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Highly Oxidized Graphene Quantum Dots from Coal as Efficient Antioxidants Lizanne Nilewski, Kimberly Mendoza, Almaz S. Jalilov, Vladimir Berka, Gang Wu, William K.A. Sikkema, Andrew Metzger, Ruquan Ye, Rui Zhang, Duy Xuan Luong, Tuo Wang, Emily McHugh, Paul J. Derry, Errol Loïc Samuel, Thomas A Kent, Ah-Lim Tsai, and James M. Tour ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01082 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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ACS Applied Materials & Interfaces

Highly Oxidized Graphene Quantum Dots from Coal as Efficient Antioxidants Lizanne Nilewski,†,┴ Kimberly Mendoza, †,¶,┴ Almaz S. Jalilov,†,1,┴ Vladimir Berka,# Gang Wu,# William K. A. Sikkema,† Andrew Metzger,† Ruquan Ye,† Rui Zhang,† Duy Xuan Luong,† Tuo Wang,† Emily McHugh,† Paul J. Derry, Errol Loïc Samuel,¶ Thomas A. Kent, †,,*,^ Ah-Lim Tsai,#,* and James M. Tour†,‡,§,* †Department

of Chemistry, ‡Smalley-Curl Institute and The NanoCarbon Center, §Department of

Materials Science and NanoEngineering, Rice University, 6100 Main Street, Houston, Texas 77005, USA; ¶Department of Neurology, Baylor College of Medicine, Houston, Texas 77030, USA; Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas 77030; ^Department of Neurology and Research Institute, Houston Methodist Hospital, Houston, Texas 77030, USA; #Hematology, Internal Medicine. University of Texas Houston Medical School, Houston, Texas 77030, USA; *Email: [email protected], [email protected], [email protected] ┴ These

authors contributed equally.

Keywords: Graphene quantum dots, antioxidant, coal, carbon nanotechnology, superoxide dismutase

1

Present address: Department of Chemistry and Center for Integrative Petroleum Research, King

Fahd University of Petroleum and Minerals, Dhahran 31261, Saudi Arabia 1 ACS Paragon Plus Environment

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Abstract Graphene quantum dots (GQDs) have recently been employed in various fields including medicine as antioxidants, primarily due to favorable biocompatibility in comparison to common inorganic quantum dots, although the structural features that lead to the biological activities of GQDs are poorly understood. Here we report that coal derived GQDs and their poly(ethylene glycol)-functionalized derivatives serve as efficient antioxidants, and we evaluate their electrochemical, chemical, and in vitro biological activity.

There is great interest in the development of synthetic antioxidants that can rival the therapeutic efficiency of metalloenzymes in disorders associated with excessive oxidative stress. Under normal physiological conditions, oxidative stress is effectively balanced by naturally occurring low levels of antioxidants and various enzymes including superoxide dismutase (SOD), catalase, vitamin A, coenzyme Q10, glutathione, vitamin E, uric acid, ascorbic acid, and riboflavin, among others.1 However, the lack of clinical evidence for therapeutic benefit suggests that these antioxidants are unable to cope with the overwhelming production of reactive oxygen species (ROS) during extreme levels of oxidative stress, particularly if given after injuries such as traumatic brain injury, stroke, or heart attack.2 Recently, many other carbon-based materials, such as carbon nanodots, GQDs, nanocarbons, and carbon nanotubes with free radical scavenging ability and antioxidant properties, have also been reported.3-8 We have previously characterized a carbon nanomaterial derived from harsh oxidation of single-walled carbon nanotubes, namely poly(ethylene glycol) (PEG)-functionalized hydrophilic carbon clusters (PEG-HCCs).9-13 The PEG-HCCs have no remaining tubular structure as shown 2 ACS Paragon Plus Environment

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from the complete loss of radial breathing modes in the Raman spectra, so they are similar to narrow oxidized graphene nanoribbons. These show exceedingly high SOD-mimetic activity due to their multiple active sites, and are also nontoxic even at very high doses. Nonetheless, seeking to replace the nanotube-derived nanoparticles, due in large part to the perceived toxicity of the parent nanotubes, we synthesized graphene quantum dots (GQDs) from coal, PEGylated them, and studied their electrochemical, chemical and biological activities. We chose coal as a carbon source due to its abundant availability, inexpensive cost, structural features of graphitic regions, and to build off of our previous work on deriving fluorescent GQDs from coal.14,15 However, the GQDs synthesized in this study are not fluorescent, we found that modulating the reduction potential to impart antioxidant activity to these GQDs resulted in loss of fluorescent properties. So they are interesting because of the reduction potential modulation. Through our studies, we determined the key aspects needed to render carbon nanoparticles with the requisite properties16 for usefulness in biological applications related to oxidative stress.

