Silver Inlaid with Gold Nanoparticles: Enhanced Antibacterial Ability

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Silver inlaid with gold nanoparticles: enhanced antibacterial ability coupled with the ability to visualize antibacterial efficacy Qing Li, Fei Lu, Hongli Ye, Kun Yu, Bitao Lu, Rong Bao, Yang Xiao, Fangyin Dai, and Guangqian Lan ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.8b00931 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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ACS Sustainable Chemistry & Engineering

Silver inlaid with gold nanoparticles: enhanced antibacterial ability coupled with the ability to visualize antibacterial efficacy Qing Li a,1, Fei Lua,b,1, Hongli Yea, Kun Yua, Bitao Lua,, Rong Bao c, Yang Xiaod, Fangyin Daia,b,

Guangqian Lana,b*

a

College of Textile and Garments, Southwest University, No.2 Tiansheng Road, BeiBei District,

Chongqing 400715, China

b

Chongqing Engineering Research Center of Biomaterial Fiber and Modern Textile, No.2 Tiansheng

Road, BeiBei District, Chongqing 400715, China

c

The Ninth People’s Hospital of Chongqing, No. 69 Jialing Village, Beibei District, Chongqing,

400715, China

d

Sericulture and Agri-Food Research Institute of Guangdong Academy of Agriculture Science, No.

133 Yiheng Road, Dongguan Zhuang, Tianhe District, Guangzhou Province, 510610, China

*Corresponding author: College of Textile and Garments, Southwest University, Chongqing 400715, China. Phone: +8613594005200; E-mail: [email protected] 1

Equally contributed.

ABSTRACT: Silver nanoparticles (Ag NPs) are widely used against bacteria, but further applications are restricted by their cytotoxicity, and the antibacterial efficiency cannot be measured because of the risk 1

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of development of multidrug resistance (MDR) with use of antibiotics. An alloy nanostructure of gold nanoparticles (Au NPs) inlaid on Ag NPs was synthesized using egg white protein (denoted here as Au-Ag NPs), exhibited enhanced antibacterial effect, and can visually indicate the antibacterial efficacy by fluorescence. Interestingly, the fluorescence recovered after antibacterial action. Au-Ag NPs showed enhanced antibacterial effect, due to higher reactive oxygen species (ROS) generation than Ag NPs, after adhering to the surface of bacteria, suggesting that the silver content in Au-Ag NPs can be tuned to reach high antibacterial activity with low cytotoxicity. Au-Ag NPs visualized bacteria by fluorescence changes, which makes the antibacterial process clear and allows the dosage of antibacterial agents to be controlled accurately, which can prevent MDR. Efficient antibacterial activity coupled with the ability to visualize bacterial processes allow Au-Ag NPs to be a potential application in medicine and biosensing. Key words: alloy nanoparticles, visualization, fluorescence, egg white, non-toxic, antimicrobial agent INTRODUCTION Multidrug resistant (MDR) bacteria risen with the widely applied of antibiotic [1-4], there is an urgency in searching for new strategies to design safe and effective antibacterial materials. Ag NPs have been extensively investigated because of their strong and broad-spectrum antibacterial ability, which shows the potential as weapons against bacteria, especially against MDR bacteria [5-8]. However, the cytotoxicity of Ag NPs often restricted their practical applications. For example, antibacterial activity is usually enhanced by raising the silver content, but this increases cytotoxicity, which raises concerns for their use as safe drugs [5, 9-12]. Studies have been conducted to address the possibility of improving Ag NPs, although, to date, the challenges have not been overcome. One potential approach to decrease Ag NP toxicity is to form an alloy with Au; this approach has been used to treat Daphnia magna and may reduce their environmental impact as reported by Li et al. [13]. Au NPs decorated with Ag NPs might be used to treat bacterial infections in a safe and efficient manner; however, the cytotoxicity of these nanoparticles was not tested on mammalian cells. Wang et al. [12] designed a Au–Ag alloy structure with a hollow inward porous wall (i.e. an Au–Ag nanocage). This novel nanocage structure had broad-spectrum antibacterial properties by the induction of ROS generation, destruction of cell membrane, and induction of cell apoptosis. Through the use of this nanostructure, the antimicrobial property of Ag NPs was greatly enhanced; however, the cytotoxicity concern was not eliminated. Hu et al. [14] reported a novel tri-metallic core/shell nanostructure using Ag-Pt nanodots grown epitaxially on Au nanorods (Au@PtAg NRs). This kind of silver nanomaterial possesses a strong composition dependence, showing obvious antibacterial activity with increased silver content, but the high silver content remains a potential cytotoxic hazard. Thus, a novel strategy that decreases the silver content of Ag NPs, increases antibacterial activity, and reduces cytotoxicity, is of great importance for the practical treatment of antibacterial infections. Having a 2


