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Biomedical Potential of Ultrafine Ag/AgCl Nanoparticles Coated on Graphene with Special Reference to Antimicrobial Performances and Burn Wound Healing Yazhou Zhou, Ru Chen, Tingting He, Kai Xu, Dan Du, Nan Zhao, Xiaonong Cheng, Juan Yang, Haifeng Shi, and Yuehe Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03021 • Publication Date (Web): 11 Apr 2016 Downloaded from http://pubs.acs.org on April 25, 2016

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ACS Applied Materials & Interfaces

Biomedical Potential of Ultrafine Ag/AgCl Nanoparticles Coated on Graphene with Special Reference to Antimicrobial Performances and Burn Wound Healing Yazhou Zhou,†,§, Ru Chen,‡ Tingting He,‡ Kai Xu,† Dan Du,§ Nan Zhao,† Xiaonong Cheng,† Juan Yang,†,* Haifeng Shi,‡ and Yuehe Lin,§,* †

School of Materials Science and Engineering, ‡Institute of Life Sciences, Jiangsu University,

Zhenjiang 212013, PR China, and

§

School of Mechanical and Materials Engineering,

Washington State University, Pullman, WA 99164-2920, USA. KEYWORDS : Ag/AgCl, graphene, oxidative radicals, antimicrobial properties, burn wound healing

ABSTRACT: Recent years, researchers have proven the release of silver ions (Ag+) from the silver nanoparticles (Ag NPs) significantly affects their toxicity to bacteria and other organisms. Due to the difficulty in maintaining a steady flux of high concentration of Ag+, it’s still challenging to develop a highly efficient, stable and biocompatible Ag NPs based antimicrobial material. To circumvent this issue, we developed a new Ag-based bactericide through the fabrication of sunlight-driven and ultrafine silver/silver chloride anchored on reduced graphene

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oxide (Ag/AgCl/rGO). This stable Ag/AgCl nanophotocatalyst with negligible releasing of Ag+ generated high amount of oxidative radicals, killing the bacteria, thus achieved both high bactericidal efficiency and stability. Moreover, functionalization of the nanomaterial with poly(diallyldimethylammonium chloride) (PDDA) has highly adsorptive capacity, which can capture the bacteria and possibly enhance the bactericidal activity. In vivo histopathological studies showed that the Ag/AgCl/rGO nanomaterial could obviously promote the regeneration of epidermis, which indicated the good biomedical potential of Ag/AgCl/rGO nanomaterial in burn wound healing.

1. INTRODUCTION As a broad-spectrum antimicrobial agent, silver metal has been used by mankind for about 7000 years.1 Ag not only can be used in disinfection of bacterial contamination, but also has potential for treatment for antibiotic resistant bacterial infections. The application of silver nanoparticles (Ag NPs) in medical and consumer products includes burn ointments, household antiseptic sprays, wound dressings and antimicrobial coatings for medical devices to efficiently prevent infection.2 Releasing silver ions (Ag+) from Ag NPs and their soluble complexes contributes to the toxicity of these antimicrobial materials.3 A challenge in the application of Ag NPs is to keep a steady flux of high concentration Ag+ from Ag NPs which is crucial in its antibacterial function.4 Moreover, the instability of Ag NPs, due to its aggregation in solution and oxidization, significantly affects its antibacterial function.5 It has been reported that various polymers, carbon materials and silicon nanowires were used to address this problem.5-7 However, it is still challenge to improve the stability of Ag NPs while maintaining the high antibacterial activity. Therefore, our research goal is to develop a new Ag-based material with efficient antibacterial


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function, high stability and good biocompatibility, which has a good biomedical potential in burn wound healing. Recent studies report that Ag/Ag halide nanomaterials have shown an excellent plasmonic photocatalytic performance in the degradation of pollutants through oxidation with oxidative species under visible-light irradiation.8 Ag/Ag halide nanomaterials also showed a high stability due to their large bandgaps, leading to strong resistance towards corrosion induced by surrounding environments.9 Based on these properties, it is possible that Ag/Ag halide nanomaterials may have antibacterial function through generating oxidative radicals, meanwhile achieving stability. However, only a few examples of using the Ag/Ag halide based material as an antibacterial agent have been demonstrated to date, wherein the obtained Ag/Ag halide NPs have big sizes, wide size range, and the extremely low content of Ag0.10 Therefore, increasing the content of Ag0 and reducing the particle size could probably improve the antibacterial activity and stability of Ag/Ag halide NPs. Graphene oxide (GO), a novel cousin of graphene provides fertile opportunities for the construction of GO-based hybrid nanocomposites owing to its abundant oxygen-containing functional groups such as hydroxyl, epoxide, carbonyl, carboxyl etc.11-13 So far, the GO-based nanostructures have been developed as advanced functional materials for potential applications in electronics,14 plasmonics,15 catalysis,16 batteries,17 photovoltaic,18 biochemical sensing,19 drug delivery,20 and more recently in biomedicine.21 Recent reports showed that the Ag/Ag halide/GO nanomaterial exhibited highly efficient and stable photocatalytic activities related to pure Ag/Ag halide NPs. The role of GO in enabling control of the particle shape and decrease of size, thereby contributing to distinctly enhance the photocatalytic activities, has been demonstrated.22,23


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In this paper, we demonstrate for the first time a long-term and highly efficient antibacterial nanomaterial made of reduced graphene oxide (rGO) sheets coated with the high-quality ultrafine silver/silver chloride (Ag/AgCl). The synthetic method of this nanomaterial is simple and green, which can effectively avoid potential environmental and biological risks. The results showed that the size of Ag/AgCl NPs in nanomaterial is ultrafine (~ 4 nm), meanwhile the surface mole ratio of the metallic Ag to Ag+ in nanomaterial is very high (2.11:1). The Ag/AgCl/rGO nanomaterial not only possessed an excellent bactericidal properties against both gram (+) and gram (-) bacteria, but also exhibited extremely stability. The investigation might open up new possibilities for the development of sunlight-driven nanophotocatalyst as a longterm and highly efficient bactericidal material. Finally, an in vivo histopathological study is also conducted on mice to investigate the potential application of Ag/AgCl/rGO nanomaterial in burned wound healing. 2. MATERIALS AND METHODS 2.1. Synthesis of Ag/AgCl/rGO nanomaterial. GO with the size of 3-7 µm was prepared from purified natural graphite by the modified Hummers method, resulting in a colloidal suspension of GO sheets with a concentration of 1 mg mL-1. Briefly, 800 mg of 25 wt% poly(diallyldimethylammonium chloride) (PDDA) solution was added into the 50 mL of prepared GO solution. After vigorous stirring for 30 min, PDDA could be adsorbed onto the surface of GO sheets. Then, AgNO3 solution (0.03 M, 10 mL) was added into PDDA and GO mixed solution. The suspension was magnetically stirred to form a homogeneous mixture within 30 min, after which a milky dispersion contained Ag/AgCl/GO nanomaterial was obtained. The mixture contained 100 mg glucose was transferred into the Teflon-lined autoclave and heated at 140 oC for 5 h. The resulting Ag/AgCl/rGO nanomaterial was washed repeatedly with DI water,


