Ag NPs Based Mussel

Research Article Cite This: ACS Sustainable Chem. Eng. 2019, 7, 12083−12097

pubs.acs.org/journal/ascecg

A Mussel Mimetic, Bioadhesive, Antimicrobial Patch Based on Dopamine-Modified Bacterial Cellulose/rGO/Ag NPs: A Green Approach toward Wound-Healing Applications Moumita Khamrai,† Sovan Lal Banerjee,† Saikat Paul,§ Anup Kumar Ghosh,§ Priyatosh Sarkar,† and Patit Paban Kundu*,†,‡

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Advanced Polymer Laboratory, Department of Polymer Science & Technology, University of Calcutta, 92 Acharya Prafulla Chandra Road, Kolkata-700009, India ‡ Department of Chemical Engineering, Indian Institute of Technology (IIT) Roorkee, Uttarakhand-247667, India § Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India S Supporting Information *

ABSTRACT: A mussel mimetic transdermal patch was prepared using bacterial cellulose (BC), a green resource derived from Glucanoacetobacter xylinus (MTCC7795). To impart the mussel mimetic property, dopamine (DOPA), a catechol-containing compound, was used to modify the isolated BC via an amidation reaction between the carboxylated BC and DOPA, and the end product was successively characterized by 1H NMR and FTIR analysis. The free hydroxyl group of the DOPA moiety of DOPA-modified BC (BC-DOPA) was utilized to prepare BC-DOPA/rGO/Ag NPs, a composite film incorporating reduced graphene oxide/silver nanoparticles (rGO/Ag NPs). The antimicrobial action of the prepared film was determined against both Gram-positive ( Staphylococcus aureus and Lysinibacillus fusiformis) and Gram-negative ( Escherichia coli and Pseudomonas aeruginosa) bacteria. The bactericidal property of the composite film was determined using the zone of inhibition (ZOI) method and live−dead assay (DAPI−PI analysis). The morphological transformation of bacteria upon the application of the composite film was observed through SEM analysis. The cell compatibility of the composite film over the NIH 3T3 fibroblast cell line was assessed through an XTT assay. The in vitro wound-healing assays over the NIH 3T3 cell line and A549 human lung epithelial cell line reveal that the presence of rGO and Ag NPs in the composite film accelerates the wound-healing process. KEYWORDS: Bacterial cellulose, Mussel mimetic, Antimicrobial action, Wound healing

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devices.15,16 Cellulose can be obtained from various resources, such as cotton,17 wood,18 and hemp.19 Among all of these, the cellulose obtained from the bacterial resources is highly significant due to its structural crystallinity, ultrafine nanofibril network structure, biocompatibility, oxygen permeability, high porosity, etc. This bacterial cellulose can be prepared in divergent shapes with excellent water absorption capacity, which helps in its utilization in widespread biological applications, including transdermal patch applications.20−22 The first report on the synthesis of the bacterial cellulose from the extracellular matrix of Acetobacter xylinum was given by Brown in 1886.23 After that, many bacterial strains were used to synthesize bacterial cellulose, among which Glucanoacetobacter xylinus24 and Acetobacter xylinus25 were the most common microbial resources to prepare microbial cellulose.

INTRODUCTION

In the past few years, interest in cellulose nanofibers has gradually increased due to their extensive array of applications, including separator membranes,1,2 drug delivery systems,3,4 green composites,5,6 biosensors,7 gas sensors,8 solar cells,9 electrical devices,10 energy storage devices,11 and electrochemical actuators.12 Cellulose is a sustainable biopolymer classified as a polysaccharide. The building blocks of the cellulose polymer are the long chains of glucose monomers, joined together by β-1,4-glycosidic linkages. It is mostabundant in nature and easily obtained from a wide range of living resources, like microorganisms and plants. Cellulose can also be used as a renewable ingredient to produce nanofillers for the synthesis of nanocomposites.13,14 Due to its distinct physicochemical properties and nanostructure, including biocompatibility, non-cytotoxicity, high water absorption capacity, structural crystallinity, economical viability, and diverse shape with high porosity, this natural material is very useful for production of human healthcare and medical © 2019 American Chemical Society

