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BIOMAC 4927 1–9

International Journal of Biological Macromolecules xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

pH-responsive release behavior and anti-bacterial activity of bacterial cellulose-silver nanocomposites

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Wei Shao a,∗,1 , Hui Liu a,1 , Xiufeng Liu b , Haijun Sun c , Shuxia Wang a , Rui Zhang a,∗∗ a

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College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, PR China College of Life Science, Nanjing University, Nanjing 210093, PR China Advanced Analysis and Testing Center, Nanjing Forestry University, Nanjing 210037, PR China

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a r t i c l e

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a b s t r a c t

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Article history: Received 26 December 2014 Received in revised form 19 February 2015 Accepted 20 February 2015 Available online xxx

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Keywords: Bacterial cellulose Silver nanoparticles pH sensitive release Anti-bacterial activity

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1. Introduction

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Bacterial cellulose (BC) has been extensively explored as some of the most promising biomaterials for biomedical applications due to their unique properties, such as high crystallinity, high mechanical strength, ultrafine fiber network structure, good water holding capacity and biocompatibility. However, BC is lack of anti-bacterial activity which is the main issue to be solved. In the study, BC-Ag nanocomposites were prepared in situ by introducing silver nanoparticles (AgNPs) into BC acting as the templates. The BC and as-prepared BC-Ag nanocomposites were characterized by several techniques including scanning electron microscope, Fourier transform infrared spectra, ultraviolet-visible absorption spectra, X-ray diffraction and thermogravimetric analyses. These results indicate AgNPs successfully impregnated into BC. The releases of Ag+ at different pH values were studied, which showed pH-responsive release behaviors of BC-Ag nanocomposites. The anti-bacterial performances of BC-Ag nanocomposites were evaluated with Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 9372 and Candida albicans CMCC(F) 98001, which frequently causes medical associated infections. The experimental results showed BC-Ag nanocomposites have excellent anti-bacterial activities, thus confirming its utility as potential wound dressings. © 2015 Published by Elsevier B.V.

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Bacterial cellulose (BC) is a polysaccharide produced by several microorganisms, particularly Acetobacter xylinus. BC is becoming a promising biopolymer for several applications in the paper and food industries, sewage purification, acoustics and optics and medical fields due to its unique properties, such as high crystallinity, high mechanical strength, ultrafine fiber network structure, good water holding capacity and biocompatibility [1–3]. Moreover, BC has recently been studied in biomaterials fields such as a cartilage scaffold, DNA separation medium, dental implants, nerve regeneration and vascular grafts, artificial skin and wound dressing for wounds and burns [4–8] because its three-dimensional network structure enables it to incorporate antibiotics or other drugs and support their controlled release.

∗ Corresponding author. Tel.: +86 25 85427024; fax: +86 25 85418873. ∗∗ Corresponding author. Tel.: +86 25 85427183; fax: +86 25 85418873. E-mail addresses: [email protected] (W. Shao), [email protected] (R. Zhang). 1 These authors contributed equally to this work.

In the processing of skin tissue repair, the moisture environment provided by the dressing has been shown to promote ulcers healing and reduce pain of patients [9]. The intrinsic properties of BC mentioned above make it an attractive novel wound-dressing material. However, lack of anti-bacterial activity of BC is the main issue to be tackled, which is critical to provide a bacteria-proof barrier to the outside world in wound-healing [10]. Bacterial adhesion is the first step in the development of wound infections. Once bacteria attach to a wound dressing, a multistep process starts leading to the formation of a complex, adhering microbial community that is termed a biofilm. Once a biofilm has formed, it is very difficult to treat clinically because the bacteria on the interior of the biofilm are protected from phagocytosis and antibiotics. Thereby, an alternative strategy is required to control infections. Since bacterial adhesion to biomaterial surfaces is the essential step in the process of infections, modifications to BC surfaces are considered [11,12]. Silver is known to be a powerful antimicrobial agent with effective broad-spectrum against a large number of Gram-positive and Gram-negative microorganisms, many aerobes and anaerobes, and several antibiotic resistant strains that has been used since ancient times [13]. Recent technical innovations facilitate the incorporation of silver-based materials to commercial formulations with

http://dx.doi.org/10.1016/j.ijbiomac.2015.02.048 0141-8130/© 2015 Published by Elsevier B.V.

