An Efficient and Benign Antimicrobial Depot Based on Silver-Infused

Apr 13, 2017 - to construct an efficient and benign antimicrobial depot. The system exhibited much enhanced broad-spectrum antibacterial activity comp...
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An Efficient and Benign Antimicrobial Depot Based on Silver-Infused MoS2 Fangfang Cao,†,‡ Enguo Ju,†,§ Yan Zhang,†,§ Zhenzhen Wang,†,§ Chaoqun Liu,†,§ Wei Li,†,§ Yanyan Huang,†,§ Kai Dong,†,§ Jinsong Ren,*,† and Xiaogang Qu*,† †

State Key Laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡ University of Science and Technology of China, Hefei, Anhui 230029, P. R. China § Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China S Supporting Information *

ABSTRACT: Silver nanoparticles (AgNPs) have been used as a broad-spectrum antimicrobial agent, whose toxicity originates from the localized release of Ag+ ions. However, the residual AgNPs core could generate potential risk to humans and waste of noble metals. Herein, we infused the cysteine-modified molybdenum disulfide with minimum Ag+ ions and coated with a layer of cationic polyelectrolyte to construct an efficient and benign antimicrobial depot. The system exhibited much enhanced broad-spectrum antibacterial activity compared with an equivalent amount of silver nitrate, owing to its increasing accessibility of released Ag+ to the cell walls of microorganisms. More importantly, the antibacterial system could be successfully applied to treat wound infection, while retaining high antibacterial activities, exhibiting negligible biotoxicity and avoiding the waste of Ag. KEYWORDS: antibacterial depot, molybdenum disulfide, silver nanoparticles, wound infection, negligible biotoxicity

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risks for humans, owing to the fact that the excess AgNPs core could lead to argyria to humans, such as spasms, gastrointestinal disorders, and even to death.5,13 To circumvent the problem of the overuse of AgNPs, fabrication strategies that focus on high antibacterial efficiency and minimum Ag+ instead of entire AgNPs core are required. Recently, Richter, A. P. et al. have successfully employed the silver-infused lignin core to form a biodegradable and green alternative to silver nanoparticles to mitigate the potential environment hazard.13 Additionally, the size controllable lignin particles could be synthesized through a high-yield and scalable manufacturing approach.19 However, the risks of excess silver for humans still narrowed its in vivo therapeutic window. Therefore, strategies are urgently needed with efficient antibacterial activity against pathogenic bacteria, while maintaining the biocompatibility to humans and reducing the waste of noble metal Ag. Ultrathin two-dimensional (2D) nanomaterials have garnered enormous interests on account of their unique electronic, physical, and chemical properties. They have been widely exploited in various fields, such as catalysis, photonics, electronics, energy storage, sensing, and diagnoses.20−28 Particularly, layered molybdenum disulfide (MoS2) is gaining

ilver nanoparticles (AgNPs) have become one of the most fascinating commercialized nanomaterials in textiles, food storage, containers, and personal care, since they exhibit broad-spectrum antimicrobial activity together with a reduced tendency to evoke microbial resistance from antiquity to the present.1−6 AgNPs are presumed to disturb the essential bacterial cell functions through two dominating mechanisms. The first mechanism, namely the interaction of released silver ions (Ag+) with proteins and enzymes, results in a serious structural deformation of the cell membrane. The second possibility is the production of high reactive oxygen species (ROS), which perturbs the cell metabolism. 5,7−11 In spite of the fact that the exact antimicrobial mechanism is still being debated, there is no doubt that the localized release of Ag+ ions from the core of AgNPs at the cell walls of microorganism contributes to the toxicity of these nanomaterials.12,13 Consequently, the design of ideal AgNPs to increase the quantity of released Ag+ and further enhance the antibacterial activity of AgNPs has remained a great challenge in this field. Up to now, tremendous efforts have been devoted to preparing AgNPs with different shapes,14 sizes,15 surface coatings,16,17 and surface charges for use in both aerobic and anaerobic conditions to promote the release of Ag+ from AgNPs.12,18 Nevertheless, the accessibility of silver-based antibacterial agents in actual therapy suffers from an intractable limitation. After intended use, the AgNPs have © 2017 American Chemical Society

Received: January 16, 2017 Accepted: April 13, 2017 Published: April 13, 2017 4651