Received: February 27, 2019 Revised: May 13, 2019 Published: June 9, 2019 12083

DOI: 10.1021/acssuschemeng.9b01163 ACS Sustainable Chem. Eng. 2019, 7, 12083−12097

Research Article

ACS Sustainable Chemistry & Engineering

film doped with the antibiotic fusidic acid (FA). The BC was isolated from the Gluconacetobacter sucrofermentans B-11267 strain.41 Due to the strong antibacterial action of the drugloaded film, the authors claimed its wound-healing activity. Hernane et al. reported the wound-healing property of BC/ collagen-based hydrogels, where the presence of the collagen accelerates the wound-healing process.42 In an another report, Kaisheng et al. reported a BC film incorporating silver sulfadiazine (SSD) that was employed as an in vivo wounddressing material applied to rat models. Histological data showed that the presence of the SSD in the BC film accelerates the epithelialization process.43 In any of the above cases, the skin adhesion property has not been addressed, although it is an important property for the preparation of the wounddressing patch. The presence of the DOPA during the patch formation can overcome this limitation. Strength, flexibility, and the electrical conductivity are other issues that also have been taken care of in our synthesized formulation. As mentioned earlier, the flexibility and toughness are prime factors for a transdermal patch system, along with the antimicrobial and biocompatibility issues. To impart the toughness to the system, reduced graphene oxide (rGO) is incorporated in the formulation. The chemically modified form of graphene oxide has achieved a great deal of attention due to its broad spectrum of applications. The chemically reduced form of GO can compose a variety of graphene-based products that retain excellent thermal, electrical, and mechanical properties.44−46 A classical example is graphene oxide crosslinking by polyallylamine and divalent anions, resulting in the elevated mechanical stability of materials like paper.47,48 The hydroxyl group of the GO also participates in the reduction process of Ag+ ions to generate Ag NPs.49 In this study, we have developed a mussel mimetic, antibacterial wound-healable transdermal patch system derived from a green source of bacterial cellulose. Bacterial cellulose was isolated from G. xylinus (MTCC7795). After that, it was surface modified with DOPA. A composite transdermal patch having Ag NPs/rGO was prepared via green reduction of the Ag+ and GO with the DOPA-grafted BC. The formed patch was charaterised with different characterization techniques, such as FTIR, FESEM, HRTEM, AFM, UV−vis, XRD, and Raman spectroscopic analyses. The mechanical property of the film was determined with tensile strength measurement. The antimicrobicity and cytotoxicity of the prepared patch were analyzed, and finally the patch was employed for an in vitro wound-healing study.

