Comparisons between Graphene Oxide and Graphdiyne Oxide in

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Biological and Medical Applications of Materials and Interfaces

Comparisons between Graphene Oxide and Graphdiyne Oxide in Physicochemistry Biology and Cytotoxicity Tingting Zheng, Yu Gao, Xiaoxiao Deng, Huibiao Liu, Jian Liu, Ran Liu, Jingwei Shao, Yuliang Li, and Lee Jia ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06804 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018

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ACS Applied Materials & Interfaces

Comparisons between Graphene Oxide and Graphdiyne Oxide in Physicochemistry Biology and Cytotoxicity

Tingting Zheng1‡, Yu Gao1‡, Xiaoxiao Deng1, Huibiao Liu2, Jian Liu1, Ran Liu1, Jingwei Shao1, Yuliang Li2, Lee Jia1*

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Cancer Metastasis Alert and Prevention Center, and Pharmaceutical Photocatalysis of State Key

Laboratory of Photocatalysis on Energy and Environment, College of Chemistry; Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Fuzhou University, Fuzhou 350002, China. 2

CAS Key Laboratory of Organic Solids, Beijing National Laboratory for Molecular Sciences

(BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.

*Corresponding author: [email protected]; [email protected] (Lee Jia) ‡

These authors contributed equally to this work.

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Abstract Graphdiyne (GDY) and graphene (G) are regarded as two promising two-dimensional (2D) carbon-based materials, which have unique planar structure and novel electronic properties. Differences between the two carbon allotropes in their physicochemistry biology and cytotoxicity have never been explored. Here, we chemically functionalized the surface of the two carbon allotropes using similar oxidation processes, and compared their physicochemistry, biology and mutagenesis. Graphene oxide (GO) and graphdiyne oxide (GDYO) showed similarities in their size, morphology, and physical spectral characteristics, excepting the differences in sp- and sp2hybridization and FT-IR. GDYO was well soluble in various media. In contrast, GO was only soluble in H2O, but kinetically aggregated in 0.9%NaCl, PBS and cell media within 24 h incubation when its concentrations increased. GO nanoparticles adhered and aggregated to the surface of human hepatocyte membrane, resulting in cell membrane ruffle, methuosis and apoptosis. Adhesion of GO to cells caused cell stress and induced reactive oxygen species (ROS). In contrast, GDYO did not adhere to cell membrane to produce the related consequences. Both GDYO and GO showed in vivo mutagenesis potential but no erythrocyte-killing effect, and both were antioxidant and bioequivalent at binding to single-stranded DNA and doxorubicin, causing fluorescence quenched. The present study significantly enriches our existing knowledge of GO/alkene and GDYO/alkyne chemistry.

Keywords: graphene oxide, graphdiyne oxide, solubility, cytotoxicity, antioxidant

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1. INTRODUCTION The serendipitous discovery of fullerenes in 1985 launched a new era in synthetic carbon allotropes1. Synthetic carbon allotropes have attractive architectures and superior material properties such as ultra-high charge-carrier mobility and a huge capability for carrying current2. Among the carbon allotropes family, graphene is the most well-studied material with structural uniform that synthesized by exfoliated from graphite with scotch tape3-5. As the latest carbon allotrope, graphdiyne has recently emerged6-8, and sparked tremendous interests in its potential applications in energy storage9,10, electrocatalytic activity11-13 and rapid heterogeneous electron transfer14,15. Graphdiyne is composed of benzene rings and butadiyne linkages in its structure6. All benzene ring connects with each other by butadiyne linkages (–C≡C–C≡C–) to form 18-C hexagon units with sp2 and sp hybridized carbon atoms16,17. The addition of the carbon-carbon triple bonds to graphdiyne (the resulting product of graphene) may change physicochemical and biological properties of graphene to a certain degree. However, the speculation has not been proven yet. In view of its remarkable electronic, mechanical and thermal properties, graphdiyne is hoped to offer a better alternative. Nonetheless, further realizing applications of graphene and graphdiyne in many fields still require many fundamental researches. In the present study, we did head-to-head comparisons between graphene oxide (GO) and graphdiyne oxide (GDYO) in their physicochemical, biological and mutagenesis properties with the hope that the well-controlled comparisons could provide clear information about the true advantages and disadvantages of the two unique carbon allotropes, define characters of alkenes and alkynes, and guide the future applications of the two carbon allotropes. 2. EXPERIMENTAL SECTION 2.1. Synthesis of GO and GDYO 3



