graphene oxide-CuO nanocomposite films

Journal Pre-proof Development and antibacterial activities of bacterial cellulose/graphene oxide-CuO nanocomposite films Yan-Yan Xie, Xiao-Hui Hu, Yan-Wen Zhang, Fazli Wahid, Li-Qiang Chu, Shi-Ru Jia, Cheng Zhong

PII:

S0144-8617(19)31124-5

DOI:

https://doi.org/10.1016/j.carbpol.2019.115456

Reference:

CARP 115456

To appear in:

Carbohydrate Polymers

Received Date:

21 June 2019

Revised Date:

2 October 2019

Accepted Date:

7 October 2019

Please cite this article as: { doi: https://doi.org/ This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier.

Development and antibacterial activities of bacterial cellulose/graphene oxide-CuO nanocomposite films Yan-Yan Xie,1,2 Xiao-Hui Hu,1,2 Yan-Wen Zhang,1,2 Fazli Wahid,1,2 Li-Qiang Chu,3 Shi-Ru Jia,1,2 Cheng Zhong1,2,*

1

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State Key Laboratory of Food Nutrition & Safety, Tianjin University of Science and

Technology, Tianjin, P.R. China 2

University of Science and Technology, Tianjin, P.R. China 3

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College of Chemical Engineering and Materials Science, Tianjin University of Science and

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Technology, Tianjin 300457, P.R. China

Correspondence: Cheng Zhong, Tianjin, 300457, China

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*

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Key Laboratory of Industrial Fermentation Microbiology, (Ministry of Education), Tianjin

Fax: 86-22-60602298; Tel: +86-22-60601268; E-mail: [email protected];

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[email protected];

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Highlights

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A novel nanocomposite film incorporating GO-CuO nanohybrids into BC was achieved.

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The BC/GO-CuO composite showed a superior activity against gram-positive bacteria.

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Antibacterial activity was attributed to cell membrane damage and ROS generation.

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The BC/GO-CuO film exhibited good biocompatibility towards mice fibroblast

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cells.

Abstract

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The absence of antibacterial activity in bacterial cellulose (BC) restricts its applications

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in the biomedical field. To introduce antimicrobial properties into BC, we studied the synthesis, structures, and antimicrobial properties of a novel nanocomposite film comprising

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BC, graphene oxide (GO), and copper-oxide (CuO) nanosheets. The nanocomposite film was synthesized by incorporating GO-CuO nanohybrids into BC matrix through homogenized blending. The CuO nanosheets, with a length range between 50 nm-200 nm and width range between 20 nm-50 nm, which were uniformly growed on the GO along with even distribution

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of GO-CuO nanohybrids on the surface of the cellulose fibers. The nanocomposites displayed better antimicrobial activity against gram-positive than gram-negative bacteria. BC/GO-CuO nanocomposites showed higher antibacterial activity than BC/CuO. We also elucidated the mechanisms of antimicrobial activity of the nanocomposites. Further, the nanocomposites

exhibited biocompatibility towards mice fibroblast cells. The nanocomposites might serve as an excellent source for development of antimicrobial materials. Key words: Bacterial cellulose; GO-CuO nanohybrid; BC/GO-CuO nanocomposites;

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Antibacterial properties

1. Introduction Bacterial infections are still a major threat to public health that may cause enormous socio-economic problems, even though there has been great advancements in medical technologies and health-care fields (Ng et al., 2014). Additionally, conventional antibiotics can lead to the emergence of a variety of drug-resistant pathogens. To deal with this problem,

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scientists are focusing on developing novel antibacterial materials. Several materials,

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including nanoparticles (NPs) and polymer-based antibacterial materials, have been

developed to treat bacterial infections (Ng et al., 2014; Fazli Wahid, Zhong, Wang, Hu, &

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Chu, 2017). Bacterial cellulose (BC) based-nanocomposites have attracted considerable

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research interest. BC is generally produced by Gluconacetobacter xylinus, a gram-negative bacteria (Shao et al., 2017). It has excellent properties, including desirable mechanical

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strength, high purity, good water-retention, high specific surface area, biodegradability, biocompatibility and low toxicity (Khattak et al., 2019; Wang, Lu, & Zhang, 2016).

