Stable Nanocomposite Based on PEGylated and Silver Nanoparticles

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Stable Nanocomposite Based on PEGylated and Silver Nanoparticles Loaded Graphene Oxide for Long-Term Antibacterial Activity Rongtao Zhao,⊥,† Min Lv,⊥,§ Yang Li,† Mingxuan Sun,† Wen Kong,† Lihua Wang,§ Shiping Song,§ Chunhai Fan,§ Leili Jia,† Shaofu Qiu,† Yansong Sun,*,‡ Hongbin Song,*,† and Rongzhang Hao*,† †

Institute of Disease Control and Prevention, AMMS, Beijing 100071, P. R. China Department of Science and Technology, AMMS, Beijing 100850, P. R. China § Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, P. R. China ‡

S Supporting Information *

ABSTRACT: The increasing occurrence of antibiotic-resistant pathogens, especially superbugs, is compromising the efficacy of traditional antibiotics. Silver nanoparticles (AgNPs) loaded graphene oxide (GO) nanocomposite (GO-Ag) has drawn great interest as a promising alternative antibacterial material. However, GO-Ag nanocomposite often irreversibly aggregates in physiological solutions, severely influencing its antibacterial capacity and practical application. Herein, a PEGylated and AgNPs loaded GO nanocomposite (GOPEG-Ag) is synthesized through a facile approach utilizing microwave irradiation, while avoiding extra reducing agents. Through PEGylation, the synthesized GO-PEG-Ag nanocomposite dispersed stably over one month in a series of media and resisted centrifugation at 10 000×g for 5 min, which would benefit effective contact between the nanocomposite and the bacteria. In contrast, GO-Ag aggregated within 1 h of dispersion in physiological solutions. In comparison with GO-Ag, GO-PEG-Ag showed stronger bactericidal capability toward not only normal Gram-negative/positive bacteria such as E. coli and S. aureus (∼100% of E. coli and ∼95.3% of S. aureus reduction by 10 μg/mL nanocomposite for 2.5 h), but also superbugs. Moreover, GO-PEG-Ag showed lower cytotoxicity toward HeLa cells. Importantly, GO-PEG-Ag presented long-term antibacterial effectiveness, remaining ∼95% antibacterial activity after one-week storage in saline solution versus 99%), silver nitrate (AgNO3, cat #03449, Bio Reagent, >99%), sodium hydroxide (NaOH, cat #S5881, reagent grade, ≥98%), and phosphoric acid (cat# 438081, ACS reagent, ≥85 wt % in H2O) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Ultrapure water (Milli-Q, 18 M Ω·cm resistivity from a Millipore system; ZMQS50F01, Millipore, Bedford, MA, U.S.A.) was used in the experiments. Membrane filters (cat# VVLP04700, 0.1 μm) for suction filtration were from Millipore. Gram-positive bacteria (S. aureus, ATCC29213 and methicillinresistant S. aureus, MRSA) and Gram-negative bacteria (E. coli, ATCC25922, E. coli carrying blaNDM‑1, and E. coli carrying mcr-1) were preserved in our laboratory. E. coli and S. aureus without special notes in this paper refer to the reference strains. Synthesis of GO and GO-PEG. GO was prepared from natural graphite flakes by a previously reported synthesis method.41 GO-PEG was synthesized according to a previous method39,42 with some modification. In brief, 25 mg as-prepared GO was dispersed in 50 mL of ultrapure water followed by ultrasonic treatment (SBL-14DT, SCIENTZ, Ningbo, China) for 30 min at 25 °C to form a GO dispersion of 0.5 mg/mL. Then, 100 mg PEG was slowly added to the dispersion under ultrasonic treatment for another 15 min to make the reaction solution mix completely. Next, 50 mg and 100 mg EDC·HCl were serially added to the solution in 20 min intervals under continuous ultrasonic treatment. The pH of the mixture was adjusted to ∼6, after which it was stirred at 800 rpm on a magnetic stirrer (RCT basic, IKA) for more than 16 h at room temperature. The GO-PEG reaction solution was purified by suction filtration with a 0.1 μm Millipore filter, and the filter cake was resuspended in ultrapure water by ultrasonic treatment for 10 min. The above purification steps were repeated at least 3 times for adequate removal of unreacted reagents. The obtained GO-PEG was lyophilized in a vacuum freeze drier (FD2; Boyikang, Beijing, China). Synthesis of GO-PEG-Ag and GO-Ag. An silver-ammonia ([Ag(NH3)2]OH) solution was prepared as follows: 84.9 mg AgNO3 was dissolved in 8 mL of ultrapure water, and the AgNO3 solution was titrated with 1 wt % ammonia solution until the precipitate completely disappeared. The volume was adjusted up to 12.5 mL with ultrapure water to form a 40 mM [Ag(NH3)2]OH 15329

DOI: 10.1021/acsami.7b03987 ACS Appl. Mater. Interfaces 2017, 9, 15328−15341

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ACS Applied Materials & Interfaces

