ZnO composite dressing

Accepted Manuscript Title: Enhanced antibacterial and wound healing activities of microporous chitosan-Ag/ZnO composite dressing Author: Zhong Lu Jingting Gao Qingfeng He Jie Wu Donghui Liang Hao Yang Rong Chen PII: DOI: Reference:

S0144-8617(16)31105-5 http://dx.doi.org/doi:10.1016/j.carbpol.2016.09.051 CARP 11578

To appear in: Received date: Revised date: Accepted date:

8-7-2016 5-9-2016 15-9-2016

Please cite this article as: Lu, Zhong., Gao, Jingting., He, Qingfeng., Wu, Jie., Liang, Donghui., Yang, Hao., & Chen, Rong., Enhanced antibacterial and wound healing activities of microporous chitosan-Ag/ZnO composite dressing.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.09.051 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Enhanced antibacterial and wound healing activities of microporous chitosan-Ag/ZnO composite dressing Zhong Lu a, Jingting Gao a, Qingfeng He *b, Jie Wu a, Donghui Liang a, Hao Yang a, Rong Chen *a

a

School of Chemistry and Environmental Engineering, Key Laboratory for Green Chemical

Process of Ministry of Education, Wuhan Institute of Technology, Xiongchu Avenue, Wuhan 430073, PR China b

Third Hospital of Wuhan, Pengliuyang road, Wuhan 430060, PR China

*Corresponding author: E-mail address: [email protected] (R. Chen) [email protected] (Q. F. He)

Highlights 

Ag/ZnO nanocomposite-chitosan dressings were successfully prepared.



The addition of Ag/ZnO increased the antibacterial activity of the dressing.



Chitosan-Ag/ZnO composite dressing showed enhanced wound healing capacity.

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Chitosan-Ag/ZnO composite dressing showed low cytocompatibility.

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Abstract Nano Ag/ZnO hybrid material has been considered to be a promising nanocomposites for biomedical application because it has enhanced antibacterial activity and low cytotoxicity. Here a sponge-like nano Ag/ZnO-loaded chitosan composite dressing was first synthesized via preparing a chitosan sponge by lyophilization process, followed by the incorporation of Ag/ZnO nanocomposites into chitosan sponge. The porosity, swelling, blood clotting and in vitro antibacterial activity against drug-sensitive and drug-resistant pathogenic bacteria were evaluated. The results demonstrate that the prepared composite dressing shows high porosity and swelling as well as enhanced blood clotting and antibacterial activity. Cytocompatibility test evaluated in vitro illustrates the very low toxic nature of the composite dressing. Furthermore, the in vivo evaluation in mice reveals that the chitosan-Ag/ZnO composite dressing enhances the wound healing and promotes re-epithelialization and collagen deposition. These results strongly support the possibility of using this novel chitosan-AgZnO composite dressing for wound care application. Keywords: Ag/ZnO nanocomposite; chitosan dressing; antibacterial activity; wound healing; in vivo test