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Therefore, it is used in a variety of fields, such as food packaging, water treatment, papermaking, and biomedicine (de Oliveira Barud et al., 2016; Santos et al., 2015; Shi, Zhang, Phillips, & Yang, 2014; Svensson et al., 2005). However, BC lacks antibacterial properties,

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restricting its applications in the biomedical field. Various antibacterial agents such as chitosan (Lin, Lien, Yeh, Yu, & Hsu, 2013), benzalkonium chloride (Wei, Yang, & Hong, 2011), metal (Biliuta & Coseri, 2019; Wu et al., 2014) and metal oxide (Janpetch, Saito, & Rujiravanit, 2016) nanostructures have been introduced to endow BC with antibacterial properties.

Graphene oxide (GO), a two-dimensional nano-plate-like material, possess unique physiochemical properties, making it an attractive material in various biomedical applications (Ahmad, Fan, & Hui, 2018; Feng, Zhang, Shen, Yoshino, & Feng, 2012; Tuan Hiep et al., 2015; Yan et al., 2013). GO has abundant functional groups, including hydroxyl, carboxyl, and epoxide, on its surface, which make it a good candidate to adsorb metals or other

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inorganic precursors (D. Fu, Han, Chang, & Dong, 2012). Various metal and metal oxide nanostructures have been loaded on GO for improving its antibacterial activity. For instance,

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Cui and Liu, (Cui & Liu, 2015) reported GO/Ag nanocomposites with improved antibacterial

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properties. The nanocomposites showed better antibacterial activity than Ag-NPs alone.

enhanced antibacterial properties.

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Recently, another study (Y. Li, Yang, & Cui, 2017) reported GO/CuO nanocomposites with

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Metal oxide nanostrucures, particularly CuO nanostrucres, have attracted considerable attention for its potential application in the biomedical field. It possesses photocatalytic and

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photovoltaic properties due to the narrow band gap in its crystal structure (J. Li et al., 2011). Recent attempts emphasizing the development of new antibacterial nanocomposites based on CuO nanostrucures and polymers (F. Wahid, Wang, Lu, Zhong, & Chu, 2017) have been carried out. Recently, Araújo et al. (Araujo et al., 2018) reported BC nanocomposites with

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CuO nanostrucures that showed antibacterial activity against tested bacterial strains. In a previous study, our group (L.-P. Liu et al., 2017) developed BC/GO/TiO2 nanocomposite films for antibacterial and photocatalytic applications. The nanocomposite showed strong photocatalytic activity while antibacterial activity was displayed only after near-UV irradiations. Therefore, in this study, we report the synthesis, characteristics, and

antimicrobial activity of a novel nanocomposite film incorporating GO-CuO nanohybrids into BC matrix. The physicochemical properties, including morphology and structures, were investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) equipped with energy dispersive X-ray spectroscopy (EDS). The antimicrobial activity against gram-positive and gram-negative

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bacteria was evaluated. Bacterial morphology and intracellular reactive oxygen species (ROS) were assessed to understand the antibacterial mechanism of the nanocomposite films.

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Furthermore, the cytotoxicity of the BC/GO-CuO nanocomposite films toward mouse

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embryonic fibroblast cells was also evaluated.

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2. Materials and methods

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2.1. Materials

Gluconacetobacter xylinus (CGMCC No. 2955) was screened from a traditional Chinese

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drink by our group and preserved in China General Microbiological Culture Collection Center with the registration number 2955. GO nanosheets were purchased from XFNANO Ltd. (Shanghai, China). Copper chloride dihydrate and sodium hydroxide were obtained from Aladdin Co. Ltd. (Shanghai, China). Yeast extract, glucose, tryptone, acetic acid and

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disodium phosphate were purchased from Oxoid Ltd. (United Kingdom). All other chemicals were of analytical grade and were used without any further purification. Distilled water was used for all experiments.

2.2 Methods 2.2.1 Preparation of BC slurry

BC membrane was obtained following the method described by Yang et al., (Yang et al., 2016). Briefly, a single colony of G. xylinus was incubated in a 200 mL sterilized culture medium containing glucose (25 g/L), yeast extract (7.5 g/L), peptone (10 g/L), and Na2HPO4·12H2O (10 g/L) at pH 6.0 and incubated in a shaker incubator at 30 °C for 24 h. The formed bacterial suspension was inoculated into 200 mL fermentation medium and

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incubated at 30°C for 7 days. The obtained cellulose membrane was rinsed with deionized water several times to remove the bacterial cells and other impurities adhered to its surface.

