Functionalized Nano-MoS2 with Peroxidase ... - ACS Publications

Nov 16, 2016 - The PEG-MoS2 NFs combine the catalysis with NIR photothermal effect, .... different modifications and their application for H 2 O 2 and...
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Functionalized Nano-MoS2 with Peroxidase Catalytic and Near-Infrared Photothermal Activities for Safe and Synergetic Wound Antibacterial Applications Wenyan Yin,*,†,# Jie Yu,†,‡,# Fengting Lv,§ Liang Yan,† Li Rong Zheng,† Zhanjun Gu,*,† and Yuliang Zhao*,†,∥ †

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China ‡ Key Laboratory of Polymer Science and Technology, School of Science, Northwestern Polytechnical University, Xi’an, Shaanxi 710129, China § Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China ∥ Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China, Beijing, 100190, China S Supporting Information *

ABSTRACT: We have developed a biocompatible antibacterial system based on polyethylene glycol functionalized molybdenum disulfide nanoflowers (PEG-MoS2 NFs). The PEG-MoS2 NFs have high near-infrared (NIR) absorption and peroxidase-like activity, which can efficiently catalyze decomposition of low concentration of H2O2 to generate hydroxyl radicals (·OH). The conversion of H2O2 into ·OH can avoid the toxicity of high concentration of H2O2 and the ·OH has higher antibacterial activity, making resistant bacteria more vulnerable and wounds more easily cured. The PEG-MoS2 NFs combine the catalysis with NIR photothermal effect, providing a rapid and effective killing outcome in vitro for Gram-negative ampicillin resistant Escherichia coli (Ampr E. coli) and Gram-positive endospore-forming Bacillus subtilis (B. subtilis) as compared to catalytic treatment or photothermal therapy (PTT) alone. Wound healing results indicate that the synergy antibacterial system could be conveniently used for wound disinfection in vivo. Interestingly, glutathione (GSH) oxidation can be accelerated due to the 808 nm irradiation induced hyperthermia at the presence of PEG-MoS2 NFs proved by X-ray near-edge absorption spectra and X-ray spectroscopy. The accelerated GSH oxidation can result in bacterial death more easily. A mechanism based on ·OH-enhanced PTT is proposed to explain the antibacterial process. KEYWORDS: MoS2, peroxidase-like activity, wound disinfection, photothermal therapy

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bacteria. In addition, H2O2 has been widely used as a reagent for disinfection. However, traditional medical concentrations of H2O2 (volume ratio: 0.5−3%) hamper wound healing and even damage normal tissues during bacterial disinfections.20−23 It has been reported that nanomaterials, including V2O5,11 Fe3O4,12 and graphene quantum dots,20 have the peroxidase-mimic ability and can be used to assist H2O2 for antibacterial

nfectious disease caused by bacteria becomes a fatal issue for human beings currently.1 Numerous of traditional control strategies that depend on antibiotics, metal ions, and quaternary ammonium ions suffer the drawbacks of ecologically noxiousness, high cost, and environmentally hazardousness.2−4 Moreover, the increasing resistance of bacteria poses serious threat to public health.5,6 Promoted by nanotechnology, versatile nanomaterials such as metal nanostructures,7−9 metal sulfide/oxide or their nanocomposites,10−12 functionalized polymers,13,14 and carbon nanomaterials15−19 have potential for defeating the drug-resistant © 2016 American Chemical Society

Received: August 29, 2016 Accepted: November 16, 2016 Published: November 16, 2016 11000

DOI: 10.1021/acsnano.6b05810 ACS Nano 2016, 10, 11000−11011

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Figure 1. (A) Schematic illustration of PEG-MoS2 as a combined system for peroxidase catalyst-photothermal synergistic eliminating of bacteria. (i) PEG-MoS2 was captured by bacteria; (ii) PEG-MoS2 catalyze decomposition low concentrated H2O2 to generate ·OH to damage the cell walls integrity; (iii) 808 nm laser irradiation causes hyperthermia, which accelerates GSH oxidation. (B) TEM image, (C) FT-IR spectrum, (D) XPS survey plot, and (E) UV−vis−NIR spectrum of the PEG-MoS2.

application.20−22 For example, graphene quantum dots are capable of catalyze low concentrated H2O2 to generate hydroxyl radicals (·OH), which has higher antibacterial activity than H2O2, while avoiding toxicity of higher concentration of H2O2. More importantly, as opposed to natural enzymes, peroxidaselike nanomaterials can prevent protein denaturing or protease digestion.11,22 Unfortunately, the further applications of peroxidase-like nanomaterials are still limited by (1) the intrinsic cytotoxicity of many reported peroxidase-like nanomaterials are still one of the most important issues;24,25 (2) especially, exposure to low concentrations of H2O2 still lead to the bacteria defense against the oxidative stress produced by higher concentrations of H2O226,27 and endospores from certain Gram-positive bacteria could even repel the H2O2;28 (3) as reported, single-modal antibacterial process base on nanomaterials is difficult to totally eradicate the resistant bacteria high efficiently.29 The combination multiple antibacterial therapeutic modalities become a promising approach to enhance antibacterial efficiency, possibly inducing potent synergy effect, decreasing the dose of drugs.17 Thus, exploring new biocompatible peroxidase-like nanomaterial equipped with multiple antibacterial capabilities is of great importance.