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1. Introduction Chitosan, a bio-copolymer comprising glucosamine and N-acetylglucosamine, is the alkaline deacetylated products of chitin. It has properties of non-toxic, biocompatible, biodegradable, moisture retentive and inexpensive (Dash, Chiellini, Ottenbrite & Chiellini, 2011; Hirano, Seino, Akiyama & Nonaka, 1990; Ravi Kumar, 2000). It also possesses activities of hemostasis, wound healing acceleration and scar prevention (Ravi Kumar, 2000). Due to these good properties, chitosan has been one of important biomaterials for wound management in recent years (Jayakumar, Prabaharan, Sudheesh Kumar, Nair & Tamura, 2011). Different forms of chitosan used for wound care have been reported, such as hydrogels (Sudheesh Kumar et al., 2012), films (Ma, Qin, Li, Zhao & He, 2014), scaffolds (Zakhem, Raghavan, Gilmont & Bitar, 2012) and sponges (Han, Dong, Su, Yin, Song & Li, 2014). However, chitosan does not show any antibacterial activity at neutral pH, which limited its use in infected wound care. To improve the antibacterial effect, some external antibacterial agents such as antibiotics have been incorporated into chitosan dressings (Denkbas, Ozturk, Ozdemir, Kececi & Agalar, 2004; Mi et al., 2002; Öztürk, Ağalar, Keçeci & Denkbas̨ , 2006; Pulat, Kahraman, Tan & Gümüşderelioğlu, 2013). However antibiotic resistant bacteria tend to spread epidemically in hospitals and often lead to wound infections (Pîrvănescu, Bălăşoiu, Ciurea, Bălăşoiu & Mănescu, 2014; Qiu et al., 2011; Robson, 1998), which reduces the efficacy of antibiotic-loaded chitosan dressings in application of infected wound care. Currently, a lot of researches are focusing on developing more effective antimicrobials to treat wound infected with multidrug-resistant bacteria (MDR). As one of the highly stable and nontoxic inorganic antibacterial materials, nano ZnO has

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been reported to be incorporated into chitosan dressing (Sudheesh Kumar et al., 2012; Vicentini, Smania & Laranjeira, 2010). However, the limited antibacterial activity of ZnO makes these composite dressings less effective in treating wounds infected with MDR. The bactericidal activity of Ag nanoparticles (AgNPs) against drug-sensitive and drug-resistant pathogenic bacteria has been studied by our group and many scientists and it was proved to be a powerful weapons against MDR (Ansari, 2011; Li, Rong, Zhao, Li, Lu & Chen, 2013; Lu, Rong, Li, Yang & Chen, 2013; Nanda & Saravanan, 2009; Panacek et al., 2006; Percival, Bowler & Dolman, 2007; Rai, Deshmukh, Ingle & Gade, 2012). Currently, AgNPs-loaded chitosan dressings have been reported to be effective in treatment of wounds by our and other groups (Ambrogi et al., 2014; Liang, Lu, Yang, Gao & Chen, 2016; Lu, Gao & Gu, 2008; Ong, Wu, Moochhala, Tan & Lu, 2008). However, significant evidence also has been reported in relation to the cytotoxicity and genotoxicity of AgNPs to human normal cells (Asharani, Mun, Hande & Valiyaveettil, 2009; Li et al., 2012; Marambio-Jones & Hoek, 2010). In order to make use of AgNPs safely, the method that supporting AgNPs on metal oxides such as ZnO is an effective way to reduce the amount of AgNPs in the mean time without jeopardizing its functionality (Agnihotri, Bajaj, Mukherji & Mukherji, 2015; Lu, Liu, Gao, Xing & Wang, 2008; Patil et al., 2014; Shah, Manikandan, Ahmed & Ganesan, 2013). In addition, these Ag/ZnO composites may inhibit the growth of bacteria synergistically due to the strong interaction between Ag and ZnO (Agnihotri, Bajaj, Mukherji & Mukherji, 2015; Matai, Sachdev, Dubey, Kumar, Bhushan & Gopinath, 2014; Patil et al., 2014; Zhang et al., 2014). At present, the physical mixture of AgNPs and ZnO had been incorporated into chitosan to form the chitosan/Ag/ZnO blend film (Li, Deng, Deng, Liu & Li, 2010) and

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chitosan/poly(ethylene glycol)/ZnO/Ag nanocomposites (Liu & Kim, 2012). However, there has not report on the incorporation of Ag/ZnO nanocomposites into chitosan to form the Ag/ZnO loaded chitosan dressing. In this study, a novel microporous Ag/ZnO-loaded chitosan dressing was prepared for the first time via the incorporation of Ag/ZnO nanocomposites into chitosan sponge. The antibacterial activity as well as wound healing ability were evaluated in vitro and in vivo.