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ACS Nano prominence due to its ultrathin direct bandgap, “S−Mo−S” sandwich structure, special components, and high specific surface area.28−38 Notably, the stacked structures of bulk threedimensional (3D) crystalline MoS2 with strong covalent bonding between Mo and S as well as weak van der Waals interactions between adjacent layers of the MoS2 could provide an easy strategy to obtain single- or few-layered MoS2.29,36 Additionally, molecules could be easily modified to the surface of MoS2 by chemical functionalization or physisorption in a simple sonication method.28,31−33 Besides, Mo is an essential trace element for several enzymes in cells, and S is a common biological element, which makes MoS2 an excellent candidate for biological applications.34 Furthermore, the high specific surface area of layered modified MoS2 has made it a promising candidate in drug and gene delivery, while the abundant functional groups modified on MoS2 could adsorb and release metal ions.28,33−38 Therefore, concerning its appropriate exfoliation, functionalization, biocompatibility as well as its high metal ions loading capacity, we envision that the infusion of minimum Ag+ into MoS2 could be an alternative for antimicrobial metallic AgNPs with the ability to improve the antibacterial efficiency as well as reduce the toxicity and avoid the waste of Ag+. Herein, we constructed an efficient and benign antimicrobial depot by integrating Ag+ and modified MoS2 nanosheets as well as cationic polyelectrolyte (designed as PDDA-Ag+-Cys-MoS2). Superior to previous antimicrobial agents, the antimicrobial depot demonstrated increased affinity to pathogens and much improved antibacterial efficiency as well as reduced biotoxicity and avoidance of the waste of noble metal. Consequently, the in vivo benign antimicrobial depot was successfully applied in the treatment of wound infection.

Scheme 1. Design of the PDDA-Ag+-Cys-MoS2 Depot for Antibacterial Applicationsa

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(a) General procedure for the preparation of the benign and efficient antibacterial depot PDDA-Ag+-Cys-MoS2 by infused minimum Ag+ ions covered with PDDA cationic polyelectrolyte on the surface of Cys modified MoS2-PEG.(b) The efficient antibacterial activity of PDDAAg+-Cys-MoS2 in vitro. (c) The PDDA-Ag+-Cys-MoS2 used in wound disinfection in vivo.

RESULTS AND DISCUSSION The concept of Ag+ ions infused MoS2 as an efficient and benign antimicrobial depot is illustrated in Scheme 1. “Benign” was used to demonstrate that our system could reduce the side effects in vivo compared with previous antibacterial agents AgNPs. PDDA-Ag+-Cys-MoS2 was synthesized with a four-step approach. First, we synthesized PEGylated MoS2 nanosheets with good water solubility (PEG-MoS2). Owing to the fact that the MoS2 contained abundant S and could form strong covalent bond with Ag+, the release of Ag+ could be suppressed, and correspondingly the bacteriostatic efficacy would be declined. Previous studies have indicated that the weakly bound Ag+ adsorbed in the low affinity region (enriched in carboxylic and aliphatic hydroxyl groups) was released during application.13 Thus, we introduced above weak-affinity functional groups to facilitate the release of Ag+. Second, we modified PEG-MoS2 with cysteine (Cys) to provide more binding sites for Ag+. The functionalization not only favored the adsorption of more Ag+ but also facilitated the release of Ag+. Third, we used Cys-MoS2 loaded minimum Ag+ instead of entire AgNPs core (Ag+-CysMoS2) to reduce the potential side effect and avoid the waste of Ag. Simultaneously, the negatively charged few-layer Cys-MoS2 was assembled into a microsized Ag+-delivery depot through electrostatic interaction. Finally, to enhance the adhesion of the Ag+ depot to the microbial cell surface, the Ag+-Cys-MoS2 was infused with a layer of cationic polyelectrolyte (PDDA-Ag+Cys-MoS2). In the present work, the water-soluble few-layer MoS2-PEG nanosheets were first synthesized according to previous reports with some modifications.29,36 Then the MoS2-PEG was