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antimicrobial properties [14]. The biocidal activity of silver is related to the biologically active silver ion released from silver base materials. In general metallic silver appears to be inert and have no anti-bacterial action. Silver ions (Ag+ ), however, bind to and react with proteins and enzymes, thereby causing structural changes in the bacterial cell wall and membranes, leading to membrane permeability damage, cellular disintegration and death of the bacterium [15]. Dispersing metal nanoparticles in a solvent is a difficult issue because they are tendency to aggregate due to their high specific surface area [14,16]. The outstanding properties of Ag nanoparticles (AgNPs) including anti-bacterial activity will lose if they aggregate. The synthesis of metallic nanoparticles with controlled shape, size and dispersion stability is a challenging research area due to their potential in biomedical, electronics, optical, sensors and catalysis sectors [17–19]. Thereby, the strategy for preventing aggregation is the controlled deposition of metal nanoparticles through hybridization with a nanoporous material as the template to ensure a well-defined spatial distribution. The formed nanoparticles in their network effectively inhibit the aggregation for longer periods and can be extracted into water whenever they are required for usage [20]. Moreover, the formed nanocomposites are highly suitable for biomedical applications because of their good biocompatibility and great anti-bacterial activity. Cellulose has been considered to be a suitable template for the fabrication of metal nanoparticles through its three dimensional and porous structures and certain nano-pore size distribution. ZnO nanoparticles have been successfully synthesized through a facile polyol method using BC as templates and the resulted nanocomposites show good mechanical properties and high photocatalytic activity in the degradation of methyl orange [21,22]. Cellulose/copper composites by depositing copper on cellulose fibers were studied and the obtained composites could be used for food packaging applications due to their excellent antifungal properties [23]. BC/silica nanocomposites with improved mechanical properties were achieved using BC as templates [24]. BC-Ag composites have been successfully developed through by hydrothermal synthesis and the composites exhibited significant anti-bacterial rates [25]. Most of these studies utilized the electrostatic interactions between metallic ions and dipole moments of cellulose molecules. However, the interactions are normally weak between the anchor metallic ions and cellulose fibers with a low yield and non-uniform distribution of metallic nanoparticles, resulting in uncontrollable release of metal nanoparticles [10]. In this paper, BC-Ag nanocomposites were prepared by the chemical reduction with BC acting as templates. BC was immersed in the silver nitrate solution that silver ions could penetrate into BC through the porous structure and bound to its microfibrils probably via electrostatic interactions. After reduction in aqueous NaBH4 , silver ions were reduced to form AgNPs on the microfibrils of BC. This method can form a stable and strong interaction between uniformly distributed AgNPs and BC fibers, which avoids the overquick release of Ag+ and therefore reduce the toxicity. The releases of Ag+ at different pH values were studied. The anti-bacterial activities of the obtained BC-Ag nanocomposites were investigated by Gram-negative Escherichia coli (E. coli) ATCC 25922, Gram-positive Staphylococcus aureus (S. aureus) ATCC 6538, Bacillus subtilis (B. subtilis) ATCC 9372 and yeast Candida albicans (C. albicans) CMCC(F) 98001, respectively.

