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Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 32946−32954
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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*,†
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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 ‡ CAS Key Laboratory of Organic Solids, Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *
ABSTRACT: Graphdiyne (GDY) and graphene are regarded as two promising two-dimensional 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 GDY oxide (GDYO) showed similarities in their size, morphology, and physical spectral characteristics, excepting the differences in sp- and sp2hybridizations and Fourier transform infrared spectroscopy. GDYO was well soluble in various media. In contrast, GO was only soluble in H2O, but kinetically aggregated in 0.9% NaCl, phosphate buffered saline, and cell media within 24 h incubation when its concentrations increased. GO nanoparticles adhered and aggregated to the surface of a human hepatocyte membrane, resulting in cell membrane ruffle, methuosis, and apoptosis. Adhesion of GO to cells caused cell stress and induced reactive oxygen species. In contrast, GDYO did not adhere to the 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, thus causing fluorescence quenching. The present study significantly enriches our existing knowledge of GO/alkene and GDYO/ alkyne chemistry. KEYWORDS: graphene oxide, graphdiyne oxide, solubility, cytotoxicity, antioxidant
1. INTRODUCTION
carbon triple bonds to GDY (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, GDY is hoped to offer a better alternative. Nonetheless, further realizing the applications of graphene and GDY in many fields still requires many fundamental research studies. In the present study, we did head-to-head comparisons between graphene oxide (GO) and GDY 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
The serendipitous discovery of fullerenes in 1985 launched a new era in synthetic carbon allotropes.1 Synthetic carbon allotropes have attractive architectures and superior material properties such as ultra-high charge-carrier mobility and a huge capability for carrying current.2 Among the carbon allotrope family, graphene is the most well-studied material with structural uniformity which is synthesized by exfoliating it from graphite with a scotch tape.3−5 As the latest carbon allotrope, graphdiyne (GDY) has recently emerged,6−8 and sparked tremendous interests in its potential applications in energy storage,9,10 electrocatalytic activity,11−13 and rapid heterogeneous electron transfer.14,15 The GDY structure is composed of benzene rings and butadiyne linkages.6 All benzene rings connect with each other by butadiyne linkages (−CC−CC−) to form 18-C hexagon units with sp2- and sp-hybridized carbon atoms.16,17 The addition of the carbon− © 2018 American Chemical Society
Received: May 4, 2018 Accepted: September 4, 2018 Published: September 4, 2018 32946
DOI: 10.1021/acsami.8b06804 ACS Appl. Mater. Interfaces 2018, 10, 32946−32954
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ACS Applied Materials & Interfaces
respectively. A 5 mg/mL MTT stock solution (Sigma, USA) was diluted by a RPMI-1640 medium (without serum and phenol red, Sigma, USA) 10 times. The cell medium was replaced with an equal amount of MTT diluent in every well and incubated for another 4 h in the dark. Then, the supernatant was removed, and 100 μL of DMSO was added 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 (LDH) Release Detection. The LDH release assay was used for detecting the cell membrane integrity, using a cytotoxicity detection kit (Beyotime, China). The LO2 cells were grown on 96-well plates, and the 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, following the instructions. The plate was gently shaken on a platform for 30 min in the dark, followed by recording the absorbance at 490 nm by a microplate reader. The values were expressed as a percent of absorbance relative to positive control wells that were treated by a LDH-releasing agent. 2.9. Reactive Oxygen Species (ROS) Measurement. ROS generation in cells was measured using a ROS assay kit (Beyotime, China). LO2 cells (4 × 105) were coincubated with different concentrations of GDYO and GO at 37 °C after 24 h, were washed three times and re-suspended in HBSS containing 10 μM of dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min, and then detected by flow cytometry and confocal microscopy. Intracellular ROS generation was denoted by the fluorescence intensity of DCF, which was produced by the non-fluorescent dye (DCFH-DA) when undergoing ROS-mediated oxidation. 2.10. Micronucleus Assay. The mutagenesis potential of GDYO and GO was preliminarily detected by the micronucleus test. SPF Kunming mice (males; Wu’s experimental animal, China) with a weight of 25−35 g were used to carry out the mutagenic assay. The mice were divided into four groups of five in each group and kept in the same environment. The animals in negative and positive control groups were administered normal saline and cyclophosphamide (CP; 10 mg/kg) intravenously once a day for 5 days. The GO group and GDYO group mice were disposed with GO or GDYO solution in the same way, corresponding to the total doses of 20 mg/kg. The followup operations referred to those reported previously by Liu et al.20 2.11. Fluorescence Detection. The fluorescence measurements were performed using a F-7000 fluorescence spectrometer (Hitachi, Japan), and the excitation and emission slits used in this experiment were all 5 nm and the voltage of the photomultiplier was 600 V. Fluorescence intensities of DOX samples were recorded in the range of 500−700 nm (λex = 488 nm). Changes in fluorescence intensities of dye-labeled single-stranded DNA (ssDNA) were excited at 490 nm and collected in the range of 500 to 600 nm. 2.12. Total Antioxidant Capacity Assay. A 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 acidic conditions, the total antioxidant capacity was recorded by matching the absorbance changes at a wavelength of 593 nm. We prepared FRAP-working solution following the operating instructions, added 180 μL of the solution to every well of a 96-well plate, and mixed it up with 15 μL of different concentrations of GDYO and GO. After 72 h of incubation, 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, where * denotes a statistical significance versus the control and # denotes a statistical significance between GDYO and GO.