modified with Cys for subsequent absorption of Ag+ ions and coated with cationic polyelectrolyte poly(diallyldimethylammonium chloride) (PDDA). The transmission electron microscopy (TEM) images and scanning electron microscopy (SEM) images showed that the morphology of MoS2 changed from the nanosized layers to microsized depot, which was assembled by negatively charged few-layer Cys-MoS2 through electrostatic interaction with Ag+ (Figure 1a and Figure S1, Supporting Information). The atomic force microscopy (AFM) images showed that the MoS2-PEG had an average height of 1.466 nm, which indicated that the morphology of as-prepared PEG-MoS2 was nanosheet (Figure S2). Simultaneously, the energy dispersive spectroscopic (EDS) mapping imaging showed the distribution of Mo, S, and Ag in the PDDA-Ag+-Cys-MoS2 nanoparticle (Figure 1b and Figure S3). The X-ray photon spectroscopy (XPS) indicated that the Cys was covalently attached to the MoS2-PEG (Figure 1c). The Mo 3d peaks of Cys-MoS2 shifted to higher binding energies by ∼0.5 eV with respect to corresponding peaks in MoS2-PEG, which corroborated that the chemical environment of the MoS2 was indeed changed. The Mo(IV)/Mo(VI) ratios measured from the XPS spectra of samples MoS2-PEG and Cys-MoS2 were found to be 6.08 and 5.41, respectively. The Mo(IV)/ Mo(VI) ratio decreased with slightly increasing oxidation level.39−41 While the CO bonds characteristic of Cys ligands appeared in conjugated samples (287.6 eV), these results from XPS further verified the presence of Cys ligands on the MoS2PEG and were accordant to thiol functionalized materials.32,42,43 The grafting of the ligand was also confirmed by 4652

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Figure 1. (a) High-magnification TEM image of MoS2-PEG, Cys-MoS2, Ag+-Cys-MoS2, and PDDA-Ag+-Cys-MoS2 (from left to right) under different magnification. (b) Dark-field TEM image of PDDA-Ag+-Cys-MoS2 and corresponding EDS element mapping of Mo L-edge, S Kedge, Ag L-edge. (c) XPS spectra of MoS2-PEG and Cys-MoS2. (d) DRIFT spectra of Cys, MoS2-PEG and Cys-MoS2. (e) Zeta-potential of the synthesized MoS2-PEG, Cys-MoS2, Ag+-Cys-MoS2, and PDDA-Ag+-Cys-MoS2. (f) Cumulative silver ion release profiles from PDDA-Ag+-CysMoS2 samples.

PDDA-Ag+-Cys-MoS2 to the microbial cell surface. Correspondingly, the zeta-potential changed from −33.9 mV to 26.5 mV and finally achieved 36.32 mV (Figure 1e), indicating the successful adhesion of Ag+ and PDDA. The amount of Ag+ delivered in the PDDA-Ag+-Cys-MoS2 depot was about 30.48 μg/mg as determined by ICP-MS. The binding energies of Ag 3d5/2 at 367.6 eV and Ag 3d3/2 at 373.4 eV were assigned to Ag (I), which further confirmed the successful infusion of Ag+ in the PDDA-Ag+-Cys-MoS2 depot (Figure S6). These results were also supported by a number of previous studies.48−51 The specific function of the MoS2 in this system was loading amounts of Ag+ and realizing the maximal release of Ag+ by easy modification. Comparing with other 2D materials, MoS2 is gaining prominence due to the following features. First, the most important advantage was that the high specific surface area of layered modified MoS2 and abundant functional groups such as carboxylic, thiol, hydroxyl groups could adsorb and release metal ions. Second, the modification of MoS2 was easy.

diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy (Figure 1d). DRIFT revealed the exposed S−H band from free Cys at 2555 cm−1.This peak became absent in the Cys-MoS2 , indicating that the thiol moiety had been successfully conjugated to the MoS2-PEG nanosheets via the S−H bond.32,44−47 Additionally, compared to the X-ray diffraction (XRD) pattern of Cys and MoS2-PEG, the CysMoS2 showed a sharp characteristic peaks at 19.0°, 28.5°, 32.3°, and 34.6°, which could be attributed to the (110), (211), and (100 + 101) crystal planes of Mo3S4 (JCPDS card no. 270319), L-cysteine (JCPDS card no. 32-1636), and MoS2 (JCPDS card no. 37-1492), respectively, indicating that the covalent modification changed the crystalline of pristine MoS2PEG (Figure S4). The loading amount of cysteine in Cys-MoS2 was 63.43%, suggested by the thermogravimetric analysis (Figure S5).44,46 Afterward, the obtained MoS2-PEG was infused with Ag+ and coated with a layer of cationic polyelectrolyte PDDA to enhance the adhesion of these 4653