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collected, and dried in an oven at 40 oC overnight. The Ag/AgCl NPs were synthesized at the same experimental condition but in the presence of PDDA or GO alone, which were used as control experiments. The Ag NPs and Ag/rGO materials were synthesized according to our previous work.6 2.2. Characterization. The morphology and microstructure of products were observed by a JEOL 2011 transmission electron microscope (TEM) and high-resolution TEM (HRTEM) at an accelerating voltage of 200 kV. The crystal structure of the products was characterized by a Philips 1730 powder X-ray diffractometer with Cu Kα radiation (λ = 1.5406 Å). The X-ray photoelectron spectroscopy (XPS) measurements were performed by a Thermo ESCALAB 250 spectrophotometer. The energy dispersive X -ray spectroscopy (EDS) analysis was performed on a JEOL-6460 scanning electron microscope (SEM) equipped with an EDS detector. Atomic force microscopy (AFM) images were taken by using a MFP-3D (America) instrument. The UVVis absorption spectrum was taken by using a Shimadzu UV-2550 spectrophotometer. The Ag content in Ag/AgCl/rGO nanomaterial was determined by an inductively coupled plasma optical emission spectrometer (ICP-OES) (VISTA-MPX, Varian Inc.). The zeta-potential was read on a JS94H Micro-electrophoresis systems. 2.3. In vitro antimicrobial properties evaluation. We evaluated the antimicrobial properties of the Ag/AgCl/rGO nanomaterials against the gram (-) bacteria Escherichia coli (E. coli, DH5a) and the gram (+) bacterium of Staphylococcus aureus (S. aureus, ATCC26085). Note that the following experiments processed under the light (Phillips 30 W lamp). The antimicrobial properties of Ag/AgCl/rGO nanomaterials were investigated by in vitro evaluation according to our previous work,6 including inhibition zone testing, growth inhibition, and time kill assay. The biocompatibility and cytotoxicity of nanomaterials were evaluated in vitro on human hepatic


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carcinoma (HepG2) cell line using MTT method. The details of experimental procedures can be seen in Supporting Information. 2.4. Sliver release: The sliver release was tested according to the previous work,24 which can be used to evaluate the stability of materials. 10 mL of 3 mg mL-1 of Ag/AgCl/rGO aqueous solution was transferred into dialysis tube (Spectra/Por Biotech; cellulose ester; MWCO 100,000) and immersed in 30 mL of ultrapure water. The dialysis was carried out under slow stirring with a magnetic stirrer at 37 °C. After a given time interval, the concentration of Ag+ was measured by an ICP-OES (VISTA-MPX, Varian Inc.). The AgNO3, Ag NPs, Ag/rGO, Ag/AgCl and Ag/AgCl/GO were used as control experiments. 2.5. Analysis of OH•. The formation of OH• from the Ag/AgC/rGO aqueous solution under the daylight could be detected by a photoluminescence (PL) technique using terephthalic acid as a probe molecule. Terephthalic acid readily reacted with OH• to produce highly fluorescent product, 2-hydroxyterephthalic acid. The intensity of the PL signal at 425 nm of 2hydroxyterephtalic acid was in proportion to the amount of OH•. PL spectra of the generated 2hydroxyterephthalic acid were measured on an Edinburgh Ins FLS920 spectrophotometer. After light irradiation every 20 min, the reaction solution was filtrated to measure the increase of the PL intensity at 425 nm excited by 315 nm light. The terephthalic acid, AgNO3, Ag NPs, Ag/rGO, Ag/AgCl and Ag NPs/GO were used as control experiments under the same process. 2.6. Cell morphological change. SEM was used to observe the morphological changes of the tested bacteria treated with materials. The details can be seen in our previous work.6 2.7. Cell-binding experiment. We designed an experiment to directly observe the interaction between bacteria and Ag/AgCl/rGO nanomaterial. In details, following incubation, E. coli and S. aureus bacteria were washed and re-suspended in 100 µl phosphate buffered saline (PBS) to a


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concentration of 1 x 1010 cfu mL-1. 100 µl each of bacteria re-suspended well with rotary agitation in 100 PBS solutions which containing 5 mg L-1 ethidium bromide (EB) and Ag/AgCl/rGO nanomaterial, and transferred into the sterile 96-well plates. The ﬁnal concentrations of Ag/AgCl/rGO nanomaterial were 20 mg L-1 and 40 mg L-1, respectively. 100 µl of bacteria at 1 x 108 cfu mL-1 or Ag/AgCl/rGO nanomaterial were also dispersed in 100 µl PBS as further controls. Wells were kept still at 37 °C. Each 30 min, wells were imaged using chemiluminescence imaging system (Clinx ChemiScope 2850). The experiments were repeated three times and similar results were noted in each replicate. 2.8. In vivo evaluation of burn wound healing by Ag/AgCl/rGO nanomaterial using an animal model. Animals: eighteen healthy male ICR mice, seven weeks old and weighting 18-20 g, were obtained from Animal Laboratory Center in Jiangsu University. All mice were fed on a standard diet and water under normal housing conditions. Experimental design and treatment of animals were carried out in strict accordance with the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ of the Institute of Laboratory Animal Resources, National Research Council (SCXK 2013-0011). All surgeries were performed under chloral hydrate, and all efforts were made to minimize suffering. 2.8.1. Experimental design. Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.5 g kg-1), and the dorsal skin was depilated. The dorsal area of each mouse was exposed for 3 s to copper stamp (3 mm in diameter) heated over a 600 W heater for 5 min to form the superficial second degree burn wound (burn area: a diameter of 3 mm round). The mice were randomly divided into three groups: negative control group which was not burned (n=6); the left-side wounds treated with sterilized deionized water were served as the positive control group, and the right-side wounds were treated with 100 µL of 10 mg L-1 Ag/AgCl/rGO aqueous