2. Experimental section 2.1 Materials Chitosan (degree of deacetylation ≥ 95%, mean molecular weight of 179 kDa) and pharmaceutical grade zinc oxide (ZnO) was purchased from Aladdin (China). Silver nitrate (AgNO3) was purchased from ABCR GmbH & Co. KG (Germany). 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich. Acetic acid (CH3COOH), absolute ethanol (CH3OH) and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co. (China). All the chemicals were used without further purification. The bacteria strains of Staphylococcus aureus (S. aureus, ATCC 9118), Escherichia coli (E. coli, CCTCC AB 93154), Pseudomonas aeruginosa (P. aeruginosa, CCTCC AB 93066) and cell strains of human normal hepatocyte L02 were obtained from China Center for Type Culture Collection (CCTCC). Methicillin-resistant S. aureus (MRSA), drug-resistant E. coli (DREC) and drug-resistant P. aeruginosa (DRPA) were obtained from Third Hospital of Wuhan. Luria-Bertani (LB) medium was used in growing and maintaining all the bacteria. Dulbecco’s minimum essential medium (DMEM) was used for the cell culture. 5

2.2 Preparation of Ag/ZnO nanocomposites The deposition-precipitation method was used to synthesize Ag/ZnO nanocomposites (Zhong et al., 2015). Typically, 0.05 g of ZnO was firstly dispersed in 99.5 mL of deionized water, then 0.5 mL of AgNO3 solution (50 mmol/L) was added into ZnO suspension under vigorous stirring, followed by adjusting pH of the mixture to 8.0 by NaOH solution. Then the mixture was continued stirred at 80 °C for 4 h. After cooling down to room temperature, the final product was collected by centrifugation and washed with alcohol and deionized water for 5 times, then dried in a vacuum oven at 60 ºC overnight to obtain Ag/ZnO composite with 5.1 wt% Ag content. 2.3 Preparation of chitosan-Ag/ZnO composite dressing Chitosan solution was prepared by dissolving 1 g of chitosan in 50 mL 1% (v/v) acetic acid solution with continuous stirring under room temperature, then the solution was poured into Teflon plates and kept at -20 °C overnight, followed by lyophilization at -60 °C using freeze dryer (Christ ALPHA 1-4/LD plus) to obtain the primary chitosan sponges. Then the primary sponges were immersed in 1.25 mol/L ammonia solution for 2 hours to neutralize the residual acetic acid and rinsed extensively in distilled water. Finally, the neutral sponges were immersed in 0.1, 0.2, 0.5 and 1.0 mg/mL of Ag/ZnO solution respectively under sonication for 1 h, then the sponges were freeze-dried to obtain chitosan-Ag/ZnO composite dressings and named as CS-Ag/ZnO-0.1, CS-Ag/ZnO-0.2, CS-Ag/ZnO-0.5 and CS-Ag/ZnO1.0, respectively. For comparison, pure chitosan sponge was also prepared in the same way but without immersion in Ag/ZnO solution and named as CS, or immersion in 0.5 mg/mL of ZnO solution and named as CS-ZnO-0.5, respectively. 6

2.4 Characterization The as-prepared Ag/ZnO composites were characterized using X-ray diffraction (XRD, Bruker axs D8 Discover, Cu Ka = 1.5406 Å), scanning electron microscopy (SEM, Hitachi S-4800, operating at 5 eV), transmission electron microscope (TEM, Philips Tecnai 20, accelerating voltage of 200 kV) and energy dispersive X-ray spectrum (EDX). The surface and interior of as-prepared chitosan-Ag/ZnO dressings were characterized by XRD and SEM. The content of Ag/ZnO in CS-Ag/ZnO-0.5 dressing was determined by inductively coupled plasma-mass spectrometry (ICP-MS, Agilent 7700). The dressing piece was treated with 20 % (v/v) nitric acid solution at 100 °C for overnight, then the mixture was filtered and the content of Ag and Zn in the filtrate was determined. 2.5 Porosity measurement The porosity of the prepared dressing was determined by using the reported method (Sudheesh Kumar et al., 2012). The pre-weighted dressing was immersed in absolute ethanol until it was saturated, then taken out and the final weight was noted. The porosity (P) was calculated by the eq 1. 𝑃=

