Pharmaceutical Intermediate-Modified Gold Nanoparticles: Against

May 22, 2017 - White and black scale bars are 100 μm and 1 cm, respectively (Ly, .... the General Hospital of People's Liberation Army with patient's...
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Pharmaceutical Intermediate-Modified Gold Nanoparticles: Against Multidrug-Resistant Bacteria and Wound-Healing Application via an Electrospun Scaffold Xinglong Yang,†,‡,§,⊥ Junchuan Yang,‡,∥,⊥ Le Wang,‡ Bei Ran,‡ Yuexiao Jia,‡ Lingmin Zhang,‡ Guang Yang,∥ Huawu Shao,*,† and Xingyu Jiang*,‡ †

Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan 610041, China CAS Center for Excellence in Nanoscience, CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology, ZhongGuanCun BeiYiTiao, Beijing 100190, China § University of Chinese Academy of Science, Beijing 100049, China ∥ National Engineering Research Center for Nano-Medicine, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China ‡

S Supporting Information *

ABSTRACT: Remedying a multidrug-resistant (MDR) bacteria wound infection is a major challenge due to the inability of conventional antibiotics to treat such infections against MDR bacteria. Thus, developing wound dressings for wound care, particularly against MDR bacteria, is in huge demand. Here, we present a strategy in designing wound dressings: we use a small molecule (6-aminopenicillanic acid, APA)-coated gold nanoparticles (AuNPs) to inhibit MDR bacteria. We dope the AuNPs into electrospun fibers of poly(ε-caprolactone) (PCL)/gelatin to yield materials that guard against wound infection by MDR bacteria. We systematically evaluate the bactericidal activity of the AuNPs and wound-healing capability via the electrospun scaffold. APA-modified AuNPs (Au_APA) exhibit remarkable antibacterial activity even when confronted with MDR bacteria. Meanwhile, Au_APA has outstanding biocompatibility. Moreover, an in vivo bacteria-infected wound-healing experiment indicates that it has a striking ability to remedy a MDR bacteria wound infection. This wound scaffold can assist the wound care for bacterial infections. KEYWORDS: gold nanoparticles, pharmaceutical intermediate, antibacterial activity, electrospun nanofibers, wound healing fight against MDR bacteria via a straightfoward route. We modify AuNPs using small molecules, such as 4,6-diamino-2pyrimidinethiol (DAPT),11 non-antibiotic amines,12 and Nheterocyclic molecules,13 by in situ synthesis without further AuNP modification. These AuNPs can inhibit both laboratorystrain (i.e., antibiotic-sensitive) bacteria and clinical MDR isolates. We use molecules that serve as the main structural components of β-lactam antibiotics, such as 6-aminopenicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA), and 7aminodesacetoxycephalosporanic acid (7-ADCA) to modify the

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kin serves as a protective barrier between internal organs and the rest of the world.1 When damaged, it could lose its protective effects, which may lead to micro-organism invasion and severe infection. Multidrug-resistant (MDR) Gram-negative bacteria-associated wound infection can be very harmful and even fatal due to few available drugs against MDR Gram-negative bacteria.2 Several antibacterial nanomaterials (such as silver,3,4 zinc oxide,5 titanium dioxide,6 tellurium, and copper oxide nanoparticles7) can be effective against MDR bacteria-caused wound infection.8,9 These nanomaterials, however, can be cytotoxic or hemolytic.10 Thus, we set out to explore wound dressing materials that can protect against MDR bacteria. Our group has developed a series of antibacterial gold nanoparticles (AuNPs) coated with small organic molecules to © 2017 American Chemical Society

Received: February 22, 2017 Accepted: May 22, 2017 Published: May 22, 2017 5737

DOI: 10.1021/acsnano.7b01240 ACS Nano 2017, 11, 5737−5745

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Figure 1. Schematic illustration of the synthesis of antibacterial AuNPs and application for wound healing.

Figure 2. Characterization of AuNPs. (A) TEM images of Au_APA, Au_ACA, and Au_ADCA. (B) Chemical structures of small molecules. (C) Particle sizes and ζ-potential of AuNPs.

surfaces of AuNPs. Because these molecules form the main structural cores of β-lactam antibiotics, we call them “antibacterial intermediates”. The synthetic routes to prepare β-lactam antibiotics usually start with the antibiotic structural core, followed by various steps of modication.14 Moreover, numerous bacterial infections become resistant to these antibiotics. We wonder if any of the main molecular moieties of β-lactam antibiotics can become effective against MDR bacteria. Thus, we try to employ these antibiotic intermediates in antibacterial AuNP preparation instead of complicated and time-consuming synthesis of antibiotics using antibiotic synthesis.15

Electrospinning can process polymeric solutions or melts into continuous fibers with diameters down to a few nanometers.15−20 Different electrospinning nanofibers can serve as wound dressings with various components, including poly(vinyl alcohol) (PVA), polyacrylonitrile (PAN), poly(vinyl acetate) (PVAc), chitosan, polyurethane (PU), polylactic acid (PLA), poly(ε-caprolactone) (PCL), and so on.16,21 As reported, PCL/gelatin coelectrospun nanofibrous scaffolds are suitable for tissue engineering, which have enough pores to ensure the exchange of liquids and gases with the external environment.22,23 PCL is a hydrophobic polymer with high toughness that mimics the property of the extracellular matrix (ECM) in tissue. Gelatin is a natural biopolymer, and it is 5738