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Graphdiyne (GDY) was synthesized by hexaethynylbenzene as the method reported previously7. GDY oxide (GDYO) was synthesized from GDY powder by H2O2 and H2SO4. GDY (50 mg) was gradually stirred in 30% H2O2 solution (1 mL) and 98% H2SO4 (2.5 mL) under an ice-water bath for 1 h. Then 50 mL double distilled water was added into the reaction mixture to stop oxidization, followed by dialysis (cutoff, 3500) for 3 days to remove mixed acid, dispersing into water under sonication for about 4 h to form a homogeneous brown aqueous solution for storage. Graphene oxide (GO) was made by an improved Hummer’s method using graphite flake as starting material18. A mixed acid of H2SO4/H3PO4 (120:13.5 mL) and KMnO4 (6 g) as the oxidizing agent were gradually added to the graphite flake (about 1 g) under an ice-water bath, after stirred at 50℃ for 12 h. Finally, the reaction was cooled to room temperature and stopped by 30% H2O2. The oxidized product washed by repeated centrifugation, and sonication exfoliated for about 2 h to form a homogeneous suspension used for further experiments. 2.2. Measurement and Characterization X-ray photoelectron spectra (XPS) survey scan were measured by ESCALAB 250 X-Ray photoelectron spectrometer (Thermo Scientific, USA). Raman spectra were recorded by the inVia Reflex Raman spectrometer (Renishaw, UK), at resolution of 2 cm-1 by using the 532 nm line of an argon ion laser as the excitation source. Fourier transform infrared spectroscopy (FT-IR) spectra were collected by an AVATAR 360 FT-IR spectrophotometer (Nicolet, USA) in 4000 - 400 cm-1 region using KBr tablets. The morphologies analysis of the GDYO and GO films were observed on the transmission electron microscopy (TEM) images, which were recorded by the field emission Hitachi HT7700 transmission electron microscopy (Hitachi, Japan). Atomic force microscopy (AFM) images were collected on the Bruker MultiMode V8-SPM (Bruker, Germany) with a ScanAsyst model. The 4


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size and zeta potential of GDYO and GO were recorded by Zetasizer Nano ZS (Malvern, UK). 2.3. Solubility and Stability of GDYO and GO The solubility and stability of GDYO and GO were tested when challenged by different physiological solutions at room temperature (about 25℃), including double distilled water, dimethyl sulfoxide (DMSO), phosphate buffer (PBS, containing NaCl about 137 mM, pH= 7.4), 0.9% NaCl (154 mM) and the commonly-used cell medium DMEM (containing NaCl about 109 mM, with 10% FBS). The dynamic changes in the size of GDYO and GO within 24 h were recorded by a Zetasizer Nano ZS (Malvern, UK). 2.4. Cell Culture LO2 cells were maintained in DMEM medium (HyClone, USA) as we described previously19. 2.5. Dynamic Morphologic Changes of LO2 Cells About 8× 104 LO2 were preincubated in a culture dish (NEST Biotechnology, China) overnight. Then cells were co-incubated with GDYO or GO (100 µg/mL). The dishes were placed in a live cell chamber with 5% CO2 and 37℃. Real-time imaging was collected by a Leica TCS SPE confocal microscopy (Leica, Germany), The images of morphologic changes were collected every 5 min for 12 h. 2.6. Apoptosis Assay Annexin V-FITC and PI apoptosis detection kit (Genview, USA) was used for detecting apoptotic and necrotic cells. The cells were co-incubated with a dose range of GDYO and GO for 24 h at 37℃. The GDYO or GO treated cells were digested and suspended in 500 µL of binding buffer, stained with Annexin V-FITC and PI. After stained in the dark for 15 min, the cells were immediately analyzed in BD FACSAria flow cytometry (BD Biosciences, USA). 5