LB plates followed by incubation at 37 °C overnight. Inhibition zones were visible as transparent halos on the LB plates. Investigation of Instant Antibacterial Activity. To compare the instant antibacterial activity of the nanocomposite and its components, antibacterial activity was assayed as described previously.8 E. coli cells (including the reference strain and two superbugs respectively carrying the blaNDM‑1 and mcr-1 genes) and S. aureus cells (including the reference strain and MRSA) were inoculated with GO, PEG, bare AgNPs (∼10 nm), GO-Ag, and GO-PEG-Ag in sterile 0.9% NaCl. The final concentration of bacterial cells was 107 CFU/ml, and the final concentration of each material was 5 μg/mL for E. coli, 10 μg/ mL for S. aureus, and 0 μg/mL for the control groups. The mixtures were incubated for 2.5 h at 37 °C in a shaking incubator, diluted in a 106-fold gradient in 100 μL of 0.9% NaCl, and spread uniformly onto LB plates, which were incubated at 37 °C overnight. The CFU were counted, and the percentage reduction was obtained in comparison to control groups. Each treatment was carried out three times in parallel. Investigation of Long-term Antibacterial Activity. We investigated the change of antibacterial effectiveness of the nanocomposites after storage in physiological solutions for different times. First, GO-PEG-Ag and GO-Ag nanocomposites were dispersed in a series of tubes containing 0.9% NaCl at a final concentration of 100 μg/mL. The tubes were kept without shaking for different times. At 0 h, 1 h, 2 h, 4 h, 8 h, 16 h, 1 d, 2 d, and 1 week, nanocomposite samples were transferred from the tubes into a bacterial suspension with a final concentration of 107 CFU/ml, at 5 μg/mL for E. coli and 10 μg/mL for S. aureus. After incubation for 2.5 h, bacterial suspensions were diluted in a gradient and cultured on LB plates to calculate the survival rate (%) using the method used for instant antibacterial activity testing. Time-Dynamic Antibacterial Test. A time-dynamic antibacterial test was used to reveal the antimicrobial effectiveness of GO-PEG-Ag over time. E. coli and S. aureus cells (107 CFU/ml) were inoculated with different concentrations of GO-PEG-Ag; 2, 5, and 10 μg/mL for E. coli, and 5, 10, and 15 μg/mL for S. aureus. A blank control group was treated with 0 μg/mL of GO-PEG-Ag. Bacteria were sampled every half hour from 0 to 4 h, diluted in a gradient, and cultured on LB plates to calculate the survival rate using the method used for instant antibacterial activity testing. TEM Characterization of Bacterial Cells. E. coli and S. aureus cells were inoculated with 5 μg/mL and 10 μg/mL of GO-PEG-Ag in a 0.9% NaCl solution at 37 °C for 2.5 h. The bacterial cells were processed for TEM as previously reported.8 In brief, the cells were collected and fixed with 3% glutaraldehyde at 4 °C for 2 h. The cells were washed twice with PBS, postfixed with 1% of osmic acid at 4 °C for 1 h, and washed twice with PBS. The samples were dehydrated through a concentration series of ethanol (50%, 70%, and 90% for 15 min each, 100% for 15 min twice). The cells were embedded in Epon/ Araldite resin at 65 °C for 15 h. The samples were cut into ultrathin sections and stained with 4% uranyl acetate and 0.2% lead citrate in the dark. After drying, the sections were observed by TEM (H-7650; HITACHI). Protein Leakage Analysis. Quantitative detection of protein leakage from bacteria was carried out referring to previous method.9 In brief, 109 CFU/ml of E. coli was inoculated with 2 μg/mL and 5 μg/ mL of GO-PEG-Ag in 30 mL of a 0.9% NaCl solution, and 109 CFU/ ml of S. aureus was treated with 5 μg/mL and 10 μg/mL of GO-PEGAg. Control groups without GO-PEG-Ag were included. The mixture was incubated in a shaker at 37 °C for 2.5 h and then centrifuged at 10 000×g at 4 °C. The supernatant liquid was immediately concentrated to 2 mL through vacuum lyophilization, the protein concentration of each sample was determined with an Enhanced BCA Protein Assay Kit (cat# P0010; Beyotime, Jiangsu, China) using a Multimode detection platform (Molecular Devices, SpectraMax i3x). Determination of Cellular Total Reactive Oxygen Species (ROS). The level of bacterial ROS induced by GO-PEG-Ag was detected by DCFH-DA assay (Reactive Oxygen Species Assay Kit, cat# S0033; Beyotime). Briefly, E. coli cells and S. aureus cells at a final concentration of 108 CFU/ml were inoculated with 10 μM of DCFHDA in 5 mL of 0.9% NaCl solution at 37 °C for 30 min for probe

solution. The as-prepared GO-PEG was dissolved in 20 mL of water to form a 0.25 mg/mL GO-PEG dispersion. One milliliter of [Ag(NH3)2]OH solution was slowly dropped into the mixture under ultrasonic treatment for 10 min. The solution was placed into the microwave synthesizer (SP, CEM, Matthews, NC, U.S.A.) and heated up to 105 °C with a 2 min hold under high-speed stirring, and then quickly cooled to 50 °C by compressed air flow followed by natural cooling to room temperature. The purification and lyophilization of GO-PEG-Ag were conducted using the same procedure as that used for GO-PEG. The synthesis method of GO-Ag was similar to that of GO-PEG-Ag. Characterization. Atomic force microscopy (AFM) was used to characterize the sheet size and height of GO and GO-PEG with a Bruker Dimension Icon atomic force microscope using a ScanAsyst-Air tip. Fourier-transform infrared (FTIR) spectra were recorded with a PerkinElmer Spectrum 100 spectrometer at a wavenumber range of 4000−800 cm−1 and at a resolution of 1 cm−1. UV−vis absorption spectra were recorded from 200 to 600 nm using a Shimadzu UV-2550 spectrometer. X-ray diffraction (XRD) patterns in a range of 5° < 2θ < 80° were obtained with a Bruker D8 Advance X-ray diffractometer using Cu Kα X-ray radiation (λ = 0.154 06 nm), at a scan rate of 2°/ min with a voltage of 40 kV. Thermal gravimetric analysis (TGA) was conducted using a NETZSCH STA 449F3 thermogravimetric analyzer with a synthetic air flow rate of 100 mL/min at a heating rate of 5 °C/ min. Transmission electron microscopy (TEM)/High-resolution TEM (HRTEM) was carried out on a PEI Tecnai G2 F30 field emission instrument operated at an accelerating voltage of 300 kV. Samples for TEM/HRTEM characterization were prepared by placing a drop of ethanol containing nanomaterials onto a carbon-coated copper grid and dried in air before characterization. The size distribution and Zeta potential of the nanomaterials were measured using a dynamic light scattering and Zeta potential instrument (Malvern, Zetasizer Nano ZS) using 0.05 mg/mL of sample in H2O. Assay of the Content of AgNPs in GO-PEG-Ag by Atomic Absorption Spectrometry (AAS). The AgNP content (%) in GOPEG-Ag was determined with an atomic absorption spectrometer (Z2000, HITACHI, Japan) after oxidizing the AgNPs of GO-PEG-Ag to Ag+. In brief, GO-PEG-Ag was dispersed in 20 mL of a mixture of H2SO4 and HNO3 (3:1). The mixture was digested in a microwave digestion instrument (MARS, CEM, Matthews, NC, U.S.A.) via a three-stage heating up to 210 °C and a 30 min hold. Next, the acid mixture was evaporated by heating. After cooling, the sample was resuspended in ultrapure water and analyzed by AAS according to the AgNO3 (GBW(E) 080462, purchased from National Institute of Metrology, China) reference curve. Bacterial Culture. All bacteria used in this paper were first cultivated overnight on LB plates at 37 °C in a bacteriological incubator. Then, a single colony was transferred into fresh LB medium and incubated to the log phase in a shaking incubator at 37 °C. The bacteria were harvested by centrifugation and washed twice with sterile saline solution (0.9% NaCl) to remove the medium constituents and other residual chemical macromolecules. 8 The bacterial cells suspended in 0.9% NaCl were quantified by measuring the optical density at 600 nm. Minimal Inhibitory Concentration (MIC) Test. MICs of all materials were determined by the microdilution method reported previously43 with slight modification. Briefly, log-phase E. coli and S. aureus cells (105 CFU) were seeded in 96-well microtiter plates containing different concentrations of materials and incubated at 37 °C for 18 h. The final concentrations for the series of materials were from 0 to 16 μg/mL with 1 μg/mL of step, 32 and 64 μg/mL. Each test was repeated 3 times. The MIC value is defined as the lowest concentration of material at which no visible bacterial growth can be observed. Agar Diffusion Test. The agar diffusion test was used to visually present the antibacterial activity of nanocomposites, according to a previously reported method.26 Bacterial suspensions (107 CFU/ml) of E. coli and S. aureus were spread onto LB agar plates using sterile cotton swabs to form a uniform carpet. Ten microliters of GO-PEGAg, GO-Ag, and NaCl were carefully dropped onto the surfaces of the 15330