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ACS Nano biocompatible.24 Thus, blending PCL with gelatin can prepare electrospun fibrous scaffolds that are both tough and biocompatible.22,25 Here, we employ antibacterial intermediate-modified AuNPs as antibacterial active ingredients and PCL/gelatin nanofibers as the scaffolds to achieve biocompatible antibacterial wound dressings against MDR bacterial wound infection. Many nanomaterials are blended with electrospun nanofibrous mats by immersion26 and plasma method.27 These methods typically result in inhomogeneous coating of nanomaterials onto fibers. To solve this problem, nanomaterials coelectrospinning with electrospun solutions may apply to our strategy.25,28 AuNPs modified with antibacterial intermediates can kill Gram-negative bacteria and MDR strains. Moreoever, electrospun polymeric fibers with the AuNPs can be efficient wound dressing materials for bacterial infection treatment (Figure 1). This kind of AuNP shows effective antimicrobial activity and outstanding biosecurity. The electrospun nanofibrous mats incorporating the antibacterial AuNPs show adequate wound healing property in vivo against bacterial wound infection.

Table 1. Antibacterial Activities Indicated with MIC (μg mL−1) laboratory antibiotic-sensitive strains

Au_APA Au_ACA Au_ADCA

clinical MDR isolates

E. coli

K. pneumoniae

P. aeruginosa

MDR E. coli

MDR K. pneumoniae

2.5 32 16

2.5 32 16

5 >64 >64

5 32 32

5 >64 32

of antibiotics (Table S1). Au_APA could also inhibit the proliferation of MDR E. coli and MDR K. pneumoniae (Figure 3A). By contrast, Au_ACA and Au_ADCA show negligible

RESULTS AND DISCUSSION Synthesis and Characterization of Antibiotic Intermediate-Capped AuNPs. We prepare antibiotic intermediate-capped AuNPs via reduction of tetrachloroauric acid (HAuCl4) by sodium borohydride (NaBH4) in the presence of antibiotic intermediates as protective agents in methanol. We characterize the morphology of AuNPs by transmission electron microscopy (TEM). We find that these AuNPs are well-dispersed, and the diameters of Au_APA, Au_ACA, and Au_ADCA are similar (≈3 nm). The hydrodynamic sizes are about 5 nm. The ζ-potential shows that these particles are negatively charged (Figure 2). The morphology of these NPs that were stored for almost 7 months is not changed, which demonstrates the high stability of the NPs (Figure S1). We employ Fourier transform infrared spectroscopy (FTIR) to confirm whether Au_APA NPs are capped with the small molecule (6-APA) (Figure S2). The spectra of 6-APA and Au_APA reveal overlapping peaks characteristic at wavenumbers of 1770 cm−1 (CO stretching) and 1620 cm−1 (amide I bond). FTIR analysis confirms that AuNPs have been modified by the small molecule (6-APA). Antibacterial Activity of Small-Molecule-Modified NPs. We test the antibacterial activities of antibiotic intermediate-capped AuNPs by minimal inhibitory concentration (MIC) via a microbroth dilution method. First, we choose Gram-positive bacteria Staphylococcus aureus and Gramnegative bacteria Escherichia coli as a microbial model to evaluate the antibacterial activities of these AuNPs. Au_APA, Au_ACA, and Au_ADCA are active against Gram-negative bacteria but inactive against Gram-positive bacteria. This difference may due to the fact that Gram-positive bacteria have thicker cell walls, which may influence the access of Au_APA, Au_ACA, and Au_ADCA into bacteria.29 The antibacterial activity of Au_APA is better than that of Au_ACA and Au_ADCA. Next, we evaluate the MIC of these AuNPs against other species of bacteria and clinical MDR strains (Table 1). Au_APA has antibacterial activity against Pseudomona aeruginosa and Klebsiella pneumoniae, which is more effective than Au_ACA and Au_ADCA. We also utilize clinically isolated MDR strains to test the antibiotic capabilities of AuNPs. These MDR strains are isolated from the patient’s urine or saliva and resist a series

Figure 3. Antibacterial activity of Au_APA NPs. (A) Optical density at 600 nm (OD600nm) of different bacterial suspensions treated and untreated with Au_APA(+): E. coli, 2.5 μg mL−1; MDR E. coli, 5 μg mL−1; K. pneumoniae, 2.5 μg mL−1; MDR K. pneumoniae, 5 μg mL−1; P. aeruginosa, 5 μg mL−1. (B) LB liquid medium turbidity assays and (C) bacteriostatic rate corresponding to B based on bacteria count. The original bacterial concentration is 1 × 106 CFU mL−1.

antibacterial activity against clinical MDR isolates. We note that Au_APA NPs show the best antibiotic activity even against clinical MDR isolates. Thus, we perform the following studies with Au_APA. We investigate Au_APA NPs with different ratios of 6-APA and Au to obtain optimized antibacterial activity. TEM images indicate that these AuNPs have similar sizes (Figure S3). The MIC of the small molecule (6-APA) itself is high, especially for MDR bacteria (>250 μg mL−1). NPs (Au/APA = 1.4:1) show the best antibacterial abilities among the NPs, which is the ratio we choose to synthesize the Au_APA NPs (Table S2). So the synergy of small molecules 5739

