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Flexible Amoxicillin Grafted Bacterial Cellulose Sponges for Wound Dressing: in Vitro and in Vivo Evaluation Shan Ye, Lei Jiang, Jimin Wu, Chen Su, Chaobo Huang, Xiufeng Liu, and Wei Shao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16680 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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Flexible Amoxicillin Grafted Bacterial Cellulose Sponges for Wound Dressing:

in Vitro and in Vivo Evaluation

Shan Ye1, Lei Jiang1, Jimin Wu1, Chen Su1, Chaobo Huang1, Xiufeng Liu2* and Wei Shao1,3* 1College

of Chemical Engineering, Nanjing Forestry University, Nanjing

210037, P. R. China 2State

Key Laboratory of Natural Medicines, Department of Biotechnology of

TCM, China Pharmaceutical University, Nanjing 210009, PR China 3Jiangsu

Key Lab for the Chemistry & Utilization of Agricultural and Forest

Biomass, P. R. China

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ABSTRACT In this study, we report the design and fabrication of a novel biocompatible sponge with excellent antibacterial property, making it a promising material for wound dressings. The sponge is formed by grafting amoxicillin onto regenerated bacterial cellulose (RBC). It was observed that the grafted RBC could enhance the antibacterial activity against fungus, Gram-negative and Gram-positive bacteria. The morphology of strains treated with the grafted RBC and fluorescent stain results further demonstrated the antibacterial ability of the fabricated sponge. Moreover, cytocompatibility test evaluated in vitro and in

vivo illustrates the non-toxicity of the prepared sponge. More importantly, the wound infection model reveals that this sponge can accelerate the wound healing in vivo. This work indicates the novel sponge has the huge potential in wound dressing application for clinical use.

Keywords: bacterial cellulose, sponge, antibacterial, cytocompatibility, wound dressing

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INTRODUCTION Skin plays a very important role in retaining the balance of electrolytes and preventing invasion from microorganisms.1 Once skin is injured, wound dressings influencing the healing process of a wound, serve a very important purpose in wound care, especially to traumatic, thermal and chronic wounds.2 In general, an ideal wound dressing should combine several factors: protecting wound from bacterial infection, providing gas exchange, absorbing exudates, keeping a moist environment to enhance epithelial regrowth, and be easy to remove without any pain.3 Most importantly, it should be non-toxic and nonallergenic. In recent years, biomaterial-based wound dressings have been widely explored and applied.4-6 Bacterial cellulose (BC) is biosynthesized mainly by the acetic acid bacterium Gluconacetobacter xylinus. So it can obtain very high purity without any complicated extraction process or harsh chemical treatment.7 BC exhibits a

distinctive three-dimensional porous structure consisting of high aspect ratio

nanofibers.8 Because of its unique micromorphology, it shows excellent properties, such as high porosity, high permeability, high crystallinity, great mechanical strength, large surface area, high water holding capacity, good biocompatibility and nontoxicity.9-11 Based on these great advantages, BC has wide applications in diverse fields such as fuel cells, oil absorbents, catalyst industry, food and paper industry.12-13 Beside these areas, BC has been recently explored in the biomedical applications such as drug delivery, scaffolds

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for cartilage tissue engineering, dental implants, bone regeneration, skin therapy, artificial skin, regenerative medicine, artificial blood vessel and wound care product.14-16 Among these, BC is considered to be an excellent wound dressing material due to its high water retention capability, high porosity with a nanofibrous 3D network.17 However, BC itself does not have any inherent antibacterial activity, limiting the possibilities of applications in many biomedical areas. Therefore, it is crucially important to functionalize BC with antibacterial agent to make it have great potentials in the wound dressings to prevent from any infections during the wound healing process. Amoxicillin (-amino-p-hydroxybenzyl penicillin, AM) is a p-hydroxy analogue of ampicillin, which is a partial hydrophilic -lactam antibiotics.18 It has broader spectrum antibacterial activity and less effect on the interaction with food comparing to penicillin. It can bind to -lactam receptor proteins that involved in the synthesis and destruction of cell wall,18 therefore it is widely used for the treatment of infectious diseases in hospitals. However, AM can be easily degraded and lose its antibacterial activity consequently.19 Furthermore, AM has been limited its use in clinical because drug resistance will appear after a long time of using antibiotics, resulting in the emergence of superbugs.20 Therefore, limiting the release of AM is of significant importance. As we all known, BC presents a large amount of hydroxyl groups on its surface that makes it suitable for functionalizing with various materials.21 This study focuses on the fabrication of AM grafted onto RBC sponges with non-