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reagent where the antibacterial ability could be visually observed using naked eye would help significantly in picking the correct dose of reagent to use. Bio-conjugated gold nanomaterials have interested many researchers for the properties of sensing and detection of bacteria [15-17]. For example, Paresh et al. [18] modified gold nanoparticles with anti-salmonella antibodies that can specifically and easily identify S. typhimurium. A colorimetric change, from pink to a bluish color, was observed after mixing the gold nanoparticles with S. typhimurium. This mainly occurs due to changes in inter-particle interactions as a result of the aggregation of these nanoparticles on bacterial surface. Although this colorimetric detection is simple, this method may not be practical for day-to-day detection of bacteria. In addition, the aggregation of nanoparticles may lead to the reproduction in bacterial contamination. Paresh et al. [19] researched a Rh-6G modified antibody-conjugated, popcorn-shaped technique with Surface Enhanced Raman Scattering (SERS), and based on Au NPs, to detect MDR Salmonella DT104, in which very low concentrations of bacteria were recognized; however, the SERS signal is subject to interference. Wang and Irudayaraj [20] studied amine-modified gold nanorods, which can simultaneous detect of S. typhimurium and E. coli within 30 min according to the changes in local surface plasma resonance (LSPR), at a low concentration of 102 CFU/mL. However, before LSPR becomes a commercially common technique, more robust LSPR substrates must be developed. Although the above studies on gold nanoparticles have contributed greatly to the development of multiple bacteria sensors, they are all hampered by one or more factors that limit their practical application. Furthermore, no relevant approach has been developed that combines both bacterial sensing and killing, which contributes to controlling the progress of visual detection of antibacterial effects. In view of the cytotoxicity challenges faced in utilizing Ag NPs, and the wide use of gold nanoparticles in sensing bacteria, we aimed to design a new Ag-based antibacterial nanostructure prepared using egg white [21-23] with Au NPs inlaid epitaxially on Ag NPs. This nanostructure possesses large specific surface area, which can prevent them from aggregating and lead to greater levels of ROS generation, thereby enhancing their antibacterial activity and decreasing cytotoxicity by reducing the overall amount of silver. In addition, Au NPs reduced by egg white have a high red fluorescence, and owing to the inlaid structure, the fluorescence properties of Au NPs can be inherited well in Au-Ag NPs. Therefore, Au-Ag NPs have high antimicrobial effect, exhibit red fluorescence, and can thus be used for both the sensing and killing of bacteria. Additionally, they make the antibacterial activity visual to the naked eye, through which the dose of Au-Ag NPs can be regulated accurately.

EXPERIMENTAL SECTION 3



Materials. If not specifically specified, all chemicals were obtained from Sigma-Aldrich Co. (Shanghai, China), and used as received. New Zealand white rabbits (two months old) were obtained from the Animal Laboratory Center of Third Military Medical University. Animal experiments were approved by the National Center of Animal Science Experimental Teaching (ASET) and were performed according to institutional ethical use protocols. Preparation of Au-Ag NPs. 0.6 mL of HAuCl4 (10 mg/mL), 5 mL of egg white, and 4 mL of NaOH (4 mg/mL) were mixed with 16.4 mL of deionized water in sequence. The mixed solution was stirred for 5 min and cultured in a 100°C water bath for 10 min. Finally, 0.4 mL of AgNO3 (10 mg/mL) was added immediately and dropwise. Ag NPs and Au NPs were produced by the similar method as controls. For preparation of Ag NPs, 5 mL of egg white and 4 mL of NaOH (4 mg/mL) were added to 17 mL of deionized water in sequence, the mixture was stirred for 5 min and cultured for 10 min in a 100°C water bath, after which 0.4 mL of AgNO3 (10 mg/mL) was added immediately and dropwise. For preparation of Au NPs, 0.6 mL of HAuCl4 (10 mg/mL), 5 mL of egg white, and 4 mL of NaOH (4 mg/mL) were mixed with 16.4 mL of deionized water in sequence. The mixed solution was stirred for 5 min, and then cultured in a 100°C water bath for 10 min, after which 0.4 mL of deionized water was added immediately and dropwise. Characterization. The morphology was determined using a transmission electron microscope (TEM, Tokyo, Japan). The Energy Dispersive X-ray (EDX) mapping was studied using a double Cs-corrected JEOL ARM200F TEM. X-ray photoelectron spectroscopy (XPS) was recorded on an X-ray photoelectron spectrometer (Shimadzu, Japan). X-Ray diffraction (XRD, Shimadzu XRD6000) analysis was used to evaluate the crystallographic structures. Fourier Transform Infrared (FTIR) were assessed on an alpha FTIR spectrometer (Karlsruhe, Germany). Absorption spectra were recorded on an UV-2550 UV−vis spectrophotometer (Hitachi, Japan). Fluorescence measurements were measured using a FluoroMax-4P spectrophotometer with excitation at 375 nm. Antimicrobial effect. The minimal inhibitory concentration (MIC) was tested on the basis of a previously reported method [5]. Samples and the S. aureus and E. coli nutrient solution (107 CFU/mL; collected at log phase stage) were cultured at 37°C in the incubator for 8 h. The final silver concentration of Au-Ag NPs was set as 3.13, 1.56, 0.78, 0.39, 0.2, 0.1, and 0.05 µg/mL, the final concentration of Ag NPs was set as 45, 40, 35, 30, 25, 6.25, 3.13, 1.56, and 0.78 µg/mL respectively. The solution was measured at OD600 to evaluate the amount of bacteria. Each assay 4