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solution (n=12). The wounds were treated once day in a paint manner. Digital photographs of the injury site were taken every few days. To assess the healing efficiency, the wound area was measured by tracing the outline of the wound after 0, 5, 10 and 14 days of topical treatment. Mice in each group were euthanized on days 0 and 10, and skin tissue samples from the wound sites were collected for histological staining and biochemical analysis. 2.8.2. Histological examination. For histological examination, the skin wound specimens were fixed in 10% buffered formalin, embedded in paraffin, and sections (3 µm thick) were stained with hematoxylin and eosin (HE) staining. The histological studies were observed by the electric light microscope. 2.8.3. Biochemical estimation of wound healing. The hydroxyproline content of the wound was determined to estimate the wound healing by Ag/AgCl/rGO nanomaterial. BioVision’s hydroxyproline assay kit was employed to measure the hydroxyproline in tissue on postwounding days 0, 14, according to the manufacturer’s instructions (BioVision, Inc. USA). 3. RESULTS AND DISCUSSION 3.1. Fabrication of the Ag/AgCl/rGO nanomaterial. The whole preparation strategy for constructing the Ag/AgCl/rGO nanomaterial includes two steps (Figure S1): the first step is fabrication of the Ag/AgCl/GO nanomaterial by adding AgNO3 aqueous solution into PDDAfunctionalized GO solution; the second step is reducing the Ag/AgCl/GO into Ag/AgCl/rGO nanomaterial using glucose under the hydrothermal process. Here, PDDA can not only provide the Cl- to form the Ag/AgCl NPs, but also be used to control the morphology and size of Ag/AgCl as surfactant. Figure S2 showed AFM images of GO and PDDA-functionalized GO nanosheets. The thickness of GO and PDDA-functionalized GO nanosheets, measured from the height profile were around 0.96 and 2.1 nm, respectively, indicated that PDDA surfactants were


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assembled onto GO sheets (Figure S2). The Ag/AgCl/rGO showed high stability in water, which can be ascribed to functionalization of PDDA surfactant (Figure S1c). XRD patterns of our nanostructures were investigated to identify the formation of Ag/AgCl nano-species. As shown in Figure 1a, distinct diffraction peaks (2θ) at 27.7° (111), 32.1° (200), 46.2° (220), 54.7° (311), 57.4° (222), 67.4° (400), 74.4° (331), and 76.6° (420) are attributed to the typical cubic phase of AgCl (JCPDS file: 31-1238). At the same time, the XRD pattern indicated that diffraction peaks at 38.1° (111), 44.3° (200), 64.4° (220), and 77.9° (311) are ascribed to the typical cubic phase of metallic Ag (JCPDS file: 65-2871). The XRD study verified the existence of AgCl and Ag0 species in the Ag/AgCl/GO nanomaterial. Through the hydrothermal reaction, metallic Ag diffraction peaks in Ag/AgCl/rGO nanomaterial increased remarkably. This suggests that some AgCl NPs were reduced to Ag NPs (Figure 1a).

Figure 1. (a) XRD patterns of Ag/AgCl/GO and Ag/AgCl/rGO nanomaterials, (b) UV-visible diffuse reflectance spectrum of the Ag/AgCl/rGO nanomaterial. UV-visible diffuse reflectance spectrum was also used to further characterize the formation of the complex, which is shown in Figure 1b. It can be seen that Ag/Ag/Cl/rGO has strong


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absorption both in the ultraviolet and visible-light regions. The absorption at 260-350 nm can be ascribed to the characteristic absorption of the AgCl semiconductor, and the strong absorption in the visible-light region can be attributed to the surface plasmon resonance of Ag0.25,26 The strong absorptions in visible region let the Ag/AgCl/rGO nanomaterial to use sunlight more efficiently.25 TEM was used to visualize and analyze the morphologies of the complex. Figure 2a showed the TEM image of the Ag/AgCl/GO. It is observed that small Ag/AgCl NPs with the 10 ± 0.5 nm sizes were uniformly attached on the surface of the GO sheets. After the hydrothermal reaction, the Ag/AgCl/GO was reduced to Ag/AgCl/rGO nanomaterial, and the morphologies can be seen in Figure 2(b-d). In the lower resolution TEM (Figure 2b,c), no particle aggregation were found on the rGO sheet, indicating the good particle dispersion in the Ag/AgCl/rGO composites. In the higher resolution TEM, the ultrafine Ag/AgCl nanocrystals with uniform size (4 ± 0.5 nm) are evenly distributed on the rGO sheets (Figure 2f). The Ag/AgCl size calculated from TEM image is much smaller than that of calculated using Scherer equation from XRD pattern. The ultrahigh dispersion of NPs on rGO sheets might affect particle size result from XRD pattern. To investigate the effects of GO sheets and PDDA on the shape and size of Ag/AgCl NPs, experiments with a series control samples were carefully designed and performed. It is very difficult to control the shape and size of Ag/AgCl synthesized in absence of the PDDA surfactant and GO sheets, and the Ag/AgCl NPs aggregated and formed large size particles.23 In contrast; the cube-like Ag/AgCl nanoparticles of 142 ± 25.3 nm were produced in the presence of PDDA, which can be seen in SEM image (Figure 2e). When the PDDA surfactant was replaced with GO sheets, sphere-like Ag/AgCl NPs of 163 ± 22.7 nm coated onto the surface of GO sheets (Figure


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2f). In addition, it was observed that the Ag NPs grew around the AgCl NPs to form the Ag/AgCl NPs (Figure 2b), which was similar with the literature.27

Figure 2. The morphology characterization of products. TEM images of (a) Ag/AgCl/GO, (b-d) Ag/AgCl/rGO synthesized in the presence of GO and PDDA, (e) SEM image of Ag/AgCl synthesized in the presence of PDDA surfactant, and (f) TEM image of Ag/AgCl/rGO synthesized in absent of PDDA. The experiments have demonstrated that both GO sheets and PDDA surfactant have effects on controlling of morphologies and decrease of size, which was summarized in Scheme 1. In the presence of GO sheets and PDDA surfactants, the Ag/AgCl NPs prepared in the first experimental step (Ag/AgCl/GO) were very small ( ~ 10 nm) (Figure 2a), which result in the ultrafine size in the final product of Ag/AgCl/rGO (~ 4 nm) after the hydrothermal reaction (Figure 2d and Scheme 1c). It is well known that the oxygen functionalities of GO sheets provided abundant sites for coating the particles and prevent them from aggregation.22,23 The long chains of PDDA surfactants attached onto the GO sheets may further prevent the particles


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from aggregation, resulting in ultrafine sized Ag/AgCl NPs. However, the PDDA surfactant used in this experiment has the optimal dosage. When the dosage of PDDA lowered to 400 mg, the NP size in product was bigger and the NP distribution was broader (12.2 ±2.7 nm), compared with the product prepared with 800 mg PDDA (Figure S3a). When the dosage of PDDA increased to 1200 mg, NP loading on the rGO sheets was lower than that of product prepared with 800 mg PDDA (Figure S3b). PDDA used to modify the GO sheets was too much to weaken the adsorption of Ag+ on the GO sheets, resulting in the lower NP loading. Therefore, the optimal dosage of PDDA is around 800 mg in our experiment.