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ACS Nano and nanoparticles gives Au_APA an outstanding antibacterial activity, which relies on the nanometer effect of AuNPs. To confirm the antibacterial activity of Au_APA NPs, we also carry out the Luria−Bertani (LB) liquid medium turbidity assay. We evaluate the antibacterial property of Au_APA for both laboratory antibiotic-sensitive strains (E. coli) and clinical MDR isolates (MDR E. coli) (Figure 3B,C). We culture E. coli and MDR E. coli in LB liquid media alone for 12 h, and the mixture become turbid, suggesting bacteria in such media rapidly proliferate. However, the medium containing Au_APA nanoparticles remains transparent, indicating few E. coli and MDR E. coli proliferated. To further investigate, we choose a commercial antimicrobial agent (ampicillin) as a comparison. The E. coli LB medium with ampicillin is pellucid, which is the same as media containing Au_APA. However, when we culture MDR E. coli with ampicillin, the mixture turns turbid within 12 h, indicating that ampicillin is ineffective to inhibit growth of MDR E. coli. These results show that Au_APA nanoparticles could prevent the bacterial growth, and the antibacterial activity of Au_APA against clinical MDR isolate is better than ampicillin. We employ commercial silver nanoparticles (AgNPs) to illustrate the superior antibacterial activity of Au_APA. The particle size of AgNPs is about 6 nm (Figure S4). We test the MIC of AgNPs (Table S3). The MIC for E. coli is 7.8 μg mL−1, which is much higher than that of Au_APA (2.5 μg mL−1). The MIC values of AgNPs for other bacteria including MDR isolate are also higher than the MIC of Au_APA. Au_APA is much more predominant than the commercial AgNPs. NP-Induced Disruption of Bacterial Cell Membranes. To understand the mechanism of the antibiotic effects of these NPs, we take E. coli as an example to evaluate the permeability of cell membranes in the presence of Au_APA. We strain samples with propidium iodide (PI), which can stain DNA or RNA specifically but cannot cross the membrane of viable cells.30 So, we can use PI to identify dead cells. We treat the suspensions of E. coli with Au_APA (10 μg mL−1) at 37 °C for 2 h and image them with PI. Fluorescence images and calculated data show that the permeability of treated E. coli increases (Figure 4A,B). To further explore antibiotic mechanisms of Au_APA NPs, we visualize the morphological change of E. coli treated with different concentrations of Au_APA using scanning electron microscopy (SEM). After E. coli was incubated for 5 h with different concentrations of Au_APA (5, 20, and 80 μg mL−1), SEM indicates that cell lysis is widespread (Figure 4C). With the increase of concentrations of Au_APA, the compromise of cell structures becomes increasingly significant (Figure S5). We also perform TEM to observe the morphological change of the bacteria clearly (Figure 4C). The cell wall is distrupted when the bacteria is treated with Au_APA. Hence, Au_APA can induce disruption to cell membrane and the lysis of bacterial cells. Biological Safety of Au_APA NPs. To investigate the biocompatibility of Au_APA, we test the hemolytic property on human erythrocytes (Figure 5). Hemolysis must be avoided when we use materials in blood-contacting applications (such as wound-healing dressing materials).31 Au_APA does not damage erythrocytes even if the concentrations of the nanoparticle dispersions are high (20 μg mL−1, 8 times that of the MIC, hemolysis rate: 2.19%). For comparison, we examine the hemolysis rate of the commercial AgNPs (Figure S6). When the concentration is 2.5 μg mL−1, the hemolysis rate

Figure 4. Observation of NP-induced disruption of bacterial cell membranes. (A) Monitoring Au_APA-induced permeability of cell membranes by propidium iodide. (B) Percentage of cells with permeable membranes. (C) Morphology of E. coli characterized by SEM and TEM images without and with Au_APA NPs treatment (concentration: 5 μg mL−1).

is as high as 11.4% (>5%). Au_APA NPs are thus suitable for a wide safety margin in blood-contacting applications. For further clinical safety evaluation, we test the potential toxicity of Au_APA using human umbilical vein endothelial cells (HUVECs) and NIH 3T3 cells with a commercial CCK-8 kit (Figure S7). Au_APA NPs are essentially nontoxic to cells under the concentration of 20 μg mL−1 (8 times the MIC). The cell viability decreases with the increase of concentrations of Au_APA. Fabrication and Characterization of Au_APA Electrospun Nanofibers. To test the antibiotic capability of the Au_APA on a solid substrate, we prepare Au_APA electrospun nanofibers with coelectrospinning using PCL, gelatin, and Au_APA NPs because the electrospun fibers composed of PCL and gelatin bring superior architecture and physical properties, which might be useful to wound healing. We employ SEM imaging and X-ray photoelectron spectroscopy (XPS) to characterize the morphology of electrospun fibers composed of Au_APA, PCL, and gelatin. We use PCL/gelatin as a control. The diameters of PCL/gelatin nanofibers (control) and Au_APA electrospun nanofibers are essentially the same (Figure 6A), which indicates that the addition of Au_APA into PCL/gelatin nanofibers did not affect the structure of the electrospun fibers. TEM images of Au_APA fibers demonstrate that the distribution of Au_APA in the fiber is homogeneous (Figure 6B). The XPS confirms the presence of Au_APA NPs in the fibers (Figure S8). We verify the structures of PCL/ gelatin nanofibers and Au_APA nanofibers by FTIR (Figure 6C). Their spectra reveal overlapping peaks characteristic at wavenumbers of 2943 cm−1 (asymmetric CH2 stretching), 2840 cm−1 (symmetric CH2 stretching), 1728 cm−1 (CO stretching), 1620 cm−1 (amide I bond), and 1242 cm−1 (asymmetric C−O−C stretching). FTIR analysis of these nanofibers also confirms that AuNPs have not changed the microstructure of electrospun nanofibers. In order to investigate how the AuNPs leach out, we obtain the AuNPs release line and weight remaining after dipping in saline (Figure 6D). After 1 day, the cumulative release of Au 5740

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Figure 5. Hemolysis activity of Au_APA nanoparticles. (A) Photographs of fresh human blood incubated with different concentrations of Au_APA NPs and (B) hemolysis ratio of different concentrations of Au_APA.