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2.7. MTT Assay LO2 cells were inoculated on 96-well plates for the MTT assay to evaluate the cells metabolic activity, then cells incubation with GDYO or GO for 24 h, 48 h, 72 h at 37℃, respectively. A 5 mg/mL MTT stock solution (Sigma, USA) was diluted by RPMI-1640 medium (without serum and phenol red, Sigma, USA) for 10 times. The cell medium was replaced with equal MTT diluent in every well, and incubated for another 4 h in dark. Then removed the supernatant, 100 µL of dimethyl sulfoxide add into every well. Absorbance was collected at 490 nm by an Infinite M200 PRO microplate reader (TECAN, Switzerland). The values were expressed as the absorbance percentage with the control wells (without treatment for GDYO or GO). 2.8. Lactase Dehydrogenase Release Detection Lactase dehydrogenase (LDH) release assay was used for detecting the cell membrane integrity, using a cytotoxicity detection kit (Beyotime, China). LO2 cells were grown on 96-well plates, cells were exposed to GDYO at various concentrations for 24 h. Then 120 µL of supernatant was transferred to a blank plate, with 60 µL of substrate mix follow the instructions. The plate was gently shaken on a platform for 30 min in dark, followed absorbance was recorded at 490 nm by a microplate reader. The values were expressed as percent of absorbance relative to positive control wells that were treated by LDH releasing agent. 2.9. Reactive Oxygen Species Measurement ROS generation in cells were measured using a reactive oxygen species assay kit (Beyotime, China). 4× 105 LO2 cells were co-incubation with different concentrations of GDYO and GO at 37℃ after 24 h, were washed three times and re-suspended in HBSS containing 10 µM of DCFH-DA for 30 min, then detected by flow cytometry and confocal microscopy. Intracellular ROS generation was 6


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denoted by the fluorescence intensity of DCF, which produced by the non-fluorescent dye (DCFH-DA) when undergo ROS mediated oxidation. 2.10. Micronucleus Assay The mutagenesis potential of GDYO and GO were preliminarily detected by micronucleus test. SPF Kunming mice (males; Wu's experimental animal, China) with the weight of 25-35 g were used to carry out the mutagenic assay. The mice were divided into 4 groups of five in each group and kept in the same environment. The animals in negative and positive control group were administered normal saline and cyclophosphamide (10 mg/kg) intravenously once a day for five days. The GO group and GDYO group mice were disposed with GO or GDYO solution in the same way, corresponding the total doses of 20 mg/kg. The follow-up operations referred to the Liu et al. reported previously20. 2.11. Fluorescence Detection The fluorescence measurements on the F-7000 fluorescence spectrometer (Hitachi, Japan), The excitation and emission slits used in this experiment were all 5 nm and the voltage of photo-multiplier was 600 V. Fluorescence intensities of DOX samples were recoded range from 500 to 700 nm (λex=488 nm). Changes in fluorescence intensities of dye-labeled ssDNA were excited at 490 nm and collected range from 500 to 600 nm. 2.12. Total Antioxidant Capacity Assay Total antioxidant capacity assay kit with the ferric reducing ability of plasma (FRAP) method (Beyotime, China) can be used to assay the antioxidant property of materials in biological fluids. The antioxidants can reduce Fe3+-TPTZ (colorless) to Fe2+-TPTZ (blue) under the acidic condition, the total antioxidant capacity was recorded by matching absorbance changes at wavelength 593 nm. We 7



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prepared FRAP working solution followed the operating instructions, added 180 µL every well to a 96-well plate, and mixed it up with 15 µL different concentrations of GDYO and GO. After 72 h incubated, absorbance was recorded at 593 nm by a microplate reader. 2.13. Statistical Analysis All data are represented as the mean with the standard deviation (mean± SD). We calculated the significance in terms of ANOVA by employing GraphPad Prism 5, * denotes a statistical significance versus the control and # denotes a statistical significance between GDYO and GO. 3. RESULTS AND DISCUSSION 3.1. Oxidation and characterization of graphene and graphdiyne Graphdiyne (GDY) was synthesized as the previously reported7. Graphene oxide (GO) was made by an improved Hummer’s method using graphite flake as the starting material18. Chemical functionalization of GDY and G could enhance solubility, processability and stabilization of GDY and G. We oxidized GDY by using concentrated H2SO4 and H2O2, and oxidized G by using concentrated H2SO4/H3PO4 and KMnO4, respectively (Figure 1a). The high quality of few-layer sheets of GDYO and GO were obtained by strong sonication and centrifugation. X-ray photoelectron spectra (XPS) reveal the differences between GDYO and GO in their functional groups. The XPS survey scan of GDYO clearly show the peaks of C1s and O1s (Figure 1f). GDYO retains the skeleton of GDY, which was evident from the presence of C≡C, C=C, C–O, and C=O at 285.1, 284.5, 286.2, and 288.5 eV in the C1s spectra (Figure 1g), respectively. While the C1s peaks of GO (Figure 1k) can be mainly convoluted into three peaks at 284.6, 286.9 and 288.3 eV, which are assigned to a C1s orbital of C=C, C–O, and C=O, respectively. Although the two carbon allotropes have a similar shape of flat atomic sheets, their chemical structures are different. 8