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2. Materials and methods

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2.1. BC preparation

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BC was prepared in a static culture medium by Acetobacter xylinum GIM1.327, which was purchased from BNBio Tech Co.,

Ltd, China. The method of preparing BC was well-established and described in literature [26]. Briefly, in a static culture system enriched with polysaccharides (5 g/L peptone, 5 g/L yeast, 5 g/L glucose, 5 g/L mannitol and 1 g/L MgSO4 ·7H2 O), bacterial strain was incubated at 30 ◦ C for 5 days and was able to produce a thin layer of BC in the interface of liquid/air [27]. This layer was washed by de-ionized water and then boiled in a 0.1 M NaOH solution at 80 ◦ C for 60 min to eliminate impurities such as medium components and attached cells. BC films were further washed thoroughly with de-ionized water until pH became neutral. 2.2. Production of BC-Ag nanocomposites BC-Ag nanocomposites were produced by liquid phase redox reaction. Firstly, BC films were cut into 2 cm × 2 cm pieces and immersed in 20 mL aqueous AgNO3 solution with concentrations of 0.01, 0.02, 0.03, 0.04 and 0.05 M for 1 h, followed by rinsing with de-ionized water for three times. Secondly, the silver ion-saturated BC were reduced in 0.02, 0.04, 0.06, 0.08 and 0.1 M of 20 mL aqueous NaBH4 for 1 h and rinsed with de-ionized water for five times to remove the excess chemicals. Finally, the obtained BC-Ag nanocomposites were freeze-dried at −40 ◦ C for 10 h. 2.3. Characterization A JSM-7600F Scanning Electron Microscope (SEM) operating at an accelerating voltage of 10–15 kV was used to investigate the surface morphologies of BC and BC-Ag nanocomposites. The samples were coated with a thin layer of gold under high vacuum conditions (20 mA, 100 s). Fourier transform infrared (FTIR) spectra were recorded on a Spectrum Two Spectrometer (Perkin Elmer, USA) with the wavenumber range of 4000–400 cm−1 at a resolution of 4 cm−1 . For the transmittance readings, the samples were grinded and mixed with KBr at a 1/50 ratio (w/w). This mixture (0.1 g) was then compressed into a thin KBr disk under a pressure of 0.4 bar for 3 min. The UV–visible absorption spectra were recorded on a Lambda 950 spectrophotometer (Perkin Elmer, USA) equipped with an integrating sphere accessory for diffuse reflectance spectra over a range of 350–650 nm by using BaSO4 as the reference. XRD patterns of the samples were recorded using a Rigaku Ultima III X-ray powder diffractometer, using a Cu K␣ X-ray tube ˚ running at 40 kV and 30 mA, respecwith a wavelength of 1.5406 A, tively. The diffraction angle (2) ranged from 5◦ to 80◦ with a step size of 0. 02◦ . Thermogravimetric analysis (TG) was carried out by using a TA Instruments model Q5000 TGA. The samples were heated from 20 to 600 ◦ C with a heating rate of 10 ◦ C/min under nitrogen atmosphere. 2.4. Release of silver ion in vitro The kinetics of silver ion release was studied from the prepared BC-Ag samples. The tested nanocomposites were cut into round pieces in diameter of 10 mm. In order to determine the effect of pH on the silver ion release profiles of the nanocomposites, HEPES (Sigma) buffers with different pH values at 5.5, 7 and 8.5 were used, respectively. The prepared nanocomposites were immersed in a beaker containing 100 mL 10 mM HEPES at 37 ◦ C and sealed using PARAFILM® M. 1.5 mL solution was taken at regular time intervals (1, 2, 3, 4, 5, 6, 7, 8 and 9 d) and analyzed for the amount of Ag ion released using inductively coupled plasma mass spectrometry (ICP-MS).