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Subsequently, the membrane was treated with 0.1 M NaOH solution for several days until the

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cellulose membrane became white, followed by washing with distilled water until neutral pH value. The wet BC membranes were cut into pieces and thoroughly treated with a

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2.2.2 Fabrication of GO-CuO nanohybrid

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homogenizer (Honghua instrument factory, Jiangsu, China) to obtain a uniform slurry.

GO-CuO nanohybrid was synthesized by following a previously reported method (Y. Li

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et al., 2017). Briefly, 40 mg of graphene oxide nanosheets were added to 40 mL water, sonicated at 500 W for 12 h with occasional stirring at an interval of 30 min to prepare 1 mg/mL GO suspension. CuCl2·2H2O (2 g) was added into the GO suspension stirred for 30

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min to get an evenly mixed solution. Subsequently, the mixed solution was heated at 100 for 30 min. During the heating process, 3 mL NaOH solution with a concentration of 9.35 mol/L was slowly added into the solution. The formed precipitates were obtained by centrifugation (10,000 rpm for 10 min) followed by rinsing with deionized water. The resultant precipitates were dried in a vacuum oven at 80 °C overnight and ground to get powder.

2.2.3 Impregnation of GO-CuO nanohybrid into bacterial cellulose BC/GO-CuO nanocomposites were prepared by mixing GO-CuO aqueous solution (2.5 mg/mL, 5.0 mg/mL, and 7.5 mg/mL) with BC slurries (1% dry weight). The resulting mixture was stirred and kept at room temperature for 5 h and then filtered through a cellulose acetate membrane, followed by vacuum-drying by a sheet former (Rapid-Kӧthen RK-3A) at

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80 °C for 15 min to obtain BC/GO-CuO nanocomposite films. The proportion of GO-CuO in

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BC/GO-CuO composites was about 5%, 10% and 15% (w/w %), and the nanocomposite

films were denoted as BGCu5, BGCu10, and BGCu15, respectively. In addition, the BC/CuO

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nanocomposite films were prepared in the same way as BC/GO-CuO composite without the

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addition of GO.

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2.3 Characterization of nanocomposite films

The X-ray diffraction (XRD) were measured by a Shimadzu XRD-6100 X-ray powder

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diffractometer (Shimadzu, Japan) using Cu Kαradiation (λ = 1.5406 Å) at 40 kV with the scan range from 5° to 70° and a scan rate of 4°/min. SEM micrographs were obtained by using a SU1510 microscope (Hitachi High-Tech, Japan). All samples were coated with a thin layer of platinum prior to SEM observation. TEM, high-resolution TEM (HRTEM)

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micrographs and the selected-area electron diffraction (SAED) patterns of the sample were obtained by using JEM-2010FE transmission electron microscope (JEOL, Japan). Elemental composition was analyzed by a SU1510 scanning electron microscope equipped with energydispersive X-ray spectroscopy (EDS) device.

2.4 Antimicrobial activity of BC/GO-CuO nanocomposites

Antibacterial activity of BC/GO-CuO nanocomposites was tested by agar disc diffusion method and colony-forming unit method. The disc diffusion method was performed using Luria-Bertani (LB) solid agar medium. The antibacterial properties of the films were tested against Gram-positive S. aureus (ATCC 6538) and B. subtilis and gram-negative E. coli (DH5α) and P. aeruginosa (NCTC 2000)

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bacterial species. In brief, a single colony of bacteria was incubated in 50 mL sterilized Luria-Bertani (LB) culture medium at 37°C for 12 h. The concentration of the bacteria was

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adjusted to OD600=0.5, approximately 2.5 × 108 CFU/mL. 1 mL of the bacterial suspension

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was added to 1% solid agar medium (100 mL), followed by pouring into Petri dishes and allowed to solidify by cooling. The circular films with 9 mm diameter were sterilized by UV

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irradiation and placed on the agar plates. Subsequently, the plates were incubated at 37 °C for

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24 h, and the formed inhibition zones were measured by a Vernier caliper (Fazli Wahid et al., 2019). The experiment was repeated three times against each strain and the average values

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were recorded.