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Raman spectroscopy was used to characterize the crystallinity and number of nanosheets, and evaluate the quality and uniformity of GDYO (Figure 1h) and GO (Figure 1l). The spectrum intensity ratios of GDYO (the intensity of the peak at 1365.75 cm-1 to that at 1587.23 cm-1) and GO (the intensity of the peak at 1360.91 cm-1 to that at 1589.39 cm-1) are 0.801 and 0.859, respectively, indicating that the as-made GO and GDYO show similar high order and low defects. Fourier transform infrared spectroscopy (FT-IR) was used for confirming the hydrophilic functional groups in GDYO and GO. As displayed in Figure 1i, peaks at 3423.92 cm-1, 1717.81 cm-1 and 1224.11 cm-1 could be ascribed to the stretching vibration of –OH, –C=O and –C–O–C– in GDYO, respectively. The bands located at 1635.85 cm-1 are assigned to the skeletal vibrations of the aromatic rings. The typical C≡C stretching vibration is very weak in spectra because of the molecular symmetry. Figure 1m shows the existence of the functional groups –OH (3424.49 cm-1), C=O (1734.69 cm-1), and C=C (1638.78 cm-1) in GO. Atomic force microscopy (AFM) provides morphological information on the synthesized GDYO (Figure 1b) and GO (Figure 1c). The height of the two carbon allotropes is about 1 nm. The transmission electron microscopy (TEM) images of GDYO (left inset of Figure 1d) and GO (left inset of Figure 1e) suggest that both GDYO and GO display a nanosheet-like morphology with occasional folding. The results of dynamic light scattering (DLS) further show the sizes of GDYO (136 nm, Figure 1d) and GO (113 nm, Figure 1e). In conclusion, the morphological features of GDYO and GO are very similar, they share the unique planar structure and large surface area. 3.2. Solubility and related bioactivity Pristine graphdiyne and graphene are highly hydrophobic, and surface functionalization by adding the oxygen functional groups to their surface may improve their hydrophilicity. To examine if 9



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the above chemical functionalization of GDY and G indeed enhances their solubility, processability, and stabilization, we mixed GDYO and GO with water, dimethyl sulfoxide (DMSO), phosphate buffer (PBS, containing NaCl about 137 mM, pH= 7.4), 0.9% NaCl (154 mM) and the commonly-used cell medium DMEM (containing NaCl about 109 mM, with 10% FBS, pH 7.4), respectively, at 25℃ and observed any changes in the solubility and stabilization of the mixtures at concentrations ranging from 10 µg/mL to 300 µg/mL. As shown in figure 2a-2b, GDYO exhibited excellent solubility and stability in all the biological solutions tested. The size of GDYO measured by using Zetasizer Nano ZS did not significantly change during the 24 h observation (Figure 2b), and GDYO solutions seemed very stable. The excellent solubility of GDYO may result from its loosely-arranged structure that gives it more structural flexibility and variability to meet changes in its environments. By contrast, GO was quite stable in water and DMSO, but gradually aggregated in the PBS, NaCl and DMEM that containing rich ions or proteins (Figure 2c). Figure 2d shows the kinetic changes in the size of GO after mixed with different solutions at 50 µg/mL. After 24 h incubation with these solutions at 25℃, the size of GO increased by 10-fold compared with its original size in 0.9% NaCl, by 6-fold compared with its original size in PBS and DMEM medium. The acetylenic bonds in GDYO/alkynes, which contain both sp2- and sp- hybridized carbon, usually possess stronger electron-withdrawing property than the ethylenic bonds in GO/alkenes, which contain merely sp2- hybridized carbon21,22 (Figure 1a). Thus the acetylenic bonds in GDYO/alkynes are more likely to attract electrons in carboxyl groups, resulting in dissociation of hydrogen ion, and consequently formation of inter-molecular hydrogen bonds with H2O to stabilize GDYO solution23. From the physicochemical point of views, ion strength, ion valence and sizes of molecules in 10