Scheme 1. A Schematic illustration for the size and shape of Ag/AgCl NPs controlled by (a) PDDA surfactant, (b) GO sheets, and (c) both PDDA and GO. We have tried to confirm the Ag and AgCl structures in Ag/AgCl NPs using the HRTEM. However, only metallic Ag crystal structure was observed in Ag/AgCl/rGO nanomaterial. Spatial


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distribution and structural morphologies of the Ag and AgCl NPs cannot be clearly imaged with TEM technique (Figure S4). Exposing Ag/AgCl NPs to an electron beam with high current density for a long time reduces Ag+ to metallic Ag0.9,28 In the high-temperature reduction process, the resulting Ag atoms in each nanoparticle may nucleate and condense on the surface of NP to form individual nanograins or a closely packed shell.9

Figure 3. Typical XPS spectra of (a) C 1s and (c) Ag 3d of Ag/AgCl/GO, (b) C 1s and (d) Ag 3d of Ag/AgCl/rGO, respectively; (e) Cl 2p and (f) N 1s XPS spectra of Ag/AgCl/rGO nanomaterial.


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The formation of nanomaterial was further characterized by XPS. High resolution C 1s peaks were shown for Ag/AgCl/GO nanomaterial in Figure 3a and for Ag/AgCl/rGO nanomaterial in Figure 3b. The peak intensities of oxygen functionalities in rGO decreased remarkably (Figure 3b) after the nanomaterial was reduced by PDDA and glucose under hydrothermal condition. Nanomaterial C/O increased from 1.34 (GO) to 5.68 (rGO). Figure 3e showed the spectrum of Cl 2p, which displayed a binding energy of Cl 2p3 and Cl 2p1 at about 198.0 and 199.5 eV, respectively. Figure 3c and 3d showed the Ag 3d spectra of Ag/AgCl/GO and Ag/AgCl/rGO nanomaterial, respectively. The Ag 3d spectra consists of two individual peaks at approximately 367.9 eV and 373.8 eV, which can be attributed to Ag 3d5/2 and Ag 3d3/2 binding energies, respectively. These two bands could be further de-convoluted into two peaks, respectively, at 368.0, 366.8 eV and 373.9, 372.8 eV. The bands at 366.8 and 372.8 eV are attributed to Ag+. Those at 368.0 and 373.9 eV are ascribed to the metallic Ag0.23 The calculated surface mole ratio of the metallic Ag0 to Ag+ in Ag/AgCl/GO and Ag/AgCl/rGO nanomaterials are ca. 0.47:1 and 2.18:1, respectively. This indicated that the mass AgCl has been reduced to Ag by glucose. Furthermore, the additional component around 285.6 eV in Figure 3a and 3b is assigned to the CN bond (originate from polymer, PDDA),29 which further support the conclusion that Ag/AgCl NPs have been effectively assembled on the surface of PDDA-functionalized rGO nanosheets. To further validate the formation of Ag/AgCl nanospecies, the components of the Ag/AgCl/rGO nanomaterial were investigated with energy dispersive EDS analysis. As shown in Figure S5a, C, O, Ag and Cl elements were evidently detected, wherein the semiquantitative analysis showed that the atomic ratio between Ag and Cl elements is approximately 2.11:1, indicating that ~ 67.8% of AgCl has been reduced to Ag NPs. The results of XRD, XPS and EDS studies have shown the content of Ag0 in the Ag/AgCl nanoparticle was much higher than


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that of in the previous reports.10,22-28 The high content of Ag0 advanced the strong absorptions in visible region, that makes the Ag/AgCl/rGO nanomaterial using the sunlight more efficiently. Moreover, the EDS results also showed the high stability of Ag/AgCl/rGO because the composition of the NPs did not show signiﬁcant variation after exposure in environment for one month (Figure S5b). 3.2. In vitro antimicrobial properties evaluation. To investigate antibacterial activity of the Ag/AgCl/rGO nanomaterial, gram (-) bacterium E. coli and the gram (+) bacterium of S. aureus were used in the experiments. The antibacterial activities of Ag/AgCl/rGO nanomaterial against E. coli and S. aureus were assessed qualitatively by determining the presence of inhibition zones. After 24 h of incubation with the Ag/AgCl/rGO nanomaterial, a clear growth inhibition zones in both E. coli and S. aureus cultures was observed (Figure 4). The average radii of inhibition zones for E. coli and S. aureus bacteria treated by 50 mg L-1 Ag/AgCl/rGO nanomaterial increased to 9.7 and 11.8 mm, respectively. The experiments demonstrated antibacterial function of the nanomaterial against both gram (-) and gram (+) bacteria.

Figure 4. Photographs of E. coli and S. aureus grow around a series of concentrations of Ag/AgCl/rGO nanomaterials on the plates.


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Quantitative evaluation of antibacterial activity of the Ag/AgCl/rGO nanomaterial was carried out by studying the bacterial growth kinetics in Luria–Bertani (LB) medium.30 Briefly, the nanomaterials with various concentrations were added into the bacterial solution. Bacterial proliferation was monitored using optical density at 600 nm (OD600) to measure the turbidity of the cell suspension within 20 h. Water, rGO, Ag NO3, Ag/rGO,6 Ag NPs,31 Ag/AgCl and Ag/AgCl/GO were used as controls in the experiments (Figure S6). The growth curve showed a dose-dependent antibacterial effect of Ag/AgCl/rGO nanomaterial (Figure 5a and 5b). Compared to the growth curves of the negative control of bacterial culture without nanomaterial, the growth curves in the presence of Ag/AgCl/rGO nanomaterial showed the remarkable lag phases for both bacterial species. The growths of E. coli and S. aureus bacteria were completely inhibited by the Ag/AgCl/rGO nanomaterial at 10 and 20 mg L-1, respectively. The minimum inhibitory concentration (MIC) of the nanomaterial was tested. A comparison of antibacterial function of the Ag/AgCl/rGO nanomaterial with other antibacterial materials was also investigated (Table S1). Based on the growth inhibition experiments, we found that the MICs for Ag/AgCl/rGO nanomaterial against E. coli and S. aureus were 2 mg L-1 and 4 mg L-1, respectively (in terms of Ag element), which were much lower than that of other antibacterial materials in comparison experiments. Growth inhibition experiments alone cannot differentiate between bactericidal and bacteriostatic activity, otherwise discernible under time-kill conditions.32 Therefore, the timekilling studies were performed for the Ag/AgCl/rGO nanomaterial against bacteria within shortterm time (2.5 h) (Figure 5c and 5d). The cfus of the surviving E. coli and S. aureus after exposure to Ag/AgCl/rGO nanomaterial for various times (0-2.5 h) are shown in Figure S7. For E. coli, there is significant decline in cell density within 0.5 h as a result of exposure to 1 mg L-1