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leaching AM as novel wound dressings. The antibacterial performance of these AM grafted RBC sponges was evaluated. Our results revealed that the synthesized AM grafted RBC sponges had enhanced antibacterial efficacy compared to RBC sponges against Gram-negative Escherichia coli ATCC 25922 (E. coli) and Gram-positive Staphylococcus aureus ATCC 6538 (S.

aureus) and fungus Candida albicans CMCC(F) 98001 (C. albicans). Moreover, this wound dressing is non-toxic to HEK293 cells. In addition, the efficacy of this wound dressing was evaluated in vivo. EXPERIMENTAL SECTION Materials. AM (>98%) were purchased from Aladdin Chemical Co. Ltd. 1-butyl3-methylimidazolium chloride ([Bmim]Cl, >97%) was purchased from Meryer Chemical Technology Co. Ltd. The other chemicals used in the tests were purchased from Sinopharm Chemical Reagent Co., Ltd. All reagents were of analytical grade and used as received without further purification. Production of AM grafted RBC sponges. RBC sponges were synthesized according to previous methods.22 RBC sponges were cut into 20 mm×20 mm pieces and immersed in a previously prepared ethanol solution of 0.8 wt% (3aminopropyl)triethoxysilane (APTES) and the mixture was gently stirred at 20 rpm at 25ºC for 4 h. The modified RBC sponges were washed with ethanol to remove the unreacted APTES and other impurities. 0.02 g EDC and 0.012 g NHS were added into 15 mL MES buffer solution with concentrations of 0.47, 0.6, 0.73, 0.87 and 1 mg/mL of AM and stirred for 2 h. Then the modified RBC

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sponges were added and the mixture was gently stirred at 20 rpm at 25ºC for 12 h. The obtained functionalized RBC sponges were washed with ethanol to remove the unreacted chemicals. They were freeze-dried at -40°C for 24 h. The final sponges were named as RBC1, RBC2, RBC3, RBC4 and RBC5, respectively. The detailed procedure is listed in Figure 1. Characterization. The surface morphologies of RBC and RBC5 sponges coated with a thin layer of platinum were evaluated by a JSM-7600F Scanning Electron Microscope (SEM). X-ray energy dispersive spectroscope (EDS) and element mappings of Si, N and S were taken on SEM as well. The chemical bonds of RBC and AM grafted RBC sponges were investigated by Fourier-transform infrared spectroscopy (FTIR). Antibacterial activity. The antibacterial activity of RBC1, RBC2, RBC3, RBC4 and RBC5 sponges was performed using culture turbidity as the qualitative measure of bacterial growth against Gram positive strain S. aureus, Gram negative strain

E. coli and fungus C. albicans. The strains were cultured in Tryptone Soya Agar (TSA) plates in an incubator overnight at 37 °C. A single colony was inoculated in 20 mL of Tryptone Soya Broth (TSB) and grown statically overnight at 37 °C. Then, 100 μL of this bacterial suspension was transferred into 100 mL of TSB in a conical flask and grown in a shaker incubator at 150 rpm at 37 °C until the bacteria concentration reached 1 × 106 CFU/mL. The RBC and AM grafted RBC sponges were sterilized by ultraviolet lamp for 60 min and then added into bacteria suspension. The bacterial growth behavior was examined in the

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presence of RBC1, RBC2, RBC3, RBC4 and RBC5 sponges and the growth was monitored at an interval of 1 h for 10 h at 150 rpm at 37 °C by measuring the increase of the OD at 600 nm. The bacterial growth curves were plotted against time by measuring the optical density (OD) at 600 nm using a SHIMADZU UV 2450 spectrophotometer. All of the experiments were performed in triplicate to confirm reproducibility. The leaching effect of the fabricated sponges was investigated by disk diffusion method against E. coli, C. albicans and S. aureus. RBC, RBC1, RBC2, RBC3, RBC4 and RBC5 sponges were cut into round shapes with 1 cm diameter and sterilized. Lawns of test strains (about 1×104 CFU/plate) were prepared on TSA. The sterilized sponges were placed upon the lawns carefully. These plates were incubated at 37°C for 24 h. The inhibitory action of tested sponges on the growth of the strain was determined. SEM Observation of Bacteria. The morphologies characterizations of bacteria were observed with JSM-7600F SEM. The bacteria treated with RBC5 sponge was fixed with 4% glutaraldehyde solution for 24 h. The untreated bacteria was used as control. The samples were dehydrated with sequential treatment of 30, 50, 70, 90, and 100% ethanol for 10 min, then lyophilized. Finally, it was mounted onto an aluminum stub, coated by platinum sputter, and observed using SEM. Fluorescent-Based Cell Live/Dead. Bacterial live/dead fluorescent staining assays were performed in order to further confirm the death of bacteria. E. coli,