𝑚2 −𝑚1 𝜌𝑉

× 100%

(1)

In this equation, m1 and m2 indicate the weight of the dressings before and after immersion in alcohol respectively, V is the volume of the dressings before immersion, and ρ is the density of alcohol. Experiment was done thrice and the average values were taken. 2.6 Swelling ratio and moisture retention time The dressing pieces of same size and weight were immersed in deionized water for swelling study. After swelling for 2 h, the dressing was taken out and water that adhered on the surface

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was removed by gently blotting and immediately weighted. The degree of swelling (DS) was calculated by eq 2 (Sudheesh Kumar et al., 2012). 𝐷𝑆 =

𝑚w −𝑚0 𝑚0

(2)

In this equation, m0 and mw are the weight of dressings before and after immersion. All samples were triplicate in the experiment. In order to measure the moisture retention time of the dressing, the wet dressing was placed in a dryer at room temperature and determined the DS at every hour, then the moisture retention time was recorded as the value of the DS reduced to 1. 2.7 In vitro antibacterial study The strains of drug-sensitive S. aureus, E. coli, P. aeruginosa and drug-resistant MRSA, DREC, DRPA were used to evaluate the antibacterial activity of the dressings by an inhibition zone method. Firstly, 100 μL of 108 CFU/mL bacteria suspension was spread on LB agar plate, then the sterile dressing with the diameter of 0.9 cm (sterilized by autoclaving) was placed onto the surface of the agar. After 48 h incubation at 37 °C, the diameter of inhibition zone was measured. 2.8 In vitro cytotoxicity study In vitro cytotoxicity of the dressings was estimated by the MTT reduction assay (Hansen, Nielsen & Berg, 1989). L02 cells were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) at 37 °C and in 5% CO2. A piece of sterile dressing (weight of 0.05 g) was placed in a well of a 12-well plate, then seeded with 1 mL of cell suspension at a concentration of 1×105 cells/mL. After 48 h incubation, 0.5 mL of 5 mg/mL MTT was added into the surface of dressing and continued to incubate for 4 h. Then 1 mL of DMSO was 8

added to each well and further incubated for 10 min. The cell viability was determined by measuring the absorbance at 490 nm on a microplate reader (Multiskan MK3). The cells without dressing served as control. All the experiments were performed in triplicates. 2.9 Whole-blood clotting study The blood-clotting ability of CS and CS-Ag/ZnO-0.5 was determined by procoagulant ratio (PR) according to the literature (Ong, Wu, Moochhala, Tan & Lu, 2008) and compared with clinically available gauze and pledget. Briefly, blood was collected from human ulnar vein and stored in anticoagulated BD Vacutainer (Nouhua 9NC, containing sodium citrate). Then mixed 1 mL of blood with the sample (weight of 20 mg) and 0.1 mL of CaCl2 solution (0.2 mol/L) to initiate whole-blood clotting. Blood without samples was used as blank. The PR of the samples was calculated based on the eq 3. 𝑃𝑅 =

𝑇0 −𝑇 𝑇0

× 100%

(3)

In the equation, T and T0 are the blood-clotting time of the samples and blank, respectively. Experiment was done thrice and the average values were taken. 2. 10 Haemolysis assay The haemolytic property of CS and CS-Ag/ZnO-0.5 was evaluated according to the literature (Archana, Singh, Dutta & Dutta, 2015). Blood testing solution was prepared by diluting 2 mL of fresh blood with 2.5 mL of 0.9% saline. The dressing pieces (20 mg, about 1 cm  1 cm) were equilibrated in 4 mL saline for 30 min at 37 °C, then 0.1 mL of diluted blood was added to each sample and incubated for 60 min at 37 °C. Positive or negative controls, which did not contain dressing, were performed by adding 0.1 mL diluted blood to 4.0 mL of distilled water (100% haemolysis) or saline solution (0% haemolysis), respectively. 9

