Chitosan Wound Dressing

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Silver inlaid with gold nanoparticle/chitosan wound dressing enhances antibacterial activity and porosity, and promotes wound healing Qing Li, Fei Lu, Guofang Zhou, Kun Yu, Bitao Lu, Yang Xiao, Fangying Dai, Dayang Wu, and Guangqian Lan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01180 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017

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Silver inlaid with gold nanoparticle/chitosan wound dressing

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enhances antibacterial activity and porosity, and promotes

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wound healing

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Qing Li a,1, Fei Lua,b,1,Guofang Zhoua, Kun Yua, Bitao Lua,,Yang Xiaoc, Fangying Daia,b, Dayang

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Wua,b, Guangqian Lana,b*

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a

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b

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400715, China

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c

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College of Textile and Garments, Southwest University, Chongqing 400715, China Chongqing Engineering Research Center of Biomaterial Fiber and Modern Textile, Chongqing

Sericulture & Agri-Food Research Institute of Guangdong Academy of Agriculture Science,

Guangzhou 510610, China

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ABSTRACT: Silver inlaid with gold nanoparticles (Au-Ag NPs) prepared by using egg white with an average sized of 10 nm and homogeneous dispersion were tested and presented red fluorescence. Au-Ag NPs were loaded into chitosan as wound dressing (CS-Au-Ag). CS-Au-Ag released silver ions faster, in higher amount, and in a more durable manner than chitosan dressing loaded with silver nanoparticles with the same silver content (CS-Ag), consequently, showing enhanced antibacterial activity. Cytotoxicity tests indicated that CS-Au-Ag showed low cytotoxicity to L929 cells similar to CS-Ag. These data suggest that cytotoxicity, which restricts further application of silver NPs, can be eliminated by decreasing the silver content. CS-Au-Ag presented rich and well-distributed pores, good mechanical properties, and enhanced swelling and retention properties, contributing to keeping the wound moist in the presence of residual egg white. Altogether, our results suggest that CS-Au-Ag greatly promoted wound healing compared to CS-Ag in vivo, demonstrating that CS-Au-Ag presents great potential for wound dressing, promoting wound healing.

*Corresponding author: College of Textile and Garments, Southwest University, Chongqing 400715, China. Phone: +8613594005200; fax: +8602368251228; e-mail: [email protected] 1 Equally contributed.

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INTRODUCTION The skin is the outermost layer of the human body and the body’s largest organ. It constitutes the first barrier of defense 1, 2. Extensive and deep skin lesions can be a big threat for the human body as they may bring about the destruction of the dermis and dermal elements 3. Dermal transplantation could solve this problem, but it is limited by the scarcity of donors and risk of rejection. Moreover, the cost may be a burden for the patients 4. Wound dressing is another strategy for wound healing. Wound dressing has garnered the attention of the scientific community in recent years as it plays an important role in wound healing 5-7. Ideal wound dressing exhibits good biological compatibility, biodegradability, water absorption and retention properties, no cytotoxicity, non-stick ability, and antibacterial effects8, 9. It prevents the wound from being infected, allows gas exchange, could be removed easily, absorbs excrescent wound exudate, and remains a part of the exudate to maintain local moisture of the wound, which accelerates wound healing 10-12. In recent years, chitosan has been widely used as a wound dressing because of its good properties, including antibacterial activity, water absorption, biocompatibility, biodegradability, non-cytotoxicity, non-antigenicity, and biological membrane function 13-15. Sarhan et al. reported a honey/chitosan nanofiber wound dressing, which possesses excellent antibacterial activity, biocompatibility, and moisturizing properties 16. In Diez-Pascual et al.’s study, wound healing bionanocomposites based on castor oil polymeric films reinforced with chitosan-modified ZnO nanoparticles (NPs) were fabricated, showing excellent cytocompatibility and enhanced mechanical property 17. Although chitosan possesses many good properties, its use as an independent wound dressing is limited by low mechanical strength and the fact that chitosan presents impactful antibacterial activity only in a liquid state. Additionally, its antibacterial activity is not sufficient for wound dressing 18. Hence, chitosan is usually chosen as a supporting material loaded with antibacterial agents as a wound dressing. Many antimicrobial agents were reported, including polymeric cations 19, 20, quaternary ammonium based compounds 21, 22, antimicrobial peptides 23, photodynamic agents 24, 25 , and inorganic NPs 26, 27. Among them, inorganic NPs (NPs) (e.g., silver, platinum and gold NPs) play significant role in the development of strong antimicrobial agents, and Ag NPs