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C. albicans, and S. aureus suspensions containing 1 × 106 to 1 × 107 CFU/mL cells were used. Then incubated them with RBC5 sponges in a shaker incubator at 150 rpm at 37 °C for 2 h. Then the mixture was stained with propidium iodide and SYTO9 (LIVE/DEAD™ BacLight™ Bacterial Viability Kit, L13152) according to the instruction of the kit and imaged using a laser scanning fluorescence microscopy (Olympus, IX53). The untreated cell suspension was taken as control. Cytotoxicity tests. The HEK293 cell lines were cultured in RPMI medium supplemented with 10% fetal bovine serum, 100 mg/mL penicillin and 100 mg/mL streptomycin. The cytotoxicity was measured using the MTT assay method. 200 mL of HEK293 cells, at a density of 1×10 5, were placed in each well of a 48-well plate. Then, the cells were incubated over night at 37°C in a humidified 5% CO2-containing atmosphere. Furthermore, the media was discarded. RBC, RBC1, RBC2, RBC3, RBC4 and RBC5 sponges with the same size (5×5 mm) were placed slightly on the top of cells and then fresh media was added. Wells containing only the cells were used as control. The cells were treated for another 24 h. Then, the media containing sample was changed with fresh media and 20 mL of dimethyl thiazolyl diphenyl (MTT) was added and the incubation continued for 6 h. The medium was removed and 200 mL DMSO was added to each well to dissolve the formazan. The absorbance was measured with a test wavelength of 570 nm and a reference wavelength of 630 nm. Empty wells (DMSO alone) were used as blanks. The relative cell viability

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was measured by comparison with the control well containing only the cells. On the other hand, HEK293 cells were plated on the confocal culture dish. As cells reached to 30% confluence in all groups, then, the cells were treated with RBC and RBC5 sponges for another 24 h. Cells were fixed and stained with FITCphalloidin and DAPI. The morphologies were visualized by confocal microscopy (Leica DM2500, Germany). In vivo wound healing evaluation. Female mice weighting about 20 g and 6-8 week age were used for in vivo studies. They were randomly divided into 3 groups. Each group contained five mice. For the surgery part, all procedures were performed in the aseptic condition during the surgery and changing dressing process. The mice were anaesthetized with intraperitoneal injection of chloral hydrate (0.3 mg/kg body weight), a full thickness skin round wounds with diameter of 6 mm was created on the back of each mouse. The wounds of two groups were covered with the RBC and RBC5 sponges, respectively. And the wounds group without any covering dressing was used as control. The dressing sponges were changed every 3 days and the wounds contraction was monitored. During the changing of dressings, the area of the wound was measured and imaged. All pictures were adjusted to be the same size. The wound area closure (RC) is determined: RC (%) =(A0− A)/A0 × 100%

Eq. (1)

Where A0 is the wound area created, and A is the wound area at day of the changing of dressing.

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RESULTS AND DISCUSSION The main objective of this study was to functionalize BC with antibacterial agent to fabricate potential wound dressings. Therefore, our solution was grafting an active molecule AM onto RBC fibers. Synthesis method of AM grafted onto RBC was displayed in Figure 1. It is based on three steps: (i) aminoalkylsilane groups grafted onto RBC through Si-O-C bonds via chemical condensation method.8 (ii) the COOH of AM are treated with EDC/NHS, which led to the formation of an NHS activated ester group.23 (iii) These groups on the AM are able to react with the terminal NH2 groups of RBC, leading AM covalently linked onto RBC surface through the amidation reaction.24

Figure 1 Scheme of the RBC grafted with AM.

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Morphologies. The morphologies of the fabricated RBC and RBC5 sponges were imaged using SEM, which is shown in Figure 2. Fig. 2a shows the morphology of RBC which exhibits a nanoporous 3D web-like structure, resulting in a large surface area and high porosity. The porosities of RBC, RBC1, RBC2, RBC3, RBC4 and RBC5 sponges were shown in Figure S1. The porosities of AM grafted RBC sponges were in the range of 80%-81%, which were a little smaller than that of RBC sponge (86%). The swelling behaviors of RBC, RBC1, RBC2, RBC3, RBC4 and RBC5 sponges in PBS buffer solution were shown in Figure S2. All the sponges were saturated with PBS after 1 h. The swelling ratio of RBC was 2020% for equilibrium and a lower swelling ability was observed in the RBC1, RBC2, RBC3, RBC4 and RBC5 sponges, which could be due to the lower porosity of functionalized RBC sponges. However, the swelling ratios of functionalized RBC sponges were still at a high level which were in the range of 1100-1150%. The high porous structure and good swelling property of functionalized RBC sponges result in great absorbed exudates ability, which make the fabricated sponges to be potential wound dressings.8 Although the nanoporous 3D web-like structure can still be found in the case of RBC5 sponge, thicker fibers were observed due to the successful graft with AM (Fig. 2b). This morphology is consistent with porosity and swelling results. The Si, N and S element maps and EDS spectrum of RBC5 sponge were displayed in Fig. 2c-f, that confirmed AM was grafted onto RBC sponge via APTES. In a