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the environment of GO affect GO’s solubility24-26. We found that, in water, GO’s charge was -40.7± 1.32 mV (Figure S1a); whereas, in DMEM, its charge became more positive (-9.98± 0.66 mV), indicating the electronic shielding effect produced by the positive ions and biomolecules on GO, which weakens the repel force between the like charges of GO in its solutions27. The interruption to the well-balanced repel force between GO may dynamically result in molecular aggregation. The low stability of GO could be due to the high van der Waals forces among the GO nanosheets after reducing the negatively charged groups by addition of cationic groups26. The coordination between GO and Cu2+ has been demonstrated, which resulted in GO folding/aggregation25. Whereas, GDYO/alkynes are characteristically more unsaturated than alkenes, and its triple bond is very strong, making alkyne relatively stable6 (Figure S1b). To further study the solubility effect of GDYO and GO on cell activities, we incubated GDYO and GO with the normal human hepatocytes LO2 cultured in DMEM with 10% FBS for 12 h. The reason why we chose LO2 is because many nanomaterials tend to accumulate and dispose in liver and be metabolized and inactivated there, causing various biological and pharmacological consequences28,29. Under the confocal microscope, we observed living LO2 grown on the confocal microscopic wells (Figure 2e), and used the time lapse phase-contrast microscopy technique to continuously watch and track interactions between LO2 and GO or GDYO (100 µg/mL) for 12 h after co-incubation. Video S1 and S2 show the kinetic morphologic changes of the LO2 cells treated with the two plane nanomaterials for 12 h. GDYO did not significantly affect LO2 activity (Figure 2f). Whereas, real time imaging clearly shows adhesion of GO to the LO2 plasma membrane (Figure 2g), resulting in the membrane ruffles and fragments. The observed LO2 membrane ruffles and fragments were new changes in cell 11



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membrane that produced upon LO2 interacted with GO. The membrane structures (Figure 2g; see Video S2) did not appear as membrane vesicles. Rather, it is possible that such ruffles quickly formed after GO adhered to the cell membrane30. Computational simulation shown that GO is able to extracted lipids from the model lipid bilayer via both electrostatic and hydrophobic interactions31. The membrane ruffles may induce methuosis, a cell morphological change including large fluid-filled vacuoles derived from macropinosomes and a loss of plasma membrane integrity32. Following the above observations, we questioned if the micro-interactions between cells and GO or GDYO could affect cell viability and mechanosensing. Flow cytometry gating analysis (Figure 3a) showed that GDYO at concentrations up to 300 µg/mL induced a small portion of LO2 into apoptosis (8%, late and early apoptosis combined; gate Q2+ Q3; Figure 3a upper panel). In contrast, GO at 100 and 300 µg/mL induced 18% and 35.6% of LO2 cells into apoptosis, respectively, with a slight increase of necrosis population (Q1; Figure 3a lower panel). Figure 3b shows the quantitative analysis of LO2 viability after 24 h treatments with GDYO and GO. Figure S2a and S2b show concentration- and time-dependent effects of GDYO and GO on LO2 viability after 24, 48 and 72 h incubation. The lactate dehydrogenase (LDH) release assay, a hallmark of necrosis, confirmed no significant LDH leaked from the LO2 cells treated with GDYO from 10 µg/mL to 300 µg/mL for 24 h (Figure S3). GO at concentrations below 100 µg/mL caused LDH release from the tested cells after 24 h incubation, suggesting again GO’s potential damage to the cell membrane33. Reactive oxygen species (ROS) is a critical factor that induce cell apoptosis. ROS is generated during internal metabolism or stimulated by external factors34,35. Using confocal laser microscopy with the aid of a non-fluorescent dye (DCFH) that is converted to green fluorescent DCF dye upon its interaction with the generated ROS36, we observed no significant green fluorescence produced 12