Figure 6. Characterization and property of Au_APA electrospun nanofibers. (A) SEM image of Au_APA electrospun nanofibers and PCL/ gelatin fibers. (B) TEM image of Au_APA electrospun nanofibers. (C) FTIR spectroscopy of nanofibers (gelatin, PCL, PCL/gelatin, Au_APA electrospun nanofibers). (D) Weight remaining and cumulative release of Au_APA fibers soaking in saline.

treat each rat with E. coli, MDR E. coli, P. aeruginosa or MDR P. aeruginosa to establish a bacterial wound infection model. Then we apply Au_APA electrospun nanofibers, PCL/gelatin nanofibrous scaffolds, and gauze (traditional wound dressing) as dressings on the wounds (Figure S11). We have examined the local bacteria count in the wound area after infection with E. coli, MDR E. coli, P. aeruginosa or MDR P. aeruginosa on day 1, day 7, and day 14, respectively (Figures 7B and 8B). The local bacteria levels in wound areas have no significant difference on day 1. After 7 and 14 day treatments, the local bacteria levels of bacteria-infected wounds treated with Au_APA fibers have decreased obsviously compared with PCL/gelatin fibers and gauze. These results indicate that Au_APA fibers can reduce the local bacteria. To expressly study the wound-healing progress, we take photos of infected full-thickness wounds and examine the histological analysis of the wound healing on day 7 and day 14 postoperation, stained with hematoxylin and eosin (H&E) (Figures 7 and 8). After 14 days, the areas of all the wounds significantly decreased. The new skin of gauze and PCL/gelatin groups is similar to that in the Au_APA nanofiber group. Inflammatory cells such as neutrophils and lymphocytes emerge in all of the wounds treated with E. coli and MDR E. coli at postoperative day 7 (Figures 7A−C,G−I and 8A). Their presence indicates inflammatory response. The epithelial layer

was 20.4% of the original amount and up to 65.7% after 7 days. After 1 and 7 days, the degradation rate is 4.8 and 19.6%. Burst release occurred during the first few days because some AuNPs existed near the surface of the fibers where they could easily diffuse out without the degradation of the nanofibers. After burst release, the inside AuNPs release slowly, which needs the help of polymer degradation. Wound dressings should be mechanically robust and hydrophilic to obtain fine permeability, which are beneficial for wound healing.32 Therefore, we measure the tensile strength and contact angle of PCL/gelatin nanofibers (control) and Au_APA electrospun nanofibers. The tensile properties of PCL/gelatin nanofibers are very close to Au_APA nanofibers (Figure S9). When we place the water on the fibers, the water contact angle is zero (100% wettability) (Figure S10). Contact angle measurement reveals that the addition of Au_APA to nanofibers retains the hydrophilicity of PCL/gelatin nanofibers. All these properties of Au_APA electrospun nanofibers demonstrate that it is suitable for wound dress engineering. Animal Evaluation. To test the practical applicability of Au_APA electrospun nanofibers, we investigate its antibacterial activity in skin wound healing by a dorsal wound model of rat exposed to E. coli, MDR E. coli, P. aeruginosa and MDR P. aeruginosa. We prepare three round full-thickness wounds with diameters of 2.0 cm on the dorsal side of Wistar female rats. We 5741

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Figure 7. Animal models of wounds infected by E. coli or MDR E. coli. Histological images of skin tissues stained by H&E dissected in the postoperative day 7 (A−C,G−I) and day 14 (D−F,J−L). (M) Local bacteria count in the wound area and (N) relative the number of immune cells compared with control group (gauze). Inserted photos show the macroscopic morphologies of wounds. The control groups are treated with wound dressings of gauze (A,D,G,J), and PCL/gelatin nanofibers (B,E,H,K) and the experimental group use Au_APA electrospun nanofibers as wound dressing (C,F,I,L). The presence of Ly and Ne indicates inflammatory response. Ec and Ef are the signal of reepithelization, and they are beneficial for the formation of matured fibrous granulation tissue; ** and *** represent two levels of significant statistical differences (**P < 0.05, ***P < 0.01). White and black scale bars are 100 μm and 1 cm, respectively (Ly, lymphocyte; Ne, neutrophil; Ec, epithelial cells; Ef, elongated fibroblasts).

nanofibers possess the best wound-healing effect among various as-prepared wound dressings. We also employ PCL/gelatin fibers containing small molecules or bare AuNPs (APA fibers and Au fibers) to investigate which part of fibers plays the dominant role in wound healing (Figure S12). We observe that the effects of APA fibers and Au fibers are similar to those of gauze, which have little contributions to the re-epithelialization in bacteriainfected wounds. In order to show the superiority of Au_APA fibers, we select a Ag mat for comparison (Figure S12). The Ag mat displays nice activity on healing E. coli-infected wound. However, the Ag mat shows poor effect on MDR E. coli-treated wounds compared with Au_APA fibers. So Au_APA fibers are superior in treating MDR bacteria-infected wounds.