Results and Discussion Synthesis of GQDs and PEG-GQDs GQDs have been produced from various coal sources using sulfuric and nitric acid with sonication and heating.14 Our goal here is to optimize that procedure to give GQDs that contain many oxidation sites, specifically abundant carbonyl and quinoidal moieties, and that are electron deficient with a reduction potential in the same range as HCCs, from ~ +180 mV to −1.0 V, which imparts efficient antioxidant activity.17,18 The procedure developed to synthesize antioxidant GQDs involves heating ground anthracite or bituminous coal in a mixture of 1:1 fuming sulfuric acid with 18-24% excess

3 ACS Paragon Plus Environment


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SO3:fuming HNO3 at 60-70 °C. The reaction time differs for each type of coal due to the inherently different size of innate graphene segments within those coals.14 Accordingly, anthracite GQDs (aGQDs) require longer reaction times than bituminous GQDs (bGQDs). Detailed procedures for each are found in the Experimental Methods section of the Supporting Information. The synthesis of GQDs and PEG-GQDs from coal is shown in Figure 1. First, finely ground anthracite or bituminous coal is suspended and stirred in oleum for 30 min, followed by addition of fuming nitric acid. The reaction is then heated for 1 day (bituminous) or 7 days (anthracite). O 1. oleum 2. nitric acid 65-70 °C

O

O

O

OH

H 2N

OH O

Antracite or Bituminous

OH

O

EDC, NHS H 2O pH 4.5

GQDs

O

O

N H

n

O n

OH O

OH

PEG-GQDs

Figure 1. Synthesis of GQDs and PEG-GQDs from coal.

The resulting GQDs are dialyzed into water for at least 7 days, then separated by size using tangential flow filtration on a KrosFlo system (Spectrum Labs). The major size fraction isolated for bGQDs is 10-30 kD, and the major fraction isolated for anthracite GQDs (aGQDs) is 50-100 kD. In addition to these fractions, sizes from 5-10 kD, 10-30 kD, 30-50 kD, 50-100 kD, 100-300 kD, and 300-500 kD are isolated for each batch in small amounts. However, only the major fractions underwent evaluation, namely, 10-30 kD for bGQDs and 50-100 for aGQDs. After tangential flow separation, the extinction coefficients of the GQDs were experimentally determined to be ε700 = 0.740 mL/mg⋅cm for bGQDs and ε840 = 1.39 mL/mg⋅cm for aGQDs, and the samples were concentrated to ~ 1.0 mg/mL. The units are necessarily changed to mg⋅cm as


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GQDs are nonhomogenous and do not have a single molecular weight, and the mg refers to the estimated carbon content. The concentrated GQDs are then PEGylated using 5000 average molecular

weight

amine-terminated

methoxy-PEG

in

an

aqueous

1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimde (NHS)-mediated amide coupling reaction (Figure 1). The resulting PEG-GQDs are purified again by tangential flow filtration to remove excess PEG and byproducts, giving a final solution of PEG-GQDs in water with concentrations, of the graphitic cores, determined by UV absorbance spectra. For aGQDs, 5000 MW PEG was used and for bGQDs 1000 MW PEG was used. The difference in PEG size was chosen for the biological application of aGQDs in in vivo animal studies. Longer PEG lengths have been shown to increase circulation time in vivo,19-21 so we used 5000 MW PEG on the aGQDs for those studies, which are underway and will be published elsewhere. Characterization of GQDs and PEG-GQDs. Morphological characterization. The size and shape of the GQDs were analyzed by various microscopy techniques. Figure 2 shows transmission electron microscopy (TEM) images of aGQDs and bGQDs and high resolution TEM images of bGQDs. The bGQDs, at 3 to 5 nm, are smaller than the aGQDs, which range from ~10 to 20 nm. In comparison to HCCs, the GQDs have a different overall shape. Since HCCs are derived from longitudinal splitting of single-walled carbon nanotubes, one of their dimensions is limited by the width of the graphene nanoribbon, and this results in ribbon like shapes with sizes of ~3 nm × 40 nm.9-13 Alternatively, the GQDs are more disc-like in shape as seen by TEM.