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concentration of Ag/AgCl/rGO nanomaterial. 90.8% of E. coli was killed (Figure 5c). When the concentration was increased to 5 mg L-1, 99.3% of E. coli was killed within 0.5 h. In the case of S. aureus however, no significant decline is observed in cell density during in the 5 mg L-1 concentration of nanomaterial, otherwise remarkable under the 10 mg L-1 concentration (Figure 5d). The minimum bactericidal concentration (MBC) for the Ag/AgCl/rGO nanomaterial is much below 0.5 mg L-1 for E. coli and 2 mg L-1 for S. aureus (in terms of Ag element).

Figure 5. Bacterial growth curves in LB media with serial concentrations of Ag/AgCl/rGO nanomaterial were added to the culture of (a) E. coli and (b) S. aureus; Bacteria time-kill profiles within 2.5 h (c) E.coli in the presence of 1 mg L-1 or 5 mg L-1 of Ag/AgCl/rGO nanomaterial; (d) S. aureus in the presence of 10 mg L-1 or 20 mg L-1 of Ag/AgCl/rGO nanomaterial. The bacteria time-kill profiles of E. coli (c) and S. aureus (d) treated with Ag/AgCl/rGO were also performed in the dark environment or with OH• radical scavenger.


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3.3. Stability and biocompatibility of Ag/AgCl/rGO nanomaterial. Besides bactericidal function, stability and biocompatibility are also very important in the applications. The releasing Ag+ from Ag/AgCl/rGO nanomaterials was analyzed by ICP-OES, which can be seen in Figure 6a. AgNO3, Ag NPs, Ag/rGO and Ag/AgCl/GO were used as comparisons. As a result, the Ag+ diffusion rates of AgNO3, Ag NPs and Ag/rGO are much higher than those of Ag/AgCl/GO and Ag/AgCl/rGO. In particular, there is no detectable Ag+ (< 1 µg L-1) in 1 mg L-1 of Ag/AgCl/rGO aqueous solution after exposure in environment for two weeks, and only the trace of Ag+ was detected after 120 d, indicated the extreme stability of Ag/AgCl/rGO nanomaterial. It has been proven that the migration of photo-excited electrons away from the AgCl core prevents photoreduction of AgCl to Ag, leading to high stability of Ag/AgCl NPs.9 Furthermore, the high content of Ag NPs can significantly enhance the stability of Ag/AgCl/rGO nanomaterial because of its strong absorption of visible-light.8,9,25 The chemical and environmental stability of rGO sheet also improved the stability of the Ag/AgCl NPs. The biocompatibility and cytotoxicity of the Ag/AgCl/rGO nanomaterial were evaluated in vitro on the HepG2 cell line. MTT assay was used to determine cell viability. The results shown in Figure 6b demonstrated that Ag/AgCl/rGO nanomaterial has a dose-dependent cytotoxicity to HepG2 cells within 24 h incubation. However, it has a good biocompatibility under the low concentration (< 5 mg L-1), meanwhile great bactericidal activity against bacteria. Further studies of the toxicity and biocompatibility of Ag/AgCl/rGO nanomaterial in human cells are need.


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Figure 6. (a) Dissolved Ag+ concentrations for Ag-based materials were detected by ICP-OES within air exposure time, (b) Cell viability measurements carried out by using MTT colorimetric assay of human HepG2 cell line in the presence of Ag-based materials; (c) PL spectra of Agbased materials in a 5 × 10−4 M basic solution of terephthalic acid under visible light irradiation at a fixed 90 min; (d) PL spectral of Ag/AgCl/rGO nanomaterial change with irradiation time. 3.4. Antimicrobial mechanism studies. The results from the comparison of antibacterial function showed that among the other Ag-based materials listed in the review by M. Epple,33 Ag/AgCl/rGO nanomaterial exhibits the highly efficient and extremely stable antibacterial function. As that mentioned early, the Ag+ released from the Ag based material contributes to their antibacterial function. However, the ICP-OES results implied that the antibacterial function


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of Ag/AgCl/rGO might be not the releasing Ag+. Ag/AgCl nanomaterial is an efficient photocatalyst for degrading pollution by generating oxidative species under visible light irradiation.34,35 The Cl atoms formed in the core of AgCl, and oxygen radicals (e.g., O2-, O22-, O•) formed through reduction of O2 (from solution) with photogenerated electrons around the Ag.9 Some oxygen radicals may further combine with the H+ to generate the OH• radicals. It was proven that the oxygen radicals, OH• in particular could directly attack the covalent bond of biomolecules on the cell membrane and wall, resulting in cell death.36, 37 Therefore, we consider that the oxidative radicals generated by the Ag/AgCl NPs maybe contribute to the highly efficient antibacterial activity. Based on these theories, PL technique, together with terephthalic acid as a probe was used to investigate the OH• generated by the effect of Ag/AgCl/rGO nanomaterial.38 Terephthalic acid, AgNO3, Ag NPs, Ag/rGO, Ag/AgCl and Ag/AgCl/GO were used as controls in the experiments. As shown in Figure 6c, the remarkable PL peaks were observed at about 425 nm for Ag/AgCl/GO and Ag/AgCl/rGO nanomaterials with the concentration of 1 mg L-1 after illumination for 90 min. In contrast, there is no detectable PL signal for AgNO3, Ag NPs, Ag/rGO and Ag/AgCl materials at 1 mg L-1, but weak PL peaks were found at 20 mg L-1 (Figure 6c). Moreover, the releasing OH• radicals gradually increased with prolongation of exposed time (Figure 6d). PL results proved that OH• radicals were generated by Ag/AgCl NPs after illumination, and the Ag/AgCl/rGO nanomaterial is the most active. To investigate the effects of oxidation radicals on bactericidal activity of Ag/AgCl/rGO nanomaterial, time-killing studies were performed in the experiments. Without illumination, only 12.7% of E. coli bacteria were killed after 2.5 h by Ag/AgCl/rGO nanomaterial at 1 mg L-1 (Figure 5c). With the addition of tBuOH, an efficient OH• radicals quencher, the bactericidal efficiency of E. coli was