in the wound treated with Au_APA electrospun nanofibers compared with gauze and PCL/gelatin nanofibers demonstrates that Au_APA electrospun nanofibers show better woundhealing ability than gauze and PCL/gelatin nanofibers even against MDR bacterial wound infection (Figures 7C,I and 8A). The wounds treated with Au_APA nanofibers are smaller than other wounds at postoperative day 7, which correlates well to the histological images. The major steps in the process of wound healing are reepithelialization and formation of granulation tissues.33,34 Migration and proliferation of fibroblasts and epidermal cells are the signal of re-epithelization. Elongated fibroblasts are helpful for the formation of mature fibrous granulation tissue.35 Elongated fibroblasts, epithelial cells, and a horny layer emerge in all wound dressing groups at postoperative day 14 (Figures 7D−F,J−L and 8A). However, in PCL/gelatin groups and gauze groups, we can still observe more inflammatory cells compared with that in Au_APA fibers groups, which validates that these group maintain inflammatory response (Figures 7N and 8C). Inserted photos also show that wounds treated with gauze or PCL/gelatin nanofibers are bigger than wounds treated with Au_APA nanofibers. Thus, Au_APA electrospun

CONCLUSIONS Our study illustrates a strategy for curing full-thickness wounds infected by MDR bacteria via an electrospun scaffold containing pharmaceutical intermediate-capped AuNPs (Au_APA). The Au_APA we used can withstand bacteria and display excellent biocompatibility. This work can broaden antibiotic screening space and the applications of AuNPs, which 5742

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Figure 8. Animal models of wounds infected by P. aeruginosa or MDR P. aeruginosa. (A) Histological images of skin tissues stained by H&E dissected in the postoperative day 7 and day 14. (B) Local bacteria count in the wound area and (C) relative the number of immune cells compared with control group (gauze); ** and *** represent two levels of significant statistical differences (**P < 0.05, ***P < 0.01). White and black scale bars are 100 μm and 1 cm, respectively (Ly, lymphocyte; Ne, neutrophil; Ec, epithelial cells; Ef, elongated fibroblasts). obtain the MDR isolates from Beijing You’an Hospital with patient’s consent, and it is approved by the local Ethics Committee. Microbe Preparation for SEM and TEM. We treat E. coli with different concentrations of Au_APA (5, 20, and 80 μg mL−1) at 37 °C for 4 h on a shaking bed at 200 rpm. We fix these NP-treated bacterial samples with 2.5% glutaraldehyde and 30, 50, 70, 80, 90, 95, and 100% (v/v, in water) ethanol dehydration for SEM. TEM samples undergo fixing with 2.5% glutaraldehyde and 0.1% osmic acid, dehydrating with graded ethanol, and further cutting of superthin slices and staining with 2% uranyl acetate and 0.2% lead citrate. Cytotoxicity Assay of Au_APA. We cultivate human umbilical vein endothelial cells (HUVECs) and NIH 3T3 cells in Dulbecco’s modified Eagle’s medium (DMEM), which contains 10% fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin. HUVECs and NIH 3T3 cells grow overnight on a 96-well culture plate (roughly 10 000 cells per well). We feed different concentrations (1.25, 2.5, 5, 10, 20, and 40 μg mL−1) of Au_APA cells in the 96-well plate. After a 24 h incubation, we wash the cells with culture medium and stain them with 10 μL of the cell counting kit at 37 °C for 2 h. After 2 h of incubation, a microplate reader (Tecan infinite M200) gives us the optical density of the cells at 450 nm. Hemolysis Assay of Au_APA. We perform the hemolysis assay using fresh human blood, and the blood sample is from the General Hospital of People’s Liberation Army with patient’s consent. We collect the erythrocytes via centrifugation at 1500 rpm for 15 min and wash three times with saline. We prepare the stock dispersion by mixing 3 mL of centrifuged erythrocytes into 11 mL of saline. We synthesize Au_APA nanoparticle dispersions in saline at different concentrations. We add 100 μL of stock dispersion to 1 mL of Au_APA dispersions. The final hematocrit level of red blood cell (RBC) is about 4%. We mix the solutions and incubate them for 3 h at 37 °C. We measure the percentage of hemolysis by UV−vis analysis of the supernatant at 540 nm absorbance after centrifugation at 12 000

may provide an option against MDR bacterial infections. The homogeneous electrospun nanofibers are promising for use in clinical treatment of wound infection.

MATERIALS AND METHODS Synthesis of Au_APA, Au_ACA, and Au_ADCA. We stir the mixture of the small molecule (0.14 mmol, dissolved in 10 mL of methanol, 50 μL of triethylamine, and 30 mg of Tween 80) and HAuCl4·3H2O (0.1 mmol, dissolved in 15 mL of methanol) for 10 min in the ice−water bath and add NaBH4 (0.3 mmol freshly dissolved in 5 mL of methanol) dropwise with vigorous stirring. The color of the solution turns brown immediately. We decrease the stirring speed and keep stirring the solution for another hour in an ice−water bath. We eliminate the solvent in vacuum at 40 °C by rotary evaporators. We add an appropriate volume of deionized water into the residue, dialyze (14 kDa MW cutoff, Millipore) for 48 h with deionized water, sterilize it through a 0.22 μm filter (Millipore), and store it at 4 °C for use. We synthesize Au_ACA, Au_ADCA, and different ratios of Au_APA using the same method. Characterization. We determine the concentrations of NPs and elemental sulfur with inductively coupled plasma analysis (ICP, Elmer Optima 5300 DV). We observe the morphologies of these NPs and fibers using a Tecnai G2 20 ST TEM from the American FEI Company. We obtain ζ-potential values of AuNPs with a Zetasizer Nano ZS (Malvern Instruments). Antimicrobial Test. We test the antibacterial activities of the AuNP microtiter broth dilution method, which determines the MIC that leads to the inhibition of microbial growth. We employ disposable Corning 96-well plates (Costar 3599) for this experiment. We dilute the NP solutions 2−128 times with 100 μL of nutrient broth inoculated with the tested microbe at a concentration of 104 CFU mL−1. We observe the MIC after 24 h of incubation at 37 °C. We 5743