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from the LO2 cells treated with the increasing concentrations (25-100 µg/mL) of GDYO for 24 h (Figure 3d-3f). The result was re-confirmed by the flow cytometry analysis (Figure 3j), showing no significant ROS signals generated from the LO2 cells treated with GDYO. However, the LO2 cells treated with GO (100 µg/mL) for 24 h showed significant green fluorescence, indicating the generation of ROS after GO treatment (Figure 3g-3i). The result was re-confirmed by the flow cytometry analysis (Figure 3j). The half-life of ROS (including 1O2, H2O2, ·OH and ·O2-) generated in biological media was very short, and the effect of transient ROS only reaches 0.02 µm as we described35,37. Therefore, the direct damaging effect of GO was likely to occur near the GO located site. Since GO appears adhered to the plasma membrane of LO2, one can assume that GO may induce production of ROS that damages the cell membrane 34,38. 3.3. In vivo mutagenesis test for GO and GDYO We then tested if GO and GDYO could induce mutagenesis in mice by using the classic micronucleus assay that is guided by government regulatory agencies for measuring formation of micronucleated polychromatic erythrocytes (MNPCEs) as an indication of mutagenesis potential, and for assessing the frequency of structural or numerical chromosome aberrations. In the test, we used saline as negative control and cyclophosphamide as the positive control. Cyclophosphamide (i.v.,10 mg/kg/day for 5 days) caused significant formation of MNPCE (51.2± 6.8 per 1000 erythrocytes) in the mice, which was trivial in the saline-treated mice. Blood GDYO and GO (i.v., 4 mg/kg/day for 5 days) induced MNPCE 7.6± 2.3 and 9.8± 3.0 per 1000 erythrocytes, respectively (Figure 4c). The MNPCE induced by GDYO and GO were significantly fewer than those induced by cyclophosphamide, but more than those induced by saline, suggesting the mutagenesis potential of GDYO and GO. The ratio of numbers of polychromatic erythrocytes (PCE) to normochromatic 13



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erythrocytes (NCE) is often used as an indication of erythrocyte-killing effect. We further calculated PCE/NCE in bone marrow smears, and found no significant differences among groups of saline, GDYO, GO and cyclophosphamide (Figure 4d), indicating that these materials have no erythrocyte-killing effect. 3.4. Fluorescent quenching and antioxidant capacities Both GDYO and GO possess large surface area and novel electronic properties owing to their unsaturated hydrocarbons and regularly-distributed cyclic C≡C and C=C bonds16,39. Double-stranded DNAs, due to their phosphate backbones are negatively-charged that protects the nucleobases from interactions with nanomaterials, cannot quench the fluorescence carried by a DNA probe40. In contrast, single-stranded DNA (ssDNA) can be adsorbed to the surface of GO or GDYO owing to the π–π stacking interactions between GO or GDYO and the nucleic acid of ssDNA, ultimately resulting in significant fluorescent quenching of the organic dyes linked to the ssDNA41,42. Indeed, the fluorescent signals of the FAM dye-labelled ssDNA (probe DNA) disappeared when the probe DNA was mixed with either GDYO or GO that can non-covalently bind to the probe DNA to quench the fluorescence via van der Waals force and π–π stacking interaction (Figure 5a). The AFM images confirms the binding between the probe DNA and GDYO or GO because the height of GDYO and GO increased from 1 nm to 3~4 nm after the probe DNA bound to GDYO (Figure 5b) or GO (Figure 5c). Addition of a target DNA oligonucleotide (i.e., the target DNA that is complementary to the probe DNA) to the mixture of the probe DNA with GDYO or GO resulted in the probe DNA far away from the GDYO or GO by competition between the probe DNA and target DNA, and restoring the fluorescence produced by the probe DNA (Figure 5a). It seems that the target DNA can combined with more probe DNA which bound to GDYO, and restore more 14