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significantly decreased and about 40.2% of the E. coli was killed under illumination. The similar results were obtained for S. aureus (Figure 5d). The results showed that oxidative radicals play a crucial role in the antibacterial function of Ag/AgCl/rGO nanomaterial. The generation of oxidative radicals by the effect of Ag/AgCl/rGO nanomaterial is highly efficient. That because (1) the high content of Ag NPs (2.11:1 for Ag:AgCl) leads to the Ag/AgCl/rGO nanomaterial use the sunlight more efficiently, and has enhanced photocatalytic activity; (2) Ag/AgCl NPs in the nanomaterial are very active because of their ultrafine sizes; Therefore, the main antimicrobial mechanism of Ag/AgCl/rGO nanomaterial should not be releasing Ag+, but generating oxidative radicals, which resulting in the highly efficient and extreme stable bactericidal function. However, the bactericidal activity of Ag/AgCl/rGO nanomaterial diminished after four-month exposure in the environment (MIC increased to 10 mg L-1 against E. coli). Besides the oxidative radicals, the rGO nanosheets also play an important role in the enhancement of the bactericidal function through their efficient adsorption. SEM was employed to evaluate the surface morphology change of the native and treated E. coli and S. aureus cells with the 1 mg L-1 of Ag/AgCl/rGO nanomaterial. After treatment with nanomaterial for 0.5 h, both E. coli and S. aureus cells were encapsulated by the Ag/AgCl/rGO sheets (Figure S8). In particular, E. coli cells enwrapped with Ag/AgCl/rGO nanosheets were seriously damaged (Figure S8c). Through the modification by PDDA, the surface charge of nanomaterial was changed from negative to positive (Zate potential is +44 mV). The oppositely charged cells were adsorbed by and/or attached on the nanomaterial based on electrostatic interaction. However, because the dry sample is necessary for SEM analysis, the SEM cannot be used to observe the true interaction between our material and cells. Here, we designed an experiment to directly observe such interaction in the liquid state. Bacterial cells were stained by using EB. The cells


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were visible with red colour under the ultraviolet illumination (302 nm). Therefore, the interaction between bacterial and nanomaterial can be observed and imaged by a chemiluminescence imaging system (Figure 7). For comparisons, E. coli cells or Ag/AgCl/rGO nanomaterial alone could remain suspended in PBS. However, in the presence of Ag/AgCl/rGO nanomaterial, cells were aggregated with Ag/AgCl/rGO sheets and then deposited on the bottom of wells. The same phenomenon was observed in the experiment of S. aureus bacteria. We speculate that due to the efficient adsorption, Ag/AgCl/rGO sheets can capture the bacteria and then Ag/AgCl NPs coated onto the rGO nanosheets can directly act on them, enhancing the bactericidal function.

Figure 7. Photographs of (a) E. coli, (b) S. aureus cells in contact with Ag/AgCl/rGO nanomaterial. The cells exhibited red colour under the ultraviolet illumination since being labelled by EB. The interaction between bacteria and nanomaterial can be observed. 3.5. In vivo evaluation of burn healing by Ag/AgCl/rGO nanomaterial. Ag, a broadspectrum antimicrobial agent, is one of the useful prophylactic or therapeutic agents for treatment the infections encountered in burns, open wounds, and chronic ulcers. Here, we


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investigated whether topical application of the Ag/AgCl/rGO nanomaterial would accelerate burn wound healing processes using an animal model. Two superficial second degree burn wounds were made on the dorsal skin of each mouse. The left-side wounds treated with water sterilized deionized water were severed as positive controls, and the right-side wounds were treated by the Ag/AgCl/rGO aqueous solution with the concentration of 10 mg L-1. Figure S9a showed the wound healing process on post-wounding days 0, 5, 10 and 14. And the wound sizes were quantified from images of wounds, which can be seen in Figure S9b. In the positive control group, the wound size increased from 2.99 mm to 3.55 mm on days 5, and then decreased progressively. The rate of wound closure in mice treated with Ag/AgCl/rGO aqueous solution was significantly faster than that of in positive control group. The burn wounds of mice in treatment of Ag/AgCl/rGO nanomaterial were completely closed on days 14. Moreover, after the wound healing with Ag/AgCl/rGO nanomaterial, the experimental mice have the negligible scab, while the mice in positive control group have the big scabs. Photomicrographs of cutaneous full-thickness biopsies (HE stained) representing skin of mice of different groups are shown in Figure 8. For the negative control group, the entire skin tissue of the healthy mouse can be clearly seen in Figure 8a. By the superficial second degree burn, the epidermis tissue of skin was fully destroyed (Figure 8b). After 10 days, the epidermis tissue of burn wound in treatment of sterilized deionized water (positive control group) was partly regenerated (Figure 8c). In contrast, the epidermis tissue of burn wound treated with the Ag/AgCl/rGO nanomaterial was completely healed after 10 days (Figure 8d). Therefore, the Ag/AgCl/rGO nanomaterial could obviously promote regenerated epidermis formation, and the effect was better than that of the positive control group. Hydroxyproline is one of the main components of collagen, which is a direct measure of the amount of collagen in tissue


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hydrolysates. Therefore, the estimation of hydroxyproline content of the wound bed can be used to estimate the collagen forming and wound healing. As shown in Figure 8e, the hydroxyproline content in the Ag/AgCl/rGO group was much higher than that of the positive control group on days 5 and 10 after burn, and eventually returned to the normal levels of unburned skin on days 14 post-wounding, indicating that the Ag/AgCl/rGO nanomaterial can promote collagen growth and healing burn wound. Even through the Ag/AgCl/rGO showed the biomedical potential in burn wound healing, the more in vivo studies are still needed, such as immunotoxicity of this material. It is proven that the immunotoxicity of silver nanoparticles is unneglectable, which are effects on the immune system.39

Figure 8. Photomicrographs of histopathological sections representing (a) healthy mouse skin, (b) initial burned skin of mice, (c) burned skin of mice treated with water and (d) Ag/AgCl/rGO on days 10. (e) Contents of hydroxyproline in granulation tissues of skin wounds in different healing periods. 4. CONCLUSION In summary, we developed a sunlight-driven Ag/AgCl/rGO photocatalyst as a new class of antibacterial material with high antibacterial activity and high stability. Ultrafine Ag/AgCl NPs