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ACS Applied Materials & Interfaces Scheme 1. Diagram of the Preparation of the GO-PEG-Ag Nanocomposite

Figure 1. AFM images of (a) bare GO and (b) GO-PEG. Cytotoxicity Assay. The cytotoxicity of the nanocomposite was evaluated by CCK-8 assay using a commercial kit (CK-04; Dojindo, Japan). HeLa cells in DMEM (cat# 11960500; Gibco, Grand Island, U.S.A.) with 10% fetal bovine serum were seeded in a 96-well microtiter plate at a final volume of 100 μL per well, and the plate was placed in an incubator under 5% CO2, at 37 °C for 24 h. Then, 10 μL of different concentrations of GO-PEG-Ag and GO-Ag dispersions were added at final concentrations of 0, 10, 20, and 50 μg/mL in triplicate. After further incubation for 8 and 24 h respectively, the solution was replaced by fresh medium and 10 μL of CCK-8 reagent was added per well followed by incubation for another 1 h. Finally, the optical density was determined on a Multimode detection platform (Molecular Devices, SpectraMax i3x) in absorbance mode, at 450 nm.

loading. After removal of the unloaded DCFH-DA by centrifugation, the bacterial cells were treated with different concentrations of GOPEG-Ag (2 and 5 μg/mL for E. coli, 5 and 10 μg/mL for S. aureus) for 1.5 h. Control groups without GO-PEG-Ag were included. The level of cellular total ROS was measured as the relative fluorescence intensity of the probes, which was detected in a Multimode detection platform (Molecular Devices, SpectraMax i3x) in fluorescence mode with excitation at 488 nm and emission at 525 nm. Adenosine Triphosphate (ATP) Assay. As an energy molecule, ATP reflects the metabolic activity of bacteria. The level of bacterial ATP was investigated by the firefly luciferase method using an Enhanced ATP Assay Kit (cat# S0027; Beyotime). E. coli cells and S. aureus cells were treated with different concentrations of GO-PEG-Ag in 20 mL of 0.9% NaCl solution at 37 °C for 1.5 h. The final concentrations of bacterial cells were 108 CFU/ml, and the concentrations of GO-PEG-Ag were 2 μg/mL and 5 μg/mL for E. coli, and 5 μg/mL and 10 μg/mL for S. aureus. Control groups without GO-PEG-Ag were included. After treatment, the cells were harvested by centrifugation at 4 °C and resuspended in 5 mL of lysis buffer. The samples were transferred into an ultrasonic cell disruption instrument (vcx500; Sonics) at 30% power (3 s on/5 s off) for 5 min in an icebath. The supernatant was collected and detected on a Multimode detection platform (Molecular Devices, SpectraMax i3x) in luminance mode.

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RESULTS AND DISCUSSION Synthesis and Characterization of the GO-PEG-Ag Nanocomposite. GO-PEG-Ag nanocomposite is synthesized in two main stages as shown in Scheme 1. First, GO-PEG complex is prepared through modifying GO with 8arm-PEGNH2 based on a covalent amide bond between the −NH2 group at the end of the PEG branched arm and the −COOH group at the edge of the GO nanosheet. Second, Ag+ are reduced to AgNPs on GO-PEG under microwave irradiation to form GO-PEG-Ag. 15331


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Figure 2. (a) FTIR spectra of GO, GO-PEG, GO-PEG-Ag, and GO-Ag. (b) UV−Vis absorption spectra of GO, GO-PEG, GO-PEG-Ag, and GO-Ag. The image in purple-dashed box shows the four nanomaterials in water. (c) XRD patterns of GO, GO-Ag, and GO-PEG-Ag. (d) Thermal gravimetric curves of GO, GO-PEG, and GO-PEG-Ag.

spectrum of GO, among which the peak at 229 nm corresponds to the electronic π−π* transitions of aromatic C−C aromatic bonds, and the shoulder at about 300 nm could be assigned to n−π* transitions of CO bonds.26 The spectrum of GO-PEG is similar to that of GO, and no obvious difference could be observed between the spectra of GO-Ag and GO-PEG-Ag. However, a new peak appears at ∼400 nm in the spectra of both GO-Ag and GO-PEG-Ag in comparison with those of GO and GO-PEG, which originates from the surface plasmon resonance of AgNPs,26,27 indicating the presence of AgNPs on both GO-Ag and GO-PEG-Ag nanocomposites, and the sharp peak in the range of 400−500 nm was reported to suggest a spherical shape of AgNPs.34 Meanwhile, the absorption peak of AgNPs in the visible region agreed with the color change; as shown in Figure 2b, GO-Ag and GO-PEG-Ag become brownish green after decoration with AgNPs. Most previously reported methods for reducing Ag+ into AgNPs required the addition of extra reducing agents or surfactants such as sodium borohydride,37 glucose,34 sodium citrate,35 and so forth, which might cause either toxicity or interference. In our work, neither reducing agents nor surfactants were added; the deprotonation of phenol groups (hydroxyl) on GO might provide reduction sites for the formation of AgNPs.29 This method reduces the potential risks to the environment and human health while decreasing the experimental interference factors. A continuous hydrothermal flow synthesis (CHFS) process reported by Kellici et al. (2016) was also a facile approach for the synthesis of graphene-Ag nanocomposites, with the advantages of time saving and no addition of reductants.29 However, the too high reaction temperature (more than 374 °C) in CHFS would