Recently, molybdenum disulfide (MoS2) nanomaterials have been reported as an intrinsic peroxidase-like catalyst that can be used for colorimetric detection of H2O2 and glucose.30−32 However, their peroxidase-like activity used for killing bacteria has not been involved. Notably, MoS2 have been suggested for photothermal therapy (PTT) of cancer because of their good biocompatibility33−36 and high photothermal conversion efficiency in the near-infrared (NIR) region.37−39 Meanwhile, NIR laser-induced hyperthermia based on such light absorbing nanoagents has become one of the most attractive strategies for combating bacteria.17−19,40 However, long-term exposure together with high power density of NIR laser may cause skin damage, which become a challenge in the PTT.19 Inspired by the peroxidase-like activity and the effective photothermal conversion of MoS 2 , we thus hypothesized that the combination of peroxidase-like catalytic activity and PTT may make up for the deficiency of single modal antibacterial process and show improved antibacterial activities to wound. Especially, ·OH can induce initial oxidative lesions to cell wall and membrane41 and once combined with PTT, the damaged membrane have improved permeability and sensitivity to heat,42 which could shorten the time of treatment largely and minimize the side effects of PTT. 11001

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Figure 2. Peroxidase-like catalytic activity of PEG-MoS2 NFs toward TMB after incubation for 5 min: (A) MoS2 concentration-dependent peroxidase-like activity with TMB (1 mM) and H2O2 (10 mM). Inset shows the photographs of the reaction system (a) MoS2+TMB, (b) H2O2+TMB, (c) H2O2+MoS2+TMB, (d) MoS2+OPD, (e) H2O2+OPD, (f) H2O2+MoS2+OPD, respectively. (B) H2O2 concentrationdependent peroxidase-like activity with MoS2 (33 μg mL−1) and TMB (1 mM). (C) Temperature- and (D) pH-dependent activities with TMB (1 mM), H2O2 (10 mM) and MoS2 (33 μg mL−1).

diffraction pattern with both 2H (JCPDF No. 37−1492) and 1T phase.46 The typical peaks of C−C (1047 cm−1), C−O−C (1101 cm−1), and C−H (2895 cm−1) bond vibration in the Fourier transform infrared (FT-IR) spectrum (Figure 1C) imply successful modification of MoS2 with PEG. The modification of MoS2 with PEG could prevent the nanoflowers aggregation in biofluid and render them well biocompability and water solubility.47 The dynamic light scatting (DLS) analysis reveals that PEG modified MoS2 NFs in water have narrow size distribution with an average hydrodynamic size of 334 nm (Figure S3a), while broad size distribution appears and average hydrodynamic size of MoS2 in the absence of PEG can reach 753 nm (Figure S3b), implying that PEG modification can decrease the aggregation of MoS2. We also measured the effective surface charge of the MoS2 in the presence and absence of PEG (Figure S3c). It can be found that the zeta (ζ) potential value of MoS2 without PEG modification is −58.12 mV in water, and this value decreased to −35.16 mV after PEG modification, suggesting the PEG modification neutralize part of the negative charges on the surface of MoS2. X-ray spectroscopy (XPS) analysis further proves the presence of 2H-MoS2 and 1T-MoS2 (Figure 1D, Figure S2c−f).48 Moreover, PEG-MoS2 also exhibit optical absorption from visible to NIR region (Figure 1E). The catalytic activity of the PEG-MoS2 in the presence of low concentration of H2O2 (100 μM) was verified by oxidation of terephthalic acid (TA) which reacted with ·OH, forming 2hydroxyl terephthalic acid (TAOH) with fluorescence emission at 435 nm. A remarkable fluorescence peak increase for the TA +MoS2+H2O2 suggests that ·OH were generated more effectively than that of the group without MoS2 under both

Here, we reported the development of polyethylene glycol functionalized MoS2 nanoflowers (PEG-MoS2 NFs) as a biocompatible antibacterial system for convenient, rapid, and effective wound disinfection. A mechanism based on exogenous ·OH-enhanced PTT to fight bacteria was proposed (Figure 1A). First, bacteria are exposed to the PEG-MoS2 before adding low concentration of H2O2 (Figure 1A-i). PEG-MoS2 captured by bacteria not only can drastically reduce concentration of H2O2 but also efficiently catalyze decomposition H2O2 to generate ·OH (Figure 1A-ii). Then, the exogenous ·OH interact with bacteria to induce membrane stress and damage cell walls and compromise membrane integrity, which makes bacteria more vulnerable. Following irradiation by 808 nm laser brings about hyperthermia (Figure 1A-iii). The combination of catalysis and PTT provides a rapid, remarkable antibacterial outcome to dermal wound. Importantly, we found that 808 nm laser-induced hyperthermia can effectively accelerate glutathione (GSH) oxidation, which breakdown the intercellular protection system of bacteria, thus resulting in the potent increase of antibacterial efficiency for Ampr E. coli and B. subtilis.

RESULTS AND DISCUSSION PEG-MoS2 NFs were prepared by a facile one-pot hydrothermal route. Transmission electron microscopy (TEM) (Figure 1B) and field emission scanning electron microscopy (FE-SEM) (Figure S1) reveal that the MoS2 exhibits flower-like morphology composed of individual nanoflakes with diameters of 25 nm.43 Raman spectra show that PEG-MoS2 have typical bands of 2H-MoS2 and 1T-MoS2 (Figure S2a).44,45 X-ray diffraction (XRD) spectrum (Figure S2b) shows a MoS2 crystal 11002

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Figure 3. Photographs of bacterial colonies formed by (A) Ampr E. coli and (B) B. subtilis after exposed to (I) PBS, (II) MoS2, (III) H2O2, (IV) MoS2+H2O2, (V) PBS+NIR, (VI) MoS2+NIR, (VII) H2O2+NIR and (VIII) MoS2+H2O2+NIR. Concentration: MoS2 100 μg mL−1, H2O2 100 μM; Relative bacteria viabilities of (C) E. coli and (D) B. subtilis after incubation with PBS, MoS2 (100 μg mL−1), H2O2 (100 μM), or MoS2+H2O2 for 20 min without or with 808 nm irradiation, determined by plate count method.

pH values of 4.0 and 7.0 (Figure S4). The catalytic activity was further evaluated by peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (inset in Figure 2A(a−c)) or o-phenylenediamine (OPD) (inset in Figure 2A(d−f)). The PEG-MoS2 can catalyze oxidation of TMB in the presence of H2O2 (10 mM) and a blue color was observed after reaction of 5 min.