The few deficiencies of these materials are their low water solubility, low adsorption capacity, and variable physical stability. To overcome the limitations and broaden the applications of bacterial cellulose, various derivatives of bacterial cellulose (BC) have been made by carboxymethylation, acetylation, oxidation, and silylation. Among all these modified forms, carboxymethylated bacterial cellulose (CMBC) is water-soluble with higher degrees of substitution and is used as a hemostatic material.26 CM-BC can produce flexible and transparent films with frequent polar hydroxyl and carboxyl functional groups and aerogels that are rigid with a larger surface area. However, less electrical conductivity, low microbial resistance, and weak interfibril interaction lead to poor mechanical properties, which have limited its applications in wearable or biomedical devices. The basic properties required for advanced wearable electronic systems are antimicrobial activity, biocompatibility, surface electrical conductivity, toughness, and sufficient flexibility.27,28 Recent studies revealed that electric fields can activate multiple cellular signaling pathways, which accelerate the wound-healing process.29−31 As per our knowledge, a combination of all of these properties in a single patch system is rarely reported. So we proposed to prepare a unique mussel mimetic bioadhesive transdermal composite patch system having a high level of antimicrobial activity and wound-healing activity, as well as flexibility, toughness, and conductivity, as an interesting candidate for a transdermal patch system. Here in, we have used a mussel-influenced ingredient, dopamine (DOPA), that can provide a material-independent adherent coating through oxidative self-polymerization due to the presence of lysine and 32,33 L-3,4-dihydroxyphenylalanine (L-DOPA). Karabulut et al. prepared DOPA-modified cellulose nanofibers extracted from wood and studied their mussel mimetic nature after layer-bylayer (LBL) assembly with polyethyleneimine (PEI).34 The main problem associated with the cellulose obtained from the wood resource is that cellulose remained as a composite material with lignin, hemicellulose, and waxy substrates, which has to be extracted out before the final use. This necessitates a hectic purification method that is not an issue in the case of extraction of bacterial cellulose. The presence of the DOPA in the formulated patch system enhanced the skin adherent property under wet conditions. DOPA was grafted over the CM-BC surface by employing an amidation reaction using 1ethyl-3-(3-(dimethylamino)propyl)carbodiimide and N-hydroxysuccinimide coupling catalysts among the carboxyl groups of CM-BC and amine group of the DOPA. Further, we have utilized the hydroxyl group of the DOPA as a green reducing agent for Ag+ ions to prepare Ag nanoparticles (NPs). It is a well-known fact that the presence of Ag NPs drastically enhances the antimicrobial and wound-healing properties of the system.35,36 Various cellulosic materials, such as cellulose gels, bacterial cellulose, cotton fabric, and filter paper, have been modified by incorporating Ag NP-based materials to build up noticeable antimicrobial activity.37,38 Because of the strong antimicrobial action, the presence of the Ag NPs effectively accelerates the cell proliferation by restricting the chance of inflammation and obstacles in the cell reepithelization.39 Antonio et al. recently reported the preparation of a bacterial cellulose film embedded with Ag NPs where the Ag NPs were prepared using an ultraviolet source. The BC was isolated from the Kombucha strains via a fermentation process of sweetened black tea using Acetobacter strains.40 In an another report, Revina et al. reported a bacterial cellulose

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EXPERIMENTAL SECTION

Materials. Dopamine, N-hydroxysuccinimide (NHS), 1-ethyl-3(3-(dimethylamino)propyl)carbodiimide (EDC), silver nitrate (AgNO3), and chloroacetic acid were procured from Sigma-Aldrich, and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) were obtained from Thermo Fisher Scientific. Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were procured from Gibco, Life Technologies. NIH 3T3 fibroblast and A549 human lung epithelial cell lines were purchased from the National Centre for Cell Science (NCCS), Pune, India. Microbial Culture. The lyophilized culture of G. xylinus (MTCC7795) was procured from the national facility in the Institute of Microbial Technology (IMTECH), Chandigarh, India, and revived by using Hestrin−Schramm (HS) medium. A 100 μL portion of the cell suspension with 20% glycerol added was stored at −80 °C for future use. The revived culture was further subcultured in 50 mL of HS media and incubated at 30 °C in an incubator shaker at 120 rpm. 12084