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. GDY was synthesized by hexaethynylbenzene as the method reported previously.7 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 doubledistilled water was added to the reaction mixture to stop oxidization, followed by dialysis (cutoff, 3500) for 3 days to remove mixed acid and dispersing into water under sonication for about 4 h to form a homogeneous brown aqueous solution for storage. GO was prepared by an improved Hummer’s method using graphite flakes as the starting material.18 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 stirring at 50 °C for 12 h. Finally, the reaction was cooled to room temperature and stopped by adding 30% H2O2. The oxidized product was washed by repeated centrifugation and exfoliated by sonication for about 2 h to form a homogeneous suspension which is used for further experiments. 2.2. Measurement and Characterization. X-ray photoelectron spectra (XPS) survey scans were measured by an ESCALAB 250 Xray photoelectron spectrometer (Thermo Scientific, USA). Raman spectra were recorded by the inVia Reflex Raman spectrometer (Renishaw, UK), at a 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 FTIR spectrophotometer (Nicolet, USA) in the 4000−400 cm−1 region using KBr tablets. The morphological analysis of the GDYO and GO films was observed on the transmission electron microscopy (TEM) images, which were recorded by field emission Hitachi HT7700 TEM (Hitachi, Japan). Atomic force microscopy (AFM) images were collected using the Bruker MultiMode V8-SPM (Bruker, Germany) with a ScanAsyst model. The 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 °C), including double-distilled water, dimethyl sulfoxide (DMSO), phosphate buffer saline (PBS, containing NaCl of about 137 mM, pH = 7.4), 0.9% NaCl (154 mM), and the commonly used cell medium Dulbecco’s modified Eagle’s medium [DMEM; containing NaCl of about 109 mM, with 10% fetal bovine serum (FBS)]. The dynamic changes in the size of GDYO and GO within 24 h were recorded by Zetasizer Nano ZS (Malvern, UK). 2.4. Cell Culture. LO2 cells were maintained in a DMEM medium (HyClone, USA) as described previously.19 2.5. Dynamic Morphological Changes of LO2 Cells. About 8 × 104 LO2 cells were preincubated in a culture dish (NEST Biotechnology, China) overnight. Then, the cells were coincubated with GDYO or GO (100 μg/mL). The dishes were placed in a live cell chamber with 5% CO2 and 37 °C. Real-time imaging was collected by Leica TCS SPE confocal microscopy (Leica, Germany), and the images of morphological changes were collected every 5 min for 12 h. 2.6. Apoptosis Assay. An Annexin V-FITC and PI apoptosis detection kit (Genview, USA) was used for detecting apoptotic and necrotic cells. The cells were coincubated with a dose range of GDYO and GO for 24 h at 37 °C. The GDYO-treated or GO-treated cells were digested and suspended in 500 μL of binding buffer and stained with Annexin V-FITC and PI. After staining in the dark for 15 min, the cells were immediately analyzed by BD FACSAria flow cytometry (BD Biosciences, USA). 2.7. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide (MTT) Assay. LO2 cells were inoculated on 96-well plates for the MTT assay to evaluate the cell metabolic activity, and then the cells were incubated with GDYO or GO for 24, 48, and 72 h at 37 °C, 32947
DOI: 10.1021/acsami.8b06804 ACS Appl. Mater. Interfaces 2018, 10, 32946−32954
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Figure 1. Oxidation and characterization of GDYO and GO. (a) Oxidation processes of GDYO (up) and GO (low). (b,c) AFM image of GDYO and GO, demonstrating nanosheet’s thickness ∼1 nm. Scale bar = 1 μm. (d,e) Hydrodynamic diameter of GDYO (136.6 nm) and GO (113.6 nm). The insets in d and e show the TEM images of GDYO and GO. (f,g) XPS survey scan and C 1s spectra of GDYO. (h,i) Raman and FT-IR spectra of GDYO. (j,k) XPS survey scan and C 1s spectra of GO. (l,m) Raman and FT-IR spectra of GO.