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was performed in triplicate. Au-Ag NPs were used in subsequent tests at a concentration of 0.1 µg/mL (the concentrations of Ag NPs and Au NPs control corresponding to that of Au-Ag NPs were 0.027, and 0.073 µg/mL, respectively). The antibacterial ability of these samples was evaluated using the bacterial inhibition ring test. Nutrient solution containing bacteria (106 CFU/mL; harvested at log phase stage) was equably spread on agar plates. Filter paper immersed in nanoparticle solutions (circular disk, 17-mm diameter) was positioned on agar plates and cultured in incubator at 37°C overnight. The average size of these inhibition zones was recorded to assess the antibacterial effects. Fluorescence-based live cell/dead cell method was used to determine the bacterial death [24]. To achieve this, 1.5 mL of bacterial suspension (106 CFU/mL, harvested at log phase stage) was obtained and washed with PBS. The suspension was cultured with samples at 37°C for 1 h, and bacterial suspension without treatment was used as control. 100 µL of mixture containing SYTO 9 stock and propidium iodide was mixed with the suspension and incubated for 15 min. A Leica DMI 4000B fluorescence microscope was used to visualize the samples. ROS Measurement. The 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay was performed for ROS measurement as previously described [25]. Briefly, the bacterium suspensions (E. coli and S. aureus, harvested at log phase stage) were attenuated to 2.5 × 106 CFU /mL, followed the addition of DFCH-DA at a ratio of 1:1000, the final concentration was 10 µM. The mixed solution was then cultured with continuous shaking at 37°C for 20 min. The pelleted bacteria were then washed with PBS, after which Au-Ag NPs were added, and Ag NPs possessing the same silver content were used as control. Fluorescence was evaluated on a FL4500 fluoroMax-4P spectrophotometer (Hitachi, Japan), with excitation and emission at 488 and 525 nm, respectively. Nucleic Acid Leakage. E. coli and S. aureus (collected at log phase stage) were cultured with samples for 2 h, centrifuged at 8,000 rpm for 5 min to obtain supernate, which was immediately filtrated with a 0.22 µm injector filtrator. Cellular contents are freed into the nutrient solution if the cytomembrane is broken; therefore, the presence of material that can absorb light at 260 nm was determined using a UV-vis spectrometer [26]. Morphology changes of bacteria. SEM was performed to record the morphology changes of bacteria caused by treatment with Au-Ag NPs [26]. S. aureus and E. coli (5×106 CFU/mL; harvested at log phase stage) were cultured with samples for at 37°C 2 h, and then pelleted by centrifugation at 8,000 rpm. The precipitate was washed with PBS, fixed using 2% glutaraldehyde, 5



and treated with 60, 70, 80, 90, and 100% ethyl alcohol for 15 min. The obtained dehydrated cell was dried on a clean glass sheet at room temperature, and then coated with gold for SEM analysis. Sensing of Bacterial Efficiency. S. aureus and E. coli (harvested at log phase stage) were separated from bacterial culture by centrifuging at 8000 rpm, and mixed with samples. The final concentrations of bacteria were 107, 106, 105, 104, 103, 102 and 10 CFU/mL; an Au-Ag NP solution without bacteria was used as the control, this test was taken at 37°C. Ag NPs and Au NPs were taken as controls. The fluorescence of the mixtures was evaluated using a FluoroMax-4P spectrophotometer with excitation at 375 nm, and photographed under UV light both before culturing and after culturing for 12 h at 37°C. The mixtures were obtained and smeared on agar plates, cultured at 37°C overnight to assess the bacterial-sensing property. Samples were cultured with 107 CFU/mL of bacterial suspension for 0 and 12 h to determine the zeta potential using a Zetasizer Nano Instrument (Malvern Ltd.). NPs were separated from bacteria after the 12 h incubation through centrifuging at 8,000 rpm for 10 min, the supernatant was centrifuged for 10 min at 20,000 rpm; the sediment was dispersed in deionized water to evaluate the zeta potential. Cytotoxicity Studies. Mouse skin fibroblasts (L929 cells) were incubated in DMEM containing 3% antibiotics and 10% fetal bovine serum (FBS) for 1 day. L929 cells were seeded in 96-well plates (1 × 104 cells/well) and cultured at 37°C for 24 h. The cells were then treated using Au-Ag NPs, Ag NPs, and Au NPs containing the same amount of gold. After incubation for 1, 2, or 3 days, the cell viability was tested by the MTT assay. MTT solution (10 µl, 5 mg/mL) was transferred to each well and incubated for 4 h. Finally, the formazan reaction product was then dissolved in 0.1 mL of dimethyl sulfoxide (DMSO). All solutions were then determined using a microplate reader (Thermo Fisher Scientific) at 490 nm. L929 cells cultured with samples for 1 day were visualized under light and fluorescence microscopes after staining with propidium iodide (PI) for 20 min. In Vivo Wound Healing. Rabbits were drugged using xylazine hydrochloride injection before surgical operation. On both sides of the backbone of each rabbit, four full-thickness and circular skin wounds (3 cm in diameter) were carved using a surgical scalpel. The four wounds on each rabbit were divided into four sections: model group, Au-Ag NP control group, Ag NP control group, and blank control group (n = 6). Then, 150 µL of S. aureus suspension (1.0 × 106 CFU/mL, harvested at log phase stage) was added on the wounds to infect the wounds of the model group and NPS control groups. The suppuration on the infected wounds was observed after 3 days, 100 µL of Au-Ag NPs and Ag NPs were smeared on the wounds in relevant control groups once a day, respectively, the treatment was persistently performed for 13 days. NPs weren’t used to treat the 6


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wounds of the blank control group and the model group. After treatments, the wounds were secured using gauze and crepe bandages, and rabbits were caged, cared and monitored daily. The wounds were photographed after the euthanasia of rabbits on days 3, 8, and 13. All photographs were changed with the uniform resolution and size; pixel method was employed to assess the wound contraction ratio. Histological Evaluation. After euthanasia, the treated wound with surrounding skin were collected for histological evaluation. Samples were fixed using 10% formaldehyde for 1 day, and then the samples were vertically sectioned and stained using Masson’s trichrome staining and hematoxylin and eosin (H&E) to detect collagen fibers. A DXM 1200F microscope (Nikon H600L; Germany) was used to observe the sections.

