different concentrations of GQDs. (b) Effect of GQD concentration on their •OH-scavenging activity. (c) ESR spectra of BMPO/•OOH adducts were obtained from samples containing 25 mM BMPO, 10% DMSO, 2.5 mM KO2, 0.35 mM 18-crown-6, 10 mM PBS buffer (pH 7.27), and different concentrations of GQDs after 1 min incubation. (d) Effect of GQD concentration on their O2•−-scavenging activity. (e) ESR spectra of DPPH• were obtained from samples containing 0.1 mM DPPH•, 10 mM PBS buffer (pH 7.27), and different concentrations of GQDs. Data were recorded 5 min after incubation. (f) Effect of GQD concentration on their DPPH•-scavenging activity.

Figure 3. Effects of GQDs on HUVECs (a) and protective capabilities of GQDs against damage induced by H2O2 (b) and X-rays (c). (a) Cell viability of HUVECs incubated with GQDs at various concentrations (0−100 μg/mL) for 24 h in the culture medium. (b) Oxidative stress was induced by exposing the cells to 200 μM H2O2 for 1 h. The protective effect of GQDs was assessed by exposing the H2O2-treated cells to 0− 100 μg/mL GQDs for 24 h. (c) Selected concentrations of GQDs (0−100 μg/mL) were incubated with HUVECs about 1 h before a single radiation with X-ray (10 Gy). Data shown are mean values and standard deviations from three independent experiments (n = 3). *P < 0.05 and **P < 0.01 vs H2O2- or X-ray-treated group.

hydroxyl radicals in a concentration-dependent manner (Figure S1). Superoxide anion (O2•−) is another important physiologically relevant ROS that forms through the one-electron

capacity of GQDs. Moreover, another system generating hydroxyl radicals was employed: we irradiated an aqueous suspension of TiO2 with UV light.31 Consistently, our results sufficiently demonstrated that GQDs can effectively scavenge 8692

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Figure 4. Photoinduced cytotoxicity and oxidative stress of GQDs on A549 cells. A549 cells were incubated with or without GQDs (20 μg/ mL) and exposed to blue-violet laser (405 nm, 1 W) for 5 min. Cells without laser were used as control. Cell viability was determined by CCK8 (a) and fluorescence-based LIVE/DEAD assays (b) after 24 h (scale bar: 100 μm). Intracellullar production of ROS was determined after 4 h by fluorescence microscopy (c) and flow cytometry (d) with the DCFH-DA dye. Data shown are mean values and standard deviations from three independent experiments (n = 3). **P < 0.01 vs control.

vivo.13,26,27 Next, damage models were used to evaluate the protection effect of GQDs on oxidative stress. It was clear that GQDs protected cells against H2O2-induced death in a dosedependent manner (Figure 3b). A similar phenomenon was observed in X-ray damage model (Figure 3c). Overall, these results suggest that GQDs can behave as anti-oxidants and protect cells against oxidative stress through effectively scavenging free radicals. Photoinduced Cellular Toxicity of GQDs. Since GQDs have a great potential as biological labels for cellular imaging,5−13 it is necessary to evaluate the biosafety of GQDs when exposed to laser radiation. Here, blue-violet laser (405 nm) was used because the GQDs exhibited bright green or blue color when imaged on the confocal laser scanning microscope.36 The assay of cell viability showed that laser exposure or GQDs alone was not able to significantly affect the viability of cells (Figure 4a). However, cells treated simultaneously with GQDs and laser displayed a substantial loss in viability, indicating the phototoxicity of GQDs. The fluorescence-based LIVE/DEAD assay provided further confirmation; simultaneous treatment with GQDs and light markedly reduced the cellular viability, as indicated by a significant increase in the number of PI-permeable, red fluorescent cells (Figure 4b). Further, we used DCFH-DA, a ROS fluorescence probe emitting green fluorescence upon oxidation by ROS, to measure the intracellular ROS level.37 As indicated in Figure 4c, almost no fluorescence can be seen in the GQD- or lasertreated cells. In contrast, bright green fluorescence signals can be observed in cells simultaneously treated with GQDs and laser, suggesting high intracellular ROS levels. The quantitative analysis of ROS generation determined by flow cytometry provided a consistent conclusion (Figure 4d). Extremely weak fluorescent signal displayed in cells treated with GQDs or laser, whereas a remarkably high fluorescent signal was observed for