In the colony-forming count method, the antibacterial properties of the films were tested against S. aureus and E. coli according to a modified method of Fu et al. (F. Fu et al., 2018). Briefly, one piece of the nanocomposite film (40 mg) was immersed in 20 mL of the bacterial

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suspension in PBS and the final concentration of the composite film was 2 mg/mL. The suspension was incubated at 37 °C for 5 h in a shaking incubator. Then, 0.5 mL of suspension was taken every half an hour and serially diluted with PBS solution. Subsequently, 100 µL bacterial suspension was spread on the LB agar plates in triplicate and incubated at 37

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24 h. The colonies were counted and the survival rate of bacteria was calculated as follows:

Survival rate

100%

Where Na and N0 are the number of the surviving bacteria in agar plates in the presence and absence of the composite film, respectively.

2.5 Antibacterial mechanism

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2.5.1 Membrane-permeability of BC/GO-CuO nanocomposites

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The permeability of the bacterial cell membrane was characterized by Propidium iodide (PI) fluorescence staining. PI cannot enter into the cells with intact membranes but can enter

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into the cells with broken membranes and bind to intracellular DNA to form complexes.

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Based on the above principle, a modified method of Veerman et al. (Veerman et al., 2010) was used to determine the cell membrane-permeability of the composite by detecting the

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fluorescence intensity of PI. The BC/GO-CuO nanocomposite film (40 mg) was immersed in 20 mL of the bacterial suspension and shaken at 37 °C for 5 h. Subsequently, 0.5 mL of

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suspension was removed after 3 h and 5 h. The final density of the suspension was adjusted to OD600=0.5. The cell suspension (100 µL) was mixed with PI (100 µL, 12 μg/mL) and incubated at room temperature for 5 min. Fluorescence intensity was measured at 535 nm and

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617 nm, using a multifunctional enzyme labeling apparatus (Infinite 200PRO, TECAN, Austria). All measurements were performed in triplicates.

2.5.2 Scanning electron microscopy for assessment of bacterial surface morphology The surface morphology of bacteria treated with BC/GO-CuO nanocomposite films was

examined by SEM, following a previously reported method (Irwansyah et al., 2015). Firstly,

the BC/GO-CuO nanocomposite film (40 mg) was immersed in 20 mL of the bacterial suspension. The solution was then incubated at 37°C for 5 h in a shaking incubator. The suspension with nanocomposites were dropped onto a silicon wafer, followed by fixation with 2.5% glutaraldehyde for 4 h at 4°C (untreated bacteria was taken as control). After washing with PBS solution, the samples were subjected to dehydration by treating with a

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series of ethanol solutions（30%, 50%, 70%, 80%, 90%, and 100%）for 10 min with each step. Finally, the silicon wafer was dried with nitrogen to retain the morphologies of the

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SU1510 scanning electron microscopy (HITACHI, Japan).

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bacteria deposited on the silicon wafer, sputter coated with gold and examined by using

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2.5.3 Measurement of intracellular reactive oxygen species (ROS)

2,7-dichlorofluorescein diacetate (DCFH-DA), a ROS indicator was used to determine

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the generation of ROS in S. aureus, Though DCFH-DA does not have fluorescence and can easily enter into the cell membrane and be hydrolyzed to DCFH by a cellular esterase. DCFH

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cannot pass through the cell membrane allowing ease in the loading of fluorescent probes into the cell. Subsequently, DCFH reacts with intracellular ROS to produce highly fluorescent dichlorofluorescein (DCF) (Guo et al., 2010). Moreover, the fluorescence intensity is

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proportional to the amount of ROS. Herein, S. aureus suspension with an initial OD of 0.05 was treated with BC/GO-CuO nanocomposites at 37 °C, 120 rpm for 5 h. The final concentration of BC/GO-CuO nanocomposites membrane in the bacterial suspension was 2.0 mg/mL and 5.0 mg/mL, respectively. 1 106 to 2 107 cells were washed twice with PBS buffer solution and centrifuged for collection. The collected cells were resuspended in DCFH-DA solution (10 µmol/L) and incubated at 37°C in the dark for 20 min. Then, the

solution was mixed upside down at every 3-5 min for proper mixing of the DCFH-DA fluorescent probe with the bacterial solution. Then the bacterial solution was washed twice with phosphate buffered saline in dark condition. Subsequently, 200 µL of bacteria solution was transferred to a 96-well cell culture plate, and the fluorescence intensity of each well was measured at the excitation wavelength and emission wavelength of 488 nm and 525 nm,

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respectively, by a multi-functional enzyme labeling instrument (Infinite 200 PRO, TECAN,

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Austria).