Figure 1. (a) Conceptual representation of preparing Au-Ag NPs. (b, c) TEM micrographs and (d) EDX mappings (red = gold, blue = silver). (e) FTIR spectra (red= egg white, black = Au-Ag NPs). (f) Full XPS spectrum of Au-Ag NPs. RESULTS AND DISCUSSION 7



Characteristics. Figure 1a showed a schematic outline of the preparation of Au-Ag NPs. Several small Au NPs were inlaid on a larger Ag NP, which composed of the Au-Ag NPs (Figure 1b). According to the TEM micrographs (Figure S1), the size of Au NP and Ag NP were about 5 nm and 10 nm, respectively. The isotropic nature of these NPs may be due to the capping by molecules of egg white that prevents particle agglomeration [27]. The space lattice of Au NP and Ag NP in Au-Ag NPs was showed in Figure 1c as being 0.2206 and 0.2113 nm, respectively, which assigned to the (111) crystal plane spacing of the face-centered cubic gold and silver, respectively, while it isn’t identical with the (111) lattice spacing of Au (0.235 nm) and Ag (0.236 nm), suggesting the involvement of egg white protein in the preparation of alloy Au-Ag NPs [28]. This inlaid structure can be supported by the EDX mappings. As the EDX mapping images shown in Figure 1d, Ag existed in the interior (blue), and Au was observed distributing across the entire particle (red), indicating an inlaid structure with Au NPs inlaid on Ag NPs. As shown in Figure 1e, asymmetrical stretching vibrations of C=O stretching (1653 cm-1) and N–H bending (1540 cm-1) showed the existence of amide bonds. In the spectrum of Au-Ag NPs, a peak around 3200–3500 cm-1, which corresponding to –OH and –NH stretching vibration, was wider than that of egg white. The peaks at 1037 cm-1 showed some difference in their spectra, indicating the structural changes of egg proteins. These results confirmed the interplay between egg white protein and Au-Ag NPs. The XPS spectrum analysis clearly illustrates compounding between nanoparticles and egg white protein, and between Au0 and Ag0 in alloy Au-Ag NPs. Figure S2a and Figure S3a showed the full scanned spectra of Au NPs and Ag NPs, respectively. In the spectrum of Au NPs (Figure S2b), two peaks at 88.4 eV and 84.4 eV were visualized respectively corresponding to Au 4f5/2 (Au (I)) and Au 4f7/2 (Au (0)); and in the spectrum of Ag NPs (Figure S3b), two peaks at 374.2 eV and 367.8 eV were visualized respectively assigning to Ag 3d3/2 (Ag (0)) and Ag 3d5/2 (Ag (I)). A peak at 399.6 eV observed for Au NPs in Figure S2c and a peak at 399.7 eV observed for Ag NPs in Figure S3c were both corresponding to the N1s, and showed the presence of egg white protein. Meanwhile, peaks for Au NPs (Figure S2d) and Ag NPs (Figure S3d) both at 166.2 eV could support the presence of the S2p. The sulfur element resided in the cysteine of protein, and the S2p may consist in the formation of Au−S and Ag−S bonding. The XPS spectrum of Au-Ag NPs also illustrates compounding between Au0 (83.78 eV and 87.48 eV) and Ag0 (373.48 eV and 367.38 eV), which are peaks in the full scanned spectra (Figure 1f), and are shown in detail for Au and Ag separately in Figure 2a and b. Compounding between Au0 and Ag0 might be formed at the S-containing interior and exterior of the protein scaffold via metal−S binding [28], this could be supported by peaks at 399.4 eV (Figure 2c) and 167.2 eV (Figure 2d), which suggesting the involvement of the N1s and the S2p, respectively. These data therefore strongly support the existence of egg white protein and the compounding of Au0 and Ag0 in alloy Au-Ag NPs [29]. XRD analysis was determined to assess the crystal pattern of Au-Ag NPs. Figure 2e showed the spectrum with small peaks at 37.95°, 44.33°, 64.32°, and 77.22°, which respectively assigned to (111), (200), (220), and (311) planes, highly 8


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matched the Joint Committee on Powder Diffraction Standards (JCPDS) file no. 65-8424, which indicated the existence of alloy Au-Ag NPs. Figure 2f showed the absorption spectrum of Au-Ag NPs from 250 to 500 nm, which can be explained by the π-π* transition of the C=O bands of amide linkage in protein. Figure 2f showed the spectrum of Au-Ag NPs with a peak at 622 nm and the fluorescent image with red color under 375 nm excitation, due to the small size of Au NPs [30], and the change in color from pale yellow (in daylight) to red (in UV-light) provides Au-Ag NPs with potential applications in biosensing.

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Figure 2. XPS spectra of (a) Au 4f, (b) Ag 3d, (c) N1s, and (d) S2p. (e) XRD spectrum analysis. (f) UV– vis absorption spectrum (black) and fluorescence emission spectrum (red). Insets: the pictures of Au-Ag NPs in sunlight and 375 nm UV-light. Antimicrobial effect. When the silver content of Au-Ag NPs reached the MIC (0.39 and 0.78 µg/mL against E. coli and S. aureus, respectively), the growth of the bacteria was strongly inhibited (Figure 3a). The MIC values for the Au-Ag NPs were nearly 60 fold lower against E. coli and 40 fold lower against S. aureus than those of Ag NPs (25 and 30 µg/mL against E. coli and S.

aureus, respectively) (Figure 3b). The MIC values also showed higher antimicrobial efficiency of these NPs, one possible explanation for why S. aureus are more resistant to them, may be due to their harder and thicker membranes than those of E. coli [14]. The antimicrobial activities were tested using a zone inhibition assay, are shown in Figure 3c. There was a larger inhibition zone for Au-Ag NPs against E. coli and S. aureus (36.4 mm and 35.3 mm in average diameters, respectively) compared with Ag NPs (24.5 mm and 23.8 mm in average diameters, respectively), suggesting enhanced antibacterial activity for Au-Ag NPs. This conclusion can be supported by fluorescence micrographs of bacteria after incubation with Au-Ag NPs or Ag NPs (Figure 3d). Under fluorescence microscopy, live bacterial cells with complete membranes showed green, while dead cells with broken membranes presented red. Bacteria were all dead after incubation with Au-Ag NPs, showing clear red fluorescence compared to the control groups. In contrast, the fluorescence micrographs of bacteria incubated with Ag NPs showed both red and green fluorescent signals, indicating that some bacteria still survived. These data confirm the increased antimicrobial effect of Au-Ag NPs, which can be due to the catalytic action of Au NPs in Au-Ag NPs. The ability to visualize antibacterial progress provides Au-Ag NPs with the possibility to be applied in medical and biosensing settings.