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are the most widely used of inorganic NPs 28-30. Practical applications of chitosan/Ag NPs wound dressing are often restricted by agglomeration of Ag NPs 31, 32, which is toxic to wounded cells and tissues. Recently, many studies reported that using egg white as a reducer and stabilizer to prepare homogeneous-dispersed metal NPs with steerable shape and size 33, 34 endowed particles some distinctive properties such as enhanced radiation effects 35 and high fluorescence characteristics 36. For example, in Lu et al.’s study, homogeneous-dispersed gold−silver nanoclusters with an average size of 5 nm were prepared by using egg white 36, showing “synergy”-enhanced fluorescence for cyanide sensing, cell imaging, and temperature sensing. Thus, Ag NPs with homogeneous dispersion and small size can be prepared by using egg white as an antibacterial agent to load chitosan dressing. In addition, egg white is a transparent colloid substance rich in proteins. It possesses important functional properties such as gel forming properties, emulsification, foaming property, antibacterial ability, and biocompatibility 35, 37, 38. Egg white loaded on the wound dressing can improve the water absorption and retention properties of the wound dressing, keeping the wound moist to promote wound healing and allowing easy dislodgment. In view of the superiority of chitosan as a wound dressing supportive material and the finer reducibility and stability of egg white for preparing homogeneous NPs, we aimed to fabricate a competitive wound dressing with superb antibacterial properties, water absorption and retention properties, non-stick property, no cytotoxicity, and biocompatibility. In this study, silver inlaid with gold NPs were prepared by using egg white and loaded into chitosan dressing. This chitosan-silver-gold wound dressing presented red fluorescence because of the super small size of contained NPs, released silver ions persistently, and the silver ions level was higher than that of chitosan-silver wound dressing, which contained the same silver, thereby enhancing its antibacterial activity. Moreover, this wound dressing was non-toxic to L929 cells because of the homogeneous dispersion of Ag NPs. In addition, because of residual egg white, this chitosan wound dressing exhibited good mechanical properties, which maintained complete form, and enhanced water absorption and retention properties, contributing to keeping the wound moist and making the wound dressing nonstick. MATERIALS AND METHODS Materials. Chitosan (molecular weight: 10,000–30,000 Da, degree of acetylation: 85–95%) was purchased from Kelong Chemical Reagent (Chengdu, China). Auric chloride acid (HAuCl4), silver nitrate (AgNO3), and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich Company (Shanghai, China). Acetic acid was purchased from Chongqing Chuan Dong Chemical Co. (Chongqing, China). New Zealand white rabbits (20 weeks of age) were purchased from the Animal Laboratory Center of Third Military Medical University. All animal experiments and care were in compliance with institutional ethical use protocols and were approved by the National Center of Animal Science Experimental Teaching (ASET) at the College of Animal Science and Technology (CAST) in the Southwest University of China, in accordance with the college’s “Guide for the Care and Use of Laboratory Animals.” All the reagents were used as obtained without further purification. Synthesis of CS-Ag-Au. Briefly, 0.3 mL of 1% (w/w) HAuCl4 solution and 2.5 mL egg white were added to 8.2 mL deionized water. The solution was stirred for 5 min, followed by the addition of 1 mL of 0.4% (w/w) NaOH solution. The mixture was stirred for another 5