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with the high content of Ag0 have been achieved under the effects of GO sheets and PDDA surfactant. In vitro study, the Ag/AgCl/rGO nanomaterial showed the excellent bactericidal properties. The highly efficient oxidative radical generation by the effect of Ag/AgCl/rGO nanomaterial contributed best to bactericidal performance. In addition, functionalizing nanomaterial with PDDA greatly increased its adsorptive capacity that also enhanced its bactericidal activity. Furthermore, Ag/AgCl/rGO nanomaterial is very stable, which results in a long shelf-life of this antibacterial agent. In vivo histopathological study, the burned wound treated with Ag/AgCl/rGO nanomaterial showed a fast healing rate and epidermis formation regeneration. Therefore, Ag/AgCl/rGO nanomaterial has been proven to have efficient antibacterial performance and benefic influence on the burn wound healing. ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Experimental section of antimicrobial activity test in vitro evaluation, AFM images of GO nanosheets and PDDA/GO nanosheets, photographs of products, HRTEM, TEM, EDS, growth inhibition of comparisons, images of bacteria colonies, images of burn wounds (PDF) AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected] (J. Yang); [email protected] (Y. Lin)

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT We gratefully acknowledge the funding of this project by National Science Foundation of China (51572114), Six Personnel Peak Project (2009137) of Jiangsu Province, and YL and DD acknowledge the financial support from a WSU start-up grant. We would also like to thank X. Ji, K. Chen and L. Pan of analytical instruments of Jiangsu University Analysis and Test Center for their help with the TEM, SEM, XPS and PL studies. REFERENCES (1) Alexander, J. W. History of the Medical Use of Silver. Surg. Infect. 2009, 10 (3), 289-292. (2) Lee, J. S.; Murphy, W. L. Functionalizing Calcium Phosphate Biomaterials with Antibacterial Silver Particles. Adv. Mater. 2013, 25 (8), 1173-1179. (3) Xiu, Z. M.; Zhang, Q. B.; Puppala, H.L.; Colvin, V. L.; Alvarez, P. J. J. Negligible ParticleSpecific Antibacterial Activity of Silver Nanoparticles. Nano Lett. 2012, 12 (8), 4271-4275. (4) Kittler, S.; Greulich, C.; Diendorf, J.; Köller, M.; Epple, M. Toxicity of Silver Nanoparticles Increases During Storage Because of Slow Dissolution Under Release of Silver Ions. Chem Mater 2010, 22 (16), 4548-4554. (5) Kong, H.; Jang, J. Antibacterial Properties of Novel Poly(methyl methacrylate) Nanofiber Containing Silver Nanoparticles. Langmuir 2008, 24 (5), 2051-2056. (6) Zhou, Y.; Yang, J.; He, T.; Shi, H. F.; Cheng, X.; Lu, Y. Highly Stable and Dispersive Silver Nanoparticle–Graphene Composites by a Simple and Low-Energy-Consuming Approach and Their Antimicrobial Activity. Small 2013, 9 (20), 3445-3454. (7) Lv, M.; Su, S.; He, Y.; Huang, Q.; Hu, W.; Li, D.; Fan, C.; Lee, S. T. Long-Term Antimicrobial Effect of Silicon Nanowires Decorated with Silver Nanoparticles. Adv. Mater. 2010, 22 (48), 5463-5467. (8) Wang, P.; Huang, B.; Qin, X.; Zhang, X.; Dai, Y.; Wei, J.; Whangbo, M. H. Ag@AgCl: A Highly Efficient and Stable Photocatalyst Active Under Visible Light. Angew. Chem. Int. Ed. 2008, 47 (41), 7931-7933.


26

Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(9) An, C.; Peng, S.; Sun, Y. Facile Synthesis of Sunlight-Driven AgCl:Ag Plasmonic Nanophotocatalyst. Adv. Mater. 2010, 22 (23), 2570-2574. (10) Nocchetti, M.; Donnadio, A.; Ambrogi, V.; Andreani, P.; Bastianini, M.; Pietrellac, D.; Latterini, L. Ag/AgCl Nanoparticle Decorated Layered Double Hydroxides: Synthesis, Characterization and Antimicrobial Properties. J. Mater. Chem. B 2013, 1 (18), 2383-2393. (11) Geim, A. K.; Novoselov, K. S. The Rise of Graphene. Nat. Mater. 2007, 6 (3), 183-191. (12) Park, S.; Ruoff, R. S. Chemical Methods for the Production of Graphenes. Nat. Nanotechnol. 2009, 4, 217-224. (13) Dreyer, D. R.; Park, S.; Bielawski, C. W.; Ruoff, R. S. The Chemistry of Graphene Oxide. Chem. Soc. Rev. 2010, 39 (1):228-240. (14) Park, Y. J.; Lee, S. K.; Kim, M. S.; Kim, H.; Ahn, J. H. Graphene-Based Conformal Devices. ACS Nano 2014, 8 (8):7655-7662. (15) Zhou, Y.; Cheng, X.; Du, D.; Yang, J.; Zhao, N.; Ma, S. Graphene–Silver Nanohybrids for Ultrasensitive Surface Enhanced Raman Spectroscopy: Size Dependence of Silver Nanoparticles. J. Mater. Chem. C 2014, 2 (33), 6850-6858. (16) Zhou.; Y, Yen, C. H.; Fu, S.; Yang, G.; Zhu, C.; Du, D.; Wo, P. C.; Cheng, X.; Yang, J.; Wai, C. M.; Lin, Y. One-Pot Synthesis of B-Doped Three-Dimensional Reduced Graphene Oxide via Supercritical Fluid for Oxygen Reduction Reaction. Green Chem 2015, 17 (6): 3552-3560. (17) Zhu, J.; Yang, D.; Yin, Z.; Yan, Q.; Zhang, H. Graphene and Graphene-Based Materials for Energy Storage Applications. Small 2014, 10, 3480-3498. (18) Zhou, Y.; Yang, J.; Cheng, X.; Zhao, N.; Sun, L.; Sun, H.; Li, D. Electrostatic SelfAssembly of Graphene-Silver Multilayer Films and Their Transmittance and Electronic Conductivity. Carbon 2012, 50 (12), 4343-4350. (19) Mustafa, B.; Burak, Z. B.; Mustafa, S. Y.; Mehmet, V. Y. Smart-Polymer-Functionalized Graphene Nanodevices for Thermo-Switch-Controlled Biodetection. ACS Biomater. Sci. Eng. 2015, 1 (1), 27-36. (20) Weaver, C. L.; LaRosa, J. M.; Luo, X.; Cui, X. T. Electrically Controlled Drug Delivery From Graphene Oxide Nanocomposite Films. ACS Nano 2014, 8 (2), 1834-1843.