All solutions were centrifuged at 1000 rpm for 5 min. The absorbance at 545 nm of the supernatant was measured, and the haemolysis was calculated as follow: Haemolysis =

𝑂𝐷𝑠𝑎𝑚 −𝑂𝐷𝑛𝑒𝑔 𝑂𝐷𝑝𝑜𝑠 −𝑂𝐷𝑛𝑒𝑔

(4)

In this equation, ODsam, ODneg and ODpos are the adsorptions of sample, negative control and positive control, respectively. All the experiments were performed in triplicate. 2.11 In vivo animal experiment In vivo animal study was approved by the animal experimental center of Wuhan University. Twenty BALB/c mice, which were about 20 g of weight and 8 weeks of age, were evenly divided into five groups. On the day of wounding, each mouse was anaesthetized by aether, then a partial thickness wound with a length of 7 mm was created on the back. After that the prepared wounds of four groups were tightly covered with CS-Ag/ZnO-0.5, CS-ZnO-0.5, CS and ZnO ointment gauze (Winguide Huangpo Pharmaceutical, Shanghai), respectively. Mice with bare wound were kept as negative control. The dressing materials were changed at every day. During the changing of dressings, the area of the wound was measured and photographs were taken. The healing ratio (HR) is defined by formula 4. 𝐻𝑅 =

𝑆0 −𝑆 𝑆0

× 100％

(4)

In this equation, S0 and S are the wound area at the day of wound created and charging of dressing, respectively. For histological examination, skin tissue samples were excised at the day 10 after injury and then fixed with 10% formalin. After staining with hematoxlin-eosin (H&E) and picrosirius red (PSR) independently, the samples were observed by an optical microscope (Leica DMI3000B). 10

Exudate from wound on day 3 and day 5 after surgery was collected using sterile swabs and cultured in 2 mL of LB broth at 37 °C for 4 h. Subsequently 100 μL of the suspension was spread on LB agar plate followed by keeping at 37 °C overnight, then the colonies were observed.

3. Results and discussion 3.1 Composition and morphological analysis The phase, chemical composition and structure of the as-prepared Ag/ZnO composites were examined by XRD, SEM, TEM and EDX. Fig 1a shows the XRD patterns of the sample. The diffraction peaks that labelled with “●” could be readily indexed to ZnO (JCPDS 792205). Due to the relatively low Ag content in the composite, only peak in the 2 of 38.2 marked with “■” could be indexed to Ag (JCPDS 89-3722). The morphology of the asprepared Ag/ZnO composites was studied by SEM and TEM. As illustrates in Fig. 1b, a large quantity of rod-like structures with a typical length of 100-400 nm and a width of about 50200 nm were obtained. From Fig. 1c it is observed that Ag nanoparticles with a diameter of 10-30 nm are homogeneously deposited on the surface of ZnO nanorods. The EDX spectrum was also performed to analyze the elemental compositions of Ag/ZnO. The presence of Zn, O and Ag signals in Fig. 1d is attributed to Ag/ZnO nanocomposites. These results indicative of the successful formation of Ag/ZnO composites. The chemical composition and structure of the as-prepared Ag/ZnO composite dressing were examined by XRD and SEM. From Fig. 2a it is seen that the characteristic peaks of chitosan and ZnO are present in the surface and interior spectra of CS-Ag/ZnO dressing. Besides, the peak at 38.2 indicates the presence of Ag in the dressing. These data reveals 11