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min; subsequently, the solution was incubated in 100℃ water for 2 min and 0.2 mL of 1% (w/w) AgNO3 solution was then added slowly within 1 min with stirring. The solution was allowed to cool to room temperature naturally. The color turned yellowish, inferring the formation of Au-Ag NPs. Next, 0.1 mL acetic acid solution and 0.4 g of chitosan powder were added to the mixture in this order, and the mixture was stirred for an additional 15 min to homogenize the solution. Finally, the mixed solution was frozen at −20℃for 1 day, followed by freeze-drying for 48 h. Using the same methods, a chitosan and Ag NPs composite (CS-Ag) was prepared by using 0.2 mL of 1% (w/w) AgNO3 solution. A chitosan and Au NPs composite (CS-Au) was prepared by using 0.3 mL of 1% (w/w) HAuCl4 solution. A chitosan and egg white polymer (CS-EW) was prepared without HAuCl4 and AgNO3 and a pure chitosan sponge (CS) was prepared without egg white, HAuCl4, and AgNO3. Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) was performed on a Hitachi 8100 transmission electron microscope. A typical sample was prepared by dropping 10 µL of the nanoparticle solution onto a holey carbon TEM grid, followed by wicking away of the solution. The grid was subsequently dried in air and imaged. Scanning electron microscopy (SEM) For SEM analysis, the samples were lyophilized to remove their water content. The lyophilized samples were mounted onto sample holders using conductive tape and then sputter-coated with platinum using an E-1045 High Resolution Sputter Coater (Hitachi, Tokyo, Japan) at 30 mA to improve their conductibility. The structural morphology of the lyophilized samples was observed using a scanning electron microscopy (SEM; S600, Japan) at an accelerating voltage of 15 kV. Characterization. Fluorescence emission spectra were obtained using a FluoroMax-4P spectrophotometer (FL4500, Hitachi, Nagoya，Japan). The chemical bonds and chemical groups of CS-Au-Ag were evaluated by Fourier-transform infrared spectroscopy (FTIR; BRUKER alpha, Bruker, Karlsruhe, Germany). X-ray photoelectron spectroscopy (XPS) experiments were performed using a Kratos AXIS Ultra DLD X-ray Photoelectron Spectrometer (Shimadzu, Nagoya, Japan). Mechanical properties. The mechanical properties of the samples were measured by using a universal material testing instrument on a Shimadzu Autograph AGS-X machine fitted with a 5N load cell. After freeze-drying, samples were cut into cubes measuring 30 × 6 × 2 mm. All the samples were subjected to tensile tests at a constant rate of 20 mm/min. The same sample was tested repeatedly five times. Characterization of porosity. The liquid displacement method was used to evaluate the porosity of the samples. Volumes of samples were measured using Vernier calipers. Samples were dried in the oven at 45℃ for 24 h and weighted (Wd), after which the samples were soaked in a volume of ethanol for 24 h at room temperature to allow the ethanol to permeate through the pores of the sample. Next, the samples were weighted (Ww) after being hung under gravity for 30 s. The same sample was tested three times. The porosities of the samples

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were calculated by using the following equation: % =

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Evaluation of swelling and retention properties. The swelling and retention abilities of the samples were tested by using the gravimetric method. Samples were cut into a cylindrical

× %

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and V represent the density of ethanol and sample volume, respectively.


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shape with a diameter of 10 mm and a thickness of 5 mm. After weighting (Wb), the samples were dried in the oven at 37℃for 24 h; then, the samples were immersed in phosphate buffer saline (PBS) solution at 37℃ and harvested to be weighted (Wa) at different times. The same sample was tested three times. The swelling ratio was determined using the following

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equation: %& % =

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For retention tests, samples (PBS saturated) were incubated at 37℃ and weighed (Wc) at each time point. Before the tests, samples were dried and weighted (Wd), having the same sizes. The same sample was tested three times. The retention ratio was calculated by using the