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ACS Nano rpm for 15 min. Saline is the negative control, and pure water (18.2 MΩ·cm) is the positive control. We perform hemolysis assay of AgNPs using the same method. We calculate the percentage of hemolysis with the following formula:

expressed as the number of colony-forming unit per wound by standard plate count methods.

ASSOCIATED CONTENT S Supporting Information *

hemolysis (%) = (A S − AN)/(AP − AN) × 100%

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b01240. Antibacterial activities of different materials; TEM images of AuNPs after 7 months storage in 4 °C; FTIR of 6-APA and Au_APA; TEM images, particle sizes, and ζpotential of Au_APA NPs with different molar ratio of 6-APA and Au element; TEM images and particle sizes of commercial AgNPs; morphology of E. coli characterized by SEM images with Au_APA NP treatment; hemolysis rate of commercial AgNPs; cytotoxicity of Au_APA NPs on HUVECs and NIH 3T3 cells tested by CCK-8; XPS, stress−strain curve, and contact angle measurement of PCL/gelatin and Au_APA electrospun nanofibers; photograph of wound model under the treatment of different dressings; animal models of wounds infected by E. coli or MDR E. coli (PDF)

where AS is the absorbance resulting from addition of AuNPs (Au_APA) to the erythrocyte suspension, AN is the absorbance following the addition of saline as a negative control, and AP is the absorbance following the addition of deionized water as a positive control. Fabrication of Au_APA Electrospinning Sheet. We obtain the solutions of polycaprolactone (PCL, MW = 80000, Sigma-Aldrich) and gelatin (type A, 300 bloom) for electrospinning. We dissolve PCL (30% w/v) and gelatin (10% w/v) in hexafluoroisopropanol (HFIP), stir for 4 h followed by the addition of HFIP solution of Au_APA (final concentration: 7.8 μg/mg) to the mixture, and stir for another 4 h. The electrospinning device consists of a 5 mL standard syringe and a high-voltage power supply (Spellman, SL150). The voltage between the needle and grounded collector is 17.0 kV, and the distance between is 8.0 cm. We collect the nanofibrous sheets on an aluminum foil adhered onto a grounded rotating mandrel. We dry the fibers in a vacuum oven at room temperature for 0.5 h. We prepare APA fibers (final concentration: 6.4 μg/mg) and Au fibers (final concentration: 9.7 μg/mg) using the same method. Au_APA Release. We determine Au release behavior of the Au_APA nanofibers by ICP (Elmer Optima 5300 DV). We place a small piece of nanofiber mat (≈23.9 mg) in a centrifuge tube and add 5 mL of saline into the tube as release medium. We agitate the tube to ensure complete immersion of the nanofiber and then incubate the mixture at 37 °C. Weight Loss. We analyze the degradation properties by measuring the mass remaining of the dry sheets after degradation. The solution we used is saline. Tensile Strength Measurement of Au_APA Electrospun Nanofibers. We determine tensile properties of the electrospun membrane at room temperature. Rectangular (1 cm × 10 cm, thickness ≈ 39 μm) specimens were cut from nanofibrous sheets and used for mechanical studies. We mount the ends of the rectangular specimens on mechanical gripping units of the tensile tester vertically. We apply a load of 10 N for tensile measurements with a speed of 20 mm min−1 to get the tensile strengths of the nanofibers. Contact Angle Measurement. We study the hydrophilicity of the electrospinning sheets by sessile drop water contact angle measurement using a video contact angle system. We use saline for drop formation. Animal Wound Model and Histological Analysis. We use Sprague−Dawley (SD) female rats (Beijing Vital River Laboratory Animal Technology Co. Ltd., China) aged at 8 weeks with an average weight of 250 g to evaluate the effect of wound dressings. We randomly assign the rats to two groups on the basis of the materials (PCL/gelatin, Au_APA electrospinning sheets, and gauze) and bacterial species (E. coli and MDR E. coli). Every group has six parallel samples. We anesthetize the rats by injecting chloral hydrate 3.5% (10 mL kg−1) in the abdominal cavity. We prepare three fullthickness round 2.0 cm diameter wounds by removing dorsal flank skin from the anaesthetized rats. After infecting the wounds with E. coli or MDR E. coli (1 × 10 8 CFU mL−1, 200 μL) for 30 min, we cover the materials on the dorsal wounds of each rat and fix with gauze and a skin stapler (Manipler AZ MSNI, Japan). We then keep the rats individually. For histological analysis, we gather wound tissues from each group of rats at postoperative day 7 and day 14. We put the tissues in 4% paraformaldehyde solution. We obtain isometric continuum cut sections using a microtome in vertical planes of each fixed tissue and stain them with H&E. We obtain animal models of wounds infected by P. aeruginosa or MDR P. aeruginosa using the same process. The bacteria count is