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2.5. Anti-bacterial activity

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The anti-bacterial activities of the nanocomposites were investigated against Gram-negative E. coli ATCC 25922, Gram-positive S. aureus ATCC 6538, B. subtilis ATCC 9372 and yeast C. albicans CMCC(F) 98001 by two methods: plate count method and disk diffusion method, respectively. BC-Ag nanocomposites and BC (the control) were cut into round shapes with 10 mm diameter and sterilized by ultraviolet lamp for 30 min. Plate count method: The sterilized samples were put into the tank containing 60 mL bacterial suspension with the concentration of 4 × 107 CFU/mL and incubated at 37 ◦ C for 1 h under a gentle stirring at 20 rpm. The samples were taken out and the bacteria adhered to each sample was removed to sterile glass beakers containing 10 mL sterile de-ionized water by ultrasonication for 5 min. 100 ␮L of the sonication suspension and 10−1 , 10−2 and 10−3 dilutions were plated out on TSA plates and incubated overnight at 37 ◦ C. The colonies were counted on the following day. The total number of bacteria in 100 ␮L of bacterial suspension was obtained for each concentration and hence the total number of bacteria as colony forming units (CFU)/cm2 attached to the tested sample as CFU was obtained. The experiments were carried out in triplicate to confirm reproducibility. Anti-bacterial ratio (R) was calculated with the following formula:

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R=

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N 0 − N1 × 100% N0

(1)

where N0 is the number of attached bacteria on BC and N1 is the number of attached bacteria on BC-Ag nanocomposites. Disk diffusion method: Lawns of test bacteria (1 × 105 CFU/plate) were prepared on TSA. The sterilized samples were then carefully placed upon the lawns and BC was used as control. The plates were placed in a 37 ◦ C incubator for 24 h. Then inhibitory action of tested samples on the growth of the bacteria was determined by measuring diameter of inhibition zone.

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3. Results and discussion

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3.1. Surface morphology

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Table 1 Average crystallinity index, Tmax and residue percentage of BC and BC-Ag nanocomposites. Samples

Crystallinity index (%)

Tmax (◦ C)

Residue (%)

BC BC-Ag0.01 BC-Ag0.02 BC-Ag0.03 BC-Ag0.04 BC-Ag0.05

89.2 83.4 82.9 81.2 79.3 77.3

363.2 286.4 269.7 260.6 256.6 261.1

6.3 45.8 55.5 61.8 63.9 64.5

For BC-Ag nanocomposites (curves b-f in Fig. 2A), the shifted peak decreased to a lower value, this indicates the presence of strong interactions between the OH groups of BC and AgNPs. BC is an oxygen-rich carbohydrate (polysaccharide) consisting of anhydroglucose units joined by an oxygen linkage to form a linear molecular chain [3]. When BC immerses in aqueous AgNO3 , most of the incorporated Ag+ were bound to BC macromolecules probably via electrostatic interactions, attributing to the electron-rich oxygen atoms of polar hydroxyl and ether groups of BC which are expected to interact with electropositive transition metal cations and strangely immobilize the metal ions [31,32]. 3.3. Optical properties The UV–vis spectroscopy is a reliable tool for examining the formation of metallic nanoparticles. Fig. 2B shows UV–vis absorption spectra of BC and BC-Ag nanocomposite. For BC (curve a), it does not exhibit any feature absorption in the UV–visible range. In the case of BC-Ag nanocomposite (curves b–f), a significant absorption at the wavelength of 414 nm can be observed, which is typical of the surface plasmon resonance of metallic AgNPs with sizes ranging from 2 to 100 nm [33], confirming the existence of silver in the nanocomposite [3]. It also can be seen that the shoulder peak becomes more prominent with higher AgNPs loadings. This could be due to the fact that AgNPs are more aggregated at higher concentrations, which is consisted with SEM images (Fig. 1). 3.4. Thermal properties

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Fig. 1 shows SEM micrographs of BC and BC-Ag nanocomposites and EDS image of BC-Ag composites. Fig. 1A shows the morphology of BC which exhibited a nanoporous three-dimensional network structure with a random arrangement of ribbon-shaped microfibrils without any preferential orientation. Therefore, the nanosized pores between microfibrils in BC can act as templates for nucleation and growth of AgNPs. In the case of BC-Ag nanocomposites, a denser network structure with clearly well dispersed spherical AgNPs (white spots) in the BC is illustrated in the BC-Ag nanocomposites (Fig. 1B–G). AgNPs were displayed as white spots which can be easily found in the nanocomposites. Moreover, the aggregations of AgNPs in the BC can be observed with increasing AgNPs loadings. EDS analysis of BC-Ag nanocomposite further confirmed the existence of silver in the BC (Fig. 1H).