3. RESULTS AND DISCUSSION
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. FT-IR was used for confirming the hydrophilic functional groups in GDYO and GO. As displayed in Figure 1i, peaks at 3423.92, 1717.81, and 1224.11 cm−1 could be ascribed to the stretching vibration of −OH, −CO, 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 CC 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 CC (1638.78 cm−1) in GO. 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 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
3.1. Oxidation and characterization of Graphene and GDY. GDY was synthesized as previously reported.7 GO was prepared by an improved Hummer’s method using graphite flakes (G) as the starting material.18 The 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 (Figure 1a). The high quality of few-layered sheets of GDYO and GO was obtained by strong sonication and centrifugation. XPS reveal the differences between GDYO and GO in their functional groups. The XPS survey scan of GDYO clearly shows the peaks of C 1s and O 1s (Figure 1f). GDYO retains the skeleton of GDY, which was evident from the presence of CC, CC, C−O, and CO at 285.1, 284.5, 286.2, and 288.5 eV in the C 1s spectra (Figure 1g), respectively. However, the C 1s 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 C 1s orbital of CC, C−O, and C O, respectively. Although the two carbon allotropes have a similar shape of flat atomic sheets, their chemical structures are different. 32948
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Figure 2. Solubility and stabilization of GDYO and GO. (a,b) Photos of GDYO (10−300 μg/mL) and its size growth (50 μg/mL) when incubated in various solutions (25 °C, 24 h). GDYO was soluble and stable in all solutions. (c,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 ionic strengths. (e− g) 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 °C, showing the adhesion and aggregation of GO on the membrane of LO2 (g). Scale bar = 20 μm.
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 ions and consequently the formation of inter-molecular hydrogen bonds with H2O to stabilize GDYO solution.23 From the physicochemical point of view, ion strength, ion valence, and sizes of molecules in the environment of GO affect GO’s solubility.24−26 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 solutions.27 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 the addition of cationic groups.26 The coordination between GO and Cu2+ has been demonstrated, which resulted in GO folding/aggregation.25 However, 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 consequences.28,29 Under the confocal microscope, we observed living LO2 grown on the confocal
light scattering 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, and they share the unique planar structure and large surface area. 3.2. Solubility and Related Bioactivity. Pristine GDY and graphene are highly hydrophobic, and surface functionalization by adding the oxygen functional groups to their surface may improve their hydrophilicity. To examine if the above chemical functionalization of GDY and G indeed enhances their solubility, processability, and stabilization, we mixed GDYO and GO with water, DMSO, 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 °C and observed any changes in the solubility and stabilization of the mixtures at concentrations ranging from 10 to 300 μg/mL. As shown in Figure 2a,b, GDYO exhibited excellent solubility and stability in all 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 the 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. In contrast, GO was quite stable in water and DMSO but gradually aggregated in PBS, NaCl, and DMEM which contained rich ions or proteins (Figure 2c). Figure 2d shows the kinetic changes in the size of GO after being mixed with different solutions at 50 μg/mL. After 24 h of incubation with these solutions at 25 °C, the size of GO increased by 10fold compared with its original size in 0.9% NaCl and by 6-fold compared with its original size in PBS and DMEM media. The acetylenic bonds in GDYO/alkynes, which contain both sp2- and sp-hybridized carbons, usually possess stronger 32949
DOI: 10.1021/acsami.8b06804 ACS Appl. Mater. Interfaces 2018, 10, 32946−32954
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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) 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) 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.
to 300 μg/mL induced a small portion of LO2 into apoptosis (8.4%, late and early apoptosis combined; gate Q2 + Q3; Figure 3a upper panel). In contrast, GO at 100 and 300 μg/mL induced 18.22 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 treatment with GDYO and GO. Figure S2a,b shows 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 to 300 μg/mL for 24 h (Figure S3). GO at concentrations below 100 μg/mL caused LDH to release from the tested cells after 24 h incubation, suggesting again GO’s potential damage to the cell membrane.33 ROS is a critical factor that induces cell apoptosis. ROS is generated during internal metabolism or stimulated by external factors.34,35 Using confocal laser microscopy with the aid of a nonfluorescent dye (DCFH) that is converted to a green fluorescent DCF dye upon its interaction with the generated ROS,36 we observed no significant green fluorescence produced 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
microscopic wells (Figure 2e) and used the time lapse phasecontrast microscopy technique to continuously watch and track interactions between LO2 and GO or GDYO (100 μg/ mL) for 12 h after coincubation. Videos S1 and S2 show the kinetic morphological changes of the LO2 cells treated with the two plane nanomaterials for 12 h. GDYO did not significantly affect LO2 activity (Figure 2f). However, 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 the new changes in the cell membrane which were produced upon the interaction of LO2 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 membrane.30 Computational simulation shows that GO is able to extract lipids from the model lipid bilayer via both electrostatic and hydrophobic interactions.31 The membrane ruffles may induce methuosis, a cell morphological change including large fluid-filled vacuoles derived from macropinosomes and a loss of plasma membrane integrity.32 Following the above observations, we questioned if the microinteractions between cells and GO or GDYO could affect cell viability and mechanosensing. Flow cytometry gating analysis (Figure 3a) showed that GDYO at concentrations up 32950
DOI: 10.1021/acsami.8b06804 ACS Appl. Mater. Interfaces 2018, 10, 32946−32954
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Figure 4. Mutagenesis potential of GDYO and GO. (a,b) are representative photos of mouse bone marrow cells (Giemsa staining) taken from in vivo mouse micronucleus assay showing polychromatic erythrocytes with micronucleus (MNPCE), PCE, and 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; iv) and CP (10 mg/kg/day for 5 days; iv). Significantly different from CP and saline: **P < 0.01, ***P < 0.001, n = 5. (d) PCE/NCE, the ratio is indicative of the erythrocytekilling effect of the tested molecules. No significant difference was found between saline and GDYO, GO or CP group.