AUTHOR INFORMATION Corresponding Authors

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

Xingyu Jiang: 0000-0002-5008-4703 Author Contributions ⊥

X.Y. and J.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank the Ministry of Science and Technology of China (2013YQ190467), Chinese Academy of Sciences (XDA09030305) and the National Science Foundation of China (81361140345, 51373043, 21535001) for financial support. REFERENCES (1) Priya, S. G.; Jungvid, H.; Kumar, A. Skin Tissue Engineering for Tissue Repair and Regeneration. Tissue Eng., Part B 2008, 14, 105− 118. (2) Levy, S. B.; Marshall, B. Antibacterial Resistance Worldwide: Causes, Challenges and Responses. Nat. Med. 2004, 10, S122−S129. (3) Xiu, Z. M.; Zhang, Q. B.; Puppala, H. L.; Colvin, V. L.; Alvarez, P. J. J. Negligible Particle-Specific Antibacterial Activity of Silver Nanoparticles. Nano Lett. 2012, 12, 4271−4275. (4) Bing, W.; Chen, Z. W.; Sun, H. J.; Shi, P.; Gao, N.; Ren, J. S.; Qu, X. G. Visible-Light-Driven Enhanced Antibacterial and Biofilm Elimination Activity of Graphitic Carbon Nitride by Embedded Ag Nanoparticles. Nano Res. 2015, 8, 1648−1658. (5) Schwartz, V. B.; Thetiot, F.; Ritz, S.; Putz, S.; Choritz, L.; Lappas, A.; Forch, R.; Landfester, K.; Jonas, U. Antibacterial Surface Coatings from Zinc Oxide Nanoparticles Embedded in Poly(N-isopropylacrylamide) Hydrogel Surface Layers. Adv. Funct. Mater. 2012, 22, 2376− 2386. (6) Brunet, L.; Lyon, D. Y.; Hotze, E. M.; Alvarez, P. J. J.; Wiesner, M. R. Comparative Photoactivity and Antibacterial Properties of C-60 Fullerenes and Titanium Dioxide Nanoparticles. Environ. Sci. Technol. 2009, 43, 4355−4360. 5744

DOI: 10.1021/acsnano.7b01240 ACS Nano 2017, 11, 5737−5745

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Characterization and in vivo Applications. Acta Biomater. 2015, 26, 97−104. (26) Jaiswal, A. K.; Kadam, S. S.; Soni, V. P.; Bellare, J. R. Improved Functionalization of Electrospun PLLA/Gelatin Scaffold by Alternate Soaking Method for Bone Tissue Engineering. Appl. Surf. Sci. 2013, 268, 477−488. (27) Shi, Q.; Vitchuli, N.; Nowak, J.; Caldwell, J. M.; Breidt, F.; Bourham, M.; Zhang, X. W.; McCord, M. Durable Antibacterial Ag/ Polyacrylonitrile (Ag/PAN) Hybrid Nanofibers Prepared by Atmospheric Plasma Treatment and Electrospinning. Eur. Polym. J. 2011, 47, 1402−1409. (28) Ding, Y.; Wang, Y.; Su, L. A.; Bellagamba, M.; Zhang, H.; Lei, Y. Electrospun Co3O4 Nanofibers for Sensitive and Selective Glucose Detection. Biosens. Bioelectron. 2010, 26, 542−548. (29) Albers, S. V.; Meyer, B. H. the Archaeal Cell Envelope. Nat. Rev. Microbiol. 2011, 9, 414−426. (30) Zamai, L.; Falcieri, E.; Marhefka, G.; Vitale, M. Supravital Exposure to Propidium Iodide Identifies Apoptotic Cells in the Absence of Nucleosomal DNA Fragmentation. Cytometry 1996, 23, 303−311. (31) Dobrovolskaia, M. A.; Clogston, J. D.; Neun, B. W.; Hall, J. B.; Patri, A. K.; McNeil, S. E. Method for Analysis of Nanoparticle Hemolytic Properties in vitro. Nano Lett. 2008, 8, 2180−2187. (32) Zahedi, P.; Rezaeian, I.; Ranaei-Siadat, S. O.; Jafari, S. H.; Supaphol, P. A Review on Wound Dressings with an Emphasis on Electrospun Nanofibrous Polymeric Bandages. Polym. Adv. Technol. 2009, 21, 77−95. (33) Reynolds, L. E.; Conti, F. J.; Lucas, M.; Grose, R.; Robinson, S.; Stone, M.; Saunders, G.; Dickson, C.; Hynes, R. O.; Lacy-Hulbert, A.; Hodivala-Dilke, K. Accelerated Re-epithelialization in Beta(3)Integrin-Deficient Mice is Associated with Enhanced TGF-Beta 1 Signaling. Nat. Med. 2005, 11, 167−174. (34) Xie, Y. Y.; Zhang, W.; Wang, L. M.; Sun, K.; Sun, Y.; Jiang, X. Y. A Microchip-Based Model Wound with Multiple Types of Cells. Lab Chip 2011, 11, 2819−2822. (35) Akasaka, Y.; Ono, I.; Yamashita, T.; Jimbow, K.; Ishii, T. Basic Fibroblast Growth Factor Promotes Apoptosis and Suppresses Granulation Tissue Formation in Acute Incisional Wounds. J. Pathol. 2004, 203, 710−720.