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ACS Sustainable Chemistry & Engineering Preparation of the Growth Medium. HS medium containing of 2% glucose, 0.5% peptone, 0.27% disodium phosphate, 0.15% citric acid, 0.5% yeast extract, and 2% agar (pH 6.0) was prepared and sterilized by autoclaving at 121 °C for 15 min at 15 psi pressure. The sterile media was inoculated with G. xylinus from the stock culture and incubated at 30 °C for 72 h under static conditions. Synthesis and Purification of Bacterial Cellulose. G. xylinus strain was used for the isolation of bacterial cellulose. For the isolation, the freshly growing young culture of bacteria was inoculated into a 250 mL Erlenmeyer flask containing HS medium and incubated for 7 days at 30 °C. After proper incubation, at the air−liquid interface of the Erlenmeyer flask, a white gelatinous pellicle (bacterial cellulose) was observed. The pellicle was isolated and washed properly for purification. The pellicle was again mixed properly with 1% of NaOH solution and boiled for 1 h at 80°C to clean away the adherent bacterial cells. The base-treated pellicle-like bacterial cellulose was further neutralized by washing repeatedly with sterile distilled water. Finally, the purified cellulose was dried properly at room temperature and stored for the subsequent experiments. Synthesis of Carboxymethylated Cellulose. carboxymethylated bacterial cellulose (CM-BC) was synthesized by using the following protocol. First, the BC was crushed and 2 g of the crushed bacterial cellulose was dispersed in 100 mL of distilled water in a 250 mL jacketed glass reactor equipped with magnetic stirrer and connected to a thermostated bath and gas-purging system. Then 20 mL of 30 wt % sodium hydroxide aqueous solution was added, and the reactor was kept at room temperature with continuous stirring. Then 15 mL of chloroacetic acid was added to the reaction mixture using a dropping funnel under vigorous stirring in the presence of inert atmosphere (N2). The reaction was continued for 6 h at 60 °C. The reaction mixture was collected and dialyzed using 10 kDa membrane until the pH was neutral. Degree of Substitution of Carboxymethylated Bacterial Cellulose. Acid−base titration was performed to check the degree of substitution of CM-BC. A completely dried sample of CM-BC (0.1 g) was mixed with 50 mL of distilled water. After that, 30 mL of hydrochloric acid solution at a concentration of 0.105 mol/L was added into it and the solution was left for 30 min with continuous stirring. The titration reaction was carried out using 0.095 mol/L NaOH solution in the presence of 1−2 drops of phenolphthalein solution. The consumed volume of NaOH solution at the end of the titration was recorded as V, and the degree of substitution (DS) of CM-BC was measured by using the equation

the addition, the reaction was allowed to continue for 3 h at room temperature (35 °C) with subsequent addition of 200 mL of deionized water. The aqueous suspension was heated to 80 °C for 30 min, followed by addition of 400 mL of water to the system. To reduce the residual permanganate salt, 15 mL of H2O2 was added slowly to the mixture. Rapid bubbling appeared as a result of the redox reaction, and the reaction mixture turned yellow from dark brown, indicating the reduction of the permanganate salt to soluble manganese ions. The suspension was centrifuged at 8000 rpm to separate out the formed GO. The centrifugation process was carried out four times using 300 mL of deionized water to complete the removal of the impurities. After the centrifugation process, the obtained material was again suspended in 30 mL of deionized water and dialyzed for 5 days using 3500 Da dialysis membrane. During the dialysis, water was changed at the end of each day. The final product was obtained after freeze-drying. Preparation of BC-DOPA/rGO. To prepare the dispersion of reduced GO/BC-DOPA (BC-DOPA/rGO) composite, 2 g of BCDOPA was dispersed in 20 mL of deionized water under N2 atmosphere with vigorous stirring. After homogenization, 0.2 g of the GO was dispersed in the aqueous dispersion of BC-DOPA. The pH of the solution was maintained at 8. After that, the solution was refluxed at 80 °C with continuous stirring for 3 h to reduce the GO by the free hydroxyl group of DOPA. Upon completion of the reaction, the obtained solution was centrifuged and then filtered under vacuum followed by washing with deionized water five times until the filtrate gets neutralized. The obtained solid sample was dried under vacuum for 48 h at 40 °C. Synthesis of BC-DOPA/rGO/Ag NPs. To obtain the Ag NPdoped rGO/BC-DOPA composite, the previously explained process was repeated in the presence of 5 mL of 0.01 M solution of AgNO3. The whole reaction was carried out under nitrogen atmosphere to prevent the oxidation of AgNO3. After the reaction, a blackish brown colored solution was obtained. The formation of the Ag NPs was confirmed through a UV−vis study. To obtain the composite film, the prepared solution was cast on a Teflon-coated Petri plate and allowed to dry at 40 °C for 3 days. To study the Ag+ ion release from the film, the composite film (2 g) was dipped into 50 mL of PBS 7.4 buffer solution for 24 h under mild shaking at 37 °C. After the predetermined time intervals (6, 8, 12, and 24 h), 5 mL of the solution was withdrawn and sodium borohydride (NaBH4) solution of 1 mg/mL concentration was added into the solution in ice-cold conditions to reduce the released Ag+ ions. The same amount of the PBS buffer was added into the system to maintain the total volume. The absorbance of the formed mild-brown-colored solution was studied using UV−vis analysis. The amount of the released Ag+ ions was calculated from the previously plotted calibration curve. Characterization. The chemically modified bacterial cellulose was analyzed with 1H NMR analysis using a Bruker Av 3000 Supercon NMR system operated at 400 MHz. In case of the carboxylated cellulose, NMR analysis was carried out using D2O as the deuterated solvent, whereas in case of DOPA-modified BC, DMSO-d6 was used as the deuterated solvent. To determine the presence of functionality before and after the chemical modifications, Fourier-transform infrared spectroscopic (FT-IR) analysis was carried out using a model Alpha instrument (Bruker) in ATR mode at a scanning range from 4000 to 500 cm−1. A total of 12 scans were applied at a resolution of 4 cm−1, and the whole analysis was carried out at room temperature. The change in the crystallinity of the bacterial cellulose upon the chemical modification was determined using an X-ray diffractometer (PANalytical). During the analysis, all the results were collected using a copper X-ray source (λmax = 1.54 Å) with a 2θ of 0°−80° and at a fixed scan rate of 10°/min. The formation of Ag NPs was monitored by using a UV−vis spectrophotometer system (Optizenview, Mecasys), carried out at a wavelength range of 200− 700 nm using a nanodrop cell. The Raman spectra of the formed GO and the composite films were collected using a Jobin Yvon Horiba spectrometer (model T64000) at an excitation wavelength of λmax = 514 nm. As an excitation source, an argon−krypton mixed-ion gas laser (model 2018 RM, Make Spectra Physics) was used. The