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Figure 2. TEM images of (a) aGQDs and (b) bGQDs. HRTEM images of bGQDs (c) and (d).

The disk shape of GQDs is also confirmed by atomic force microscopy (AFM) where a dispersion of GQDs in water is deposited on a clean silicon substrate. Figure 3 shows AFM data for aGQDs, bGQDs, and PEG-aGQDs made using 5000 MW PEG (PEG5000-aGQDs). It appears that some GQDs aggregate during the deposition process to form larger particles and stacked particulates. It is also possible that many of the GQDs always exist as multilayered particles since the synthetic processes applied might not exfoliate the components of coal down to single layers. However, both the non-aggregated and aggregated GQDs lay flat on the surface with slightly larger appearance than those seen by TEM due to the lateral size of the AFM tip affecting the observed dimensions. Restrictions due to the AFM tip also prevent the acquisition of higher resolution images; therefore, the shape and more exact size of GQDs can be observed in the HRTEM images above. The height 6 ACS Paragon Plus Environment

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profiles of the aGQDs show that GQDs imaged via AFM are a few layers of graphene with heights ranging from 2-4 nm, close to the height of HCCs of 1.9 nm as previously reported.22 PEG5000aGQDs shown in Figure 2b display a larger lateral size but similar height, due to the PEG addends. AFM of the bGQDs shows large aggregates of the small particles with heights from 2-5 nm. GQDs were also analyzed by Raman spectroscopy (Figure S1), the Raman spectrum shows a characteristic graphene G peak at ca. 1600 cm-1 and a strong D peak at ca. 1400 cm-1. A D+D' peak at ca. 2800 cm-1 is also observed, which results from the vacancies and interstitial and substitutional atoms in the GQD lattice resulting from the oxidation process.23 These suggest the defective nature of GQDs and are consistent with our previous GQD studies.14

Figure 3. AFM characterization of (a) aGQDs (b) PEG5000-aGQDs and (c) bGQDs. Height profile of particles are shown below each image.

Initially, it was thought that the physical properties of the highly-oxidized antioxidant carbon nanomaterials, both the HCCs and GQDs, might be very similar to graphene oxide (GO). However,



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comparison and investigation into the structural features and the electrochemical and antioxidant activity of each material reveals that the HCCs and GQDs have different properties than GO, and because of these difference the GQDs are better antioxidants.24 A representative comparison of the structure of GO to GQDs is shown in Figure 4.

Figure 4. Schematic representation of the difference in the π-conjugated structure and oxygen functional groups between GO and GQDs. GO has a dynamic structure with several epoxides25 while GQDs have more C-C bonds.

XPS comparison of GO, HCCs, and GQDs. The GQDs contain different kinds of oxygen functional groups when compared to those in GO (Figure S2). These features are quantified using X-ray photoelectron spectroscopy (XPS), as outlined in Table 1, and all following characterization data is collected using GO (EMD Merck prepared using KMnO4 in acid) and HCCs, aGQDs, and bGQDs made in our lab. Notably, HCCs and GQDs possess significantly more carboxylic acid groups than GO, as well as fewer carbon-oxygen single bonds than GO. Additionally, the XPS data in Table 1 also illustrates the effect of PEGylation on the GQDs, where the PEG-GQD samples show a large increase in C-O bonds and a decrease in free carboxylic acids as they are


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converted to amides. The XPS spectra for C 1s and O 1s peaks of GO, HCCs, and GQDs are shown in Figure S2, Figure S3 and Figure 5. The differences in the C-O peaks between GO and the HCCs/GQDs is evident, as well as the differences in carboxylic acid peaks and the effect of PEGylation. The extent of PEGylation on the aGQDs was also studied by thermogravimetric analysis (TGA), shown in Figure 6. From the TGA data, it can be calculated that the PEG5000aGQDs are ~ 40-50% PEG by weight.