that the Ag/ZnO nanocomposites had been loaded in the surface and interior of chitosan dressing. Fig. 2b-2d present the surface and cross-section SEM photographs of chitosanAg/ZnO dressing. It is seen that both surface and interior of chitosan dressing show interconnected porous structure with pore size in the range of 50-150 μm. Moreover, Ag/ZnO nanorods are observed in the surface and interior of dressing. The ICP-MS analysis shows that the dressing of CS-Ag/ZnO-0.5 contain 2.42 wt% of Ag/ZnO, and Ag content in Ag/ZnO composite is 4.73 wt%, which is closed to the theoretical Ag content (5.1 wt%) in the dressing. 3.2 Porosity, swelling ratio and moisture retention time Porosity, swelling ratio and moisture retention time of the chitosan-Ag/ZnO and pure chitosan dressings were analyzed and shown in Fig. 3. The pure chitosan dressing shows the porosity of 93% of the total dressing volume. The presence of Ag/ZnO nanorods slightly reduces the porosity to 81-88% (Fig. 3a), which are slightly higher than that of reported chitin hydrogel/nano ZnO bandage (60-70%) (Sudheesh Kumar et al., 2013) and chitosan hydrogel/nano ZnO (75-85%) (Sudheesh Kumar et al., 2012). The high porosity of the dressings is helpful to absorb more exudate from a wound surface, and reduce the wound infection caused by the exudates. Further, the presence of large volume of porosity is also helpful for the transfer of nutrients and oxygen to the cells which attached on the dressings. Swelling ratio analysis reveals that at day 1 the pure chitosan dressing has the swelling ratio of 26 whereas Ag/ZnO incorporated composite chitosan dressing shows the swelling ratios in the range of 21-24. The swelling ratio of the pure chitosan dressing is slightly increased to 28 after 1 week storage, whereas Ag/ZnO dressing increases to 23-26. 12

Furthermore, the moisture retention time of the all dressing was about 13-14 days. The moist environment of wound can promote wound healing and reduce scar formation, in the meantime the dressing can be removed painlessly (Winter, 1963). 3.3 In vitro antibacterial activity, cytotoxicity and hemocompatibility The antibacterial activity of the chitosan-Ag/ZnO dressings against drug-sensitive and drugresistant pathogenic bacteria tested by an inhibition zone method is shown in Fig. 4a. The pure chitosan dressing do not show obviously antibacterial activity against all strains. The chitosan-Ag/ZnO dressings with different Ag/ZnO content appeare obviously antibacterial activities towards drug-sensitive E. coli, S. aureus and P. aeruginosa. The dressings with higher Ag/ZnO content such as CS-Ag/ZnO-0.5 and CS-Ag/ZnO-1.0 show toxicity toward drug-resistant DREC and MRSA No obvious inhibition zone is observed for the chitosanAg/ZnO dressings against DRPA. Cell viability of the prepared chitosan-Ag/ZnO composite dressings on L02 cells is shown in Fig. 4b. After 72 h incubation, the pure chitosan dressing obviously enhanced the growth of L02 cells. Although the cell viability treated with chitosan-Ag/ZnO composite decreased with the increase of Ag/ZnO content in dressing, the viability treated with CS-Ag/ZnO-0.1 and CS-Ag/ZnO-0.2 was still higher than that of control. CS-Ag/ZnO-0.5 and CS-Ag/ZnO1.0 show 94% and 66% cell viability, respectively. These results indicate the low cytotoxicity of chitosan-Ag/ZnO composite. The haemolytic property of CS dressing was further determined and the haemolysis is 1.79%, which showed negligible haemolytic property according to American Society for Testing and Materials (ASTM F 756-00, 2000). The addition of Ag/ZnO composite slightly 13