reduction of molecular oxygen. When overproduced, superoxide anion can denature enzymes, oxidize lipids, and fragment DNA. Here, we employed non-enzymatic and enzymatic superoxide anion generating systems to investigate the superoxide anion-scavenging capacity of GQDs.32 In the nonenzymatic system, a typical four-line ESR spectrum with relative intensities of 1:1:1:1 was observed, indicated the formation of radical adducts between O2•− and the spin trap, BMPO (Figure 2c,d).33 However, the radical’s ESR signal intensity was dramatically reduced when GQDs were introduced. Similar concentration-dependent effects were observed in the xanthine/xanthine oxidase system (Figure S2). A 10 μg/mL of GQDs displayed excellent scavenging capacity against O2•−, similar to that of SOD. Reactive nitrogen species is another group of free radicals that can alter cellular function. The stable, nitrogen-centered free radical, DPPH•, is often used to assess anti-oxidant activity of substances.34 We found that GQDs could also quench these reactive nitrogen species (Figure 2e,f), and the scavenging properties of GQDs for DPPH• was similar to those observed for quenching ROS. GQDs Protect HUVECs against Oxidative Stress. Reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide, singlet oxygen and hydroxyl radicals, are generated as byproducts of cellular metabolism. Oxidative stress occurs when the production of ROS, exceeds the anti-oxidant capacity of cellular anti-oxidants in a biological system, resulting in oxidative damage to lipids, proteins, and DNA.35 We speculated that GQDs, having excellent free radical-scavenging ability, would be able to protect cells against oxidative damage. First, we evaluated the biocompatibility of as-prepared GQDs using the CCK-8 assay. No obvious adverse effects were observed after exposure to 100 μg/mL GQDs (Figure 3a), which confirms the good biocompatibility of GQDs and agrees well with our and others’ previous investigations in vitro and in 8693

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Figure 5. Generation of several free radicals by photoexcited GQDs. (a) ESR spectra obtained from samples containing 10 mM 4-oxo-TEMP, 10 mM PBS buffer (pH 7.27), and different concentrations of GQDs. (b) ESR spectra obtained from samples containing 25 mM DEPMPO, 10 mM PBS buffer (pH 7.27), and different concentrations of GQDs. (c) ESR spectra obtained from samples containing 100 μM CPH, 10 mM PBS buffer (pH 7.27), and different concentrations of GQDs. (d) ESR spectra obtained from samples containing 10 mM POBN, 10 mM PBS buffer (pH 7.27), and different concentrations of GQDs. The control (black) represents both the sample containing spin probe alone under light, and the sample containing spin probe and GQDs before exposure to light. All the spectra were recorded after 5 min of irradiation with the filtered (400 nm) light.

superoxide anion) effectively decreases the ESR signal of 1O2 (Figure S3b). This suggests that superoxide anion is involved in the generation of singlet oxygen, implying that electron transfer is an intermediate step for generation of singlet oxygen by photoexcited GQDs. It is notable that with increasing concentration of SOD over 0.2 U/mL, the ESR signal no longer changed and remained decreased by one-half compared to control. This observation implies that superoxide anion is also involved in the production of singlet oxygen. Based on these results, we speculate that singlet oxygen is generated by photoexcited GQDs via both energy-transfer and electrontransfer pathways. Next, we used the spin-trapping agent, DEPMPO, to detect superoxide anion and hydroxyl radical.39 Upon exposing GQDs to light, the characteristic spectrum of the DEPMPO/•OH adduct appeared (Figure 5b), and the signal increased as the concentration of GQDs increased. BMPO is another spin trap frequently used to detect superoxide anion and hydroxyl radical. As expected, a BMPO/•OH signal was observed during irradiation of GQDs in the presence of BMPO (Figure S4). Since these adducts, BMPO/•OH and BMPO/•OOH, have overlapping ESR spectra, we used SOD (a specific scavenger for superoxide anion) and DMSO (a specific scavenger for hydroxyl radical) to further investigate whether superoxide anions are involved in this ESR signal (Figure S4). Addition of 0.5 U/mL SOD partially inhibits the ESR intensity observed during photoexcitation of GQDs. A residual, four-line spectrum