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Figure 2. Viability analyses of (a) E. coli and (b) S. aureus. SEM images of (c) E. coli samples and (d) S. aureus samples. The actual concentrations of AgNO3 and AgNPs followed the equation of C (AgNO3 or AgNPs) = (x × 30.84 μg Ag+/mg × M (AgNO3 or AgNPs))/ 107.87 g/mol, where C (AgNO3 or AgNPs) represents the actual concentration of AgNO3 or AgNPs, x represents the concentration of PDDAAg+-Cys-MoS2, and M (AgNO3 or AgNPs) represents the molar mass of AgNO3 or AgNPs. The concentrations of PDDA, Cys-MoS2, and Ag+Cys-MoS2 were equal to PDDA-Ag+-Cys-MoS2. The bacteria samples 1−7 were prepared by incubating bacteria with PBS, AgNO3, AgNPs, PDDA, Cys-MoS2, Ag+-Cys-MoS2, and PDDA-Ag+-Cys-MoS2, respectively.

analyzed their Ag+ content with ICP-MS. The amount of released Ag+ as a function of time was plotted in Figure 1f, which showed that 68.75% of the Ag+ was released within the 3 days. In contrast, our PDDA-Ag+-Cys-MoS2 depot exhibited ultrahigh Ag+ release rather than AgNPs and showed even 2 orders of magnitude higher than AgNPs, which has been particularly studied (Table S1).12 Then the potential of the PDDA-Ag+-Cys-MoS2 as antibacterial agents was evaluated by spread plate method and a growth-inhibition assay in liquid medium, respectively (Figure 2 and Figures S7 and S8). Seven different samples of PDDA-Ag + -Cys-MoS 2 and AgNO 3 solutions with an equivalent amount of Ag+ ion were investigated to study the antibacterial activities against drugresistance Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. It can be seen that the PDDA-Ag+-CysMoS2 exhibited particularly higher antibacterial activity against E. coli and S. aureus than equal amount of AgNO3 and AgNPs. This phenomenon was accordant with the fact that the PDDAAg+-Cys-MoS2 could release large amounts of Ag+ ions at the cell walls of microorganisms, whereas the toxicity of Ag+ from AgNO3 was considerably decreased by biological effect as well as the release of Ag+ from AgNPs was determined by the surroundings and the total released Ag+ quantity was little.5,12,13 Thus, PDDA-Ag+-Cys-MoS2 depot could reduce the waste of Ag and replace the commercialized antimicrobial agent AgNPs.

The functionalization of carboxylic, thiol, and hydroxyl groups could be realized only by a simple sonication method. Third, the unreleased Ag+ would be reserved in this system for a longterm in the form of Ag−S, which significantly decreased their toxicity due to the lower release of Ag+ and potentially limited their short-term environmental impact. Fourth, the strategy to obtain single- or few-layered MoS2 was simple. The preparation was only through a solvo-thermal approach without further exfoliation. Last but not the least, MoS2 was biocompatibility due to the fact that the Mo is an essential trace element for several enzymes in cells and S is a common biological element. Simultaneously, Kurapati et al. have reported that the MoS2 nanosheets exhibited an enhanced biocompatibility and a better biodegradability through in vitro degradation and cellular toxicity studies. Thus, the safety use of MoS2 made it a better candidate for biomedical applications in comparison with other carbon or 2D nanomaterials.40 If the MoS2 was replaced by other 2D materials, then more issues would be concerned, such as the modification, absorption and release of ions, disposal, exfoliation, biocompatibility, and biodegradation. Therefore, we envisioned that the MoS2 was superior to other 2D materials. To test whether the infusion of Ag+ into MoS2 could improve the antibacterial activity as well as avoid the waste of precious metals through fully utilizing the Ag+ compared to that of AgNPs, we investigated the release of Ag+ in water and 4654