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,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) 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.
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 (Figures 3g−3i). The result was reconfirmed by the flow cytometry analysis (Figure 3j). The halflife of ROS (including 1O2, H2O2, •OH and •O2−) generated in biological media was very short, and the effect of transient 32951
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ACS Applied Materials & Interfaces ROS only reaches 0.02 μm as we described.35,37 Therefore, the direct damaging effect of GO was likely to occur near the GOlocated site. Because 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 the 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 the negative control and CP as the positive control. CP (iv, 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 (iv, 4 mg/kg/day for 5 days) induced MNPCE of 7.6 ± 2.3 and 9.8 ± 3.0 per 1000 erythrocytes, respectively (Figure 4c). The MNPCEs induced by GDYO and GO were significantly fewer than those induced by CP, but more than those induced by saline, suggesting the mutagenesis potential of GDYO and GO. The ratio of numbers of PCEs to normochromatic erythrocytes (NCEs) is often used as an indication of the erythrocyte-killing effect. We further calculated PCE/NCE in bone marrow smears and found no significant differences among groups of saline, GDYO, GO, and CP (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 CC and CC bonds.16,39 Double-stranded DNAs, because of their phosphate backbones are negatively charged which protects the nucleobases from interacting with nanomaterials and cannot quench the fluorescence carried by a DNA probe.40 In contrast, 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 ssDNA.41,42 Indeed, the fluorescent signals of the FAM dyelabeled ssDNA (probe DNA) disappeared when the probe DNA was mixed with either GDYO or GO that can noncovalently bind to the probe DNA to quench the fluorescence via van der Waals force and π−π stacking interaction (Figure 5a). The AFM images confirm the binding between the probe DNA and GDYO or GO because the height of GDYO and GO increased from 1 to 3−4 nm after the probe DNA bound to GDYO (Figure 5b) or GO (Figure 5c). The 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 combine with more probe DNA, which bound to GDYO, and restore more 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 biosensing.43,44 GDYO has been used as the reducing agent because of its low reduction potential and highly conjugated electronic structure.45 We used the FRAP assay to evaluate the antioxidant capacities of GDYO and GO by examining their capacity of reducing ferric to ferrous as indicated by the 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 plateau 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 CC and CC 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 in GDYO under the physiological isosmotic concentrations. GDYO seems to be more biosafe and less cytotoxic. 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.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b06804. Detailed oligonucleotide sequences (probe DNA and target DNA), the charge of GO and GDYO, the effect of GDYO on cell LDH release, concentration- and timedependent effects of GDYO and GO on LO2 viability, the reduction reaction degree of GDYO and GO (PDF) Kinetic morphological changes of the LO2 cells treated with GDYO for 12 h (AVI) Kinetic morphological changes of the LO2 cells treated with GO for 12h (AVI)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected],
[email protected]. 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 §
These authors contributed equally to this work.
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DOI: 10.1021/acsami.8b06804 ACS Appl. Mater. Interfaces 2018, 10, 32946−32954
Research Article
ACS Applied Materials & Interfaces Author Contributions
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L.J. conceived the idea. T.Z., Y.G., and L.J. analyzed the data. T.Z., X.D., H.L., J.L., R.L. and J.S. conducted the experiments with assistance from Y.G. Y.L. and L.J. supervised the work. Notes
The authors declare no competing financial interest.
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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, and 21790050; the Natural Science Foundation of Fujian Province (2016J06020); Fujian Development and Reform Commission project #829054 (2014; 168).
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DOI: 10.1021/acsami.8b06804 ACS Appl. Mater. Interfaces 2018, 10, 32946−32954