cells treated with photoexcited GQDs. Therefore, the phototoxicity of GQDs can be ascribed to a burst of ROS in cells. Mechanisms of ROS Generation Induced by Photoexcited GQDs. To understand the mechanism of ROS generation induced by photoexcited GQDs, ESR spectroscopy was used to directly detect and identify the ROS generated by GQDs exposed to the filtered (400 nm) light. Here, we selected 4-oxo-TEMP as a spin trap for characterization of singlet oxygen38 as several groups have reported that GQDs exhibit a 1 O2 generation and can be a candidate of photodynamic therapy (PDT) agent.23−25 Figure 5a illustrates the dosedependent increase in ESR spectra obtained upon the irradiation of GQDs with 4-oxo-TEMP. The ESR signals displayed a typical 1:1:1 triplet spectrum characteristic for the 4-oxo-TEMP/1O2 adduct, with a hyperfine splitting constant, αN = 16.08 G, while no ESR signals were observed for GQDs without irradiation. This result showed that photoexcited GQDs induced the generation of singlet oxygen in irradiation time- and GQD concentration-dependent fashion (Figure 5a and Figure S3a). To confirm that the signal originated from singlet oxygen, 1 mM NaN3, a scavenger of 1O2, was added and the ESR signal almost completely disappeared, indicating that the active species generated by photoexcited GQDs ascribed to singlet oxygen (Figure S3b). Previous studies reported that GQDs generate 1O2 through energy transfer to molecular oxygen.24 In contrast, our study shows that the addition of SOD (specific scavenger for 8694

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Figure 6. Oxidation of AA and GSH by photoexcited GQDs. UV−vis spectroscopy was used to determine the amount of AA remaining (a) or the amount of GSH remaining (b) in dispersions containing irradiated GQDs (10 μg/mL). ESR spectra of AA• (c) and GS• (d) were recorded after photoexcitation for 5 min.

observed a considerable reduction of ESR signal. The results obtained using CPH and TEMPO confirm that band-to-band electron transition and charge separation is the important source of generation of ROS. In addition to active oxygen species, we also observed apparent carbon-centered free radicals during irradiation of GQDs by using POBN as a spin trap.44 Under conditions where samples contained POBN and GQDs or irradiation alone, no ESR signal was detected (Figure 5d). However, upon exposure to photoexcited GQDs, a six-line ESR signal of the POBN radical adduct was detected, which had hyperfine coupling constants αN = 15.1 G and αH = 2.5 G. This characteristic ESR spectrum indicates the generation of carboncentered free radicals, which may have come from the interaction between photoexited electron and the backbone of GQDs. Effects of Photoexcited GQDs on Anti-oxidant Defense System. It is expected that ROS overproduction will result in depletion of intracellular anti-oxidants.45,46 To explore the effect of photoexcited GQDs on AA and GSH, we conducted the following spectroscopic studies. As shown in Figure S6 and Figure 6a, the absorption peak at 265 nm, the characteristic absorption maximum of AA, was unchanged in the presence of GQDs or irradiation alone. However, the absorption peaks gradually diminished in the presence of photoexcited GQDs. These results indicate that AA is undergoing oxidation. Ellman’s reagent was then used to quantify the concentration of thiol groups in samples containing GSH. As shown in Figure S7 and Figure 6b, photoexcitation of GQDs significantly reduced the thiol groups. After 20 min irradiation, GSH was predominately oxidized after its exposure to GQDs. Moreover, the intermediate products of AA and GSH oxidation including AA• and GS• were determined by ESR.47,48 As displayed in Figure 6c, the extremely weak ESR signals of AA• indicated that the oxidation of AA by GQDs is very limited. Upon irradiation, strong ESR