Table 1. Elemental composition of nanomaterials estimated from the C 1s peak of high resolution XPS Sample

C‒C/C=C

C‒O

C=O

HO‒C=O

%

%

%

%

C/O

GO

1.9

36.0

51.3

8.1

4.6

HCC7

2.0

60.3

15.7

5.9

16.5

aGQDs

1.8

65.9

12.8

5.8

15.5

bGQDs

1.9

61.1

17.3

8.8

12.8

PEG1000-bGQD

1.6

51.0

37.3

5.2

5.4

PEG5000-aGQD

1.7

15.4

68.6

11.4

4.6



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Figure 5. High resolution deconvoluted XPS C 1s spectra of (a) aGQD, (b) bGQD (c) PEG1000bGQD, and (d) PEG5000-aGQD.


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Figure 6. TGA curves of carbon nanomaterials.

Electrochemical characterization. The GQDs were analyzed electrochemically to compare their reduction potentials to those of GO and HCCs. Based on the well-characterized antioxidant activity of HCCs,12 we are interested in synthesizing GQDs with a similar range of redox potential. The HCCs have reduction peaks starting at +180 mV with the maxima ~ −0.8 V, which makes HCCs stronger oxidants than GO by almost 1.0 V. The O2 reduction potentials overlap with that of the HCCs, and therefore the electron transfer oxidation of superoxide by HCCs is a thermodynamically favorable process.17



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Figure 7. CV of GO, HCCs, aGQDs + GO and bGQDs + GO on a glassy carbon (GC) electrode under nitrogen atmosphere. Conditions: 100 mM PBS (pH = 7.4) bare glassy carbon (GC, black line) working electrode. Scan rate: 200 mV/s.

Figure 7 shows the cyclic voltammograms (CVs) of electrode immobilized carbon nanomaterials. The technique of using electrode immobilized HCCs to estimate the electrochemically reducible electron deficient functional groups has been reported.17 Using analogous techniques, we wanted to determine the electrochemically reducible functional groups for aGQDs and bGQDs. However, due to the smaller size and higher solubility of aGQDs and bGQDs in aqueous solvent compared to HCCs, it is difficult to immobilize aGQDs and bGQDs on the electrode surface. Therefore, mixtures with GQDs and GO in 1:1 ratio by weight (GQDs:GO) are used as electrode immobilized materials in order to estimate the electrochemically reducible functionalities of GQDs. GO has been previously reported and it shows well-defined reduction properties for which the reduction onset starts at −1.0 V with the peak maxima at −1.8 V (Figure 7).17 Both aGQDs and bGQDs have a reduction peak with an onset close to −0.2 V that covers a 12 ACS Paragon Plus Environment

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wide range with peak maxima at ~ −1.5 V, which is on the positive side in comparison to GO. This shows that aGQDs and bGQDs are stronger oxidants than GO, and based on the onset peak potential still almost as strong of oxidants as are the HCCs. The differences in reduction potential between GO, HCCs, and GQDs are due to the structural differences and content of oxygen functionalities, as shown by the CV and XPS data. Although the carbon to oxygen ratio is almost the same for each material, the types of C-O bonds and oxygen-containing functional groups are different in GO than in HCCs and GQDs (Figure 4). Specifically, the CV data in combination with XPS points to the predominant quinoid and carbonyl moieties in GQDs and HCCs compared to GO. Quinones and carboxyl groups are the main oxygen-containing functional groups capable of imparting a redox potential in the range of 0.0 V to −0.7 V.18 These groups are also detected by XPS in the O 1s peak for the HCCs.