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2.6 Cell Cytotoxicity

The cytotoxicity of BC/GO-CuO nanocomposites was tested by MTT (3-(4,5-

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dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay. Mouse embryonic fibroblasts cells (NIH-3T3) were cultured in DMEM medium containing 10%

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FBS, and 1% penicillin-streptomycin (PS) mixture followed by incubation in a humidified atmosphere with 5% CO2 at 37°C. The sterilized samples were put into the 96-well plate at

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concentrations of 0.5 mg/mL, 2.5 mg/mL, 10 mg/mL and 50 mg/mL. The cells were seeded into the plate at a density of 106 cells/well and incubated at 37°C and 5% CO2. The films were removed after 24 h, and the medium was replaced with fresh medium. Then MTT

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solution (5 mg/mL) was added into each well and incubated for 4 h. Subsequently, the medium was removed and replaced with DMSO and mixed to dissolve the formazan crystals. Cells cultured in the medium were used as a negative control, and the cell survival rate was defined as 100%. The absorbance of the final solution was measured at 490 nm, using a microplate reader (Infinite 200 PRO, TECAN, Austria) (Xu et al., 2018).

3. Results and discussion

3.1 Development of BC/GO-CuO nanocomposites and the stability of CuO nanosheets in nanocomposite films In this study, we first synthesized the GO-CuO nanohybrid by an in situ method, then loaded the GO-CuO nanohybrid into the BC matrix using the homogenized blending method

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to synthesize a novel BC/GO-CuO antibacterial composite film. The schematic representation

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of the nanocomposite film development is shown in Fig. 1. The presence of GO provides

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nucleation sites for CuO nanosheets growth and increases the surface area for efficient in situ growth. Moreover, GO can keep CuO nanosheets well-dispersed in aqueous solution and

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2017; Mohammadnejad et al., 2018).

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enhance the antibacterial activity by its synergistic effect with CuO nanosheets (Y. Li et al.,

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3.2 Structure and morphology of the nanocomposite films XRD patterns of BC film, BC/GO10%, BC/GO-CuO5% and BC/GO-CuO10% were analyzed (Fig. 2). BC displayed two typical crystallization peaks at 2θ=14.9° and 2θ=23.1°

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(Fig. 2A) that could be attributed to (110) and (200) crystal planes of BC, respectively. The XRD peak at 2θ=17.2° could be assigned to the amorphous region of BC. These characteristic diffraction peaks indicated a typical Iα cellulose (Zhu et al., 2011). Except, the

characteristic diffraction peaks of BC, the additional peak at 2θ=11° in XRD pattern of BC/GO10% (Fig. 2B) could be attributed to the (002) reflection of stacked GO sheets (Ma, Zhang, Xiong, Yong, & Zhao, 2011). XRD peaks at 2θ=35.8°, 39.0°, 49.0°, 58.2°, 61.8°,

66.6°and 68.2° could be respectively indexed to (002), (111), (-202), (202), (-113), (-311) and (220) monoclinic phase of CuO (Fig. 2C, D) (JCPDS, no. 65-2309), confirming the presence of CuO-NPs in the nanocomposite films (Lanje, Sharma, Pode, & Ningthoujam, 2010). In addition, it was observed that the peak intensity of CuO increased by increasing the

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content of CuO nanosheets in the BC/GO-CuO composite films.

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Transmission electron microscopic (TEM) image of GO-CuO nanohybrid revealed a uniform

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distribution of the CuO nanosheets grown on the GO sheets (Fig. 3A, and Fig. S1). The CuO nanosheets showed a length range from 50 nm to 200 nm and a width range from 20 nm to 50

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nm. The in situ-grown CuO nanosheets on GO flakes appeared to be agglomerated rather than individual nanosheets. This could be due to the absence of a capping agent or a

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surfactant as well as the presence of the high-density nucleation sites of the GO nanosheets. As shown in the high-resolution TEM (HRTEM) images (Fig. 3B and C), the lattice fringes