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Figure 3. Antibacterial effects of NPs against E. coli and S. aureus. (a, b) MICs measurements. (c) Inhibition zones. (d) Fluorescent photographs of bacteria cultured with Au-Ag NPs; green signal and red signal represent live and dead cells, respectively. ** P < 0.01. Mechanism of Antibacterial Activity. Previous studies have reported that ROS contribute to effective antibacterial activity by destroying the bacterial membrane [14, 31-33]. The ROS generation was evaluated to clarify the mechanism of the increased antibacterial effects seen for Au-Ag NPs. As Fig 4a shown, ROS generation was higher (by approximately 50%) in both E. coli and S. aureus for Au-Ag NPs compared to Ag NPs. The increased ROS levels could therefore explain the enhanced antimicrobial ability of Au-Ag NPs versus Ag-NPs. A schematic representation summarizing this is mechanism shown in Figure 4b [34].

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Figure 4. Mechanism of the increased antibacterial ability of Au-Ag NPs. (a) ROS generation measurments, and (b) a schematic comparison of Ag NP (left) and Au-Ag NP (right) killing of S. aureus through the induction of ROS. Destruction of Bacterial Membrane. To better study the mechanism of action of Au-Ag NPs against bacteria, SEM was performed to observe bacterial morphology after treatment. Before treatment, bacteria possessed integrated membrane structures, whereas the integrity of cell membrane was broken after incubation with Au-Ag NPs, exhibiting a shriveled and colliquative morphology (Figure 5a). As a result of membrane disruption, the cytoplasmic contents could be observed around individual bacteria (Figure 5b). The release of cytoplasmic components, especially RNA, into the culture medium was easily detected by measuring the absorbance at 260 nm. The values for the medium from cultures of bacteria were low in the absence of nanoparticles, but increased significantly by over ten-fold in the present of NPs (Figure 5c). These data demonstrate that the antibacterial effects of these NPs are associated with increases in membrane permeability, as has been suggested previously [26]. Of note, the increase in bacterial cell leakage was 60% greater in bacteria treated with Au-Ag NPs than in those treated with Ag NPs, supporting the conclusion that Au-Ag NPs have higher antimicrobial activity.

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Fig 5. Morphological analysis. After incubation with Au-Ag NPs, (a) SEM and (b) TEM were performed to record the morphological changes of bacteria; the arrows with red color indicated the breakage of the

cell membrane. (c) Amount of RNA released from bacteria after incubation with Au-NPs and Ag NPs. * indicates P < 0.05.

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Figure 6. Fluorescence analysis. Photographs and fluorescence intensity measurement of solutions containing Au-Ag NPs and bacterial suspensions (a) before incubating, and (b) after incubating for 12 h. (c) Au-Ag NP solutions containing different concentrations of bacteria and cultured for 12 h were then analyzed for visualizing bacterial levels by culturing on agar plates. (d) Schematic representation of the effect of bacteria on the fluorescence of Au-Ag NPs. * indicates P < 0.05. Sensing of Antibacterial Efficiency. Multidrug resistant (MDR) bacteria usually appear as a result of overuse of antibiotics in bacterial infections, and this concern can be removed by accurately using antibacterial agents [35]. Au-Ag NPs showed high antibacterial activity, and the antibacterial efficacy could be visualized based on changes of fluorescence, allowing the dosage of antibacterial agents to be tuned accurately to avoid overuse of bacterial agents. The fluorescence intensity of Au-Ag NPs decreased in response to increasing bacteria levels (Figure 6a), especially when the concentration of added bacteria increased over 105 U/mL, at which levels wounds can be infected [36,37], the fluorescent color obviously varied from red to pale purple. The changes can be observed with the naked eye, and after the bacteria 14


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were all killed, the fluorescence recovered. The fluorescence intensity of Au NPs (Figure S4a and b) and Ag NPs showed no changes with increasing bacteria levels (Figure S5a and b), suggesting that the ability of Au-Ag NPs to visualize bacteria by fluorescence can be due to their special nanostructure. Furthermore, due to this special nanostructure, Au-Ag NPs showed higher antibacterial ability (Figure 6c) and could visually indicate bacteria by fluorescence (Figure 6d). The antibacterial efficacy could be observed via fluorescence changes to accurately tune the dosage of the antibacterial agent (Figure 6d). Au NPs showed no obvious antibacterial activity and possessed red fluorescence property that was not dependent on the presence of bacteria (Fig S4). Ag NPs possess antimicrobial activity lower than that of Au-Ag NPs, and without fluorescence (Fig S5). Additionally, the fluorescence intensity of Au-Ag NPs gradually recovered with the time of culture (Fig S6); changes of fluorescence were observed and the detection time was about 4 h. The changes of fluorescence, may be a result of the aggregation of Au-Ag NPs on bacterial surface. Au-Ag NPs showed high fluorescence as they can disperse stably in the solution and form a stable sol system. The reason may be that, according to the double-layer electrostatic repulsion energy (DLVO) theory [38-40], the double electric layer, which is formed by protein protective layer on Au-Ag NPs, can cause electrostatic repulsion. The Van der Waals attraction and the electrostatic repulsion between Au-Ag NPs reached equilibrium. When the bacteria were added into Au-Ag NPs solution, the equilibrium of this solution was broken. Living bacteria can move freely in solution, which may cause it to collide with these nanoparticles. Owing to the larger volume of bacteria compared to that of Au-Ag NPs, some of them might be attracted towards bacterial surface. Au-Ag NPs gathered on bacterial surface and no longer showed fluorescence due to the aggregation. With the increase in bacterial concentration added into the solution, more and more Au-Ag NPs were attracted and aggregated on bacterial surface; thus, the fluorescence intensity decreased. After culturing for 12 hours, Au-Ag NPs, which have been adsorbed on the surface of the bacteria, generated ROS to kill bacteria. As the bacteria were killed, the cell wall of bacteria was destructed, which lead to the release of RNA. Thus, Au-Ag NPs were no longer adsorbed on bacterial surface, and could stably disperse in the solution again. This may be because the electronegativity and repulsive force in this solution system were recovered, and the Van der Waals attraction and the electrostatic repulsion between Au-Ag NPs reached equilibrium again. Finally, this solution formed a new stable sol system, and the fluorescence of Au-Ag NPs returned to original intensity. This assumption is supported by zeta potential changes (Figure S7), the death of bacteria resulted the decrease of negative potential; after incubation with bacteria for 12 h, the negative potential of Au-Ag NP group significantly decreased by 51% (for E. coli) and 38% (for S. aureus). 15