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word, AM was successfully grafted onto RBC sponges combining SEM and EDS results. a

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Figure 2 SEM images of RBC (a) and RBC5 (b) sponges, element mapping pictures of Si (c), N (d) and S (e) and EDS analysis (f) of RBC5 sponge. FTIR analysis. The successful grafting AM onto RBC sponge was further confirmed by FTIR analysis, as shown in Figure 3. For the IR spectrum of RBC (Figure 3a), it was quite similar to that of BC (Figure S1a). There is a board band between 3500 and 3200 cm-1 corresponding to the intramolecular

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hydrogen bond and the hydroxyl group.25 The peaks at 1163 cm-1 and 1061 cm1

are attributed to the C-O asymmetric bridge stretching and the C-O-C

pyranose ring skeletal vibration of RBC, respectively.22 It can be seen that there is no chemical reaction occurred during the dissolution and regeneration processes because the chemical structures of BC and RBC are the same. In the case of AM (Figure S3b), there are two characteristic bands at 3461 cm−1 and 3175 cm−1, that can be assigned to phenol OH stretching and amide N-H, respectively.26 The other main characteristic peaks observed at 3,020 cm−1 (benzene ring C-H stretching), 1,776 cm−1 (β lactam C=O stretching), 1,688 cm−1 (amide I, C=O stretching) and 1,520 cm−1 (benzene ring C=C stretching) can be found on the FTIR spectra.27-28 All characteristic peaks of both AM and RBC are present in the FTIR spectra of the AM grafted RBC. Besides, two new characteristic absorption bands centered around 1480 and 765 cm -1 appear which are assigned to N-H bend C-N stretch combination band and stretching vibration of Si-O-Si, respectively.8, 29 This indicates that AM was successfully grafted onto RBC sponges.

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Figure 3 FTIR spectra of RBC (a) and AM grafted RBC sponges: RBC1 (b), RBC2 (c), RBC3 (d), RBC4 (e) and RBC5 (f). Bacterial Growth kinetics. The growth kinetics were monitored by measuring optical density value at 600  nm (OD600) at different time intervals. Figure 4 shows the kinetics of antibacterial activity of AM grafted RBC sponges against the tested strains in terms of cell viability. The bacteria treated with functionalized RBC sponges grew quite slower than the control. However, a noticeable difference in the growth rate was displayed among the tested strains. For E. coli strain (Figure 4A), the growth curve was found to be predominantly affected by the initial additive amount of AM into the functionalized RBC sponges. Along with the increase of the initial additive amount of AM, the growth of E. coli is inhibited more and more severely for the functionalized RBC sponges. The bacterial growth can be completely inhibited by RBC5 sponge with the highest initial additive amount of AM. In the case of C. albicans strain (Figure 4B), the inhibitory efficiency of functionalized RBC sponges is primarily dependent on its initial additive amount of AM. Functionalized RBC sponges can slightly inhibit the bacterial growth while the bacterial growth can’t be inhibited completely even treated with RBC5 sponge. As to S. aureus (Figure 4C), the bacterium cells stopped growing treated with RBC1, RBC2, RBC3, RBC4 and RBC5 sponges. Overall, the synthesized functionalized RBC sponges can effectively kill the strains. Besides, disk diffusion method was carried out with RBC and functionalized RBC sponges against E. coli, C.

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albicans and S. aureus to check for the leaching effect of the fabricated sponges. The images of inhibition zones were shown in Figure S5. No inhibition zones were observed around the tested sponges after 24 h inhibition, which proves that no AM leaches out from the prepared functionalized RBC sponges. Thus, in this study the fabricated functionalized RBC sponges displayed excellent antibacterial performance without any AM leaching.

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Figure 4 Bacterial growth curves of AM grafted RBC sponges: control (a), RBC1 (b), RBC2 (c), RBC3 (d), RBC4 (e) and RBC5 (f) against E. coli (A), C.

albicans (B) and S. aureus (C). Live/dead assay. The bacteria treated with AM grafted RBC sponges were stained with LIVE/DEAD BacLight Bacterial Viability Kit (L13152) for 15 min and then observed with a fluorescence microscope (OLYMPUS IX53, Japan). The live bacteria with intact cell sponges can stain fluorescent green, while the dead bacteria with damaged membranes can stain fluorescent red.30 Figure 5 demonstrated that all the E. coli cells without any treatment (the control) were green (alive), and only a few cells (