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of the dark spots on GO with lattice spacing of 0.253 nm and 0.232 nm could be assigned to the (002) and (111) lattice planes of CuO nanosheets, respectivly (Islam, Chakraborty, Roy, Das, & Acharya, 2018; Zhou, Wang, Xu, & Li, 2006). Moreover, the selected-area electron

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diffraction (SAED) pattern (Fig. 3D) also shows the two major crystalline planes of the ring patterns, indexed as the (002) and (111) rings of of CuO nanosheets, in agreement with the XRD results. Energy-dispersive X-ray spectroscopy (EDS) analysis in SEM confirmed the presence of CuO in the BC/GO-CuO film (Fig. S2). As shown in Fig. 3E1, the original BC film was white and translucent. The SEM micrograph of BC film showed a three-dimensional entangled network structure of the

nanofibers. The diameter of the nanofibers was found to be 30-100 nm (Fig. 3E2). The crosssectional micrographs of the BC membrane revealed a layered structure of the nanofibers (Fig. 3E3). The brown color of the BC/GO-CuO membrane could be due to the incorporation of GO-CuO nanohybrids (Fig. 3F1). Natural porous structures of BC were observed to be filled with a large number of GO-CuO nanohybrids with a uniform distribution of GO-CuO

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nanohybrids on the surface of the BC/GO-CuO membrane (Fig. 3F2). The cross-sectional micrograph of BC/GO-CuO membrane exhibited a more compact structure compared to the

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BC film (Fig. 3H3).

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The stability of the BC/GO-CuO nanocomposite film was investigated by soaking it in different solutions with different pH. For the purpose, 100 mL of 0.1 M HCl (acidic), 0.1 M

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PBS (neutral), and 0.1 M NaOH ( alkaline ) solutions, each was used. The loss of CuO

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nanosheets in BC/GO-CuO nanocomposite films was observed after soaking for 24 h (Fig. S3). The weight loss of CuO nanosheets in 0.1M HCl was 95.23%, wherein the weight loss of

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CuO nanosheets in 0.1M NaOH and PBS solution was found to be 1.48%, and 0.16%, respectively.

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3.3 Antibacterial activity

The antimicrobial activity of nanocomposites against gram-positive (S. aureus, B.

subtilis) and gram-negative (E. coli, P. aeruginosa) was measured using agar disc diffusion assay. The results are shown in Table 1 and Fig. S4. Original BC did not show any activity against tested bacterial strains, which was in agreement with previous studies (Shahmohammadi Jebel & Almasi, 2016; Yang et al., 2016). BC/GO10% also did not produce

prominent inhibition zone against the tested bacterial strains. It could be due to the presence of GO that exerts its antibacterial effect mainly through direct contact inhibition mechanism (S. B. Liu et al., 2011). In contrast, BC/GO-CuO showed antimicrobial activity against all strains. In addition, as shown in Table 1, increasing the content of GO-CuO in the composite, the diameter of the inhibition zone was increased. The diameters of inhibition zones produced

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by the composite material were in the order from large to small: B. subtilis > S. aureus > E. coli > P. aeruginosa, revealing a stronger antimicrobial activity of BC/GO-CuO composites

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against the gram-positive bacteria. The results were similar to previously reported research

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(Almasi, Jafarzadeh, & Mehryar, 2018). This could be attributed to the different cell wall

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Gopalakrishnan, & Natarajan, 2013).

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structures of gram-positive and gram-negative bacteria (Anitha, Brabu, Thiruvadigal,

Furthermore, the antibacterial properties of BC, BC/CuO10%, and BC/GO-CuO10%

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nanocomposite films were quantitatively evaluated by the colony-forming unit method. As shown in Fig. 4, it was observed that the original BC films did not affect the viability of the tested bacterial strains. However, the number of viable S. aureus was found to be reduced to 27.3% and 48.3% by incubation with BC/GO-CuO10% and BC/CuO10% nanocomposite films