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equation: % =

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Antibacterial activity. The inhibition zone method was carried out to evaluate the antimicrobial activity, and S. aureus and E. coli were used. After bacteria suspension (105 CFU/mL) were uniformly spread on agar plates, the sterilized samples of chitosan nanomaterials (circular disks, diameter of 17 mm) were placed on the agar plates and incubated for 12 h at 37℃, the diameter of the zone of inhibition around each disc was measured to evaluated the antibacterial effects. The bacterial death assay was conducted by using fluorescence-based cell live/dead method. One milliliter of the bacterial suspension (107 CFU/mL) was collected and washed with PBS three times, the suspension was treated with CS-Au-Ag and CS-Ag (each weights 0.1 g), and untreated bacteria were used as control. After culture at 37℃ for 1 h, 100 µL of fluorescent dyes were added to the solution and incubated for 20 min; live and dead bacteria were observed under an inverted fluorescence microscope (Leica DMI 4000B). Silver ion release. To test the release of silver ions, samples of CS-Au-Ag and CS-Ag (each sample weighed 0.1 g) were immersed in 30 mL of deionized water at 25℃ for 2, 4, 8, 16, 24, 48, and 72 h, respectively. The exudate was collected and filtered, and then analyzed by using an inductively coupled plasma mass spectrometer (ICP-MS, Thermo Electron Corporation). Cell morphology observation. The morphological changes of E. coli and S. aureus cultured with CS-Au-Ag and CS-Ag were recorded by SEM and TEM 39. Bacterial suspensions (107 CFU/mL) were incubated with CS-Au-Ag and CS-Au for 2 h at 37℃, centrifuged at 8000 rpm, and washed with PBS, following by fixation with 2.5% glutaraldehyde. The cells were dehydrated by sequential treatment with 60, 70, 80, 90, and 100% ethanol for 15 min and were coated on a sheet glass to dry at room temperature. Obtained samples were coated with gold for SEM. Cytotoxicity assay. Mouse skin fibroblast cells (L929 cells) were used to evaluate the cytotoxicity of CS-Au-Ag. Before the tests, the samples were cut into small wafers (diameter: 5 mm), UV-sterilized for 2 h, and immersed in 5 mL of serum-containing medium (2 mg/mL) at 37℃for 12 h. The cells (1 × 104 cells/well) were seeded in 96-well plates and incubated in atmospheric humidity (5% CO2, 95% air, 37℃) for 24 h, and then treated by using the sample supernatants. Untreated cells used as controls. The plates were cultured for 24, 48, or 72 h and tested by MTT assays. Formazan crystals were dissolved with dimethylsulfoxide (DMSO) after 4 h. The absorbance of all solution was evaluated at 490 nm on a microplate reader (Multiskan MK3). In vivo study. In vivo wound healing was evaluated by using New Zealand white rabbits.

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Full-thickness skin excision wounds measuring 3 cm in diameter were created on the dorsum of anesthetized rabbits. The first wound acted as a control without treatments, and the other wounds were covered with CS-Au, CS-Ag, CS-Au-Ag, CS-EW, or nothing. After surgery, the conditions of the wound surface on the dorsum of the rabbits were inspected daily. The rabbits were euthanized on days 3, 7, 11, and 15, and the wounds were photographed. All images were adjusted to be the same size and resolution, the pixel method was used to measure the wound contraction rate. The tissue specimens containing the entire wound and circular normal skin were harvested, fixed in 10% buffered formalin for 24 h, paraffin embedded, and sectioned vertically. After staining with hematoxylin and eosin (H&E), all samples were observed by using a DXM 1200F microscope (Nikon H600L; Germany).

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Fig. 1. Conceptual illustration of the preparation for CS-Au-Ag and use as a wound dressing. Using egg white to reduce HAuCl4, we obtained Au-Ag NPs with an average size of 5 nm, following the addition of Ag+. CS-Au-Ag wound dressing was prefabricated by compounding chitosan and Au-Ag NPs solution. RESULTS AND DISCUSSION Characterization of CS-Au-Ag. As shown by the TEM micrographs in Fig. 2a, Au NPs with an average size below 5 nm overlaid on top of a larger Ag NPs, having an average diameter of 10 nm as shown in Fig. 2b, and the space lattice was observed as being 0.2122 and 0.2231 nm (Fig. 2b). This silver inlay of gold NPs can be conceptually illustrated as shown in Fig. 2c. The small size could be considered as being characteristic of heterogeneous


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NPs. These small NPs showed red fluorescence under UV light because of the inlaid Au NPs with a size below 5 nm. The fluorescence of NPs has garnered the attention in the sense field40, 41. Thus, the red fluorescence endows Au-Ag NPs the potential of been applied in biosensing. The peak of Au-Ag NPs was slightly weaker than that of Au NPs peak as observed in Fig. 2d-f. This can be explained by the fact that the presence of Ag NPs induced the quenching effect of Au-Ag NPs. CS-Au-Ag inherited the red fluorescence well, presenting a slightly weaker red fluorescence slight than CS-Au (Fig. 1g, h).

Fig. 2. Transmission electron micrographs of Au-Ag NPs (a, b), and conceptual image of Au-Ag NPs (c). Micrographs of Au-Ag NPs (left) and Au NPs (right) under day light (d), the corresponding images under UV light (e), and the fluorescence spectra of Au-NPs and Au-Ag NPs (f). The micrographs of CS-Au-Ag and CS-Au under day light and UV light (g), and the fluorescence spectrum of CS-Au-Ag and CS-Au (h). Micrographs of insert shows the corresponding images.