is seen having intensities of 1:2:2:1. This spectrum is characteristic for the BMPO/•OH adduct. Addition of 10% DMSO leads to a typical ESR spectrum for the BMPO/•OOH adduct with intensity ratios of 1:1:1:1. The above results demonstrate that both the hydroxyl radical and superoxide anion are generated during photoexcitation of GQDs. Next, we investigated the mechanism of hydroxyl radical and superoxide anion generation. Recently, several groups have reported that GQDs have a natural band gap because of quantum confinement and that the band gap of the GQDs increases as the size of the GQDs decreases.27 It is well known that absorption of light having energy similar to the band gap of a semiconductor results in charge separation and creation of electron−hole pairs, which can react with surrounding molecular oxygen or water to form various ROS.40 The electrons trapped transiently on the surface or on the next-tosurface defects can react with the adsorbed oxygen molecules forming O2•− and hole can react with the H2O molecules forming •OH. To examine the oxidizing activity of photogenerated holes, we used CPH, an often-used hole scavenger which can be oxidized to form CP-nitroxide radicals (CP•) with a typical ESR spectrum with intensity ratios of 1:1:1.41−43 As shown in Figure 5c, CPH itself is ESR silent. Upon addition of GQDs exposed to the filtered (400 nm) light, a strong triplet signal appeared with hyperfine splitting constant αN = 16.2 G. The signal intensity increased in a concentration-dependent manner. Additionally, the spin label TEMPO was selected for characterizing the electrons generated by photoexcited GQDs. TEMPO with stable triplet ESR spectrum can be reduced to give a hydroxyl amine (TEMPOH), which is ESR silent. Samples were purged with nitrogen gas (N2) to avoid the reaction between molecular oxygen and the photogenerated electron. As shown in Figure S5, the ESR signal intensity of TEMPO was unchanged for samples that had not been exposed to irradiation or for irradiated TEMPO alone. However, within 5 min of irradiation in the presence of TEMPO and GQDs, we 8695

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Figure 7. Effects of GQDs on the lipid peroxidation in liposomes. (a) Lipid peroxidation was inhibited by 100 μg/mL GQDs. The samples contained 30 mg/mL EYPC liposomes, 0.2 mM 15N-PDT, and 25 mM AAPH with (right) or without (left) GQDs. (b) Lipid peroxidation was enhanced by 100 μg/mL GQDs and irradiation with 400 nm bandpass filtered light. The samples contained 30 mg/mL EYPC liposomes, 0.2 mM 15N-PDT with (right) or without (left) GQDs. The progressive increases in peak-to-peak signal intensity (and accompanying progressive narrowing of line width) in each panel are due to time-dependent oxygen consumption resulting from lipid peroxidation.

signals of AA• were observed, illustrating that exposure to light accelerates the oxidation of AA by GQDs. Similarly, upon irradiation, a typical DMPO/•SG ESR spectrum (αN = 15.3 G and αH = 16.2 G, which is different from DMPO/•OH) was seen (Figure 6d), indicating the oxidation of GSH by photoexcited GQDs. Therefore, under condition of light exposure, GQDs could accelerate the oxidation of critically important cellular anti-oxidants, AA and GSH. Effect of GQDs on Lipid Peroxidation in Liposomes. The above studies demonstrate that GQDs can effectively scavenge free radicals, whereas upon exposure to light they promote the significant generation of free radicals. Under physiological conditions, those free radicals can react with the polyunsaturated fatty acids of lipid membranes, inducing lipid peroxidation, which has been associated with a variety of disease states.49 We measured the effects of GQDs on lipids using ESR oximetry and multilamellar liposomes, consisting of EYPC.50 We used liposomes contained in closed capillaries to estimate the rate of lipid peroxidation by monitoring oxygen consumption (ESR oximetry). That spin label, 15N-PDT, exhibits a narrower line width and higher peak intensity as the concentration of oxygen decreases (Figure S8a). No oxygen depletion was observed for samples containing liposomes and PBS or 100 μg/mL GQDs within 30 min (Figure S8b,c). However, a sharp spectral peak, increasing with time, for 15N-PDT appeared after adding 500 mM AAPH (Figure 7a), a strong inducer of lipid peroxidation. The increase in the ESR peak intensity for 15N-PDT results from AAPHinduced lipid peroxidation with concomitant consumption of oxygen. In the presence of both GQDs and AAPH, much smaller peak heights in the ESR signal was observed, suggesting the significant arrest of lipid peroxidation caused by GQDs. In contrast, photoexcited GQDs resulted in a significant increase in oxygen consumption (Figure 7b), even faster than AAPH on the same time scale, whereas irradiation alone resulted in a limited decrease in oxygen levels. These results illustrate that photoexcited GQDs induced lipid peroxidation to a high degree, an observation consistent with the observed photoxicity of GQDs.