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for 0.5 h, respectively (Fig. 4A). Moreover, after 5 h of incubation, the S. aureus was found

to be completely eradicated. The analysis revealed that the number of viable E. coli was

around 80% even after 5 h of incubation with the two films (Fig. 4B). The results showed that BC/GO-CuO nanocomposite films displayed stronger antibacterial properties towards the gram-positive bacteria than gram-negative bacteria. The results were found to be consistent

with that of the agar disc diffusion assay. Moreover, the inhibitory activity of BC/GOCuO10% on S. aureus was better than that of BC/CuO10% films. The enhanced antibacterial activity may be due to the well-dispersion of CuO nanosheets over GO and the synergistic antibacterial effect of CuO nanosheets and GO. The results were further confirmed by the live/dead bacterial staining assay using two

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fluorescent dyes—Hoechst 33342, that produces blue color stained live bacteria, and propidium iodide, that produces red color stained dead bacteria. As shown in Fig. S5, it was

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observed that the number of red color stained bacteria in control were lower (Fig. S5A) as

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compared to those being incubated with the BC/GO-CuO10% nanocomposite (Fig. S5B), indicating its antimicrobial efficiency against S. aureus. This indicated that the bacterial cell

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wall and membrane were disrupted after coming in contact with the composite films

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(Irwansyah et al., 2015). On the other hand, only a few cells of E. coli were labeled red (Fig. S5C, D), revealing a stronger efficiency of the composite film against the gram-positive

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bacteria. This result was consistent with that obtained by the agar plate diffusion method and the colony counting method.

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3.4 Antibacterial mechanism

The cell membrane integrity was characterized by the change of fluorescence intensity

of samples after PI staining. As shown in Fig. 5A, the fluorescence intensity of S. aureus

bacteria after incubation with 2.0 mg/mL and 5.0 mg/mL BC/GO-CuO10% nanocomposite films for 5 h was 2.18 and 3.26 times higher than that of the control group, respectively.

Wherein, the fluorescence intensity for E. coli treated with same conditions was found to be 1.05 and 1.26 times higher than that of the control group, respectively. The results indicated that BC/GO-CuO composite membrane could disrupt the membrane integrity of S. aureus and E. coli. Moreover, the damage was observed to be increased with increased concentration of BC/GO-CuO. This could probably be due to the interaction of the sharp GO nanosheets

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and CuO-NPs, with the lipid membrane of the bacteria cells changing their permeability

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The morphological changes of bacteria after incubation with the nanocomposite films

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were investigated using Scanning electron microscopy (SEM) to understand the mechanism of antimicrobial activity of the BC/GO-CuO nanocomposite films, As shown in Fig. 5C and

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D, the control group of S. aureus and E. coli showed clear edges and smooth surfaces. On the

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contrary, cellular deformation, surface collapse, surface perforation, and roughness were found in treated group incubated with BC/GO-CuO nanocomposite films for 5 h (Fig. 5 E and

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F). This indicated that BC/GO-CuO composite material disrupted the bacterial cell wall and membrane, which was also supported by the live/dead bacterial staining assay. In addition, TEM technology were used to characterize the S. aureus, and E. coli intracellular sections of different samples. As shown in Fig. S6, GO-CuO composites adhere to the surface of the

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bacterial cells.

To determinate intracellular ROS generation of S. aureus bacteria, DCFH-DA was used

in the present study as a visual indicator for the oxidative status. The fluorescent intensity of DCFH is positively correlated with the content of intracellular ROS (Ji et al., 2017). As shown in Fig. 6, the content of ROS in the bacteria treated with 2 mg/mL BC/GO-CuO

nanocomposite films were found to be increased significantly (almost 2.6 times more than the control group). It has been reported that the increase of intercellular ROS can mediate oxidative damage to membrane lipids, proteins, and DNA, etc. (Dizaj, Lotfipour, BarzegarJalali, Zarrintan, & Adibkia, 2014). However, the content of ROS in the 5 mg/mL BC/GOCuO nanocomposite films treated bacteria was 1.72 times higher than that in the control

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group. The reduction in the fluorescent intensity for 5 mg/mL composite films treated group compared to that of the 2 mg/mL composite films treated group, could be due to the

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excessive cell death in the 5 mg/mL treatment group, resulting in a decrease in ROS content.

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3.5 Cytotoxicity

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The cytotoxicity of the BC/GO-CuO10% composites film was evaluated using NIH 3T3 cells as an in vitro model. The results are shown in Fig. 7. Compared to the control group, we

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observed an increase in viability of the NIH 3T3 cells treated with the BC/GO-CuO10% composite films. Moreover, no apparent change in cell viability was observed by increasing the concentration of the nanocomposites, displaying the non-cytotoxicity property of the BC/GO-CuO nanocomposite films. The non-cytotoxicity of nanocomposite films could be

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attributed to the slow release of Cu2+ (Almasi et al., 2018; Henson et al., 2019).