increased the haemolysis to 2.81%, which also showed negligible haemolysis. So we prepared chitosan-Ag/ZnO composite dressing was considered as a nonhaemolytic materials. 3.4 Evaluation of in vitro blood clotting Blood clotting test was conducted to assess the hemostatic potential of the chitosanAg/ZnO dressings. Here CS-Ag/ZnO-0.5 was selected as sample and its blood clotting activity is shown in Table 1. Compared with gauze and pledget, the chitosan dressing can effectively shorten the clotting time. Furthermore, the incorporation of Ag/ZnO nanorods into chitosan dressing effectively shorten the blood clotting time, and the advance rate of clotting increased from 52% to 75%. The enhanced hemostatic potential of the Ag/ZnO dressing is related to its chemical composition, morphological feature and microstructure (Paul & Sharma, 2010). Firstly, cationic chitosan can absorb negatively charged red blood cell, blood fibrinogen and plasma proteins, which constitutes hemostatic nature of chitosan (Shelma, Paul, Sharma, Shelma & Sharma, 2008). Moreover, the highly porous chitosan dressings can enhance the absorption ability. The blood-clotting capability of the chitosan dressing is further enhanced by the incorporation of Ag/ZnO because Ag can denature the anticoagulant proteins and affect the intrinsic pathway of blood coagulation (Kapadia, Kristol & Spillert, 2005; Kumar, Abhilash, Manzoor, Nair, Tamura & Jayakumar, 2010). 3.5 In vivo wound healing and antibacterial effects The dressing of CS-Ag/ZnO-0.5 was chosen as sample to evaluate in vivo wound healing capacity and compared with pure chitosan dressing CS, ZnO-loaded chitosan dressing CSZnO-0.5 and ZnO ointment gauze (Fig. 6). As illustrates in Fig. 6a, wounds closure are observed in all treatment groups with 9 days except for the bare wound. More importantly, 14

the dressing of CS-Ag/ZnO-0.5 shows excellent healing effect as early as 3rd day after surgery, when compared with ZnO ointment gauze, CS and CS-ZnO-0.5. Moreover, the growing fresh skin after CS-Ag/ZnO-0.5 treatment appears smoother and leaves less scab than that of being treated by CS, ZnO ointment gauze and blank control. The extent of wound closure at different healing time is also quantified and the result is shown in Fig. 6b. Two days after surgery, the wound treated with CS-Ag/ZnO-0.5 appears significant closure to 52%, whereas the wounds treated with ZnO ointment gauze, CS and CS-ZnO-0.5 shows 17%, 29% and 42% wound closure, respectively. After 6 days of healing, the wound closure of CSAg/ZnO-0.5 treated was 92%, which was about 20-30% higher than that of the CS-ZnO and CS, and 200% higher than that of ZnO ointment gauze. The complete wound closure is observed as early as on day 7 for CS-Ag/ZnO-0.5, while it is 8 days for CS-ZnO-0.5. For pure chitosan dressing CS and ZnO ointment gauze, the time required for complete wound closure is more than 9 days. From the in vivo healing results, it is concluded that the incorporation of Ag/ZnO into chitosan dressing can obviously accelerate wound healing, especially at the initial stage. It is reported that AgNPs can decrease inflammation of wound through cytokine modulation and enhance wound healing (Tian et al., 2007). Moreover, ZnOloaded chitosan bandage enhanced wound healing was also reported (Sudheesh Kumar et al., 2012). The enhanced wound healing capacity of chitosan-Ag/ZnO dressing is probably attributed to the existence of Ag and ZnO. The antibacterial activity of the dressings on mice wounds was evaluated by culturing the exudates from wound on day 3 and day 5 after surgery and observing the colonies of each culture. From Fig. 7 it is seen that the number of bacteria from bare wound, ZnO ointment