GQDs have the potential to become potent anti-oxidants for controlling ROS-induced cell damage, after exposure to 405 nm laser, these same GQDs elicited phototoxicity, as demonstrated by increased intracellular ROS levels and cell death. We have used ESR techniques to directly detect and identify the generation of singlet oxygen, hydroxyl radical, and superoxide anion induced by photoexcited GQDs. This enabled us to design a series of experiments exploring the origin of ROS induced by photoexcited GQDs and provide a systematic elucidation of the mechanism by which free radicals are generated by photoexcited GQDs. Our proposed mechanism is that singlet oxygen is generated by photoexcited GQDs via energy-transfer and electron-transfer pathways; hydroxyl radical and superoxide anions arise from creation of electron−hole pairs and charge separation, respectively, upon reacting with surrounding molecular oxygen or H2O. Additionally, we have identified a potential mechanism explaining the phototoxicity of GQDs: upon photoexcitation, GQDs accelerate the oxidation of ordinarily non-enzymatic anti-oxidant biomolecules (for example, ascorbate or glutathione) and also accelerate lipid peroxidation. These results demonstrate the dual properties of GQDs as anti-oxidants and pro-oxidants upon irradiation. This deeper understanding of the dual properties of GQDs will help us better define conditions for appropriate therapeutic use of GQDs. The intrinsic anti-oxidant activity of GQDs suggests these might have great potential for treating clinical conditions associated with oxidative stress. On the other hand, ROS such as singlet oxygen, have been shown to kill cancerous cells or harmful bacteria. Thus, our finding that exposure to light can cause GQDs to generate high concentrations of singlet oxygen, i.e., behave in a pro-oxidant manner, suggests that GQDs could be adapted as antibacterial or anticancer treatments.

MATERIALS AND METHODS Materials. Graphite was purchased from Alfa Aesar (Ward Hill, MA). Egg yolk phosphatidylcholine (EYPC) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). The LIVE/DEAD viability/ cytotoxicity kit was purchased from Invitrogen (Carlsbad, CA). 5tert-Butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), and Cell Counting Kit-8 (CCK-8) kit were obtained from Dojindo Laboratories (Kumamoto, Japan). 1Hydroxy-3-carboxy- 2,2,5,5-tetramethylpyrrolidine (CPH) and 2,2,6,6tetramethylpiperidine-1-oxyl (TEMPO) were purchased from Alexis, Enzo Life Sciences, Inc. (Farmingdale, NY). 4-Oxo-2,2,6,6-

CONCLUSIONS The GQDs we prepared were capable of scavenging a series of free radicals. Such GQDs should be useful for protecting cells against the oxidative damage resulting from in vitro ROSgenerating models. However, although our findings show that 8696