Meanwhile, yellow color from oxidized OPD also implies the catalytic activity. Furthermore, the catalytic activity was proved to be dependent on the concentrations of PEG-MoS2 (Figure 2A) and H2O2 (Figure 2B), temperature (Figure 2C), and pH values (Figure 2D), similarly to other reported peroxidase-like nanomaterials.12,20,49 The catalytic mechanism was verified by 11003

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ACS Nano steady-state kinetics method (Figure S5). The Michaelis− Menten constant (Km) and maximal reaction velocity (Vmax) are obtained by Lineweaver−Burk equation. As listed in Table S1, the results imply the PEG-MoS2 have good affinity to H2O2 substrate superior to that of horseradish peroxidase (HRP). The parallel-line plots suggests a typical Ping Pong kinetic mechanism (Figure S5e,f).30 Meanwhile, the photothermal absorbance behavior was examined by irradiating 808 nm laser to the PEG-MoS2 aqueous solutions. In Figure S7a,b, the temperature elevation increases with the increased concentration of the PEG-MoS2. The photothermal conversion efficiency is calculated to be ∼43.72% (Figure S7c,d and the Supporting Information). For antibacterial therapy, high concentrations of H2O2 can induce side effects to normal tissues and even delay wound healing.20 However, ·OH have higher antibacterial ability than the H2O2 and PEG-MoS2 with effective peroxidase-like activity can catalyze low concentration of H2O2 to generate ·OH. We evaluated the antibacterial ability of PEG-MoS2 in the presence of H2O2 against both Ampr E. coli and B. subtilis. Plate counting method was used to determine the antibacterial ability (Figure 3A−D). As compared to (I) PBS, (II) MoS2 and (III) H2O2 groups in Figure 3A,B, even with the assistance of PEG-MoS2, the bacteria survival rates are still above 46% and 13% for Ampr E. coli and B. subtilis, respectively, for the (IV) MoS2+H2O2 when kept the H2O2 at 100 μM, indicating that peroxidase catalyst alone cannot effectively eliminate the two bacterial strains with a low concentration of H2O2 (Figure 3C,D). Moreover, dose-dependent antibacterial ability was further evaluated by measuring optical density at 600 nm (OD600). Without PEG-MoS2, a higher concentration of H2O2 was needed for efficiently eliminating Ampr E. coli (Figure S8a), and the PEG-MoS2 alone exhibit negligible antibacterial effect toward Ampr E. coli (Figure S8b). After treated with H2O2, the PEG-MoS2+H2O2 group with various concentrations still cannot kill the E. coli totally even though the relative bacterial viabilities decrease to 58−35% (Figure S8c). To improve the antibacterial effect against Ampr E. coli and B. subtilis and further avoid the toxicity from high concentrated H2O2, PTT was introduced to construct peroxidase-like catalysis/PTT synergetic antibacterial system. Survival rates of the Ampr E. coli and B. subtilis were also determined by plate counting method (Figure 3A−D). As expected, when the bacteria were incubated with MoS2+H2O2 for 10 min and then exposure to the 808 nm laser for 10 min, the bacteria viabilities can be apparently reduced, and the bacteria inactivation percentages are 97% and 100% for Ampr E. coli and B. subtilis, whereas MoS2+NIR group can only be kept at 81% and 93%, respectively. Moreover, when the H2O2 concentration was kept at 100 μM, PEG-MoS2 concentration-dependent antibacterial behaviors under 808 nm irradiation were evaluated (Figure 4). The Ampr E. coli inhibition rate up to 89% was achieved by treatment with 150 μg mL−1 of PEG-MoS2 and then irradiation by 808 nm laser with a final temperature of 50 °C (MoS2+NIR group), while increasing to 99% for the PEG-MoS2+H2O2+NIR group. Moreover, we also evaluated if the external heating could kill the bacteria. Ampr E. coli were cultured in LB at 50 °C for 12 h by using a water bath, which is the same temperature in above NIR laser-induced PTT experiment. Compared with the control group cultured at 37 °C, it is clear that external heating of Ampr E. coli at 50 °C did not cause the bacterial death based on the plate counting method (Figure S9). These results in Figure 4 and Figure S9 indicate that the Ampr E. coli were not

Figure 4. Relative bacterial viability of Ampr E. coli incubated with different concentrations PEG-MoS2 NFs with or without H2O2 (100 μM) under 808 nm laser irradiation.

efficiently killed by heat whatever it comes from MoS2 or external heating. In addition, the response of B. subtilis shows more sensitive toward the synergetic antibacterial treatment, which could be attributed to the different chemical compositions between Gram-positive and Gram-negative bacterial cell walls (Figure 3D). To further decipher the antibacterial behavior, field emission scanning electron microscopy (FE-SEM) was used to investigate the Ampr E. coli and B. subtilis. As shown in Figure 5A-a, Ampr E. coli is rod-shaped with smooth surface. After treatment with H2O2 (Figure 5A-b) or PEG-MoS2 (Figure 5Ac) for 20 min, a few disruptions occurred on the cell walls,

Figure 5. FE-SEM images of (A) Ampr E. coli and (B) B. subtilis treated with (a) PBS, (b) H2O2 (100 μM), (c) MoS2 (100 μg mL−1), (d) MoS2+NIR, (e) MoS2+H2O2, and (f) MoS2+H2O2+NIR. Irradiation time: 10 min. 11004

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Figure 6. (A) Measurements of O2− generation of PEG-MoS2 at different exposure time. (B) Loss of GSH plot after incubation with PEGMoS2 at different time intervals. (C) Loss of GSH plots measured at 20 and 50 °C in water bath at different time. (D) Loss of GSH plots heated by water bath and NIR 808 nm irradiation, respectively, for 20 min at 50 °C. Concentration of MoS2 in (B, C, D) is 100 μg mL−1.