AFM displayed that GO and GO-PEG are composed of monodispersed layers (Figure 1). The bare GO (Figure 1a) sample presents a height of about 1 nm, which is the feature of fully exfoliated single-layer GO.38 GO-PEG (Figure 1b) presents a lateral size (∼450 nm) similar to that of GO, but is about 1.5 nm thicker. The increase in height of GO-PEG could be attributed to the coverage of PEG on the basal plane of GO, which also indicates the formation of PEGylated GO nanocomposite. FTIR spectroscopy was carried out to characterize the functional groups of the nanomaterials. As shown in Figure 2a, the spectrum of GO suggests the existence of −OH (3392 cm−1), CO (1730 cm−1), CC (1619 cm−1), and C−O−C (1057 cm−1) functional groups.34,35 The abundance of oxygencontaining functional groups gives GO good hydrophilicity7 and ease of modification with functional materials.14 The spectra of GO-PEG and GO-PEG-Ag are similar to each other; when compared to those of GO and GO-Ag, respectively, there appear new, sharp peaks at about 2870 cm−1, due to −CH2− vibrations of the PEG chains, and at about 1635 cm−1, from the characteristic stretching vibration of CO on amide group, suggesting successful covalent binding of PEG onto GO. The decrease in stretching vibrations of CO in GO-PEG, GOPEG-Ag, and GO-Ag might be due to the covalent bond between −NH2 of PEG and −COOH of GO or the electrostatic interaction/chemical bond between AgNPs and −COOH.34 GO, GO-PEG, GO-Ag, and GO-PEG-Ag were further characterized by UV−Vis absorption spectroscopy. As shown in Figure 2b, two characteristic peaks are observed in the 15332


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Figure 3. (a and b) TEM images of GO-PEG-Ag at different magnifications. Size distributions of (c) AgNPs on GO-PEG-Ag and (d) GO-PEG-Ag. (e) HRTEM image of AgNPs on GO-PEG-Ag. (f) SAED pattern of GO-PEG-Ag.

Ag, the amount of residual above 700 °C could be mostly assigned to the AgNPs27 of the nanocomposite. According to TGA, the component contents of the GO-PEG-Ag composite are GO ≈ 43 wt %, PEG ≈ 35 wt %, and AgNPs ≈ 27 wt %. Additionally, the content of AgNPs in GO-PEG-Ag was quantitatively analyzed by AAS (Table S1 of the Supporting Information) after microwave digestion; the result, ∼28 wt %, was close to that of TGA. The results of TGA and AAS of GOAg are presented in Figure S1 and Table S1; the content of AgNPs in GO-Ag nanocomposite according to AAS is ∼27 wt %. This indicates that the contents of AgNPs, the main antibacterial agents, of GO-Ag and GO-PEG-Ag are nearly equal, and thus, that it is meaningful to compare the antibacterial activity of the two composites at the same concentrations. TEM was used to investigate the morphology of GO-PEGAg (Figure 3). The GO-PEG-Ag nanocomposite presents as a single-layer GO basis, as shown in Figure 3a,b. The AgNPs, observed as dark spots, are separate from each other, spherical in shape, and uniformly attached on the entire surface of the nanocomposite at high density. The morphology of GO-Ag is similar to that of GO-PEG-Ag, as shown in Figure S2a. The size range of the AgNPs on GO-PEG-Ag, measured on ∼2000 AgNPs, is 5−10 nm, with a quite small average diameter of 7.06 ± 2.54 nm (Figure 3c), which is very close to that on GO-Ag (7.32 ± 2.55 nm) (Figure S2b). The antibacterial capacity of AgNPs is size-dependent; smaller size is usually associated with stronger antibacterial capacity.25 The size distribution of the GO-PEG-Ag nanocomposite, analyzed with a nanoparticle size analyzer, was on the scale of several hundreds of nanometers, with an average of 444.5 ± 123.2 nm (Figure 3d). The average size of GO-Ag was 459.5 ± 148.3 nm, as shown in Figure S2c. HRTEM images (Figure 3e) clearly show that the AgNPs have a consistent, spherical shapein accordance with the UV−Vis absorption spectroscopic analysisand well-formed polycrystalline structure with a lattice fringe of 0.235 nmthe same as

cause the decomposition or loss of both oxygen-containing groups and grafted polymer on GO, which might restrain its application for the synthesis of the graphene-Ag nanocomposites with good stability in physiological solutions. The crystal structures of GO, GO-Ag, and GO-PEG-Ag were characterized through XRD, and the results are given in Figure 2c. The sharp peak at about 2θ = 10.9° is characteristic of GO and represents the (0 0 2) crystalline plane with the interlayer spacing of 0.809 nm, which is probably due to the high degree of exfoliation30 and interlayer H2O trapped between the hydrophilic GO layers.27 For GO-PEG-Ag and GO-Ag, the patterns are similar, except the broad weak peak at 2θ = 21.5° of GO-PEG-Ag nanocomposite, which might be assigned to noncrystalline diffraction from PEG. The peaks observed at about 2θ = 38.2°, 44.3°, 64.5°, and 77.6° are respectively attributed to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) crystalline planes of the face-centered cube of AgNPs,27 which confirms the presence of AgNPs in both GO-Ag and GO-PEG-Ag nanocomposites. Moreover, the d value corresponding to the (1 1 1) plane of AgNPs is 0.235 nm. Compared with GO, the peak at 2θ = 10.9° disappears in GO-Ag and GO-PEG-Ag, which could be explained by the anchoring of AgNPs, preventing the GO layers from stacking.26 The content of each component in the nanocomposite was assayed using the TGA as displayed in Figure 2d. For GO, two main steps of thermal decomposition are observed. The first mass loss between 175 and 225 °C is derived from the decomposition of the oxygen-containing groups on the GO surface, the second loss observed between 430 and 510 °C could be attributed to the decomposition of the graphitic portion of GO.27 The functionalization of GO by PEG and AgNPs leads to small shifts of the decomposition temperature in the curves of GO-PEG and GO-PEG-Ag. For GO-PEG, three steps of thermal decomposition are observed, among which a new intense mass loss between 250 and 420 °C might be from the decomposition of the grafted PEG. For GO-PEG15333


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Figure 4. Digital images of 100 μg/mL of (1) GO, (2) GO-PEG, (3) GO-PEG-Ag, and (4) GO-Ag in (a) H2O and (b) 0.9% NaCl after centrifugation at 10 000×g for 5 min.