DS = [162m/(m − 58B)] where B, indicating the molar weight of NaOH reacted with the carboxyl group, is equal to (0.095 V − 0.105 × 30) × 10−3 and m is the weight of CM-BC. Fabrication of Dopamine-Modified CM-BC (BC-DOPA). Catechol moieties were conjugated onto the carboxylate functional groups in BC backbones via amidation. For this, the purified CM-BC (1 g) was dissolved in 100 mL of phosphate buffer solution (PBS, pH 6.0) with vigorous stirring. Subsequently, EDC (1.63g) and NHS (0.98 g) were mixed and dissolved properly in the PBS buffer and subsequently added to the CM-BC solution for activation of the carboxylic acid functional groups of CM-BC. The system temperature was maintained at 4−5 °C. Activation of the carboxylate group was continued for 2 h, followed by the dropwise addition of the aqueous solution of dopamine to the reaction system with vigorous stirring by maintaining an inert atmosphere. The amidation reaction was continued for 1 day at room temperature. The final product was separated via dialysis for 2 days by using 10 kDa dialysis membrane followed by freeze-drying of the solution. Graphene Oxide Synthesis (GO). We prepared the GO by using the modified Hummers method.50 To prepare the GO, in a typical synthesis method, 3.0 g of graphite and 1.5 g of sodium nitrate were mixed properly into 70 mL (98%) of sulfuric acid under ice-cold conditions with vigorous stirring, which was maintained for 60 min. After that, about 10.0 g of potassium permanganate (KMnO4) was added cautiously to the reaction mixture under cold conditions. After 12085


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Scheme 1. Schematic Representation of the Modification of Bacterial Cellulose with DOPA and Preparation of the Bioreduced rGO/Ag NPs/BC-DOPA Complex