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Figure 3. (a) Photographs of wound on the mice from the seven groups at different times during the therapeutic process. The mice samples 1−7 were treated with PBS buffer, AgNO3, AgNPs, PDDA, Cys-MoS2, Ag+-Cys-MoS2, and PDDA-Ag+-Cys-MoS2, respectively. (b) The bacteria separated from wound tissue were cultured on agar plates. Inserts are the wound tissue. (c) Number of the surviving bacteria in the wound tissue of each sample. Error bars are taken from three mice per group. (d) Photomicrographs showing section of skin tissues with H&E staining. The skin tissues samples 1−7 were treated with PBS buffer, AgNO3, AgNPs, PDDA, Cys-MoS2, Ag+-Cys-MoS2, and PDDA-Ag+Cys-MoS2, respectively.

Meanwhile, the antimicrobial efficiency of PDDA-Ag+-CysMoS2 was also much higher than that of Ag+-Cys-MoS2 without PDDA coating (Figure 2a,b), which might attribute to their strong affinity to the cell walls of the pathogen and subsequently in situ increased amounts of released Ag+.13 Their viabilities were also analyzed by a live/dead assay (Figures S9 and S10). To investigate the changes of bacterial morphology caused by the antibacterial system, SEM was employed to observe E. coli and S. aureus before and after various nanoparticles treatment. As can be seen in Figure 2c, both untreated E. coli and E. coli cells treated with control nanoparticles were typically rod-shaped with smooth and intact cell walls, which also certified that the equal control

nanoparticles, even AgNPs, showed negligible toxicity against bacteria. In contrast, after the treatment with PDDA-Ag+-CysMoS2, the bacterial surface got rough and wrinkled since it could release numerous Ag+ upon adherence to the bacteria due to the electrostatic attraction. As for S. aureus cells, the results were similar to that of E. coli cells (Figure 2d). In the control groups, the S. aureus cells were spherical-shaped and smooth. However, after treatment with PDDA-Ag+-Cys-MoS2, S. aureus cells became rough and damaged. In brief, all these in vitro experiments demonstrated that our antimicrobial depot with minimum Ag+ possessed strong antibacterial properties against both Gram-positive and Gram-negative bacteria, which showed competitive advantage over AgNPs. To assess the 4655

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ACS Nano activity of our designed antimicrobial depot with minimum Ag+ in vivo, the injury model was fabricated on the back of mice. The back of Balbc mice (6−8 weeks) was slashed and injected with 1 × 106 of methicillin-resistant Staphylococcus aureus (MRSA) cells to construct the infected wound model. The mice were divided into seven groups: treated with PBS, PDDA, AgNO3, AgNPs, MoS2-PEG, Cys-MoS2, Ag+-Cys-MoS2, and PDDA-Ag+-Cys-MoS2, respectively. During the whole therapeutic process, the wounds treated with the PDDA-Ag+-CysMoS2 did not appear erythema, and they formed scabs after therapy. The wounds in the other six groups showed different levels of erythema (Figure 3a). To assess the bactericidal effect, we excised the wound tissues to quantify the number of bacteria on them (Figure 3b,c). From the grown colonies, we could see that the PDDA-Ag+-Cys-MoS2 led to the most effective wound antibacterial therapy. The control groups were also tested for infection treatment. Moreover, we further evaluated the wound healing progress by hematoxylin and eosin stained sections (H&E staining). As shown in Figure 3d, a large amount of inflammatory cells and fragmentary epidermal layer appeared on the wound after 3-day treatments with the control groups, while the intact epidermal layer as well as less inflammatory cells emerged on the wound treated with PDDA-Ag+-Cys-MoS2 dressing for 3 days. Thus, it can be concluded that PDDA-Ag+Cys-MoS2 exhibited the best antibacterial effect and woundhealing effect among various silver antibacterial agents. Though all the results forcefully demonstrated the excellent antibacterial effect of PDDA-Ag+-Cys-MoS2 depot, the in vivo biosafety of this antibacterial system should also be taken into account. Indeed, no appreciable abnormalities or damages of major organs from the mice (without infection) were observed even 3 days after PDDA-Ag+-Cys-MoS2 injection with an applied concentration of 15 μg/mL as well as even at a high concentration of 50 μg/mL (Figure 4), which indicated that