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SEM. The structure and surface morphology of the chitosan nanocomposites were measured by SEM to evaluate the influence of the NPs. Pure chitosan composite without NPs exhibited a mixed and disorderly surface, with non-uniform fragment and pores ranging from 20 to 50 µm in size (Fig. 3a). After the addition of egg white, chitosan-egg white composite showed fluctuant and random surface structure, while the size of the pores increased to 50– 100 µm (Fig. 3b). When the chitosan composite was added of Au NPs, Ag NPs, or Au-Ag NPs, the surface structure of the chitosan nanocomposites became smooth, the pore distribution and shape were regular, and showed a favorable porous structure, and CS-Au-Ag showed the most uniform and regular surface structure of them (Fig. 3e, f). The pores of CS-Au ranged from 50 to 100 µm in size (Fig. 3c) and those of CS-Ag and CS-Au-Ag were 100–150 µm in size (Fig. 3d-f). The increased pore size and better surface structure of CS-EW, CS-Ag, CS-Au, and CS-Au-Ag was attributed to the presence of egg white. The better pore size and surface structure of the nanocomposites relative to the chitosan-egg white may be explained by the interaction between NPs and chitosan. Further, the optimal pore size and surface structure of CS-Au-Ag is attributable to the increased interaction resulting from the incorporation of Au-Ag NPs into chitosan. These results are related to the increased water absorption and retention, porosity, and mechanical properties of CS-Au-Ag.

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Fig. 3. Scanning electron micrographs of CS (a), CS-EW (b), CS-Au (c), CS-Ag (d), and CS-Au-Ag (e, f). FTIR. Fig. 4a shows the FTIR spectra of chitosan and the nanocomposites. The peaks at 2925 cm-1 and 2885 cm−1 are assigned to stretching vibration of the C–H from the methylene and methyl of chitosan, respectively. The peaks at 1655 cm−1 and 1605 cm−1 are assigned to the carbonyl stretching –C=O and the deformation vibration of N–H from chitosan (amide I, amide II band). Another peak is attributed to the vibration of C–O–C at 1078 cm−1 of chitosan. There is an overlap of the amide-III band with in-plane deformational modes of O–H bond around 1450–1250 cm−1, and C–O vibrations of esters around 1330–1050 cm−1. In the spectrum of chitosan, the peaks around 3200-3500 cm−1 are attributed to stretching vibration of O–H and N–H, while the peaks in the spectrum of chitosan compounded with egg white


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(around 3200–3500 cm−1) become wider. This result may be attributed to –OH stretching vibration from –COOH groups, suggesting that the structural changes were caused by the interaction between egg white and chitosan. In the spectrum of chitosan compounded with nanoparticles, all the absorbance bands were similar to those of chitosan compounded with egg white. In addition, the two peaks in the spectrum of nanocomposites assigned to the vibration of the –C=O and N–H blue shifted to 1636 cm−1 and 1558 cm−1; this reason may be explained by the interaction of nitrogen atoms of primary amine groups and amide groups with nanoparticles, which reduces the carbonyl stretching –C=O and deformation vibration intensity of N–H. This result proves that the nanoparticles altered the structure of chitosan. XPS. XPS spectrum analysis is shown in Fig 4b-d. The peaks in full scanned spectra and the peaks for Au and Ag clearly illustrated that the composite was well compounded between Au0 (84.38 eV and 88.08 eV) and Ag0 (374.08 eV and 367.58 eV), suggesting the existence of Au-Ag NPs in this chitosan composite. The interactions, including the Van der Waals force and coordination bond, existed between Ag NPs, Au NPs, or Au-Ag NPs and chitosan in the composite sponge. Chitosan possesses a strong affinity (hydrogen bonds and van der Waals forces) for nanoparticles due to the presence of –NH2 and –OH groups; as a result, Au-Ag NPs were stably anchored by chitosan 42, 43.