Figure 5. Digital images of 100 μg/mL of (1) GO, (2) GO-PEG, (3) GO-PEG-Ag, and (4) GO-Ag in (a) H2O, (b) 0.9% NaCl, (c) PBS, (d) MH broth, and (e) LB broth after the storage for the indicated time.

microwave irradiation for heating to improve the reaction condition through the high-frequency oscillation, which significantly increases effective contact between Ag+ and GO sheets contributing to the uniform and high-density distribution of nucleation centers of AgNPs on the GO-PEG surface. Coupled with the advantages of microwave irradiation for uniform and rapid heating, the AgNPs cores grow synchronously, and the reduction reaction time is reduced to about 3 min greatly shortening the growth time of AgNPs, which is much shorter than that of previous hydrothermal methods (30 min to 1 h).26,35 Moreover, this method is energy-saving, and the pressurized and closed state of the reaction avoids contact of the reactants with the outside air, preventing the formation of the silver oxide byproduct on the surfaces of the AgNPs.33

the XRD resultscorresponding to the (1 1 1) plane of AgNPs.34 The typical polycrystalline diffraction features of AgNPs on GO-PEG-Ag are also indicated by selected area electron diffraction (SAED) (Figure 3f), presenting visible diffraction rings mixed with some diffraction dots.34 These results suggest that GO acts as the basic skeleton of GO-PEGAg, providing anchoring sites for AgNPs, while preventing their agglomeration.37 In addition to the benefit from the support by GO, such good morphology of AgNPs might be owing to the improved preparation method. We used [Ag(NH3)2]OH solution as the precursor of AgNPs, which is superior to the use of silver nitrate, to slowly release Ag+ from the [Ag(NH3)2]OH complex facilitating the control of size and uniformity of AgNPs.33,44 Furthermore, we utilized single-mode 15334


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Figure 6. Reduction percentage of bacteria treated with GO-PEG-Ag, GO-Ag, AgNPs (∼10 nm), GO, and PEG for 2.5 h. (a) Reference strain of E. coli, E. coli carrying blaNDM‑1, and E. coli carrying mcr-1 treated with 5 μg/mL materials, (b) reference strain of S. aureus and MRSA treated with 10 μg/mL materials. **P ≤ 0.01, versus GO-PEG-Ag. Error bars represent the standard error of three parallel experiments.

Stability of the Nanomaterials in Various Media. The instability of GO-based nanomaterials in physiological solutions is a considerable limitation to their biological application.21 We investigated the stability of the four nanomaterials dispersed in various media. GO, GO-PEG, GO-PEG-Ag, and GO-Ag were dispersed in H2O and 0.9% NaCl solution at the concentration of 100 μg/mL, followed by centrifugation at 10 000×g for 5 min. As shown in Figure 4a, the four nanomaterials were well dispersed without visible aggregation in H2O after centrifugation, indicating excellent hydrophilicity. In contrast, GO (Figure 4b (1)) and GO-Ag (Figure 4b (4)) were completely precipitated in 0.9% NaCl after centrifugation, while GO-PEG (Figure 4b (2)) and GO-PEG-Ag (Figure 4b (3)) remained in stable suspension, as in H2O. To investigate the influence of storage time and media on the stability of the nanomaterials, we dispersed GO, GO-PEG, GOPEG-Ag, and GO-Ag in H2O, 0.9%NaCl, phosphate-buffered saline(PBS), Mueller−Hinton (MH) broth, and Luria−Bertani (LB) broth for different durations. There was no obvious aggregation for each nanomaterial in H2O at each time point (Figure 5a), which was consistent with the above results after centrifugation. However, GO and GO-Ag aggregated to different degrees in the other four media after 1 h, and completely precipitated after 1 week ((1) and (4) in Figure 5b−e). This aggregation might be owing to the fact that the GO surface charge is screened by electrolytes in the media.21 In comparison, GO-PEG and GO-PEG-Ag maintained a stable dispersion state for 1 week ((2) and (3) in Figure 5b−e) and even up to 1 month (Figure S3), without obvious aggregation. These results demonstrated that PEGylation of GO-based nanomaterials could effectively prevent their aggregation in a variety of media. Moreover, comparison of the stability between GO-PEG-Ag and GO-Ag at the low concentration of 10 μg/mL in NaCl solution containing bacteria revealed that GO-PEG-Ag could still disperse stably, while GO-Ag precipitated at the bottom of the tube (Figure S4a), which is expected to cause a significant difference in the contact with bacteria between GOPEG-Ag and GO-Ag. An antibacterial test confirmed that the higher stability is associated with stronger bactericidal ability (Figure S4b). After incubation of the two nanomaterials (10 μg/mL) with S. aureus, the number of bacterial colonies on LB

agar was significantly lower for the GO-PEG-Ag (∼14 colonyforming units (CFU), Figure S4b (1)) than for the GO-Ag group (∼100 CFU in Figure S4b (2)). The results of the stability test are inconsistent with those reported by Chen et al.,35 who prepared a similar GO@PEG@ Ag by use of PEG bis-NH2, which was used for directing the growth of AgNPs on GO. Although the authors did not provide detailed data on the stability of GO-PEG-Ag in physiological solutions, the intermediate product GO@PEG precipitated in H2O after centrifugation at 6000 rpm. In contrast, in our work, both GO-PEG and GO-PEG-Ag synthesized by use of 8armPEG-NH2 did not precipitate after centrifugation at a higher speed as mentioned above. These inconsistent results might be due to the differences in the structure of PEG or experimental objective. And this difference in stability might cause a significant difference in long-term antibacterial activity of the nanocomposites. Antibacterial Activity of the GO-PEG-Ag Nanocomposite. MIC is a significant parameter to evaluate the susceptibility of a given microorganism to a given antimicrobial substance. The lower the MIC is, the higher the microbial susceptibility is, and thus, the stronger the antimicrobial capacity becomes.32 The MICs of the GO-PEG-Ag and GOAg nanocomposites and their components (including GO, PEG, and AgNPs with an average diameter of ∼10 nm) for E. coli and S. aureus are shown in Table S2. The MICs of GO and PEG were undetectable at a concentration lower than 64 μg/ mL, which indicated very weak to no antibacterial ability. This also suggests that the main antibacterial ingredient of GO-PEGAg is the AgNP. The MIC of bare AgNPs (∼10 nm) was much higher than those of GO-PEG-Ag and GO-Ag nanocomposites because of the easy aggregation of AgNPs causing the reduction of active surface, and thus weakening their antibacterial effectivity.25,37 The MICs of GO-PEG-Ag were the lowest, with values of 2 μg/mL against E. coli and 5 μg/mL against S. aureus, while those of GO-Ag were 3 μg/mL against E. coli and 7 μg/mL against S. aureus. The low MICs of GO-PEG-Ag are likely due to the synergistic effect among the three constituents, where GO prevents the aggregation of AgNPs, while PEG enhances the dispersion of the nanocomposite and the contact efficiency between the nanocomposite and the bacteria. As a 15335