Electron paramagnetic spectroscopy (EPR) characterization of inherent radicals and antioxidant activity. In addition to possessing electron deficient domains of oxygen-containing functional groups, the HCCs and GQDs also contain an inherent carbon-centered stable radical.26 The intrinsic radical of the bGQDs shown in the EPR spectra in Figure 8 is similar to the radical observed for aGODs (data not shown). The EPR signal of the bGQDs is narrow, 8 gauss wide (peak-to-trough), and symmetrical, centered at g = 2.003. The maximal radical concentration of bGQDs, by double integration against a copper standard, was ~ 0.1:1 mole:mole. Although these EPR characteristics are similar to those of the PEG-HCC radical reported previously,26 the ~10% radical concentration in GQDs is quite different from the 1:1 stoichiometry of PEG-HCCs radical and indicates a possible difference in stability and functional role of the intrinsic radical in GQDs vs PEG-HCCs. The intensity of the EPR signal for the radical decreases with PEGylation of the



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bGQDs as can be seen from comparison of PEG1000-bGQDs (blue line) and PEG5000-bGQDs (red line) with bGQDs (black line) in Figure 8. The similar effect of PEGylation was observed also for aGQDs (data not shown). While the radicals present on GQDs may contribute to part of their antioxidant mechanism, a direct correlation between the presence of intrinsic radicals and antioxidant activity is not clear at this time, and further mechanistic studies are planned but are inherently difficult due to the low molar ratio of radicals and heterogeneous nature of the nanoparticles.


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Figure 8. EPR spectra of bGQDs (black line), PEG1000-bGQDs (blue line) and PEG5000-bGQDs (red line) showing the intensity of the unpaired electron on the carbon core. Inset: The SOD activity of PEG5000-aGQDs (black dots) and PEG5000-bGQDs (red dots) are shown as a turnover number (moles of superoxide consumed per mole of GQDs per second). The concentration of GQDs is 10 nM and reaction time 10 s (average ± SD from 4 independent experiments).

The SOD activity of GQDs and PEG-GQDs is analyzed using manual freeze-trap EPR measurements under steady-state conditions at pH 13 and the turnover number (TON), defined as the number of moles of consumed O2•– per mole of GQD per s, is calculated from the obtained EPR data as previously described.26 The PEGylation of GQDs had minimal effect on their activity as the TON for bGQDs and PEG5000-bGQDs is 0.12 x 106 s-1 (Figure 7 inset, red dots) and 0.1 x 106 s-1 (data not shown) respectively, and the TON for PEG5000-aGQDs is found to be 0.29 x 106 s-1 (Figure 8 inset, black dots). The previously reported TON for PEG-HCCs at pH 13 is 0.19 x 106 s-1, indicating that the superoxide quenching activity of GQDs is comparable to or better than that of PEG-HCCs.14 For comparison, the turnover number of Cu/Zn SOD, and PEG-HCCs are 0.65 x 106 and 1.05 x 106 s-1 at pH 7.7, respectively.26,27 In addition to activity with superoxide, the ability of GQDs to quench hydroxyl radicals (HO•) was investigated. This was measured by EPR detection of the change in the concentration of the BMPO-OH• radical in the presence of GQDs.28-30 As GQDs compete with BMPO to react with HO• (Figure 9), the BMPO-OH• radical peaks will decrease. The hydroxyl radical scavenging ability of PEG5000-aGQDs is shown in Figure 10. Figure 10a shows that with increasing concentrations of PEG5000-aGQDs, the EPR peak of the BMPO-OH• radical decreases, corresponding to the PEG5000-aGQDs reacting with HO•. Figure 10b depicts the comparison of



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PEG5000-aGQDs concentration vs the intensity of the BMPO-OH• radical peak, showing again that higher concentrations of PEG5000-aGQDs diminish the BMPO-OH• peak due to more efficient quenching of hydroxyl radicals. Overall, the EPR data shows very close similarities between the properties and behaviors of the GQDs to the PEG-HCCs, suggesting that the GQDs are also efficient antioxidants and mimics of SOD (Figure S4).

Figure 9. Generation of the BMPO-OH• radical by reaction of BMPO with HO•.

Figure 10. (a) EPR spectra of BMPO-OH• generated from FeSO4 and H2O2 in PBS at different concentrations of PEG5000-aGQDs. (b) Effect of PEG5000-aGQDs concentration on •OH scavenging activity.