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Fig. 4. FT-IR spectra of nanocomposites (a). X-ray photoelectron spectroscopy full scanned spectra of CS-Au-Ag (b), Au 4f (c), and Ag 3d (d). Mechanical properties. The mechanical properties of the composite materials were determined with a stretching apparatus. Fig 5a shows that the stress at the breaking point of CS, CS-EW, CS-Au, CS-Ag, and CS-Au-Ag increased in that order, while the strain of the materials decreased, correspondingly. The higher stress and lower strain for the composites


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could be explained by the content of Ag NPs, Au NPs, or Au-Ag NPs. The Young’s moduli of CS, CS-EW, CS-Au, CS-Ag, and CS-Au-Ag were 1.13 ± 0.02, 1.56 ± 0.03, 2.06 ± 0.02, 2.46 ± 0.02, and 2.89 ± 0.01 MPa, respectively (Fig 5b), compared to CS and CS-EW, thus, the Young’s modulus of CS-Au, CS-Ag, and CS-Au-Ag were higher, compared to the Nanoparticulate bioactive-glass-reinforced gellan-gum hydrogels reported by Gantar et al.44, which exhibited a Young's modulus of 1.2 MPa, CS-Au-Ag showed enhanced Young’s modulus. These results indicate that NPs acted as reinforcing filler in the chitosan sponge: the interaction between existing NPs and –NH2 and –OH of chitosan increased the interaction force of molecules42, indicating that the three nanocomposites were able to resist deformation and possessed enhanced mechanical strength as a wound dressing.

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Fig. 5. Structural characterization of nanocomposites. Mechanical behavior (a), Young’s modulus (b). * means P < 0.05. Porosity evaluation. Figure 6a shows the total porosity of CS, CS-Au, CS-Ag, CS-Au-Ag, and CS-EW. The curve of CS shows 26.7% porosity, whereas the others show higher porosity of 72.5%, 70.3%, 73.6%, and 79.3%, respectively, likely due to the foaming property of egg white, which could enhance water content and the hydrophilicity of the materials. The porosity of the chitosan composites contained Au-Ag NPs is the highest, slightly enhanced compared to the composites previously reported on the literature45. The high porosity of the dressings could benefit the absorption of the exudate from the wound surface. Additionally, high porosity is also beneficial for the transfer of nutrients and oxygen to the cells, which bind to the dressing 45.


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Fig. 6. Structural characterization of CS, CS-EW, CS-Au, CS-Ag, and CS-Au-Ag. Porosity analysis (a), micrographs of CS-Au-Ag samples placed in glass dishes before and after water swelling under Day light and UV light, respectively (b), swelling and retention ratio (c, d). * means P < 0.05. Swelling and retention properties. The swelling and retention properties of CS, CS-EW, CS-Au, CS-Ag, and CS-Au-Ag were evaluated (Fig 6b-d) by immersing them in PBS (PH 7.4) at 37℃. Specifically, the swelling ratio of the CS was about 32 times its dry weight after swelling for 3 h, while the swelling ratio of the other four composites exceeded 50 times and were about 52, 53, 57, and 61 times their respective weights, respectively (Fig 6c), all the composites reached equilibrium within about 3 h and were converted into spongy composites (Fig 6b). The swelling ratio of the chitosan composites containing egg white was enhanced and found to be higher than that of pure chitosan. This result may be explained by the presence of egg white. CS-EW, CS-Ag, CS-Au, and CS-Ag showed increased swelling ratio in order. Further, the range of increments was small, indicating that Ag NPs, Au NPs, or Au-Ag NPs incorporated in chitosan slightly enhance its swelling properties. CS-Au-Ag showed the best swelling property of these composites, possibly because of the effect of Au-Ag NPs. This result was related to the improved pore structure of CS-Au-Ag. Compared with other composite materials previously reported in the literature18, 42, 45, CS-Au-Ag fabricated in this study showed enhanced swelling property. Fig 6d shows the retention properties of the five chitosan materials. CS-EW, CS-Au, CS-Ag, and CS-Au-Ag retained PBS for a longer time than CS, and their retention ratios were > 5% after 600 min, indicating their suitability as wound dressing. This result may be attributable to the interaction between egg white and chitosan, which increased the retention ratio of chitosan. Thus, CS-Au-Ag