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Figure 7. Long-term antibacterial effectiveness of GO-PEG-Ag and GO-Ag presented as the survival rate of (a) E. coli treated with 5 μg/mL and (b) S. aureus treated with 10 μg/mL of nanocomposites for 2.5 h. Time-dynamic survival rate curves of (c) E. coli. and (d) S. aureus treated with different concentration of GO-PEG-Ag. Error bars represent the standard error of three parallel experimental data.

of its instability. GO-PEG-Ag exhibited the most powerful instant antibacterial capacity among the five types of materials against both reference strains as well as superbugs: the reduction rates for the reference E. coli, E. coli carrying blaNDM‑1, and E. coli carrying mcr-1 were all larger than 99.5%, and the reduction rates for reference S. aureus and MRSA were larger than 95.3% (Figure S5 shows the colonies of reference bacteria on LB plates after treatment with GO-PEG-Ag). For equal content of AgNPs in GO-Ag and GO-PEG-Ag, the antibacterial activity of GO-PEG-Ag was stronger than that of GO-Ag, which might be owing to the better stability and dispersity of GO-PEG-Ag in physiological solutions. And the better stability might also explain the more reduction of bacteria caused by GO-PEG-Ag than that caused by a similar GO@PEG@Ag nanocomposite (92% of E. coli and 76% of S. aureus reduction at the concentration of 10 μg/mL) reported previously.35 In addition, GO-PEG-Ag has a positive charge (16.2 ± 6.16 mV) from positively charged PEG, which enhances its contact with the negatively charged bacteria, while the negatively charged GO-Ag (−38.4 ± 8.93 mV) might electrostatically repulse the bacteria. An agar diffusion test (Figure S6) further confirmed the stronger antibacterial activity of GO-PEG-Ag; obvious inhibition halos appeared in the GOPEG-Ag group (∼2.03 cm for E. coli and ∼1.61 cm for S. aureus), which were larger than those in the GO-Ag group (∼1.49 cm for E. coli and ∼1.27 cm for S. aureus). There were no significant differences in the reduction rate between reference strains and superbugs of E. coli or S. aureus after treatment with GO-PEG-Ag, suggesting that GO-PEG-Ag had equally powerful antibacterial capacities against references strains and superbugs. However, the reduction rate for the Gram-positive S. aureus was lower than that for the Gramnegative E. coli, even when the concentration of GO-PEG-Ag for S. aureus was twice that for E. coli. This indicates that the bactericidal ability of GO-PEG-Ag is not influenced by bacterial resistance, but by the structure of the bacteria, which might be due to the fact that the antibacterial ability of nanocomposite is

result, the interaction between AgNPs and bacteria is greatly improved, leading to effective antibacterial activity of the AgNPs. The MIC for S. aureus was higher than that for E. coli, which might be caused by the stronger physical resistance of the thicker cell wall of S. aureus (Gram-positive bacteria) than that of E. coli (Gram-negative bacteria). Additionally, we determined instant and long-term antibacterial activity to evaluate the antibacterial capability of the nanocomposites. Herein, instant/long-term antibacterial activity is defined as the antibacterial activity of a nanocomposite freshly dispersed/stored for a certain period in physiological media. The instant antibacterial properties of GO-PEG-Ag, GO-PEG, GO, PEG, and bare AgNPs (∼10 nm) against reference and drug-resistant strains of E. coli (Figure 6a) and S. aureus (Figure 6b) were evaluated by the CFU counting method. The results are shown as the reduction (%) in bacterial colony count after treatment with each tested material as compared to that with a control NaCl solution. The tested E. coli strains included a reference strain (ATCC25922) and superbug strains respectively carrying blaNDM‑1 and mcr-1 genes, and were treated with the materials at a concentration of 5 μg/ mL. The S. aureus strains included a reference strain (ATCC29213) and MRSA, which were treated with 10 μg/ mL of the materials. The superbugs used are resistant to numerous antibiotics. Of particular concern, mcr-1 has emerged as the first plasmid-mediated colistin resistance gene, and colistin is considered a last-resort antibiotic against multidrugresistant bacteria at present.3 GO and PEG induced no obvious reduction in bacterial colonies of all the tested strains (Figure 6), and partial results (with negative reduction rate) even indicated the promotion of bacterial growth, which might be due to uncontrollable factors in biological experiments, such as the error caused by a nonabsolutely homogeneous bacterial suspension. And GO and PEG might contribute to bacterial proliferation by providing adsorbable platform or nutrients. In accordance with the MIC analysis, the instant antibacterial property of bare AgNPs (∼10 nm) was also very weak because 15336


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Figure 8. TEM images of (a, b, and c) E. coli and (d, e, and f) S. aureus cells. (a) and (d) are blank control groups. (b) and (c) are cells treated with 5 μg/mL and (e) and (f) are cells treated with 10 μg/mL of GO-PEG-Ag for 2.5 h, at different magnifications.