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Fig. 2A displays the FTIR spectra of BC and BC-Ag nanocomposites with different loadings of AgNPs. For BC (curve a), the FTIR spectrum was typical and the dominating signal is at 3200–3500 cm−1 , corresponding to the intramolecular hydrogen bond for 3O· · ·H-O5 and the hydroxyl group [28,29]. In the C O stretching vibration region, the peaks at 1163, and 1061 cm−1 correspond to the C O asymmetric bridge stretching and the C O C pyranose ring skeletal vibration, respectively [30].

TG is a continuous process, involving the measurement of sample weight in accordance with increasing temperature in the form of programmed heating. Typical plots of weight loss versus temperature for BC and BC-Ag nanocomposites are displayed in Fig. 2C. The maximum peak temperature (Tmax ) and the residues of them were listed in Table 1. Two significant weight loss stages below 600 ◦ C are observed in the thermal degradation curve of BC (curve a) and the total weight loss is 92.61%. The initial weight loss occurred around 110 ◦ C, which is attributed to the evaporation of absorbed moisture. Physically adsorbed and hydrogen bond linked water molecules were lost at this first stage [34]. The second weight loss occurred between 110 and 400 ◦ C which can be assigned to the thermal degradation and decomposition of BC, which involves the formation of levoglucosan, transglycosylation and free radical reaction, followed by generation of C, CO, CO2 , H2 O and combustible volatiles [35,36]. For BC-Ag nanocomposites, two significant weight loss stages are also revealed (curves b–f). However, the weight loss stages shifted to lower temperature when AgNPs loaded into BC. Tmax is decreased from 363.2 ◦ C for BC to the range of 256.6–286.4 ◦ C for BC-Ag nanocomposites. The observed decrease of Tmax might be due to the fact that AgNPs in the BC networks can catalyze CO2 elimination from polymer chains and accelerate the degradation process [37]. The residues increased with the increasing AgNPs, e.g. the residue were highly enhanced from 6.3% for BC to 64.5% of BC-Ag0.05 , which was related to the existence of AgNPs.

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Fig. 1. SEM images of BC and BC-Ag nanocomposites: pristine BC (A), BC-Ag0.01 nanocomposite (B), BC-Ag0.02 nanocomposite (C), BC-Ag0.03 nanocomposite (D), BC-Ag0.04 nanocomposite (E), BC-Ag0.05 nanocomposite (F), BC-Ag0.04 nanocomposite (G), and EDS spectrum of BC-Ag0.04 nanocomposite (H). (A–F) at magnification 8000× and (G) at magnification 15,000×.

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3.5. XRD analysis XRD is most common mean used to investigate the crystalline structure, the ratio of crystalline to non-crystalline (amorphous) regions, crystal size, the arrangement pattern of crystals and the

distance between the planes of the crystal of nanocomposites [38]. This means that structural changes induced in a crystalline material by blending with other materials can be monitored using the XRD technique [4]. In the present study, XRD analyses were carried out in order to investigate the micro-structural changes in the

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Fig. 3. XRD patterns of BC (A), BC-Ag nanocomposites (B): BC-Ag0.01 nanocomposites (a), BC-Ag0.02 nanocomposite (b), BC-Ag0.03 nanocomposite (c), BC-Ag0.04 nanocomposite (d) and BC-Ag0.05 nanocomposite (e).

index (CI ) of BC is calculated by Segal’s method, using the following equation: CI =

Fig. 2. FTIR analysis (A), UV–vis absorption spectra (B) and TG profiles (C) of BC and BC-Ag nanocomposites: pristine BC (a), BC-Ag0.01 nanocomposite (b), BC-Ag0.02 nanocomposite (c), BC-Ag0.03 nanocomposite (d), BC-Ag0.04 nanocomposite (e), and BC-Ag0.05 nanocomposite (f).