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possesses adequate swelling and retention properties to maintain the moisture content of the wound and avoid dehydration. Antibacterial activity of CS-Au-Ag. There was no inhibition zone observed for CS, CS-EW, and CS-Au as shown in Fig 7a. In contrast, the inhibition zone was clearly observed for CS-Ag and CS-Au-Ag, and a biggest inhibition zone was measured for CS-Au-Ag, with average diameters against E. coli and S. aureus of 36.2 mm and 34.3 mm, respectively, compared to 21.5 mm and 20.8 mm for CS-Ag against E. coli and S. aureus, respectively (Fig 7b), indicating that CS-Au-Ag have excellent antibacterial activity. A previous study reported that both Ag+ and Ag NPs are effective disinfectants that act by destroying the bacterial membrane 46. The obvious increase in antibacterial activity indicates the synergistic antibacterial effect of CS-Au-Ag, which can be explained by the increased Ag+ release. To validate this hypothesis, the role played by silver ion in bacterial damage was tested. As shown in Fig 7c, the silver ion release curves for CS-Au-Ag and CS-Ag in chitosan materials showed a constant release over time; the amount of released silver from CS-Au-Ag increased rapidly and was higher than that of CS-Ag over the first 48 h, after which the rate of release of silver from the two chitosan nanocomposites leveled off and identical levels were observed after 72 h. These results indicate the high-performance antibacterial effect of CS-Au-Ag.

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Fig. 7. Antibacterial analysis using E. coli and S. aureus. Antibacterial activity of CS-Au-Ag, CS-Ag, CS-Au, CS-EW, and CS (a). Average size of the inhibition zone of CS-Au-Ag and CS-Ag (b). Ag+ release analysis at 2, 4, 8, 16, 24, 48, and 72 h (c). * means P < 0.05. Live/dead assay was implemented by staining E. coli and S. aureus to confirm the antibacterial effect of CS-Au-Ag. The live bacterial cells with intact membranes appeared in green under a fluorescence microscope, and the dead cells with damaged membranes


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appeared in red. Hardly any cell survived after treatment with CS-Au-Ag for 1 h, while a large amount of bacteria survived after being treated with CS-Ag as both the red and green signals were observed as shown in Fig. 8a. The significant difference between CS-Au-Ag and CS-Ag demonstrated the enhanced antibacterial activity of CS-Au-Ag. Furthermore, SEM and TEM were carried out to visualize bacterial morphology after treatment with CS-Au-Ag (Fig. 8b). E. coli and S. aureus without treatment had intact membrane structures after incubation, whereas bacteria cultured with CS-Au-Ag lost their cellular integrity, exhibiting crinkly and colliquative morphology, demonstrated that CS-Au-Ag has high-performance antibacterial activity by releasing silver ions to destroy the membranes of bacteria cells. Furthermore, this strong antibacterial ability endows CS-Au-Ag great potential for applications as antibacterial materials or wound dressing.

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Fig. 8. Fluorescent images of S. aureus and E. coli treated with CS-Au-Ag and CS-Ag. Live cells were stained in green and dead cells were stained in red (a). SEM and TEM micrographs of S. aureus and E. coli treated with CS-Au-Ag (b). The red arrows represent breakages of bacterial cell membrane.


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Cytotoxicity of CS-Au-Ag. The cytotoxicity of CS-Au-Ag was measured by using L929 cells to assess the capability of CS-Au-Ag as a wound dressing. As shown in Fig. 9a, after culture with chitosan composites for 24 h, the viability of L929 cells was below 90%, and cell viability increased over 100% after 48 and 72 h. The cell viability of cells treated with CS-Au, CS-Ag, and CS-Au-Ag was similar. Although it was slightly lower than that of CS-EW and CS without NPs, these nanocomposites were relatively non-toxic. All these results demonstrated that CS-Au-Ag is almost non-toxic to L929 cells.