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ACS Nano tetramethylpiperidine-d16-1-oxyl (15N-PDT) was obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). Human umbilical vein endothelial cells (HUVEC) and human A549 lung cancer cells were obtained from the American Type Culture Collection (Manassas, VA). Milli-Q water (18 MΩ·cm) was used for preparation of all solutions. Other chemicals were obtained from Sigma-Aldrich (St. Louis, MO) and used as received. Synthesis of GQDs. GQDs were prepared according to previously published methods,13,26,27 with some modifications. Specifically, 1 g of graphite was mixed with 20 mL of fuming HNO3, followed by adding 100 mL of H2SO4 (98%). This mixture was sonicated for 30 min and stirred for 2 h at 110 °C. Afterward, this mixture was neutralized with NaOH, cooled, and diluted with ultra-pure water. The final products were dialyzed (using a dialysis bag with a molecular weight cutoff of 1000 Da) against pure water for further characterization and application. Characterization of GQDs. The morphology and size of the GQDs were characterized using a FEI Tecnai G-20 transmission electron microscopy (TEM) at an operating voltage of 200 kV. Atomic force microscope (AFM) images were obtained from an AFM (Nanoscope Icon, Veeco) operating in the tapping mode at room temperature. The UV−vis spectra were recorded at room temperature on a Shimadzu UV-3600 UV−vis spectrophotometer. Fluorescence spectra were acquired using an Edinburgh Instruments FLS 980 spectrophotometer. Anti-oxidant Activities of GQDs. Electron Spin Resonance Spectroscopic Measurements. Fifty microliter aliquots of either control or sample solution were put in quartz capillary tubes with internal diameters of 0.9 mm. All the ESR measurements were carried out at ambient temperature using a Bruker EMX ESR spectrometer (Billerica, MA) with 20 mW microwave power, 1 G field modulation unless otherwise stated. Hydroxyl Radical (•OH)-Scavenging Activity. Hydroxyl radicals were produced by a classical Fenton reaction (200 μM (NH4)2Fe(SO4)2/200 μM H2O2) or irradiating a TiO2 (100 μg/mL) solution with UV light (a 450 W xenon lamp coupled to 340 nm band-pass filter). The ESR spectra were recorded at 1 min after initiating the generation of •OH by exposing to H2O2 (for Fenton reaction) or UV light (for TiO2 solution). DMPO was used to trap •OH in the form of the spin adduct DMPO/•OH, and the amount of •OH was quantitatively estimated by the peak-to-peak height of the second line of the ESR spectrum. Superoxide Radical Anion (O2•−)-Scavenging Activity. The spin trap, BMPO, was used to identify superoxide anion during the ESR measurements. Two O2•− sourcesenzymatic xanthine/xanthine oxidase (Xan/XOD, XOD-catalyzed oxidation of Xan to uric acid produces O2•−) and chemical KO2 system (O2•− was generated by dissolving KO2 in DMSO solvent in the presence of crown ether) were used to verify the ability of GQDs to scavenge O2•−. This reaction was initiated by adding either XOD or KO2. DPPH•-Scavenging Activity. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH•), a stable, nitrogen-centered free radical, was used to demonstrate the ability to scavenge reactive nitrogen species by GQDs. One mM DPPH• in ethanol was mixed with a fixed volume of GQDs at different concentrations and ESR spectra were collected after exactly 5 min of incubation. The extent of scavenging ability was determined by comparing the activity of solutions containing GQDs with the activities of control groups that did not contain GQDs. Cytoprotective Effects. Cell viability was evaluated using a CCK-8 assay. The responses of HUEVEC cells to exposure to either Hydrogen peroxide (H2O2) or X-ray were used to establish the models of ROS generation. Briefly, HUVECs were seeded on 96-well plate, at a density of 8 × 103 cells per well, and cultured in a humidified 5% CO2 incubator at 37 °C for 24 h. Then the cells were treated with 200 μM H2O2. After 1 h, different concentrations of GQDs (0, 25, 50, or 100 μg/mL) were added to the cell cultures, which then incubated for 24 h. In the X-ray treated groups, GQDs were incubated with cells for 1 h before a single 10-Gy dose of X-radiation was administered. Cells not given any treatment were used as controls. Cell viability was