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Assessment of GQD protective activity in vitro. Finally, the protective capacity of PEGylated graphene quantum dots was assessed using a H2O2 rescue assay using bEnd.3 murine endothelioma cells.20 In this assay, the effect of aGQDs and bGQDs on cell viability was compared to PEGHCCs by treating bEnd.3 cells with 100 µM H2O2 for 15 min before adding solutions of the carbon nanomaterials and incubating the cells overnight. H2O2 generates secondary radicals (SO and HO•) intracellularly which PEG-HCCs are known to mitigate.31 The aGQDs and bGQDs were further tested against 100 µM H2O2 by adding the particles to cultured bEnd.3 cells in media at final concentrations of 2, 4, or 8 mg/L 15 min after initial exposure to the H2O2. The cells are then incubated overnight. The following morning, a live cell count is performed and shows that PEGGQDs of both types are capable of protecting the bEnd.3 cells against 100 µM H2O2 but with particle-dependent outcomes. Protection was dose-dependent as the concentration increased from 0 to 8 mg/L with aGQDs protecting more cells at lower concentrations than bGQDs. All levels of PEG-GQD treatment showed a statistically significant improvement in cell viability (p < 0.001) compared to H2O2 alone (Figure 11; live cell percentage of untreated controls). bGQDs afforded less protection than aGQDs at all levels (aGQD 2 mg/L vs bGQD 2 mg/L: 87.1 ± 18.3 vs 75.7 ± 11.62, p = 0.042; aGQD 4 mg/L vs bGQD 4 mg/L: 104.5 ± 14.8 vs 77.7 ± 12.1, p < 0.0001; aGQD 8 mg/L vs bGQD 8 mg/L: 102.2 ± 11.1 vs 88.8 ± 9.6, p = 0.0079). These results are consistent with the findings of SOD activity shown in Figure 8 whereby aGQDs consume more superoxide per particle than bGQDs. The comparison between PEG-HCCs and GQDs as shown in Figure 11 shows that HCCs and GQDs have similar in vitro activity and aligns with the EPR and electrochemical characterizations. As for ongoing and future biological studies including in vivo work, we envision the PEG-GQDs to be administered via intravenous or interstitial injection in



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the same manner that the previously published in vivo studies with PEG-HCCs have been performed.10-12,32

Figure 11. Cell viability following treatment with 100 µM H2O2 (H2O2) without and with PEGHCCs (H2O2 + PEG-HCC, 8 mg/L) or GQDs at different concentrations given 15 min after initial exposure and incubated overnight. PEG-HCC results are from a separate experiment. (a) Results of aGQDs given at 2, 4, and 8 mg/L 15 min after initial 100 µM H2O2 treatment. All treatment levels with aGQDs are statistically significant compared to H2O2 alone (*p < 0.001). (b) Results of bGQDs given at 2, 4, or 8 mg/L 15 min after initial 100 µM H2O2 treatment. All treatment levels with aGQDs are statistically significant compared to H2O2 alone (*p < 0.001). Results are averages of 32 samples of duplicate wells within a single experiment. Statistical analysis employed oneway ANOVA with Tukey correction.


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Conclusions In summary, highly oxidized non-toxic (per our cell assays) antioxidant carbon nanoparticles called GQDs have been synthesized from the readily available coal carbon source. These GQDs were shown to be mimics of the enzyme SOD and of our previously reported antioxidant carbon nanomaterials, HCCs. GQDs were characterized with respect to redox chemistry, size and morphology, extent of oxidation, and reactivity with various radical species. Specifically, GQDs were shown to quench both SO and HO•. GQDs were also found to convert SO into oxygen, consistent with SOD mimetics. PEG-modified GQDs (PEG-GQDs) were synthesized to impart improved solubility and biological stability and demonstrated in vitro cellular protection against H2O2, even with delayed administration.

Acknowledgements This work was funded by The Alliance for NanoHealth Grant W81XWH-0902-0139, National Institutes of Health Grants HL095820 and NS084290-01; R01-NS094535 and R21-NS084290 (T.A.K., J.M.T., A.L.T.) the Dunn Foundation (J.M.T), National Defense Science Engineering Graduate Fellowship (K.M.) and Grant No. BE-0048 from the Welch Foundation (T.A.K.).

Supporting Information: The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional materials and methods, XPS spectra and references



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