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BC films caused by the introduction of AgNPs. The XRD patterns of BC and BC-Ag nanocomposites with different AgNPs loadings are shown in Fig. 3A and B. The profile of BC (Fig. 3A) is a typical cellulose I XRD pattern and three characteristic peaks of BC are observed at 2 values of 14.46◦ , 16.62◦ , 22.66◦ corresponding to (1 1 0), (1 1 0) and (2 0 0) crystal planes of cellulose, which is consistent with previous reports [10,39,40]. For BC with different AgNPs contents (Fig. 3B), the peaks of BC become weak but they are still evident, which clearly indicates the formation of the nanocomposite and supports the SEM and FTIR structural confirmations. The XRD pattern of BC-Ag nanocomposites show characteristic four peaks at 2 values of 38.1◦ , 44.3◦ , 64.4◦ and 78.0◦ corresponding to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of the face centered cubic (FCC) structure of the metallic AgNPs that were impregnated inside BC [41]. On the other hand, the introduction of AgNPs into the BC matrix affects the crystallization behaviors of BC. The relative crystallinity

I 002 − Iam × 100% I002

(2)

where I002 is the peak intensity of the (0 0 2) lattice diffraction at 2 = 22.66◦ for BC and Iam is the intensity diffraction of amorphous fraction at 2 = 18◦ . The calculated relative CI of BC and its nanocomposites are listed in Table 1. The relative CI of BC equals 89.2%, owning a very high crystallinity that is reported by other researches [10,42,43]. After the incorporation of AgNPs, CI of BC-Ag nanocomposites slightly decreased in the range of 77.3–83.4% with the increase of AgNPs loadings. The reason could be due to the native hydrogen bonding interactions in BC microcrystalline chains were slightly disturbed by the penetration and interaction of AgNPs into the BC matrix. Taking together with SEM pictures, it further proved AgNPs were successfully prepared in the BC matrix. 3.6. Silver ion release in vitro Both intrinsic and extrinsic factors can affect wound healing process. There are many factors including oxygen, angiogenesis, protease activity and bacterial toxicity can affect the pH of the wound. The pH environment of chronic wounds had been recorded within the range of 7.1–8.9. As the wound progress toward healing the pH moves to neutral and then becomes acidic up to 5.4–5.6 [44,45]. It usually takes shorter time for wound healing in acidic environment than alkaline environment. The release of anti-bacterial silver ions can highly vary depending on the moisture content or the pH of the surrounding environment in contact with the films [46,47]. In order to evaluate the silver ion release of BC-Ag nanocomposites, the release behaviors were monitored

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Fig. 4. Cumulative release profiles of Ag+ from BC-Ag nanocomposites in 10 mM HEPES with different pH values: pH 5.5 (A), pH 7 (B) and pH 8.5 (C). ((a)–(e) are BC-Ag0.01 , BC-Ag0.02 , BC-Ag0.03 , BC-Ag0.04 and BC-Ag0.05 nanocomposites).