measured by addition of CCK-8 and then reading the optical density (OD) of each well at 450 nm. Pro-oxidant Activities of GQDs upon Photoexcitation. Cell Viability. CCK-8 and LIVE/DEAD assays were used to assess the cytotoxicity of GQDs photoexcited with blue-violet laser (405 nm, 1W). Specifically, A549 cells were seeded in 96-well plates (for CCK8) or 24-well plates (for LIVE/DEAD assay) and incubated at 37 °C in a 5% CO2 atmosphere for 24 h. Next, the culture medium, DMEM, was replaced with fresh complete medium either with GQDs or without them, after which the cultures were either exposed to laser for 5 min or not exposed to laser treatment. After irradiation, all groups of cells were incubated for another 24 h, then treated with CCK-8 or LIVE/DEAD probes to determine cell viability. Generation of Free Radicals. A 450 W xenon lamp filtered by a 400 nm bandpass filter was used as the excitation light. The spin trap 4oxo-2,2,6,6-tetramethylpiperidine (4-oxo-TEMP) was used to verify the generation of singlet oxygen. 5-(Diethoxyphosphoryl)-5-methyl-1pyrroline-N-oxide (DEPMPO) and BMPO were used to demonstrate the formation of superoxide anion and hydroxyl radical. CPH and TEMPO were used as spin probes for studying the holes and electrons generated during photoexcitation of GQDs. α-(4-Pyridyl-N-oxide)-Ntert-butylnitrone (POBN) was used to detect the carbon-centered free radicals. Control conditions ran either without GQDs or without irradiation. To further distinguish each ROS, dimethyl sulfoxide (DMSO), superoxide dismutase (SOD), and sodium azide (NaN3) were employed separately to test their scavenging effects on the ESR signal for hydroxyl radical, superoxide, and singlet oxygen, respectively. Oxidative Damage to Anti-oxidant Systems. The oxidation of ascorbic acid (AA) and glutathione (GSH) by photoexcited GQDs were monitored by UV−vis and ESR. The 450 W xenon lamp filtered by a 400 nm bandpass filter was chosen as the excitation light. To examine the effects of irradiated GQDs on AA, 10 μg/mL GQDs was mixed with 100 μM AA. During the 20 min of irradiation, samples (1 mL) were collected every 5 min and the amount of remaining AA was determined by measuring the absorbance of AA at the λmax (265 nm). The concentration of thiols, measured using Ellman’s reagent, demonstrated the effects of photoexcited GQDs on GSH. Suspensions of GQDs (10 μg/mL) were added to 50 μM GSH and illuminated with light for times varying from 0 to 20 min. Adding 100 μM DNTB to the mixtures to yielded a yellow product, which was quantitated by measuring absorbance at 412 nm. In this case, oxidized GSH (GSSG) was used as a positive control, and an AA or GSH solution without GQDs or light was used as a negative control. The loss of AA or GSH was calculated using the following formula: loss of AA% or GSH% = 100 × (absorbance of negative control − absorbance of sample)/ absorbance of negative control. Ascorbyl radicals (AA•) and glutathionyl radicals (GS•) are intermediates formed during the oxidation of AA and GSH. The ESR technique can directly detect AA•, which only has a 10 min half-life. The samples containing GQD suspensions (2, 10 μg/mL) was mixed with 5 mM AA and irradiated with light for 5 min. The ESR spectra were recorded under the following conditions: 20 mW incident microwave power; 1 G field modulation; 20 G scan width. The reaction of GS• can be monitored by trapping with DMPO to form the adduct DMPO/•SG. Therefore, we prepared a suspension containing 5 mM GSH, 50 mM DMPO, and 2 or 10 μg/mL GQDs and then illuminated it with 400 nm bandpassfiltered light for 5 min. The resulting ESR spectra were recorded using a 100 G scan width. Effects on Lipid Peroxidation. We measured the effects of GQDs on peroxidation of lipids in liposomes using ESR oximetry, which is based on the bimolecular collision of O2 with a spin label. As O2 molecules are paramagnetic, colliding the spin label with O2 produces a spin exchange between these two compounds, resulting in shorter relaxation times (both T1 and T2); the ESR spectrum of the spin label then exhibits a broader line width. Because the integrated area of the ESR spectrum is unaffected by these effects on relaxation times, this broadening of ESR spectrum line width is inevitable, accompanied by a decrease in the peak-to-peak height of the ESR signal. Since the extent of spin exchange is dependent on molecular O2 concentration, any increases or decreases in O2 concentrations will result in a concomitant 8697

DOI: 10.1021/acsnano.6b04061 ACS Nano 2016, 10, 8690−8699

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ACS Nano increase or decrease of the line width and a related decrease or increase in signal amplitude for the spin label. Therefore, a time-dependent change in line width that occurs in conjunction with a change in the amplitude of the ESR spectrum indicates continuous O2 generation or consumption, and this spectral change can be quantified.50 For our current experiments, we used the spin probe 15N-PDT, which produces a line width very sensitive to changes in the levels of O2 concentration. As lipid peroxidation reduces the concentration of O2, the line width of the 15N-PDT spin label decreases. A timedependent decrease in line width, along with an increase in the intensity of the ESR signal, indicates continuous oxygen consumption. Therefore, one can assess rate of lipid peroxidation in the samples by repeatedly measuring the spin label’s line widths or signal intensities. The preparation of liposomes and measurement of lipid peroxidation by the ESR oximetry had been reported before.50 First, the samples containing 30 mg/mL EYPC liposomes, 0.2 mM 15NPDT, 25 mM AAPH, either with or without GQDs, were sealed in each capillary tube. Oxygen consumption was measured by obtaining ESR spectra recorded at 5 min intervals for 30 min. These spectra were obtained with a 3 G scanning width, l mW microwave power, and 0.01 G field modulation. AAPH is known to strongly induce lipid peroxidation in cellular and reconstituted membranes. In addition, to determine whether photoexcited GQDs affect the extent of lipid peroxidation, samples of 30 mg/mL egg PC liposomes and 0.2 mM 15 N-PDT, with or without GQDs, were irradiated using light (λ = 400 ± 10 nm) for 5−20 min. Oxygen consumption was determined by ESR, as previously described. Statistical Analysis. Mean and standard deviation (SD) were calculated. Results were expressed as mean ± SD. Comparisons within each group were conducted by a Student’s t test. P value of