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treated and pure chitosan dressing CS treated wounds are much higher than that of CS-ZnO0.5 and CS-Ag/ZnO-0.5 treated wounds, and CS-Ag/ZnO-0.5 treated wound appears the lowest number of bacterial colonies, which should be attributed to the excellent antibacterial capacity of Ag/ZnO. The above results could explain the phenomena that there is no obvious fester on the wound treated with CS-Ag/ZnO-0.5, but it exists in bare, ZnO, CS and CS-ZnO0.5 treated wounds. 3.6 Histological observations Fig. 8 shows the histological observations of the wound tissue after 10 days of treatment. The H&E stained images show that the wounds treated with CS, CS-ZnO-0.5 and CSAg/ZnO-0.5 appear complete re-epithelialization and intact epidermis whereas the bare and ZnO ointment treated wounds have not been fully closed. A number of granulation tissue are observed on the bare, ZnO ointment and CS treated wounds (marked with black arrows). However, for CS-ZnO-0.5 and CS-Ag/ZnO-0.5 groups, the granulation tissue have been organized to fibrous connective tissue. Furthermore, the packed keratinocytes in the wounds treated with CS-ZnO-0.5 and CS-Ag/ZnO-0.5 are much denser than that of bare, ZnO ointment and CS treated wounds. The above results indicate the enhanced wound tissue repair capability of the Ag/ZnO-loaded chitosan dressing. The PSR stained images showed the extent of collagen deposition are also presented in Fig. 8, in which the collagen is stained into red. After 10 days of healing, the collagen deposition at the CS-Ag/ZnO-0.5, CS-ZnO-0.5 and CS treated wounds are much denser than that of the bare and ZnO ointment treated wounds. For the bare, ZnO ointment and CS treated wounds, there are still large and obvious light red area in the dermal layer, which indicates 16

that the wound tissues are still in the process of repair. By contrast, the derma of wounds treated with CS-ZnO-0.5 and CS-Ag/ZnO-0.5 shows uniform and dense red staining, which indicates that the collagen had filled the defective area and the necrotic tissue had been replaced by the organized fibroblast. In summary, CS-ZnO-0.5 and CS-Ag/ZnO-0.5 show the outstanding wound repairing ability, which could promote the re-epithelization, benefit the organization of granulation tissue and accordingly accelerate the wound healing process.

4. Conclusion The nano Ag/ZnO-loaded chitosan composite dressing was successfully prepared by the lyophilization and immersion method. The composite dressings show as highly as 81-88% of porosity, 21-24 of swelling ratio, 13-14 days of moisture retention time and enhanced blood-clotting capability, which are helpful in accelerating wound healing. In vitro and in vivo antibacterial studies prove the high antibacterial activities against drug-sensitive and drug-resistant pathogenic bacteria. In vivo evaluation in mice confirms that the chitosanAg/ZnO dressings appeared faster wound healing, more complete re-epithelialization and denser collagen deposition properties when compared with that of pure chitosan dressing and ZnO ointment gauze. All these results demonstrate that the prepared chitosan-Ag/ZnO composite dressing has potential application for wound care. 5. Acknowledgments This work was supported by National Natural Science Foundation of China (21371139), High-Tech Industry Technology Innovation Team Training Program of Wuhan Science and Technology Bureau (2014070504020243) and Depatrment of Education of Hubei Province 17

under the project of Science and Technology Innovation Team of Outstanding Young and Middle-aged Scientists (T201606).

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Figure 1 Characterization of Ag/ZnO nanocomposite. (a) XRD patterns, (b) SEM image, (c) TEM image, (d) EDX spectrum.

21

Figure 2 XRD patterns (a) and SEM images of surface (b, c) and cross-section (d, e) of CSAg/ZnO-1.0.

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Figure 3 Porosity (a) and swelling ratio (b) of chitosan-Ag/ZnO composite dressings.

Figure 4 Antibacterial activity (a) and cytotoxicity of chitosan-Ag/ZnO composite dressings. 23

0-4 represents CS, CS-Ag/ZnO-0.1, CS-Ag/ZnO-0.2, CS-Ag/ZnO-0.5 and CS-Ag/ZnO-1.0, respectively.

Figure 5 Photographs of wounds treated with the dressings (a) and evaluation of the wounds area closure (b).

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Figure 6 Bacteria isolated from the mice wound on day 3 and day 5 after surgery.

Figure 7 Micrographs of wound tissues stained with H&E and PSR. In figure the black arrows indicate granulation tissue and red box indicates the unrecovered wound.

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Table 1. Whole-blood clotting evaluation of the dressings Blank Clotting Time (s) Advance Rate of Clotting

Gauze

Pledget

1170 ± 80 1042 ± 18 915 ± 65 -

11%

26

22%

CS 558 ± 32 52%

CS-Ag/ZnO-0.5 295 ± 25 75%