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on a daily basis in 10 mM HEPES buffer at different pH. Acidic (pH = 5.5), neutral (pH = 7) and alkaline (pH = 8.5) conditions were chosen to further assess the effect of pH on release behavior. As can be observed in Fig. 4, the release is in many cases more rapid in the first stage and then a lower release capacity is noted. As expected, BC-Ag0.01 nanocomposites released the lowest amount of Ag ion at different pH values. And BC-Ag0.05 nanocomposites had the highest releases of Ag ion in the same conditions after 9 days of incubation (Fig. 4C). Meanwhile, higher silver ion concentrations were observed under lower pH value in HEPES buffer systems. Especially at pH 5.5 (Fig. 4A), the release silver ion concentration was the highest for BC-Ag0.05 after 9 d immersion. At pH 7, the silver ion released is much lower than from BC-Ag0.05 after 9 d incubation (Fig. 4B), which was in the middle of the released concentrations between pH 5.5 and 8.5. This suggested that the silver ion release was strongly pH dependent, and ion release rates decreased with

Fig. 5. No. of adhered bacteria on BC and BC-Ag nanocomposites: E. coli (A), S. aureus (B), B. subtilis (C) and C. albicans (D).

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Table 2 Anti-bacterial ratios and diameters of inhibition of BC and BC-Ag nanocomposites. Samples

E. coli BC-Ag0.01 BC-Ag0.02 BC-Ag0.03 BC-Ag0.04 BC-Ag0.05 a

358 359 360 361 362

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Diameter of inhibition (mm)a

Anti-bacterial ratio (%)

26.1 47.3 85.3 96.9 98.4

± ± ± ± ±

S. aureus 4.0 3.7 2.1 0.6 0.5

71.1 84.3 98.6 99.6 100

± ± ± ± ±

2.1 2.2 0.3 0.2 0.1

B. subtilis 65.2 96.4 99.2 99.8 100

± ± ± ± ±

13 0.7 0.5 0.1 0.1

C. albicans 34.4 57.4 74.4 77.5 89.6

± ± ± ± ±

3.57 10.3 8.5 3.8 0.6

E. coli 11.7 12.4 13.0 13.5 14.1

± ± ± ± ±

S. aureus 0.1 0.2 0.1 0.1 0.1

11.6 12.3 14.0 16 18.7

± ± ± ± ±

0.1 0.3 0.5 0.6 0.4

B. subtilis 11.5 17.7 19.5 23.3 24.3

± ± ± ± ±

0.3 0.4 0.8 0.8 0.8

C. albicans 12.1 13.1 14.5 15.0 16.0

± ± ± ± ±

0.2 0.2 0.3 0.1 0.1

Diameter of the zone of inhibition includes disk diameter, 10 mm.

increasing pH, which was in accordance with the previous study [48,49]. In aqueous solutions, there is a possibility of the release of Ag+ due to the oxidation by dissolved oxygen. The dissolution of AgNPs due to oxygen may be expressed by the following equation [50]:

+

4Ag(s) + O2(aq) + 4H =

4Ag+ (aq)

+ 2H2 O(l)

(3)

Based on the above equation, the release Ag+ is directly proportional to the extent of Ag oxidation. Lower pH values leads to higher H+ concentration formed in the medium, which could explain our results that more Ag+ releasing from BC-Ag nanocomposites, and consist with other researches’ works [48,51].

3.7. Anti-bacterial activity Four strains including Gram-negative E. coli ATCC 25922, Grampositive S. aureus ATCC 6538, B. subtilis ATCC 9372 and yeast C. albicans CMCC(F) 98001 were selected for anti-bacterial tested because they are usually associated with the infections during wound healing procedure [52,53]. The numbers of bacteria colonies attached to BC-Ag nanocomposites with different AgNPs loadings and to BC at 37 ◦ C after a contact time of 1 h are shown in Fig. 5A–D, respectively. The calculated anti-bacterial ratios were listed in Table 2. The BC-Ag nanocomposites performed much better than BC in reducing bacterial attachment, and it was found that bacterial adhesion decreased with increasing AgNPs content in the nanocomposites. The BC-Ag0.05 nanocomposites reduced E. coli attachment by 98.36%, S. aureus attachment by 99.98%, B. subtilis attachment by 100% and C. albicans by 89.6% compared with

Fig. 6. Comparative inhibition zones of BC and BC-Ag nanocomposites: E. coli (A), S. aureus (B), B. subtilis (C) and C. albicans (D) (in all plates (a) indicates pristine BC as the control, (b)–(f) are BC-Ag0.01 , BC-Ag0.02 , BC-Ag0.03 , BC-Ag0.04 and BC-Ag0.05 nanocomposites).