4. Conclusion

In this study, BC/GO-CuO nanocomposite films were successfully prepared by combining the GO-CuO nanohybrid and bacterial cellulose nanofiber. Structural details of the films were analyzed by XRD, SEM, and TEM. XRD studies confirmed the formation of CuO nanosheets in the GO-CuO nanohybrids. TEM micrographs revealed the formation of CuO sheets with the length range between 50 nm - 200 nm and width range between 20 nm-50 nm.

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These in situ-grown CuO nanosheets appeared to be agglomerated duo to the presence of the high-density nucleation sites of the GO sheets as well as the absence of capping agent or

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surfactant. The SEM results revealed the even distribution of the GO-CuO nanohybrids on

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the surface of the cellulose fibers. BC/GO-CuO composites were more efficient against grampositive bacteria than gram-negative bacteria and BC/GO-CuO composite films showed

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higher antibacterial activity than BC/CuO films. From the findings of the present study, we

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predicted the possible mechanism of the antibacterial effects of the nanocomposites, which could be due to (1) direct contact and interaction of GO sharp nanosheets and CuO nanorods

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with the cell membrane and lipid membrane, resulting in alteration of membrane permeability, (2) disruption of the integrity of the bacterial membrane, accompanied by cellular deformation, surface collapse, surface perforation and roughness, and (3) increased production of ROS leading to cell death. Moreover, the GO/CuO nanohybrid-decorated

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cellulose nanocomposite showed good cell compatibility in vitro. We believe that the prepared nanocomposite films could find applications in the biomedical field as well as in the field of active packaging materials.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 21576212, no. 21978219), Natural Science Foundation of Tianjin city (no.

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19PTSYJC00060), Tianjin Science and Technology Support Program (no.201805010).

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Fig. 1. The scheme for development of BC/GO-CuO nanocomposite films.

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Fig. 2. (A) XRD patterns of BC film; (B) BC/GO10%; (C) BC/GO-CuO5%; (D) BC/GO-CuO10%.

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Fig. 3. Nanostructures and chemical composition of original BC film and BC/GO-CuO film. (A) The TEM

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image of BC/GO-CuO composites film; (B-C) High-resolution TEM images of CuO nanosheets on the BC/GO-CuO film. (D) The selected-area electron diffraction (SAED) patterns of CuO nanosheets on the BC/GO-CuO film. (E1) Photograph, (E2) SEM images of surface, and (E3) cross section of original BC

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film. (F1) Photograph, (F2) SEM images of surface, and (F3) cross-section of BC/GO-CuO film.

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Fig. 4. Viability of (A) S. aureus, and (B) E. coli treated with BC, BC/GO-CuO10% and BC/CuO10% films

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for different contact times.

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Fig. 5. Effect of BC/GO-CuO composite film on the cell membrane integrity of (A) S. aureus, and (B) E.

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coli; Representative SEM images of untreated (C) S. aureus, and (D) E. coli; Representative SEM images

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enlarged image.

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of (E) S. aureus, and (F) E. coli treated with BC/GO-CuO10% films for 5 h. The inset image (d1) is an

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Fig. 6. Formation of intracellular ROS in S. aureus bacteria following incubation with BC/GO-CuO10%

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composites for 5 h.

Fig. 7. Estimation of viabilities of NIH 3T3 cells incubated with the BC/GO-CuO10% composite films through MTT assay. The control group was subjected to the same treatment without adding the film.

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Table 1. The antibacterial inhibition zones produced by original BC, BC/GO10% and BC/GO-CuO nanocomposites against different bacterial strains. Inhibition zones / mm Samples E. coli

B. sabtilis

P. aeruginosa

BC

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0

0

0

BC/GO10%

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0

0

0

BC/GO-CuO5%

16.3±0.2

12.7±0.4

27.8±0.4

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BC/GO-CuO10%

17.8±0.2

14.0±0.4

28.1±0.2

BC/GO-CuO15%

18.3±0.3

15.2±0.3

28.5±0.2

12.2±0.2 15.2±0.2

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S. aureus