Figure 7. (a) Viability of L929 cells incubated with various NPs for days 1, 2, and 3. (b) Fluorescence micrographs and light micrographs of L929 cells incubated with NPs for 1 day; green signal represent live cells, and red signal indicated dead cells. In Vivo Cytotoxicity. L929 cells were then treated with various nanoparticles for 1, 2, and 3 days to study their biocompatibility. On day 1, cell viability was under 100% for all the nanoparticle groups, as shown in Fig, 7a, and increased to over 100% on days 2 and 3. These data suggest that all of the nanoparticles used in this study were almost non-toxic on L929 cells. We also assessed dead L929 cells stained with PI using fluorescence microscopy and light microscopy (Figure 7b). Many of the live cells stained green adhered to the bottom of the petri dish, and the dead cells stained red floated free in solution. Using this assay, cell viability decreased only slightly in the presence of the three nanoparticles, indicating a negligible impact of Au-Ag NPs on cell viability. Au-Ag NPs showed almost no cytotoxicity, which can be owing to the prevention of particle agglomeration by egg white. Wound Healing. To demonstrate practical utility, the nanomaterials were tested in wound healing experiments. Wound contraction ratios and the average healing times were measured. Figure 8b shows the 16


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photographs of wound healing after treatment for 3, 8, and 13 days. On day 3, the wounds in model group showed constant and severe inflammation, the wounds in the blank group showed slight edema, and wounds in Au-Ag NPs group showed evidence of healing with approximately 35% contraction (Figure 8a). On day 8, wounds in the Au-Ag NP, Ag NP, and blank groups had become crusted, as compared to those in the model group, and the contraction of these wounds was 64%, 43%, 35%, and 6%, respectively. On day 13, wounds treated with Au-Ag NPs had complete wound re-epithelialization, whereas wounds treated with Ag NPs and wounds in the model and blank groups failed to heal completely. The average healing times and wound contraction ratios indicate that Au-Ag NPs facilitated wound healing faster than Ag NPs and the controls, this can be attributed to the excellent antibacterial property of Au-Ag NPs.

Figure 8. Wound healing analysis. (a) Wound contraction ratios. (b) Micrographs of wounds in Au-Ag NPs, Ag NPs, blank, and model groups at days 0, 3, 8, and 13. (c) Histological evaluation. The histological outcome for the untreated and treated wounds was evaluated at various time points using hematoxylin and eosin (H&E) staining (Figure 8c). Acute necrosis, edema, and inflammation cells could be surveyed in all groups on day 3. On day 8, the wounds treated with Au-Ag NPs and Ag NPs showed fibroblasts and collagen fibers, and the number of fibroblasts and collagen fibers was lower for 17



the Au-Ag NP group, indicating that this material was highly effective. Furthermore, mature granulation tissue appeared in the wounds in the Au-Ag NP group, the newborn epidermis, dermis, and microvessels could be observed, indicating that Au-Ag NPs made a more significant contribution to wound healing. On day 13, wounds treated with Au-Ag NPs had formed a complete, symmetrical and thickened epidermis; in the wounds of Ag NPs group, few inflammatory cells were visualized, and the epidermis and dermis appeared. While there were still amounts of inflammatory cells remaining in both the wounds in the blank group and the model group. These results could be ascribed to the beneficial antibacterial effects of these NPs, which prevented further infection. These data indicate that Au-Ag NPs possess high efficiency in promoting wound healing, presumably for reasons of high antibacterial activity and negligible cell cytotoxicity. CONCLUSION In summary, Au-Ag NPs, which synthesized by using egg white as reductant and protectant, showed increased antibacterial ability, and can visually indicate antibacterial process by fluorescence. Au-Ag NPs induced higher levels of ROS after assembling on the bacterial surface, resulting in destruction of the bacterial membrane to kill bacterial cells. In addition, Au-Ag NPs show red fluorescence and can indicate bacteria visually based the fluorescence intensity, furthering the application potential of Au-Ag NPs as antimicrobial agents. Au-Ag NPs exhibited low cytotoxicity, similar to that of Ag NPs, in L929 cells, demonstrating that Au-Ag NPs can reach the same antimicrobial efficiency compared with Ag NPs, but exhibiting lower cytotoxicity to cells via a reduction in the silver content. For purpose of assessing the practical application of Au-Ag-NPs, these nanoparticles were used on infected wounds, and found to significantly promote the healing of the wounds. This silver inlayed gold alloy structure therefore endows Au-Ag NPs with both safe antibacterial activity and a bacteria-sensing ability, making its antibacterial ability visual and easy to assess. The present findings suggest new opportunities for this nanomaterial in the biological imaging and sensor fields, as both an indicator and a sensor, and in the biomedical and medicine field, as an antibacterial agent.