(7) Ren, G. G.; Hu, D. W.; Cheng, E. W. C.; Vargas-Reus, M. A.; Reip, P.; Allaker, R. P. Characterisation of Copper Oxide Nanoparticles for Antimicrobial Applications. Int. J. Antimicrob. Agents 2009, 33, 587−590. (8) Li, W.; Dong, K.; Ren, J. S.; Qu, X. G. β-Lactamase-Imprinted Responsive Hydrogel for the Treatment of Antibiotic-Resistant Bacteria. Angew. Chem., Int. Ed. 2016, 55, 8049−8053. (9) Wang, Z. Z.; Dong, K.; Liu, Z.; Zhang, Y.; Chen, Z. W.; Sun, H. J.; Ren, J. S.; Qu, X. G. Activation of Biologically Relevant Levels of Reactive Oxygen Species by Au/g-C3N4 Hybrid Nanozyme for Bacteria Killing and Wound Disinfection. Biomaterials 2017, 113, 145−157. (10) Kittler, S.; Greulich, C.; Diendorf, J.; Koller, M.; Epple, M. Toxicity of Silver Nanoparticles Increases During Storage Because of Slow Dissolution under Release of Silver Ions. Chem. Mater. 2010, 22, 4548−4554. (11) Zhao, Y. Y.; Tian, Y.; Cui, Y.; Liu, W. W.; Ma, W. S.; Jiang, X. Y. Small Molecule-Capped Gold Nanoparticles as Potent Antibacterial Agents that Target Gram-Negative Bacteria. J. Am. Chem. Soc. 2010, 132, 12349−12356. (12) Zhao, Y. Y.; Chen, Z. L.; Chen, Y. F.; Xu, J.; Li, J. H.; Jiang, X. Y. Synergy of Non-Antibiotic Drugs and Pyrimidinethiol on Gold Nanoparticles Against Superbugs. J. Am. Chem. Soc. 2013, 135, 12940−12943. (13) Feng, Y.; Chen, W. W.; Jia, Y. X.; Tian, Y.; Zhao, Y. Y.; Long, F.; Rui, Y. K.; Jiang, X. Y. N-Heterocyclic Molecule-Capped Gold Nanoparticles as Effective Antibiotics Against Multi-Drug Resistant Bacteria. Nanoscale 2016, 8, 13223−13227. (14) Elander, R. P. Industrial Production of β-Lactam Antibiotics. Appl. Microbiol. Biotechnol. 2003, 61, 385−392. (15) Andrade, R. B. Total Synthesis of Desmethyl Macrolide Antibiotics. Synlett 2015, 26, 2199−2215. (16) Greiner, A.; Wendorff, J. H. Electrospinning: a Fascinating Method for the Preparation of Ultrathin Fibres. Angew. Chem., Int. Ed. 2007, 46, 5670−5703. (17) Yang, D. Y.; Lu, B.; Zhao, Y.; Jiang, X. Y. Fabrication of Aligned Fibirous Arrays by Magnetic Electrospinning. Adv. Mater. 2007, 19, 3702−3706. (18) Yang, D. Y.; Niu, X.; Liu, Y. Y.; Wang, Y.; Gu, X.; Song, L. S.; Zhao, R.; Ma, L. Y.; Shao, Y. M.; Jiang, X. Y. Electrospun Nanofibrous Membranes: a Novel Solid Substrate for Microfluidic Immunoassays for HIV. Adv. Mater. 2008, 20, 4770−4775. (19) Chen, W.; He, S.; Pan, W. Y.; Jin, Y.; Zhang, W.; Jiang, X. Y. Strategy for the Modification of Electrospun Fibers that Allows Diverse Functional Groups for Biomolecular Entrapment. Chem. Mater. 2010, 22, 6212−6214. (20) Du, Q.; Wu, J. B.; Yang, H. Pt@Nb-TiO2 Catalyst Membranes Fabricated by Electrospinning and Atomic Layer Deposition. ACS Catal. 2014, 4, 144−151. (21) Shirole, A.; Sapkota, J.; Foster, E. J.; Weder, C. Shape Memory Composites Based on Electrospun Poly(vinyl alcohol) Fibers and a Thermoplastic Polyether Block Amide Elastomer. ACS Appl. Mater. Interfaces 2016, 8, 6701−6708. (22) Zhao, Q. L.; Wang, S. W.; Xie, Y. Y.; Zheng, W. F.; Wang, Z.; Xiao, L.; Zhang, W.; Jiang, X. Y. A Rapid Screening Method for Wound Dressing by Cell-on-a-Chip Device. Adv. Healthcare Mater. 2012, 1, 560−566. (23) Dong, R. H.; Jia, Y. X.; Qin, C. C.; Zhan, L.; Yan, X.; Cui, L.; Zhou, Y.; Jiang, X. Y.; Long, Y. Z. In situ Deposition of a Personalized Nanofibrous Dressing via a Handy Electrospinning Device for Skin Wound Care. Nanoscale 2016, 8, 3482−3488. (24) Nagarajan, S.; Soussan, L.; Bechelany, M.; Teyssier, C.; Cavailles, V.; Pochat-Bohatier, C.; Miele, P.; Kalkura, N.; Janot, J. M.; Balme, S. Novel Biocompatible Electrospun Gelatin Fiber Mats with Antibiotic Drug Delivery Properties. J. Mater. Chem. B 2016, 4, 1134−1141. (25) Martin, J. T.; Milby, A. H.; Ikuta, K.; Poudel, S.; Pfeifer, C. G.; Elliott, D. M.; Smith, H. E.; Mauck, R. L. a Radiopaque Electrospun Scaffold for Engineering Fibrous Musculoskeletal Tissues: Scaffold 5745

DOI: 10.1021/acsnano.7b01240 ACS Nano 2017, 11, 5737−5745