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Page 28 of 30

(21) Bijukumar, D.; Girish, C. M.; Sasidharan, Abhilash. Nair, S.; Koyakutty, M. TransferrinConjugated

Biodegradable

Graphene

for

Targeted

Radiofrequency

Ablation

of

Hepatocellular Carcinoma. ACS Biomater. Sci. Eng. 2015, 1 (12), 1211–1219. (22) Zhu, M.; Chen, P.; Liu, M. High-Performance Visible-Light-Driven Plasmonic Photocatalysts Ag/AgCl with Controlled Size and Shape Using Graphene Oxide as Capping Agent and Catalyst Promoter. Langmuir 2013, 29 (29), 9259-9268. (23) Zhu, M.; Chen, P.; Liu, M. Graphene Oxide Enwrapped Ag/AgX(X = Br, Cl) Nanocomposite as a Highly Efficient Visible-Light Plasmonic Photocatalyst. ACS Nano 2011, 5 (6), 4529-4536. (24) Li, C.; Wang, X.; Chen, F.; Zhang, C.; Zhi, X.; Wang, K.; Cui, D. The Antifungal Activity of Graphene Oxide–Silver Nanocomposites. Biomaterials 2013, 34, 3882-3890. (25) Wang, P.; Huang, B.; Lou, Z.; Zhang, X.; Qin, X.; Dai, Y.; Zheng, Z.; Wang, X. Synthesis of Highly Efficient Ag@AgCl Plasmonic Photocatalysts with Various Structures. Chem. Eur. J. 2010, 16 (2), 538-544. (26) Yu, J.; Dai, G.; Huang, B. Fabrication and Characterization of Visible-Light-Driven Plasmonic Photocatalyst Ag/AgCl/TiO2 Nanotube Arrays. J. Phys. Chem. C 2009, 113 (37), 16394-16401. (27) Zhang, H.; Fan, X.; Quan, X.; Chen, S.; Yu, H. Graphene Sheets Grafted Ag@AgCl Hybrid with Enhanced Plasmonic Photocatalytic Activity Under Visible Light. Environ. Sci. Technol. 2011, 45 (13), 5731-5736. (28) Zhu, M.; Chen, P.; Ma, W.; Lei, B.; Liu, M. Template-Free Synthesis of Cube-Like Ag/AgCl Nanostructures via a Direct-Precipitation Protocol: Highly Efficient SunlightDriven Plasmonic Photocatalysts. ACS Appl. Mater. Interfaces 2012, 4 (11), 6386-6392. (29) Zhang, S.; Shao, Y.; Liao, H.; Engelhard, M. H.; Yin, G.; Lin, Y. Polyelectrolyte-Induced Reduction of Exfoliated Graphite Oxide: A Facile Route to Synthesis of Soluble Graphene Nanosheets. ACS Nano 2011, 5 (3), 1785-1791. (30) Regoes, R. R.; Wiuff, C.; Zappala, R. M.; Garner, K. N.; Baquero, F.; Levin, B. R. Pharmacodynamic Functions: A Multiparameter Approach to the Design of Antibiotic Treatment Regimens. Antimicrob Agents Chemother 2004, 48 (10), 3670-3676. (31) Zhou, Y.; Yang, J.; Cheng, X.; Zhao, N.; Sun, H.; Li, D. Transparent and Conductive Reduced Graphene Oxide/Silver Nanoparticles Multilayer Film Obtained by Electrical Self-


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Page 29 of 30

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Assembly Process with Graphene Oxide Sheets and Silver Colloid. RSC Adv 2013, 3 (10), 3391-3398. (32) Levin, B. R.; Udekwu, K. I. Population Dynamics of Antibiotic Treatment: A Mathematical Model and Hypotheses for Time-Kill and Continuous-Culture Experiments. Antimicrob Agents Chemother 2010, 54 (8), 3414-3426. (33) Chernousova, S.; Epple, M. Silver as Antibacterial Agent: Ion, Nanoparticle, and Metal. Angew. Chem. Int. Ed. 2013, 52 (6), 1636-1653. (34) Houas, A.; Lachheb, H.; Ksibi, M.; Elaloui, E.; Guillard, C.; Herrmann, J. M. Photocatalytic Degradation Pathway of Methylene Blue in Water. Appl. Catal. B 2001, 31 (2), 145-157. (35) Wang, P.; Huang, B.; Qin, X.; Zhang, X.; Dai, Y.; Whangbo, M. Ag/AgBr/WO3•H2O: Visible-Light Photocatalyst for Bacteria Destruction. Inorg. Chem. 2009, 48 (22), 1069710702. (36) Hou, Y.; Li, X.; Zhao, Q.; Chen, G.; Raston, C. L. Role of Hydroxyl Radicals and Mechanism of Escherichia Coli Inactivation on Ag/AgBr/TiO2 Nanotube Array Electrode Under Visible Light Irradiation. Environ. Sci. Technol. 2012, 46 (7), 4042-4050. (37) Li, Y.; Zhang, W.; Niu, J.; Chen, Y. Mechanism of Photogenerated Reactive Oxygen Species and Correlation with the Antibacterial Properties of Engineered Metal-Oxide Nanoparticles. ACS Nnao 2012, 6 (6), 5164–5173. (38) Yu, J. G.; Wang, W. G.; Cheng, B.; Su, B. L. Enhancement of Photocatalytic Activity of Mesoporous TiO2 Powders by Hydrothermal Surface Fluorination Treatment. J. Phys. Chem. C 2009, 113 (16), 6743-6750. (39) Jong, W. H. D.; Ven, L. T. M. V. D.; Sleijffers, A.; Park, M. V. D. Z.; Jansen, E. H. J. M.; Loveren, H. V.; Vandebriel, R. J. Systemic and Immunotoxicity of Silver Nanoparticles in an Intravenous 28 Days Repeated Dose Toxicity Study in Rats. Biomaterials, 2013, 34(33), 8333-8343.


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BRIEFS Our research goal is to develop a new Ag-based material with efficient antibacterial function, high stability, and good biocompatibility, which has been proved to have efficient antibacterial performance and benefic influence on the burn wound healing. SYNOPSIS


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AgCl ... - ACS Publications

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