Supporting Information. TEM micrographs of Au NPs and Ag NPs; XPS spectrum of Au NPs; XPS spectrum of Ag NPs; images of fluorescence intensity measurements and antibacterial test of Au NPs; images of fluorescence intensity measurements and antibacterial test of Ag NPs; fluorescence changes of Au-Ag NPs cultured with bacteria; negative potential analysis.

Notes The authors declare no competing financial interest. 18


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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 51703185), the Social development project of Guangdong province (No. 2017A020211015), and the Fundamental Research Funds for the Central Universities (nos. XDJK2017B041 and XDJK2017C012), and this work was also funded by National Under-graduate Training Programs for Innovation and Entrepreneurship (201710635019).

REFERENCES (1) Jonathan W. Betts, Amir S. Sharili, Roberto M. La Ragione, and David W. Wareham. In Vitro Antibacterial Activity of Curcumin–Polymyxin B Combinations against Multidrug-Resistant Bacteria Associated with Traumatic Wound Infections. Journal of Natural Products. 2016, 79, 1702-1706. (2) Hoque J , Adhikary U , Yadav V , Samaddar S , Konai MM , Prakash RG. Chitosan Derivatives Active against Multidrug-Resistant Bacteria and Pathogenic Fungi: In Vivo Evaluation as Topical Antimicrobials. Molecular Pharmaceutics. 2016, 13, 3578-3589. (3) Yinfeng Lyu, Xuan Yang, Sudeep Goswami, Bala Kishan Gorityala, Temilolu Idowu, Ronald Domalaon. Amphiphilic Tobramycin–Lysine Conjugates Sensitize Multidrug Resistant Gram-Negative Bacteria to Rifampicin and Minocycline. Journal of Medicinal Chemistry. 2017, 60, 3684-3702. (4) Xuan Yang, Sudeep Goswami, Bala Kishan Gorityala, Ronald Domalaon, Yinfeng Lyu, Ayush Kumar. A Tobramycin Vector Enhances Synergy and Efficacy of Efflux Pump Inhibitors against Multidrug-Resistant Gram-Negative Bacteria. Journal of Medicinal Chemistry. 2017, 60, 3913-3932. (5) Xiaomei Dai, Qianqian Guo, Yu Zhao, Peng Zhang, Tianqi Zhang, Xinge Zhang. Functional Silver Nanoparticle as a Benign Antimicrobial Agent That Eradicates Antibiotic-Resistant Bacteria and Promotes Wound Healing. ACS Applied Materials & Interfaces. 2016, 8, 25798-25807. (6) Liang D, Lu Z, Yang H, Gao J, Chen R. Novel Asymmetric Wettable AgNPs/Chitosan Wound Dressing: In Vitro and In Vivo EvaluationA CS. Applied Materials & Interfaces. 2016, 8, 3958-3968. (7) CD Tran, F Prosenc, M Franko, G Benzi. One-Pot Synthesis of Biocompatible Silver Nanoparticle Composites from Cellulose and Keratin: Characterization and Antimicrobial Activity. ACS Applied Materials & Interfaces. 2016, 8, 34791-34801. (8) Y Zhao, H Cao, H Qin, T Cheng, S Qian. Balancing the Osteogenic and Antibacterial Properties of Titanium by Codoping of Mg and Ag: An in Vitro and in Vivo Study. ACS Applied Materials & Interfaces. 2015, 7, 17826-17836.

19



(9) E Bolea, F Laborda, L Arola, JR Castillo. Detection and characterization of silver nanoparticles and dissolved species of silver in culture medium and cells by AsFlFFF-UV-Vis-ICPMS: application to nanotoxicity tests. The Analyst. 2014, 139, 914-922. (10) CY Flores, AG Miñán, CA Grillo, RC Salvarezza, C Vericat. Citrate-Capped Silver Nanoparticles Showing Good Bactericidal Effect against Both Planktonic and Sessile Bacteria and a Low Cytotoxicity to Osteoblastic Cells. ACS Applied Materials & Interfaces. 2013, 5, 3149-3159. (11) J Yang, N Xia, X Wang, X Liu, A Xu. One-pot one-cluster synthesis of fluorescent and bio-compatible Ag14nanoclusters for cancer cell imaging. Nanoscale. 2015, 7, 18464-18470. (12) Y Wang, J Wan, RJ Miron, Y Zhao, Y Zhang. Antibacterial properties and mechanisms of gold–silver nanocages. Nanoscale. 2016, 8, 11143-11152. (13) T Li, B Albee, M Alemayehu, R Diaz, L Ingham. Comparative toxicity study of Ag, Au, and Ag–Au bimetallic nanoparticles on Daphnia magna. Analytical and Bioanalytical Chemistry. 2010, 398, 689-700. (14) X Hu, Y Zhao, Z Hu, A Saran, S Hou. Gold nanorods core/AgPt alloy nanodots shell: A novel potent antibacterial nanostructure. Nano Research. 2013, 6, 822-835. (15) D Li, Y Dong, B Li, Y Wu, K Wang. Colorimetric sensor array with unmodified noble metal nanoparticles for naked-eye detection of proteins and bacteria. The Analyst. 2015, 140, 7672-7677. (16) OR Miranda, X Li, L Garciagonzalez, ZJ Zhu, B Yan. Colorimetric Bacteria Sensing Using a Supramolecular Enzyme–Nanoparticle Biosensor. Journal of the American Chemical Society. 2011, 133, 9650-9653. (17) W Wei, T Zhu, Y Wang, H Yang, Z Hao. Engineering a gold-specific regulon for cell-based visual detection and recovery of gold. Chemical Science. 2012, 3, 1780-1784. (18) PC Ray, SA Khan, AK Singh, D Senapati, Z Fan. Nanomaterials for targeted detection and photothermal killing of bacteria. Chemical Society Reviews. 2012, 41, 3193-3209. (19) SA Khan, AK Singh, Z Fan, D Senapati, PC Ray. Designing distance dependent SERS assay for monitoring photothermal antibacterial activity response. Chemical Communications. 2012, 48, 11091-11093. (20) Wang, C. and J. Irudayaraj. Gold Nanorod Probes for the Detection of Multiple Pathogens. Small. 2008, 4, 2204-2208. (21) R Lu, D Yang, D Cui, Z Wang, L Guo. Egg white-mediated green synthesis of silver nanoparticles with excellent biocompatibility and enhanced radiation effects on cancer cells. International Journal of Nanomedicine. 2012, 7, 2101-2107. 20