Figure 7. Photographs for the color change after GSH treatment at different time intervals under water bath at (a) 20 °C and (b) 50 °C, respectively, determined by the Ellman’s assay. The concentration of MoS2 was 100 μg mL−1. GSH without adding MoS2 was acted as control, showing a negligible color change at 20 °C and the higher temperature of 50 °C with 120 min.

With the increasing number of studies on the antibacterial behavior of 2D graphene-based materials (graphene, graphene oxide and reduced graphene oxide) since 2010, several predominant mechanisms including nanoknives, oxidative stress, and wrapping or trapping have been proposed because physicochemical properties of graphene-based materials (morphology, size, and surface functionality, and so on) might affect their antimicrobial activities.50−52 In consideration that MoS2 is a graphene-like 2D material which has gained great interest recently in the biomedical field, we explored the peroxidase-like catalysis/PTT synergetic antibacterial mechanism of the PEG-MoS2. First, we investigated the oxidative stress mediated by the PEG-MoS2. XTT (2,3-bis(2-methoxy-4nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide) assay indicates almost no superoxide anion (O•− 2 ) from the PEG-MoS2 were produced (Figure 6A),

which indicate that H2O2 or MoS2 alone only have less influence on the integrity of cell walls. However, when exposure to the MoS2+H2O2 for 20 min, the cell walls became partially wrinkled and incomplete (Figure 5A-e). Notably, after treatment with PEG-MoS2 (Figure 5A-d) or MoS2+H2O2 for 10 min and irradiation for 10 min, the bacterial surfaces of the MoS2+H2O2+NIR group show much more violent damage, indicating a stronger antibacterial ability of the synergistic system (Figure 5A-f). For killing the B. subtilis (Figure 5B), after 808 nm irradiation, the synergetic group was similar to that of Ampr E. coli, and the B. subtilis lost their integrity under the same concentration and incubation condition as the E. coli (Figure 5B-f). Consequently, all the results demonstrate that the PEG-MoS2 was a superior peroxidase-like catalysis/PTT agent with high effective synergistic antibacterial activity in the presence of low-concentrated H2O2. 11005

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ACS Nano suggesting that the antibacterial process is reactive oxygen species (ROS)-independent, which is similar to the antibacterial mechanism of graphene. Second, GSH played an important role in the bacterial antioxidant defense system, which can prevent damage to cellular components induced by oxidative stress, and it has been acted as oxidative stress indicator in cells.53,54 Thus, Ellman’s assay was introduced to determine the possibility of reactive oxygen species (ROS)-independent oxidative stress mediated by PEG-MoS2. Typically, after incubation with GSH, PEG-MoS2 show a time-dependent oxidation behavior (Figure 6B, Figure S10). The statistical loss of GSH can reach to 73.4% after addition of PEG-MoS2 (80 μg mL−1) for 6 h. Nevertheless, the GSH oxidation was inconspicuous within 4 h. Meanwhile, temperature-dependent GSH oxidations were further carried out to investigate the influence of heat (Figure 6C, Figure 7). With the existence of MoS2 (100 μg mL−1) at 50 °C in water bath, loss ratios of GSH can reach to 29.52 ± 1.14% and 62.46 ± 1.45% after incubation of 20 and 100 min, respectively, which were higher than those corresponding data obtained at 20 °C. Furthermore, hyperthermia induced by the PEG-MoS2 under 808 nm laser irradiation shows much higher GSH oxidation level than water bath group at 50 °C for only 20 min (Figure 6D). To confirm the speciation and chemical forms of Mo element of the PEGMoS2 after 808 nm treatment with or without presence of GSH, X-ray near-edge absorption spectra (XANES) (Figure 8A,B) and XPS spectra (Figure 8C) were collected. The result demonstrates that the chemical state and composition of PEGMoS2 were stable during the 808 nm laser-induced GSH oxidation procedure. Notably, MoS2 could facilitate the oxidation of organic thiols (R−SH) to yield disulfides (R−S− S−R).55 The catalytic oxidation of organic thiols into disulfides was found to be a temperature-related process.56 Thus, this finding implies a temperature-enhanced catalytic activity of GSH oxidation by the PEG-MoS2, which can explain the rapid bacterial death under NIR-induced hyperthermia. The possibly antibacterial mechanism was proposed: (1) Cell walls damage arise from the generation of ·OH from low concentration of H2O2 under the existence of the PEG-MoS2; (2) Hyperthermia induced by 808 nm laser irradiation could accelerate GSH oxidation and potently disrupt the intrinsic balance of bacterial protection environment and cause bacterial death rapidly. The understanding of the antibacterial mechanism of MoS2 plays an important role in the manipulation of highly efficient antibacterial materials for future biomedical applications. Note that, we evaluate the in vitro cytotoxicity of the PEG-MoS2 on human cervical carcinoma cells (HeLa) and human umbilical vein endothelial cells lines (HUVEC). After being incubated for 24 h with PEG-MoS2, the viability of HeLa cells was greater than 90% even the concentrations up to 250 μg mL−1 (Figure S11a). For the HUVEC cells, more than 85% of cell viability was observed at the concentration of 250 μg mL−1 after incubation with the PEG-MoS2 for 24 h (Figure S11b). Moreover, body weight is a useful indicator for the toxicity effects. In our study, PEG-MoS2 were administered to Kunming mice via tail vein injection. Mice without any injection were acted as control group. The body weight of the mice was recorded every other day for 30 days (Figure S11c). During the period of 30 days, the body weight of the mice injected with PEG-MoS2 increased gradually in a pattern similar to that of the control group, and only small weight differences between the mice of the two groups was observed, indicating the low toxicity of PEG-MoS2 in mice.