derived from a nontarget-selective physical effect,6 while traditional antibiotics usually present selectivity toward one or more biological targets.45 The highly effective antibacterial ability of GO-PEG-Ag, especially its excellent performance against the superbugs, indicates a great application potential of GO-PEG-Ag in the fight against the increasingly serious bacterial resistance problem. The long-term antibacterial effectiveness of GO-PEG-Ag and GO-Ag is presented as changes in the bacterial survival rate with storage duration. The long-term antibacterial effectiveness of GO-PEG-Ag after storage in saline solution for different durations against both E. coli (red line in Figure 7a) and S. aureus (red line in Figure 7b) was strong and stable. The survival rates of E. coli were very low, in the narrow range of 0− 3.3%, while those of S. aureus were 3.6−9.3%. However, the long-term antibacterial effectiveness of GO-Ag (blue lines in Figure 7a,b) significantly decreased after only 4 h storage; the survival rate of E. coli increased from 10.5% to 65.8% over GOAg storage time from 0 h to 1 week, while that of S. aureus even rose from 22.8% to 81.5%. Thus, after 1 week of storage, the bactericidal rate of GO-PEG-Ag against E. coli and S. aureus was respectively about 3 times and 4 times that of GO-Ag. This difference in long-term effectiveness of the two nanocomposites is much larger than the difference in their instant antibacterial activity. The decrease in antibacterial effectiveness of GO-PEGAg was ∼5% after 1 week storage in NaCl, in comparison to more than 65% for GO-Ag, indicating the long-term effectiveness of GO-PEG-Ag is significantly superior to that of GO-Ag. This is mainly caused by the difference in stability of the two nanomaterials in NaCl, as shown in Figure 5b. The excellent stability and dispersion enable effective contact between GO-PEG-Ag and bacteria, almost free from the impact of storage time, which ensures stable and efficient antibacterial activity of GO-PEG-Ag. It also revealed the positive relationship between the stability and the long-term

effect of the nanocomposite. Strangely, the time point at which the antibacterial effectiveness of GO-Ag began to rapidly decline (8 h of storage in NaCl), as shown in Figure 7a,b, was later than the time point at which GO-Ag came to obvious aggregation and complete precipitation (1 and 4 h, respectively, as shown in Figure 5b). It suggests a transition from physical sediment to irreversible aggregation, which could explain the instant bactericidal performance of GO-Ag. Despite of the physical sediment of GO-Ag in the media, it could accomplish the process of killing bacteria before the irreversible aggregation. Next, we investigated the time-dynamic antimicrobial effectiveness of GO-PEG-Ag toward E. coli and S. aureus. The survival rate of bacteria, negatively correlated with the antibacterial effectiveness, was determined after treatment with different concentrations of the nanocomposite for different time. As shown in Figure 7c for E. coli and Figure 7d for S. aureus, the survival rates of the bacteria depend on the exposure time and the concentration of the nanocomposite. The survival rates of E. coli after exposure to 2, 5, and 10 μg/mL of GOPEG-Ag for 2.5 h were 12.2%, 0.2%, and 0, respectively, while those of S. aureus after exposure to 5, 10, and 15 μg/mL of GOPEG-Ag were 18.6%, 4.7%, and 0.4% respectively. Comparison of the time-dynamic curves of E. coli and S. aureus after treatment with 5 μg/mL GO-PEG-Ag (the green lines in Figure 7c,d) revealed that the survival rate of E. coli decreased rapidly at the beginning of the exposure and decreased to below 40% at 0.5 h, while a relative plateau stage was observed at the beginning of the curve of S. aureus, the survival rate of S. aureus was still ∼95% at 0.5 h, and then gradually decreased between 1 and 1.5 h and rapidly declined between 1.5 and 2.5 h. The differences in the curves between E. coli and S. aureus might be due to their different cell wall structure and composition. GOPEG-Ag can easily destroy the thin outer structure of E. coli leading to apoptosis, while the thick and dense peptidoglycan 15337


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Figure 9. Protein leakage from (a) E. coli and (b) S. aureus suspensions treated with different concentrations of GO-PEG-Ag for 2.5 h. The level of cellular total ROS of (c) E. coli and (d) S. aureus treated with different concentrations of GO-PEG-Ag for 1.5 h. The level of ATP of (e) E. coli and (f) S. aureus treated with different concentrations of GO-PEG-Ag for 1.5 h. **P ≤ 0.01, GO-PEG-Ag groups versus control group. Error bars represent the standard error of three parallel experiments.

layer in the cell wall of S. aureus provides a buffer against GOPEG-Ag action to delay apoptosis. This suggests a longer initial bactericidal period, during which the GO-PEG-Ag begins to interact with and destroy bacteria, while some bacterial cells undergo apoptosis owing to the gradual damaging of the peptidoglycan layer. With the prolongation of treatment time, the peptidoglycan layer of the bacteria is severely damaged because of effective interaction between the nanocomposite and the bacteria, which leads to a rise in apoptosis. Moreover, after exposure to GO-PEG-Ag for more than 2.5 h, the decrease in survival rate of both E. coli and of S. aureus gradually stabilized, which might be due to the decrease in live bacteria and the screening of the nanocomposite by bacterial residues. On the basis of our combined findings, we propose that 2.5 h is a reasonable treatment duration for GO-PEG-Ag. Antibacterial Mechanisms of GO-PEG-Ag. To explore the antibacterial mechanism of GO-PEG-Ag, TEM was used to investigate the interaction between the nanocomposite and bacteria. As shown in Figure 8a,d, control E. coli and S. aureus cells are of normal shape, with smooth membranes/walls and cellular integrity. In contrast, after exposure to the nanocomposite, bacterial cells were tightly wrapped by GO-PEG-Ag, the membranes/walls of E. coli (Figure 8b,c) and S. aureus (Figure 8e,f) became rough, and the cells were broken, which was accompanied by obvious cytoplasm leakage (indicated by arrows). The irreversible damage to the bacterial structures caused by the nanocomposite is likely responsible for the apoptosis of the bacterial cells. The large-scale changes in integrity of bacterial membranes/walls after treatment with GO-PEG-Ag were confirmed by confocal fluorescence microscopy (details of which are presented in “Confocal Fluorescence Microscopy” and Figure S7). We propose that the antibacterial process of GO-PEG-Ag entails the following steps: First, the excellent dispersibility together with electrostatic attraction promotes the positively charged GO-PEG-Ag nanocomposite to rapidly capture and wrap negatively charged bacteria. Then, the AgNPs and the released Ag+ exert physical