with minimum Ag+ and unreleased Ag+ existed in a form of Ag−S, we hypothesized the potential risks of toxicity of PDDAAg+-Cys-MoS2 should be lower than AgNPs, further indicating that the antibacterial system was superior to AgNPs. Compared with previous antimicrobials, the main features of our depot can be summarized in the following three points. First, our platform exhibited much enhanced broad-spectrum antibacterial activity compared to equivalent amount of AgNO3 solution. Second, the system could reduce the side effects in vivo because of infusing minimum Ag+ ions. Third, the waste of Ag could be avoided due to maximizing the utilization of Ag. These features have been strongly confirmed by both in vitro and in vivo antibacterial experiments. Our antibacterial platform eliminated the risks of excess silver for application in the treatment of bacterial infection. We expected that the PDDAAg+-Cys-MoS2 antibacterial depot could be a promising alternative to AgNPs. Comparing our platform with a silverinfused lignin core,13 we solved issues of different areas by the strategy of infusing minimum Ag+ to substitute AgNPs. One was about the potential risk to environment, and the other was concerned with human health as far as excess silver. Richter, A. P. et al. presented a problem that AgNPs have been recognized as a potential environmental hazard. To solve the problem of persistent nanoparticle waste and potential environmental risk, they infused the lignin with minimum Ag+. The biodegradable nanoparticles had a higher antimicrobial activity and smaller environmental impact than metallic silver nanoparticles. In this work, our mission focused on the solution of argyria and waste of Ag in biologic conditions. Our designed PDDA-Ag+-CysMoS2 enhanced the antibacterial activity and reduced the side effects in vivo as well as avoided the waste of noble metal. Simultaneously, the unreleased Ag+ would exist in the form of Ag−S, which significantly decreased their toxicity due to the lower release of Ag+ and potentially limited their short-term environmental impact. Thus, these features have made our depot superior to previous antimicrobials.

CONCLUSION In summary, we have constructed an innovative antibacterial depot with cysteine-modified molybdenum disulfide loaded with minimum Ag+ ions and coated with a layer of cationic polyelectrolyte. The PDDA-Ag+-Cys-MoS2 depot exhibited a much enhanced broad-spectrum antibacterial activity, including Gram-negative E. coli and Gram-positive S. aureus, while the equivalent amount of AgNPs or AgNO3 solution showed extremely low antibacterial ability. The cationic polyelectrolyte promoted the adhesion of PDDA-Ag+-Cys-MoS2 to pathogens and, together with minimum Ag+ ions, could reduce the side effects in vivo as well as avoid the waste of Ag. Importantly, both in vitro and in vivo antibacterial experiments strongly demonstrated that the PDDA-Ag+-Cys-MoS2 depot possessed a prominent antibacterial property with negligible biotoxicity. We expected that the PDDA-Ag+-Cys-MoS2 antibacterial depot could be a promising alternative to AgNPs.

Figure 4. Pathological study of the cytotoxic effect caused by PDDA-Ag+-Cys-MoS2 in major organs (heart, liver, spleen, lung, kidney, and brain) after 3 days of treatment with an applied concentration of 15 μg/mL as well as even at a high concentration of 50 μg/mL.

this antibacterial agent resulted in negligible side effects to the mice during the antibacterial therapy. Considering that the broad-spectrum antimicrobial agent AgNPs could accumulate in blood, lungs, liver, kidneys, and brain, bringing about some adverse effects such as gastrointestinal tract damage, convulsions, argyria, or neurotoxicity,52,53 the potential toxicity of AgNPs to mammal was still debatable when applied into in vivo. In our study, the antibacterial depot was directly subcutaneously injected into the mice, and negligible biotoxicity on normal tissues was observed for 3 days at the applied dose. This biosafety of PDDA-Ag+-Cys-MoS2 was probably relevant to the fact that the released Ag+ could be bound by the proteins with thiol groups.5,12,13 Because our antibacterial depot was infused

MATERIALS AND METHODS Chemicals. Sodium molybdate (Na2MoO4·2H2O) and thioacetamide (C2H5NS) were purchased from Aladdin Reagent (Shanghai, China). L-Cysteine was obtained from Sigma-Aldrich. Poly(ethylene glycol) (average MW 200, abbreviated to PEG-200) was purchased from Acros Organics. Dialysis bags (molecular weight cut off = 1K and 3.5K) were ordered from Shanghai Sangon Biotechnology Development Co., Ltd. Fabric and dressings were purchased from a drugstore. 4656