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BC, respectively. These results indicate that BC-Ag nanocomposites have excellent anti-bacterial activities against Gram negative E. coli, Gram positive S. aureus, B. subtilis and yeast C. albicans. The anti-bacterial activities of BC and BC-Ag nanocomposites against E. coli, S. aureus, B. subtilis and C. albicans were further investigated by disk diffusion method. The prepared nanocomposites were placed on a lawn of tested bacteria in TSA, respectively. The anti-bacterial activity is measured by the clear zone of inhibition around the samples after 24 h incubation and the images are shown in Fig. 6. The results of anti-bacterial activities of prepared BC and BC-Ag nanocomposites evaluated from the disk diffusion method are given in Table 2. As expected, no inhibition zones were observed for BC as control (a), implying that BC do not have any anti-bacterial properties against the tested four stains [54]. Among the tested four bacteria, B. subtilis has maximum zone of inhibition of 24.3 mm and E. coli has minimum zone of inhibition of 14.1 mm for BCAg0.05 nanocomposites. The differences observed in the diameter of zone of inhibition may be due to the difference in the susceptibility of different bacteria to the prepared BC-Ag nanocomposites. The differential sensitivity of different stains toward AgNPs possibly depends upon their cell structure, physiology, metabolism and their interaction with the charged AgNPs [55,56]. Similar nanocomposites were performed by other researches. The cellulose-Ag nanocomposites were prepared by Li et al. [57], and their anti-bacterial results were consistent to our results in this study that the nanocomposites have better anti-bacterial activities for S. aureus than E. coli. Silver/chitosan/cellulose fibers foam composites were synthesized and excellent anti-bacterial properties against Pseudomonas aeruginosa, E. coli and S. aureus were obtained by Guibal et al. [58]. In our system, the prepared BC-Ag nanocomposites not only displayed good Gram-positive or negative bacterial resistances, but also presented excellent anti-yeast properties. The present study clearly indicates that BC-Ag nanocomposites are excellent board spectrum anti-bacterial materials against Gram negative organism, Gram positive organism and yeast. Combining all beneficial qualities, make the prepared BC-Ag nanocomposites good anti-bacterial wound dressing materials such as wound dressings and bandages, as well as in other biomedical applications. 4. Conclusions In this study, BC-Ag nanocomposites were prepared in situ by introducing AgNPs into BC acting as the templates. The SEM, EDS, UV, XRD and TG analysis of the nanocomposites confirmed the existence of AgNPs in BC matrix and dispersed uniformly. Silver ion release behaviors in different pH conditions were tested and the results showed the silver ion release were strongly pH dependent, and silver ion release rates decreased with increasing pH. Antibacterial tests revealed that the nanocomposites containing AgNPs displayed excellent anti-bacterial performances on E. coli, S. aureus, B. subtilis and C. albicans, and also showed that the anti-microbial activity of the materials depended on the silver concentration. Furthermore, this process could be expanded in the synthesis of other metal-contained bacterial cellulose nanocomposites. Among the materials prepared, BC-Ag0.05 nanocomposite proved to have the best anti-bacterial properties. Meanwhile, its high silver ion release ability could favor wound healing. Thus, BC-Ag nanocomposites are confirmed to be excellent wound healing therapy option, which can be applied as potential wound dressings, bandages and so on. Acknowledgements The work was financially supported by the National Natural Science Foundation of China (51401109), the High-level Talent Project of Nanjing Forestry University (GXL201301), the Major Program

of the Natural Science Foundation of Jiangsu Higher Education of China (14KJB430018) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The authors would like to thank the Advanced Analysis & Testing Center of Nanjing Forestry University.

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