Figure 8. (A) Mo K-edge XANES spectra of PEG-MoS2 and PEGMoS2+GSH treated with or without 808 nm irradiation. (B) The corresponding mass percentage plot fitted by XANES spectra. (C) XPS spectra of Mo 3d core-level for the PEG-MoS2 and PEGMoS2+GSH treated with or without NIR. The vertical dash line in (C) marks the feature of the Mo 3d signal.

We also evaluated healing ability in wound-infecting bacterial skin of mice using the synergetic antibacterial strategy. First, the female BALB/c mice were divided into six groups and three mice (n = 3) for each group. Then, the in vivo wound healing models were constructed. The Ampr E. coli infected wound were treated with (I) PBS (control), (II) H2O2, (III) MoS2, (IV) MoS2+NIR, (V) MoS2+H2O2, and (VI) MoS2+H2O2+NIR. Figure 9 shows the wound healing treatment groups and their corresponding histologic analyses results. Compared with control groups, the scars appeared in all treatment groups at the second day. Hematoxylin and eosin (H&E) stain results indicate the wound boundary between wound and normal tissue was obvious. At the fifth day, the keratinocytes migrated to the wound site from the normal tissue for all treatment groups, and scars became apparently smaller and even vanished for synergistic MoS2+H2O2+NIR group whereas the wound boundary and incomplete dermal 11006

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Figure 9. Photographs of Ampr E. coli infected wound treated with (I) PBS (control), (II) H2O2, (III) MoS2, (IV) MoS2+NIR, (V) MoS2+H2O2, and (VI) MoS2+H2O2+NIR at (A) the second day and (B) the fifth day and their corresponding histologic analyses. (Three mice in each group).

CONCLUSION In summary, we constructed a synergistic antibacterial system based on biocompatible PEG-MoS2 NFs, which possess both peroxidase catalytic activity and PTT efficacy. The peroxidase catalyst can decompose low concentration of H2O2 to generate ·OH, which disrupted integrity of the bacterial cell walls. By combination with the generated ·OH, 808 nm laser-induced hyperthermia from the PEG-MoS2 NFs can eliminate both Ampr E. coli and B. subtilis in vitro rapidly and effectively as compared to peroxidase catalytic process or photothermal treatment alone. The wound healing results reveal that the versatile synergy antibacterial system could be expediently used in the dermal wound disinfection in vivo. We also proposed a mechanism based on ·OH-enhanced PTT to explain the antibacterial process. The potent accelerated GSH oxidation upon 808 nm laser-induced hyperthermia in the presence of PEG-MoS2 proved by XPS and XANES breakdown the intercellular protection system and could more easily make the bacterial death. This work provides a simple, effective, and rapid way to wound disinfection using the PEG-MoS2 NFs and has a long-term impact on fighting drug-resistance and endospore-forming bacterial infections of wounds in the future.

layer were still observed for other groups. The edema and ulceration were seen except that the synergistic group even after 5 days and the wound of the synergistic group depicts a mostly entire epidermis structure. Masson’s trichrome staining was used to verify the formation of collagen fiber (blue) during the wound healing progress. At the second day, the collagen fiber lost for all the groups. At the fifth day, the wound of H2O2 treated group shows unrepaired collagen fibers, whereas the MoS2+H2O2 groups show the regeneration of collagen fibers. Particularly, the MoS2+H2O2+NIR group shows good established collagen fibers and dermal layer. Wound sizes of the synergistic group decrease rapidly in contrast to other groups (Figure S12). In addition, the antibacterial effect in vivo was further assessed by determining the number of colony forming units (CFU) in wound area using standard plate counting method (Figure S13a). Compared with other groups, the relative CFU count demonstrates that treatment with synergetic MoS2+H2O2+NIR group had an obvious bactericidal effect, resulting in the CFU count being decreased to 1.6%, whereas H2O2, MoS2+NIR, MoS2+H2O2 group can only be decreased to 66%, 37%, and 49%, respectively (Figure S13b). Thus, the synergistic antibacterial system can effectively kill bacteria, promote the scar generation to protect wound tissue, and further modulate the collagen alignment.

METHODS AND EXPERIMENTS Preparation and Characterization of PEG Modified MoS2 Nanoflowers (PEG-MoS2 NFs). MoS2 NFs were synthesized through a simple hydrothermal route. Typically, 0.50 g of polyethylene glycol 11007

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ACS Nano Km 1 1 = + v Vmax[S] Vmax