damage to bacterial cell, which cause the loss of cellular integrity and cytoplasm leakage, leading to apoptosis of the bacteria. The obvious cytoplasm leakage as observed by TEM was confirmed by quantitative analysis of protein leakage, which was taken as one of the representative indicators of cell-content leakage. For E. coli, as shown in Figure 9a, protein leakage from bacteria of the control group was 7.62 μg/mL, which increased to 50.04 and 71.68 μg/mL, about 6.6 and 9.4 times that of the control group, after treatment with 2 and 5 μg/mL of GOPEG-Ag, respectively. For S. aureus, as shown in Figure 9b, protein leakage in the control group was 12.86 μg/mL, while 5 and 10 μg/mL of GO-PEG-Ag induced increases to 54.66 and 102.61 μg/mL, about 4.3 and 8.0 times that of the control group, respectively. Protein leakage increased with increasing concentration of nanomaterial, which suggests that higher dosage of GO-PEG-Ag induces more severe cell damage in more bacteria. Except for destroying the integrity of the bacterial structure, GO-PEG-Ag also induced an increase in cellular ROS level. For E. coli (Figure 9c), after treatment with 2 and 5 μg/mL of GOPEG-Ag, ROS levels were elevated by 47% and 229% versus the control group, respectively; for S. aureus (Figure 9d), after treatment with 5 and 10 μg/mL of GO-PEG-Ag, the rises were 71% and 120%, respectively. This indicates that GO-PEG-Ag might release AgNPs or Ag+ into the cell interior through the damaged cell membrane/wall or penetrating the cells. The cellular oxidative stress injury thus induced is likely partly responsible for the apoptosis of the bacterial cells. The apoptosis of the bacteria induced by the GO-PEG-Ag nanocomposite was also investigated on the basis of changes in ATP level. As an energy molecule, ATP plays an important role in various physiological and pathological processes of cells. Usually, the level of ATP will decline when the cells undergo apoptosis or necrosis or are in a toxic state. The ATP levels of E. coli (Figure 9e) and S. aureus (Figure 9f) significantly decreased after exposure to GO-PEG-Ag. After incubation with 15338


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Figure 10. Viability of HeLa cells treated with different concentrations of GO-PEG-Ag and GO-Ag for (a) 8 h and (b) 24 h. * P ≤ 0.05, **P ≤ 0.01, GO-Ag group versus GO-PEG-Ag group. Error bars represent the standard error of three parallel experiments.

5 μg/mL of GO-PEG-Ag, the ATP level in S. aureus declined by 12.5%, which was much lower than the decline of 62.76% in E. coli, likely owing to the thicker physical structure of S. aureus. Cytotoxicity Assay. The cytotoxicity of nanocomposites to mammalian cells was investigated by evaluating cell viability using the CCK-8 assay in HeLa cells cultured with different concentrations of GO-PEG-Ag and GO-Ag nanocomposites for 8 and 24 h. GO-PEG-Ag exhibited no obvious cytotoxicity to HeLa cells: even at the high concentration of 50 μg/mL (10 times the MIC toward S. aureus), cell viability remained ∼80% after 24 h of stimulation. After incubation for 8 h (Figure 10a), the cell viabilities for the three GO-Ag groups remained ∼80%, which is slightly lower than that for the three GO-PEG-Ag groups (∼90%), which indicates that both nanocomposites have low cytotoxicity toward HeLa cells within 8 h of stimulation. After incubation for 24 h (Figure 10b), the cytotoxicity of both GO-PEG-Ag and GO-Ag at the concentration of 10 μg/mL remained low, as indicated by cell viabilities of 85.5% and 77.0%, respectively. However, the viabilities of HeLa cells treated with 20 and 50 μg/mL GO-Ag declined to 60−70%, which were much lower than those of GO-PEG-Ag groups. The results demonstrate that GO-PEG-Ag possesses better biocompatibility and a higher dose safety threshold than GO-Ag, and that the incorporation of the biocompatible PEG does not only stabilize the GO-PEG-Ag nanocomposite in physiological solutions, but also reduces its cytotoxicity.

carrying the mcr-1 gene encoding resistance to colistin, which is considered as a last resort antibiotic against multidrug resistant bacteria. Systematic comparisons of instant/long-term antibacterial activity and cytotoxicity demonstrated that GO-PEGAg displayed higher antibacterial behavior and better biocompatibility than GO-Ag. Of particular importance, the GO-PEG-Ag composite had good stability and long-term antibacterial effectiveness, which were also demonstrated as being positively related. As systematically revealed by TEM, fluorescence staining, protein leakage assay, and ATP and ROS detection, the antibacterial mechanisms of GO-PEG-Ag were evidenced as that its unique physical properties caused damage to cell integrity, production of ROS, cytoplasm leakage, and bacterial metabolism decrease resulting in bacterial die-off. The synthesized GO-PEG-Ag nanocomposite, as a type of powerful antibacterial nanomaterial, might have promising anti-infection applications, especially in the treatment of the ever-increasing resistant strains, in biomedical and public health fields.

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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.7b03987. TEM images, size distribution and TGA of GO-Ag nanocomposite; size distribution of AgNPs on GO-Ag; AAS assay of AgNPs content; MIC results, digital images of GO-PEG-Ag in various solutions after storage for one month, confocal fluorescent microscopy analysis, images of colonies on LB plates, and inhibition halo (PDF)

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CONCLUSIONS In summary, a stable nanocomposite of GO-PEG-Ag was synthesized utilizing a facile method, presenting powerful antibacterial capacity, good biocompatibility, and long-term effectiveness. The stability of the nanocomposite in physiological solutions was greatly enhanced by the modification with PEG. As a result, the GO-PEG-Ag could well disperse in various media, even after centrifugation at 10 000×g for 5 min, which would significanlty benefit practical applications. In addition, through the improved method, where silver ammonia was used as the precursor of AgNPs and microwave irradiation as the heating mode, without the addition of extra reductants and surfactants, small-sized and uniformly distributed AgNPs were formed on nanocomposite at high density in a facile manner within 3 min. The antibacterial test results indicate that the GO-PEG-Ag presented powerful antibacterial capability toward not only normal Gram-negative/positive bacteria such as E. coli and S. aureus, but also superbugs including those

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (R.H.). *E-mail: [email protected] (H.S.). *E-mail: [email protected] (Y.S.). ORCID

Lihua Wang: 0000-0002-6198-7561 Chunhai Fan: 0000-0002-7171-7338 Rongzhang Hao: 0000-0002-1527-427X Author Contributions ⊥

R.Z. and M.L. contributed equally to this work.

Notes

The authors declare no competing financial interest. 15339


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ACKNOWLEDGMENTS This work was funded by the National High Technology Research and Development Program of China (2015AA020929), National Key Research and Development Program of China (2016YFC1200700), Beijing Nova Program (Z141107001814071), Innovation Foundation of AMMS (2015CXJJ27), and National Natural Science Foundation of China (U1432116).

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Stable Nanocomposite Based on PEGylated and Silver Nanoparticles

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