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Fig. 9. Viability of cells incubated with CS-Au-Ag, CS-Au, CS-Ag, CS-EW, and CS (a). Micrographs of wounds covered with CS-Au-Ag, CS-Au, CS-Ag, CS-EW, and CS at days 0, 3, 7, 11, and 15 (b). In vivo study. CS-Au-Ag was used as a wound dressing to demonstrate its practical applicability. The other composites were used as controls. As shown in Fig. 9b, the macroscopic photographs of postoperative wound healing after covering with CS-EW, CS-Au, CS-Ag, and CS-Au-Ag for 3, 7, 11, and 15 days were observed to evaluate wound contraction at each time point. All wounds showed calluses and inflammation, and wounds without treatment became infected and showed slight edema with 21% contraction on day 3. Wounds treated with CS-EW, CS-Au, CS-Ag, and CS-Au-Ag composite sponges showed evidence of healing with about 47%, 53%, 61%, and 69% wound contraction, respectively (Fig 10a). On day 11, wounds treated with CS-Au, CS-Ag, and CS-Au-Ag were crusted compared to wounds covered with CS-EW or untreated, and the contraction of these wounds was 76%, 80%, 87%, 91%, and 97%, respectively. On day 15, complete re-epithelialization of the wound treated with CS-Au-Ag was observed, while untreated wounds and wounds treated with CS-EW, CS-Au, and CS-Ag failed to heal completely, showing 82%, 88%, 91%, and 96% contraction, respectively. The percent wound contraction and average healing time indicated that CS-Au-Ag composite promoted wound healing to a greater degree than CS-EW, CS-Au, CS-Ag, and control. Moreover, the effectiveness of CS-EW, CS-Au, CS-Ag, and CS-Au-Ag increased in this order. The specific characteristics may be due to the superb antibacterial properties, non-toxic, good swelling, and water retention capacities of CS-Au-Ag. The histological outcome of wounds treated with the different materials at various time points was evaluated by H&E staining. On day 3, all groups showed acute necrosis and edema along with increased number of inflammatory cells. Moreover, all dressing formed gels after absorbing the exudate, and thus served as an insulating layer to protect the regenerating tissue (Fig. 10b). On day 7, some fibroblasts and collagen fibers appeared on the wound treated with CS-Ag and CS-Au-Ag, and the numbers of fibroblasts and collagen fibers were lower in


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CS-Ag groups. Furthermore, the epidermis and dermis started to form in the CS-Au-Ag groups, and some microvessels were observed, implying that CS-Au-Ag presented a higher capacity in supporting wound healing. On day 11, there was still a large number of inflammatory cells remaining on the untreated wounds and wounds treated with CS-EW and CS-Au, but fewer inflammatory cells on wounds treated with CS-Ag. In addition, the epidermis and dermis started to form in the CS-Ag groups. Complete, symmetrical and thickened epidermis could be observed on the wounds treated with CS-Au-Ag on day 11, while the other four groups were still covered with an incomplete, uneven, and thin epidermis on day 15, indicating the superior efficiency of CS-Au-Ag in promoting wound healing.

Fig. 10. Wound contraction ratio (a), and micrographs of H&E-stained tissue from wounds treated with CS-Au-Ag, CS-Au, CS-Ag, CS-EW, and CS at 3, 7, 11, and 15 days (b). CONCLUSION Chitosan loaded with Au-Ag NPs prepared by using egg white presented red fluorescence, enhanced antibacterial activity by releasing silver ions faster, in higher number, and in a more durable manner compared to chitosan loaded with Ag NPs, which contain the same silver content. CS-Au-Ag showed almost no cytotoxicity like CS-Ag, indicating that CS-Au-Ag can achieve the same antibacterial effect as CS-Ag, while demonstrating a lower cytotoxicity through reduction in the silver content. CS-Au-Ag exhibited rich pores, good mechanical properties, good swelling and retention properties, and can be removed easily. In vivo animal studies demonstrated the efficiency of CS-Au-Ag in promoting wound healing. Taken together, our results indicate that the developed chitosan dressing loaded with silver-gold NPs can be further researched in the sensing or label fields because of its red fluorescence. Additionally, the enhanced antibacterial and wound healing properties make CS-Au-Ag a competitive candidate for use as an effective wound dressing. AUTHOR INFORMATION Corresponding Author Phone: +8613594005200; fax: +8602368251228; e-mail: [email protected]. Author Contributions


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The manuscript involved contributions from all authors. All authors have approved the final version of the manuscript.

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Author Contributions

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Qing Li and Fei Lu contributed equally to this work.

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Notes

The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (number

446

51703185), the Hi-Tech Research and Development 863 Program of China Grant (number

447

2013AA102507), the Fundamental Research Funds for the Central Universities (numbers

448

XDJK2017B041 and XDJK2017C012), and the Social development project of Guangdong

449

province (number 2017A020211015).

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Chitosan Wound Dressing

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