(PEG), Mw = 20 000 (Alfa Aesar) and 0.1766 g of ammonium molybdate tetrahydrate (H24Mo7N6O24·4H2O, 99%, Aladdin Industrial Inc.) were dissolved in 20 mL of deionized water (18.5 MΩ, SHROplus DI system). Then, 10 mL of aqueous solution containing 2 mmol of thiourea (TU, 98%, Aladdin Industrial Inc.) was added and stirred until a form transparent solution was formed. Then, the mixture was transferred into a Teflon-lined stainless steel autoclave and heated to 180 °C for 12 h. Finally, the black product (PEG-MoS2 NFs) was washed with ethanol and deionized water for three times then separated by centrifugation and redispersed in phosphate buffer saline (PBS) solution for further use. Characterization. Images of size and morphology of the products were obtained by a field emission scanning electron microscopy (FESEM, Hitachi High Technologies, Japan) and a TecnaiG2 T20 UTWIN transmission electron microscope (TEM). X-ray powder diffraction (XRD) analysis was carried out by a Japan Rigaku D/ max-2500 X-ray powder diffractometer with Cu Kα radiation (λ = 1.54 Å). Raman spectra were recorded by Raman spectrometer (Horiba LabRam HR 800). Ultraviolet−visible−near-infrared spectroscopy (UV−vis−NIR, Cary 5000, Agilent, USA) was used for the absorbance measurements. Photothermal effects were measured by an infrared thermal imager (E40, FLIR) and the temperature change was recorded once per 20 s, when an 808 nm NIR laser oriented perpendicularly to a 1 mL of quartz cuvette containing the dispersions of materials of various concentrations. The MoS2 before and after PEG binding dispersed in water were sonicated for 30 min, respectively. And then, dynamic light scattering (DLS) and zeta potential of the MoS2 suspensions were tested by a ZLS Particle Size Analyzer and Zeta potential/Particle system, respectively (NanoBrook Omni, Brookhaven). X-ray photoelectron spectroscopy (XPS) measurements were carried out by an ESCALab220i-XL spectrometer with a twin-anode Al Kα (1486.6 eV) X-ray source. All spectra were calibrated to the binding energy of the adventitious C 1s peak at 284.8 eV. Fluorescence (FL) spectra were collected with a Fluoroloy-3 modular spectrofluorometer (HORIBA). X-ray near-edge absorption spectra (XANES) were measured at the 14W beamline of the Beijing Synchrotron Radiation Facility (BSRF). Detection of Hydroxyl Radical (·OH). The possibility of ·OH generation from H2O2 catalyzed by the PEG-MoS2 NFs was evaluated by monitoring the change of fluorescence (FL) of 2-hydroxy terephthalic acid (TAOH) due to the oxidation of terephthalic acid (TA) in aqueous solution.20 ·OH can reduce TA to form TAOH with the maximum FL peak at 435 nm. Five groups of solutions (H2O2, TA, MoS2, TA+MoS2 and TA+H2O2+MoS2) were investigated. In detail, 0.3 mL of 10 μg mL−1 of PEG-MoS2 NFs aqueous solution, 0.3 mL of 1 mM H2O2 aqueous solution, and 0.3 mL of 5 mM TA aqueous solution were mixed with 0.3 mL of 1.0 M acetate buffer (pH = 4.0) or 0.01 M PBS buffer (pH = 7.0), and the resulting solution was diluted by distilled water to 3 mL. The final working concentrations were 1 μg mL−1, 100 μM, 500 μM, and 100 μM for the MoS2, H2O2, TA, and acetate buffer, respectively. The mixture was gently shaken and stored at 30 °C for 12 h in the dark then changes in the 435 nm fluorescence emission peak were recorded. Peroxidase-like Catalytic Activity of PEG-MoS2 NFs. The color change of peroxidase-like PEG-MoS2 NFs toward 3,3′,5,5′-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) were photographed at room temperature after 5 min of coincubation. The steadystate kinetic assay of the PEG-MoS2 NFs to TMB in the presence of H2O2 was carried out at room temperature. The final concentrations of TMB, H2O2, and MoS2 were 1 mM, 10 mM, and 33 μg mL−1, respectively. The reaction time was 5 min. The buffer solutions were 0.2 M of Na2HPO4·12H2O and 0.1 M of citric acid. The pH values were modulated from pH = 2.2 to pH = 8.0 by mixing different volumes of these two buffer solutions. The reactions were monitored in the time scan mode at 652 nm for TMB using a Hitachi U3900 UV−vis spectrophotometer. The kinetic parameters were obtained by the plot linear-fitting method based on the Lineweaver−Burk doublereciprocal equation derived from the Michaelis−Menten equation:

where v is the initial velocity, Vmax is the maximal reaction velocity, [S] is the concentration of substrate, and Km is the Michaelis constant. Preparation of Bacterial Solutions. Single colony of Gramnegative ampicillin-resistant Escherichia coli (Ampr E. coli) grown on Luria−Bertani (LB) agar plate and Gram-positive endospore-forming Bacillus subtilis (B. subtilis) grown on Beef-Peptone-Yeast (BPY) agar plate were transferred to 10 mL of LB or BPY broth at 37 °C and shaken for 6 h, respectively. The LB culture media contained 50 μg mL−1 of ampicillin. Bacteria were harvested by centrifuging (8000 rpm for 1 min), and then were washed with phosphate buffer saline (PBS, 10 mM, pH = 7.4). The supernatant was discarded and the remaining bacteria were resuspended in PBS, and diluted to an optical density of 0.1 at 600 nm (OD600 = 0.1). Dose-Dependent Antibacterial Ability of the Antibacterial System. The antibacterial abilities of H2O2, MoS2, and MoS2+H2O2 against bacteria were determined by using optical density at 600 nm (OD600). Ampr E. coli (1.0 × 106 CFU mL−1) were incubated separately with different concentrations of H2O2 (1.0 × 10−8, 1.0 × 10−7, 1.0 × 10−6, 1.0 × 10−5, 1.0 × 10−4, 1.0 × 10−3, 1.0 × 10−2, and 1.0 × 10−1 mol L−1), MoS2 NFs (3.906, 7.813, 15.63, 31.25, 62.50, 125, 250, 500, 1000 μg mL−1) without or with 100 μmol L−1 of H2O2 that were dispersed in LB culture at 37 °C under orbital shaking at a speed of 180 rpm for 12 h. The absorbance at 600 nm was recorded and the bacteria without PEG-MoS2 NFs were used as control and the culture medium without bacteria was considered as background. In Vitro Antibacterial Effects of Synergetic Peroxidase-Like Catalytic Activity and NIR Photothermal Therapy of PEG-MoS2 NFs. Plate counting method determining the number of CFU was used to check antibacterial ability of peroxidase-like PEG-MoS2 NFs. Ampr E. coli or endospores-forming B. subtilis were divided into eight groups: (I) bacteria; (II) bacteria+MoS2; (III) bacteria+H2O2; (IV) bacteria+H2O2+MoS2; (V) bacteria+NIR; (VI) bacteria+MoS2+NIR; (VII) bacteria+H2O2+NIR; and (VIII) bacteria+H2O2+MoS2+NIR. Briefly, 0.1 OD600 of the bacteria were diluted 100 times with PBS and 50 μL of this diluted bacteria was added into 48-well cell culture plates. The final concentrations of PEG-MoS2 NFs, H2O2, and bacteria were 100 μg mL−1, 100 μM, and 1.0 × 105 CFU mL−1, respectively. The total volume of solution in each well was 0.5 mL. After incubation for 20 min, 100 μL of bacterial suspension of group I−IV were spread on the agar culture plate and incubated at 37 °C for 18 h. And, after incubation for 10 min, group V−VIII were further exposed to 808 nm laser (1.0 W cm−2) for another 10 min and the temperature was monitored by the Infrared thermal imager (E40, FLIR). After the NIR treatment, the procedure was the same as group I−IV. All experiments were repeated for three times. Morphology Observation of Bacteria. After the antibacterial abilities assessment, six typical groups of the bacterial suspensions (a) PBS, (b) H2O2 (100 μM), (c) MoS2 (100 μg mL−1), (d) MoS2+NIR laser, (e) MoS2+H2O2, and (f) MoS2+H2O2+NIR laser were dropped on silicon wafers and fixed with 4% paraformaldehyde containing PBS solution for 4 h at 4 °C. Then, the bacteria were dehydrated by sequential treatments with 30, 50, 70, 80, 90, and 100% of ethanol, respectively, for 10 min. Dried bacteria were sputter-coated with gold for FE-SEM images. Detection of Superoxide Radical Anions (O•− 2 ). The possibility of O•− 2 generated from PEG-MoS2 NFs was quantified by 2,3-bis (2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. The XTT is reduced by the O•− 2 to form XTT-formazan with the maximum UV−vis absorption band at 470 nm.57 Briefly, 1 mL of 0.4 mM XTT PBS (pH = 7.0) solution was mixed with 1 mL of 100 μg mL−1 MoS2 PBS solution. The mixture was incubated in the dark at room temperature for 2−6 h under shaking (150 rpm). Then, the mixture was centrifuged at 13 000 rpm for 10 min and the supernatant was measured on a Hitachi U3900 UV−vis spectrophotometer recording the change at the absorbance of 470 nm. Ellman’s Assay. Glutathione (GSH) oxidation was examined by Ellman’s assay57,58 and all the experiments were carried out in the dark 11008

DOI: 10.1021/acsnano.6b05810 ACS Nano 2016, 10, 11000−11011

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ACS Nano and in triplicate. Ellman reagent (5,5′-dithiobis(2-nitrobenzoic acid), DTNB, Alfa) reacted with thiol groups (−SH) in GSH by cleaving its disulfide bonds (−S−S−) to obtain a yellow product (2-nitro-5thiobenzoate acid). Typically, 225 μL of bicarbonate buffer solution (50 mM, pH = 8.7) containing PEG-MoS2 NFs in a concentration of 20, 40, and 80 μg mL−1 were mixed with 225 μL of 0.8 mM GSH bicarbonate buffer solution (50 mM, pH = 8.7) in a 1.5 mL centrifuge tube. All the tubes were incubated and shaken with speed of 150 rpm at room temperature for 2, 4, and 6 h. The positive and negative control groups were 1 mM of H2O2+GSH and GSH solution, respectively. Afterward, 785 μL of 0.05 M Tris-HCl (pH = 8) solution and 15 μL bicarbonate buffer solution (50 mM, pH = 8.7) with 100 mM DTNB was added into the mixture and the MoS2 NFs were separated by centrifuging at 12 000 rpm for 10 min. The 200 μL of the centrifuged solution was then added in the 96-well culture plate, and the absorbance at 410 nm was recorded on a microplate spectrophotometer (Multiskan MK3, Thermo Scientific). Temperature-dependent GSH oxidation was carried out by Ellman’s assay at 20 and 50 °C, respectively, using water bath, and the concentration of PEG-MoS2 NFs was 100 μg mL−1. NIR 808 nm laser-induced GSH oxidation was also determined by Ellman’s assay. Briefly, 225 μL of bicarbonate buffer solution (50 mM, pH = 8.7) containing PEG-MoS2 NFs in the concentration of 100 μg mL−1 was mixed with 225 μL of 0.8 mM GSH bicarbonate buffer solution (50 mM, pH = 8.7) in centrifuge tubes. Then, the tubes were exposed to 808 nm laser for 20 min. The temperature was monitored by infrared thermal imager and maintained at 50 °C. Afterward, 785 μL of 0.05 M Tris-HCl (pH = 8) solution and 15 μL of bicarbonate buffer solution (50 mM, pH = 8.7) with 100 mM DTNB was added into the mixture and the PEG-MoS2 NFs were separated by centrifuging at 12 000 rpm for 10 min. 200 μL of the centrifuged solution was then added in the 96-well culture plate, and the absorbance at 410 nm was recorded on a microplate spectrophotometer. XANES Spectra Analysis. To verify the mechanism of GSH oxidation promoted by NIR irradiation in the presence of PEG-MoS2 NFs, the Mo K-edge XANES of the MoS2, MoS2+NIR, MoS2+GSH, and MoS2+NIR+GSH were collected. Before the experiment, the samples dispersions with NIR irradiation were exposed to 808 nm laser (1.0 W cm−2) and the temperature was kept at 50 °C for 10 min. The commercial Mo, MoO3, MoO2, and MoS2 bulk powder were chosen as the standard materials. These materials were fixed on a tape. The XANES data were recorded in fluorescence mode (using a 19elemental Ge solid detector) for all samples. After background subtraction and normalization, the data for the four samples were fitted to all model compounds. In successive fits the components that have the lowest percentage of composition were removed, and the remaining compounds were then refit until a reasonable fit was achieved. In Vitro Cytotoxicity Experiments. Human cervical carcinoma cell lines (HeLa) and human umbilical vein endothelial cells lines (HUVEC) were employed for investigation the cell viabilities. HeLa and HUVEC cells were grown in normal DMEM culture medium, respectively, with 10% of fetal bovine serum (FBS). The in vitro cytotoxicity was measured by a standard Cell Counting Kit-8 (CCK-8) assay. First, the cells were incubated in 96-well plate (about 5000 cells/ well, six wells for each concentration) for 24 h in a humidified incubator (37 °C, 5% CO2). After washing each well with PBS (0.01 M, pH = 7.4), the PEG-MoS 2 NFs solutions in different concentrations (0, 10, 25, 50, 100, and 250 μg mL−1) were added. Then, the cells were coincubated for 24 h. CCK-8 was subsequently added to each well and the plate was kept in the incubator for another 1.5 h. Finally, cell viability was evaluated by the absorbance at 450 nm of each well measured by a microplate reader (SpectraMax M2, MDC, USA). The cells without PEG-MoS2 NFs were used as control and the culture medium without cells but with CCK-8 were considered as background. The cell viability was calculated relative to the control cells. In Vivo Toxicity Study. Four-week-old Kunming mice were purchased from Vital River (Beijing, China) and then were given free