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ACS Nano Commercial AgNPs of 30 nm were obtained from Yunfu Nanotech. Co., Ltd. (Shanghai, China). Other reagents and solvents were achieved from Beijing Chemicals (Beijing, China). Ultrapure water (18.2 MU; Millpore Co., USA) was used throughout the experiment. Instruments. SEM images were obtained on a Hitachi S-4800 FESEM at a working voltage of 10 kV and working current of 10 μA. TEM measurements were carried out on a TECNAI G2 equipped with EDS at 200 kV. XRD measurements were performed on a Bruker D8 FOCUS using Cu Kα radiation. DRIFT analyses were measured on a Bruker Vertex 70 FT-IR Spectrometer. XPS data were recorded with an ESCALab220i-XL electron spectrometer from VG Scientific using 300 W Al Kα radiation. The concentrations of released Ag+ ions were analyzed on the Inductively coupled plasma-atomic emission spectrometry (ICP-AES, X Series 2, Thermo Scientific, USA). All the photos were taken with a Canon camera. Synthesis of Few-Layer PEG-MoS2 Nanosheets. PEG-MoS2 nanosheets were synthesized according to a modified solvo-thermal approach.29,36 Briefly, 30 mg of sodium molybdate (Na2MoO4·2H2O) and 60 mg of thioacetamide (C2H5NS) were dissolved in 20 mL of PEG-200 aqueous solution (50%, v/v) and transferred into a 100 mL polyphenylene-lined stainless steel autoclave at 200 °C for 24 h. The MoS2-PEG nanosheets products were washed with deionized water for 3 times, freeze-dried, and stored at room temperature. Synthesis of Cys-MoS2. Cys-MoS2 nanosheets were prepared by blending 20 mL of PEG-MoS2 nanosheets dispersion (100 mg/mL) and L-cysteine solution (500 mg/mL) under vigorous stirring and kept still for 8 h. The supernatants were dialyzed against nanopure water with a dialysis bag (MW cutoff 3.5K Da) for 3 days to purify the CysMoS2 from nonreacted species. The modified Cys-MoS2 nanosheets were freeze-dried and stored at room temperature. Synthesis of Ag+-Cys-MoS2. Cys-MoS2 nanosheets were infused with silver ions by mixing 1 mg/mL of previously prepared Cys-MoS2 suspensions with 1 mg/mL of AgNO3 solution for 8 h under dark conditions. The final volume was 40 mL. The obtained Ag+-Cys-MoS2 was centrifuged at 12000 rpm for 10 min and washed with deionized water for one time to remove the unabsorbed Ag+. Finally, the products were freeze-dried and stored at room temperature in the dark. Synthesis of PDDA-Ag+-Cys-MoS2. The as-prepared Ag+-CysMoS2 was coated with 10 mL of PDDA (1 mg/mL) for 24 h in the dark. After washing 2 times, the obtained sample was freeze-dried, stored at room temperature, and avoided light. Silver Ion Release Experiment. The release of Ag+ ions from PDDA-Ag+-Cys-MoS2 was investigated at different time points using a dialysis device.12,13 The dialyzed samples were isolated, dialyzed against nanopure water with a dialysis bag (MW cutoff 1K Da) for 3 days, and analyzed for their Ag+ content with ICP-AES. Bacterial Culture and Antibacterial Experiments. A monocolony of E. coli and S. aureus on the solid Luria−Bertani (LB) agar plate was transferred to 20 mL of liquid LB culture medium in the presence of ampicillin (200 μg/mL) and grown at 37 °C for 12 h under 180 rpm rotation. Then the bacteria were diluted with broth to 106 cfu mL−1. The obtained solution (500 μL) was mixed with asprepared nanoparticles with different concentrations for 8 h, respectively. Control experiments were performed in parallel without nanoparticles. The condition of bacteria was then studied by live/dead staining analysis, solid medium culture, and SEM image. For live/dead staining analysis, 10 μL of 10 mg/mL of FDA and 6 μL of 1 mg/mL PI were sequentially mixed with 1 mL E. coli bacteria solution in the dark for 30 min (and 4 μL of 1 mg/mL of AM and 4 μL of 1 mg/mL PI with 1 mL S. aureus bacteria solution for 30 min). The bacteria were collected by centrifugation and washed with PBS buffer for two times. They were finally imaged under fluorescence microscope. For solid medium culture, after incubation at 37 °C, the solution removed from the 24well plate was diluted 10,000 times with growth medium. 200 μL of the diluted bacteria solution was streaked on the solid medium by spread plate method. The plates were cultured at 37 °C for 12 h, and the number of colony forming units (CFUs) was counted. For SEM images of bacteria, the bacteria cultured at 37 °C were harvested by