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fluorescence signals from the mixture of GDYO and probe DNA. The drug-loading and fluorescence-quenching capacities of GDYO and GO seem compatible. When we incubated doxorubicin hydrochloride (DOX, 100 nM) with GDYO or GO (100 µg/mL) overnight at room temperature, the fluorescence signals of DOX were completely quenched (Figure 5d). The results demonstrate the loading capacities of GDYO and GO, and their potentials for biosensing43,44. GDYO has be used as the reducing agent due to its low reduction potential and highly conjugated electronic structure45. We used the ferric reducing ability of plasma (FRAP) assay to evaluate the antioxidant capacities of GDYO and GO by examining their capacity of reducing ferric to ferrous as indicated by changes in UV-vis absorbance at 593 nm. Both GDYO and GO reduced ferric to ferrous slowly and the reduction rate was dependent on GDYO and GO concentrations. The reduction reached a plateaus after 72 h reaction (Figure S4). As shown in figure 5e, GDYO and GO possess a similar antioxidant capacity probably owing to their intrinsic surface electronegativity (Figure S1a). 4. CONCLUSIONS In conclusion, GDYO and GO contain, respectively, the unsaturated but regularly-distributed cyclic C≡C and C=C bonds. Elucidating the similarities and differences in physicochemistry and biology between GDYO and GO is important for defining alkene and alkyne chemistry, and making our important choices between the two carbon allotropes for future development. This study for the first time systematically revealed the physicochemical differences between GDYO and GO, and found the ion strength-related kinetic aggregation of GO in solutions, which did not occur to GDYO under the physiological isosmotic concentrations. GDYO seems to be more biosafe and less 15



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cytotoxicity. Nonetheless, both GDYO and GO behave, bioequivalently, as a good ligand to bind biomolecules (ssDNA) and aromatic drugs via the surface hydrophobic interactions and π-π stacking between the aromatic regions and the unsaturated bonds of GDYO and GO. The data presented here significantly contribute to the era of carbon allotropes. ■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website: Detailed oligonucleotide sequences (probe DNA and target DNA), the charge of GO and GDYO, the effect of GDYO on cell LDH release, concentration- and time-dependent effects of GDYO and GO on LO2 viability, the reduction reaction degree of GDYO and GO, dynamic morphologic changes of LO2 cells when stimulated by GDYO and GO. ■ AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected] (Lee Jia) ORCID Yu Gao: 0000-0002-1137-7669 Huibiao Liu: 0000-0002-9017-6872 Jingwei Shao: 0000-0002-2060-8887 Yuliang Li: 0000-0001-5279-0399 Lee Jia: 0000-0001-6839-5545

Author Contributions LJ conceived the idea. TZ, YG, and LJ analyzed the data. TZ, XD, HL, JL, RL and JS 16


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conducted the experiments with assistance from YG. YL and LJ supervised the work. Notes The authors declare no competing financial interests. ■ ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of China (2015CB931804); National Natural Science Foundation of China (NSFC) grants U1505225, 81773063, 81273548, 81571802, 21790050; the Natural Science Foundation of Fujian Province (2016J06020); Fujian Development and Reform Commission project #829054 (2014; 168). ■ REFERENCES (1) Kroto, H.; Heath, J.; O'Brien, S.; Curl, R.; Smalley, R.; Kroto, H. W.; Smalley, J.; Kroto, H. W.; Heath, R.; O’Brien, C., C60: Buckministerfullerene. Nature 1985, 318 (6042), 162-163. (2) Hirsch, A., The Era of Carbon Allotropes. Nat. Mater. 2010, 9 (11), 868-871. (3) Geim, A. K.; Novoselov, K. S., The Rise of Graphene. Nat. Mater. 2007, 6 (3), 183-191. (4) Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Katsnelson, M. I.; Grigorieva, I. V.; Dubonos, S. V.; Firsov, A. A., Two-dimensional gas of Massless Dirac Fermions in Graphene. Nature 2005, 438 (7065), 197-200. (5) Zhang, Y.; Tan, Y. W.; Stormer, H. L.; Kim, P, Experimental Observation of the Quantum Hall Effect and Berry's Phase in Graphene. Nature 2005, 438: 201-204. (6) Jia, Z.; Li, Y.; Zuo, Z.; Liu, H.; Huang, C.; Li, Y., Synthesis and Properties of 2D Carbon-Graphdiyne. Accounts Chem. Res. 2017, 50(10): 2470-2478. (7) Li, G.; Li, Y.; Liu, H.; Guo, Y.; Li, Y.; Zhu, D., Architecture of Graphdiyne Nanoscale Films. Chem. Commun. 2010, 46 (19), 3256-3258. 17



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Figure 1. Oxidation and characterization of GDYO and GO. (a) The oxidation processes of GDYO (up) and GO (low). (b and c) AFM image of GDYO and GO, demonstrating the nanosheet’s thickness ~1 nm. Scale Bar =1 µm. (d and e) Hydrodynamic diameter of GDYO (136.6 nm) and GO (113.6 nm). The insets in d and e show TEM images of GDYO and GO. (f and g) XPS survey scan and C1s spectra of GDYO. (h and i) Raman and FT-IR spectra of GDYO. (j and k) XPS survey scan and C1s spectra of GO. (l and m) Raman and FT-IR spectra of GO.