access to standard chow and water. Fresh whole blood was obtained from the mice. All the animal procedures were in agreement with the guidelines for the care and use of laboratory animals of Ministry of Science and Technology of the People’s Republic of China’s requirements. The Animal Study Committee of the Ministry of Science and Technology of the People’s Republic of China approved the experiments. PEG-MoS2 NFs at a total dose of 15 mg kg−1 in PBS (pH 7.4) were injected into Kunming mice (n = 4) through the tail vein. Kunming mice (n = 4) with no injection of the NPs were acted as the control group. The body weight of the mice in both groups was recorded every other day for 30 days. In Vivo Mice Wound Model and Healing Process. Two kinds of antibacterial solutions including 100 μg mL−1 of PEG-MoS2 NFs and 100 μM H2O2 aqueous solutions were prepared by using sterile water. PEG-MoS2 NFs was sterilized in an autoclave before use. Female BALB/c mice (8 weeks, 18−23 g) were purchased from Vital River and divided into six groups: PBS, H2O2, MoS2, MoS2+NIR, MoS2+H2O2, and MoS2+H2O2+NIR with three mice in each group. A wound of d = 5 mm (∼78 mm2) were obtained by surgical procedure on the back of the mice after anesthesia. The wounds were infected by the bacterial suspension of ampicillin-resistant E. coli with 1 × 105 CFU mL−1. After 12 h, 5 μL of 100 μg mL−1 of PEG-MoS2 NFs and 100 μM H2O2 solutions were dropped on the wound area at the corresponding groups. After 10 min (for NIR treated groups), the wound area was irradiated with an 808 nm NIR laser (power density: 0.5 W cm−2) for 10 min. Other groups were treated with the same condition, but without the NIR laser irradiation. A homemade BandAids consist of sterile cotton covered on wound was changed with 24 h intervals. The wounds were photographed after another 24 h and 5 days. Wound tissues were collected from the mice for analysis after euthanasia. In addition, to check the antibacterial activity in vivo, subsequent to the surgical procedure for 24 h, the bacterial samples in each group were collected from the wound area using a sterile swab and then they were placed into 5 mL of LB broth and shaken for 8 h at 37 °C. 100 μL of bacterial suspension of the culture solution were spread on the agar culture plate and incubated at 37 °C for 2 days. All mice were treated according to the guidelines of the Institutional Animal Care and Use Committee. The mice were discarded according to the standard approved protocol after we finished the experiment. Skin tissues were fixed with 10% formaldehyde solution before the histology analysis. Hematoxylin and eosin (H&E) staining was used for histological analysis and Masson’s trichrome staining was employed for collagen formation assessment.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b05810. Additional experimental data as described in text (PDF)

AUTHOR INFORMATION Corresponding Authors

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

Zhanjun Gu: 0000-0003-3717-2423 Author Contributions #

W.Y. and J.Y. contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Basic Research Programs of China (2015CB932104, 2016YFA0201603, 2016YFA0202104), the National Natural Science Foundation 11009

DOI: 10.1021/acsnano.6b05810 ACS Nano 2016, 10, 11000−11011

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ACS Nano

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of China (11621505, 31751015, 21320102003), Beijing Natural Science Foundation (2162046), and Innovation Program of the Chinese Academy of Sciences (QYZDJ-SSW-SLH022).

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DOI: 10.1021/acsnano.6b05810 ACS Nano 2016, 10, 11000−11011

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DOI: 10.1021/acsnano.6b05810 ACS Nano 2016, 10, 11000−11011