centrifugation at 3000 rpm for 5 min. They were washed with PBS and then fixed in PBS containing 4% formaldehyde for 2 h. The cells were further washed with DI water, followed by dehydration using a series of ethanol. The bacteria in 100% ethanol were finally dried in a vacuum drying chamber at room temperature. Before imaging, the bacteria were sputter-coated with platinum. Mouse Injury Model. To evaluate the potential of PDDA-Ag+Cys-MoS2 for treating wound infection, the injury model was built on the back of mice. The back of Balbc mice (6−8 weeks) was slashed and injected with 1 × 106 of MRSA cells to build the infected wound model. The mice were divided into seven groups (three mice per group). The mice in different groups was treated with PBS buffer only and 15 μg/mL of AgNO3, AgNPs, PDDA, PEG-MoS2, Cys-MoS2, Ag+Cys-MoS2, and PDDA-Ag+-Cys-MoS2 on their wound, respectively. The wounds were observed and photographed, and Band-Aids were changed at 24 h intervals. After 3 days of therapy, the mice were sacrificed, and the wound tissues were harvested. The wound tissues were placed in 1 mL of sterile saline and homogenized. The obtained solutions were cultured at 37 °C overnight, and the numbers of bacteria in them were determined by plate count methods. The animal studies were conducted in compliance with the guidelines of the Institutional Animal Care and Use Committee. Histology. For histology, the mice were sacrificed, and the wound tissues were harvested after 3 days of therapy. The wound tissues treated with different nanoparticles were fixed in neutral buffered formalin, processed routinely into paraffin, sectioned into ∼4 μm, and stained with H&E. The histology was performed in the college of Basic Medical Sciences of Jilin University. The samples were examined by an Olympus BX-51 microscope in bright field. In Vivo Biosafety. Different concentrations of PDDA-Ag+-CysMoS2 were directly subcutaneously injected into the mice.52,53 The mice were divided into three groups (three mice per group) and treated with PBS buffer as well as PDDA-Ag+-Cys-MoS2 at an applied concentration of 15 μg/mL and even a high concentration of 50 μg/ mL. After observation for 3 days, the mice were sacrificed, and the wound tissues were harvested after 3 days of therapy. The wound tissues treated with different nanoparticles were fixed in neutral buffered formalin, processed routinely into paraffin, sectioned into ∼4 μm, and stained with H&E. The histology was performed in the college of Basic Medical Sciences of Jilin University. The samples were examined by an Olympus BX-51 microscope in bright field. Disposal of the Used Material and Time-Dependent Activity. From the silver ion release curve of PDDA-Ag+-Cys-MoS2, we could observed that after 2 days, our system exhibited a negligible release of Ag+ and the residual Ag+ could exist in the form of Ag−S, which significantly decreased its toxicity and potentially limited its short-term environmental impact.13,54 However, to better convert the AgNPs to a less hazardous form, the used materials were concentrated and then released after sulphidation by soaking in the sodium sulfide solution.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00343. Supporting Information including SEM images, AFM images, XRD spectra, TGA spectra, XPS spectra, disk diffusion assay, live−dead fluorescence imagesm and cumulative Ag+ release profiles are provided (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jinsong Ren: 0000-0002-7506-627X Xiaogang Qu: 0000-0003-2868-3205 4657

DOI: 10.1021/acsnano.7b00343 ACS Nano 2017, 11, 4651−4659

Article

ACS Nano Notes

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The authors declare no competing financial interest.

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DOI: 10.1021/acsnano.7b00343 ACS Nano 2017, 11, 4651−4659