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Figure 2. Solubility and stabilization of GDYO and GO. (a and b) Photos of GDYO (10-300 µg/mL) and its size growth (50 µg/mL) when incubated in various solutions (25℃, 24 h). GDYO was soluble and stable in all the solutions. (c and d) Photos of GO (10-300 µg/mL) and its dynamic size growth (50 µg/mL) when incubated in PBS, 0.9% NaCl and DMEM. The dynamic aggregation of GO in the solutions depends on its ion strengths. (e-g) show morphological changes of human hepatocytes LO2 when they incubated with the control medium DMEM (e), in the presence of 100 µg/mL GDYO (f) or GO (g). The insets amplify the morphological change of LO2 cells after 12 h incubation at 37℃, showing the adhesion and aggregation of GO on the membrane of LO2 (g). Scale Bar = 20 µm.

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Figure 3. Bioactivities of GDYO and GO. (a) Flow cytometric analysis showing effects of GDYO (upper panel) and GO (lower panel) on apoptosis of LO2 cells. Q1: necrotic cells, Q2: late apoptotic cells, Q3: early apoptotic cells, Q4: live cells. (b) Cell viability of LO2 cells treated with GDYO and GO for 24 h. Statistical difference analyzed by the ANOVA test: *P < 0.05, **P < 0.01, compared to the untreated control; #P < 0.05,

##

P < 0.01, compared to the GO groups (n = 3). (c-i) show ROS

production by LO2 cells treated with GDYO (d-f) and GO (g-i) at 25, 50 and 100 µg/mL (from left to right panels) for 24 h. The green fluorescence (c-i) represents production of ROS; Scale Bar = 20 µm. (j) shows ROS-associated fluorescence by LO2 cells treated with GDYO and GO, and measured by flow cytometry; The mean fluorescence intensity was measured from 10,000 LO2 cells. 25



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Figure 4. Mutagenesis potential of GDYO and GO. (a) and (b) are representative photos of mouse bone marrow cells (Giesa staining) taken from in vivo mouse micronucleus assay showing polychromatic erythrocytes with micronucleus (MNPCE), polychromatic erythrocyte (PCE), and normochromatic erythrocyte (NCE). (c) MNPCE numbers (mean± SD) observed from bone marrow cells of the mice treated with GDYO, GO (both 4 mg/kg/day for 5 days; i.v.) and cyclophosphamide (CP; 10 mg/kg/day for 5 days; i.v.). Significantly different from CP and Saline: **P < 0. 01, ***P < 0.001, n= 5. (d) PCE/NCE, the ratio is indicative of the erythrocyte-killing effect of the tested molecules. No significant difference was found between saline and GDYO, GO or CP group.

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Figure 5. Fluorescent quenching and antioxidant capacities. (a) Changes in fluorescence intensities of dye-labeled ssDNA alone (probe DNA, black curve), in the presence of GDYO (blue curve), GDYO and target DNA (red curve), GO (orange curve), or GO and target DNA (green curve) at λex 490 nm. Note, the fluorescence signals of dye-labeled ssDNA were quenched by GDYO or GO, and restored by the competitive target ssDNA. (b) and (c) show the AFM images of ssDNA loaded on to GDYO and GO, and the insets show thickness of the ssDNA-loaded GDYO and GO at ~3.5 nm. (d) Fluorescence intensities (λex= 488 nm) of DOX before and after loaded on to GDYO or GO by H-bonding and π-π stacking. (e) The antioxidant capacity of GDYO and GO measured by the FRAP assay: the absorbance at 593 nm is positively correlated with changes in the ferrous concentrations.

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Comparisons between Graphene Oxide and Graphdiyne Oxide in

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