Page 20 of 23

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


(22) Selvaprakash, K. and Y.-C. Chen. Detection of ricin by using gold nanoclusters functionalized with chicken egg white proteins as sensing probes. Biosensors and Bioelectronics. 2017, 92, 410-416. (23) X Yan, H Li, T Hu, X Su. A novel fluorimetric sensing platform for highly sensitive detection of organophosphorus pesticides by using egg white-encapsulated gold nanoclusters. Biosens Bioelectron. 2017, 91, 232-237. (24) Z Zhao, R Yan, X Yi, J Li, J Rao. Bacteria-Activated Theranostic Nanoprobes against Methicillin-Resistant Staphylococcus aureus Infection. ACS Nano. 2017, 11, 4428–4438. (25) Yinghua Li, Zhengfang Lin, Mingqi Zhao, Tiantian Xu, Changbing Wang, Liang Hua. Silver Nanoparticle Based Codelivery of Oseltamivir to Inhibit the Activity of the H1N1 Influenza Virus through ROS-Mediated Signaling Pathways. ACS Appl Mater Interfaces. 2016, 8, 24385-93. (26) Y Wang, J Wan, RJ Miron, Y Zhao, Y Zhang. Antibacterial properties and mechanisms of gold-silver nanocages. Nanoscale. 2016, 8, 11143-52. (27) Vilas, V., D. Philip, and J. Mathew. Biosynthesis of Au and Au/Ag alloy nanoparticles using Coleus aromaticus essential oil and evaluation of their catalytic, antibacterial and antiradical activities. Journal of Molecular Liquids. 2016, 221, 179-189. (28) Tian L, Li Y, Ren T, et al. Novel bimetallic gold-silver nanoclusters with "Synergy"-enhanced fluorescence for cyanide sensing, cell imaging and temperature sensing. Talanta. 2017, 170, 530-539. (29) D Ye, Z Zhong, H Xu, C Chang, Z Yang, Construction of cellulose/nanosilver sponge materials and their antibacterial activities for infected wounds healing. Cellulose. 2015, 23, 749-763. (30) J Zhu, JF Wang, JJ Li, JW Zhao. Specific detection of carcinoembryonic antigen based on fluorescence quenching of Au-Ag core-shell nanotriangle probe. Sensors and Actuators B: Chemical.

2016, 233, 214-222. (31) P Korshed, L Li, Z Liu, T Wang. The Molecular Mechanisms of the Antibacterial Effect of Picosecond Laser Generated Silver Nanoparticles and Their Toxicity to Human Cells. PLoS One.

2016, 11, e0160078-e0160101. (32) Q Liu, Z Zhou, G Qiu, J Li, J Xie. Surface Reaction Route To Increase the Loading of Antimicrobial Ag Nanoparticles in Forward Osmosis Membranes. ACS Sustainable Chemistry & Engineering. 2015, 3, 2959-2966. (33) J Song, H Kim, Y Jang, J Jang. Enhanced Antibacterial Activity of Silver/Polyrhodanine-Composite-Decorated Silica Nanoparticles. ACS Applied Materials & Interfaces. 2013, 5, 11563-11568.

21



(34) P Yuan, X Ding, N Gao, H Zhu, YY Yi. Au-Ag core-shell nanoparticles for simultaneous bacterial imaging and synergistic antibacterial activity. Nanomedicine. 2017, 13, 297-305. (35) Tian J, Zhang J, Yang J, et al. Conjugated Polymers Act Synergistically with Antibiotics to Combat Bacterial Drug Resistance. ACS Applied Materials & Interfaces. 2017, 9. 18512-18520. (36) Miranda O R, Li X, Garciagonzalez L. Colorimetric bacteria sensing using a supramolecular enzyme-nanoparticle biosensor. Journal of the American Chemical Society. 2011, 133, 9650-9653. (37) Thiramanas R, Laocharoensuk R. Competitive binding of polyethyleneimine-coated gold nanoparticles to enzymes and bacteria: a key mechanism for low-level colorimetric detection of gram-positive and gram-negative bacteria. Microchimica Acta. 2016, 183, 389-396. (38) Derjaguin, B. V.; Landau, L. Theory of the Stability of Strongly Charged Lyophobic Sols and of the Adhesion of Strongly Charged Particles in Solution of Electrolytes. Acta Phys. Chim. URSS.

1941, 14, 30-59. (39) Verwey, E. J. W.; Overbeek, J. T. G. Theory of the Stability of Lyophobic Colloids; Elsevier Publishing Company. The Journal of Physical and Colloid Chemistry. 1948, 51, 631-636. (40) Miklavic, S. J.; Chan, D. Y. C.; White, L. R.; Healy, T. W. J. Double-Layer Forces between Heterogeneous Charged Surfaces. Phys. Chem. 1994, 98, 9022−9032.

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For Table of Contents Use Only:

Brief synopsis: Au-Ag NPs prepared using egg white had antibacterial efficacy coupled with the ability to visualize bacterial processes.

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Silver Inlaid with Gold Nanoparticles: Enhanced Antibacterial Ability

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