Fluorescent Staining for the Bacterial Viability Assay. The viability of bacterial cells in the presence of prepared films was analyzed on S. aureus (Gram-positive bacteria) and E. coli (Gramnegative bacteria) by using fluorescent staining based on microscopic observation of cells. In brief, 1.5 × 108 bacterial cells were measured by using a 0.5 McFarland standard (Himedia) and inoculated into LB broth containing modified composite films for 3 h at 37 °C with an incubator shaker (Eppendorf) with continuous shaking. Incubated bacterial cells were collected by centrifugation at high speed for 3 min. Bacterial cells were stained with 1 μg/mL of propidium iodide (PI) for 10 min and further counterstained for 5 min with 5 μg/mL of 4′,6diamidino-2-phenylindole (DAPI) in the dark. The viability of bacterial cells was checked by observing the cells under a Nikon Eclipse Ci-L/S fluorescent microscope. Cell Viability Assay. To check the cytotoxic effect of BC-DOPA (control), BC-DOPA/rGO, and BC-DOPA/rGO/Ag NPs, a cell viability assay was performed. For this analysis, a colorimetric-based XTT assay was performed. In brief, NIH 3T3 cells were grown in DMEM supplemented with 10% (v/v) FBS and 1% antibiotic to remove the bacterial contamination. Cells were grown at 37 °C in the presence of the 5% CO2 with a moist atmosphere. For the XTT assay, BC-DOPA, BC-DA/rGO, and BC-DA/rGO/Ag NPs were placed at the bottom of a 96-well culture plate (Tarson) (CMBC-DA was used as a control sample). The plates containing films were sterilized by keeping them under UV for 1 h. Subsequently, 0.5 × 106 cells were inoculated in each well of the 96-well culture plate and incubated for 12 h. After proper incubation, the media was removed and replaced with XTT solution, and the plates were incubated at 37 °C for 4 h. Depending on the presence of live cells, a purple color with different intensities was developed. Finally, the color intensities of the samples were measured at 450 nm by using a UV-spectrophotometer. The XTT assay was repeated three times for each sample.

morphological analysis of the synthesized components was carried out using FESEM and HRTEM analyses. During the FESEM analysis (model EV018, Carl Zeiss), all the samples were platinum coated using a Q150T Plus-Turbomolecular pumped coater (Quorum Technologies Ltd.). For the high-resolution transmission electron microscopic (HRTEM) analysis, which was carried out with a JEOL JEM 2000E7 high-resolution TEM, the samples were dispersed with sonication in deionized water at a concentration of 1 mg/mL for 1 h, drop-cast over the carbon-coated copper TEM grid having a mesh size of 300, and dried at room temperature prior to the imaging. For the conductivity measurement of the composite film, the current vs voltage measurement was carried out using a Keithley 4200 SCS semiconductor parameter analyzer keeping a 1 cm distance between the two crocodile clip electrodes. Voltage is applied with a step size of 0.1 V and the corresponding current is measured. Antimicrobial Activity. The antimicrobial properties of CMBCDOPA, BC-DOPA/rGO, and BC-DOPA/rGO/Ag NPs were checked by treating Gram-positive bacterial strainsStaphylococcus aureus and Lysinibacillus fusiformisand Gram-negative strainsEscherichia coli and Pseudomonas aeruginosa. The conventional disk diffusion method was used to analyze the antimicrobial activity. For this method, the synthesized films were cut into a round shape. The sterile growth media was poured onto the sterile plates and dried properly to reduce the contamination. The test cultures of bacteria were spread properly on the surface of the growth media in an aseptic condition. Then, the round-shaped films were placed on the culture-containing plates and incubated for 24 h at 37 °C. After incubation, the circular zones of inhibition (ZOI) were checked and measured to analyze the inhibition activity. For the control study, the antibacterial activity of ciprofloxacin-doped BC-DOPA film was also studied against E. coli and S. aureus bacteria. 12086


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Figure 1. 1H NMR of (a) carboxymethylated bacterial cellulose and (b) dopamine (DOPA)-modified bacterial cellulose. In Vitro Wound-Healing Assay. The capability of the composite film to influence the wound healing of NIH 3T3 fibroblast cells and A549 human lung epithelial cell line was analyzed. For this analysis, BC-DOPA (control), BC-DOPA/rGO, and BC-DOPA/rGO/Ag NPs films were placed on the 12-well cell culture plate and sterilized under UV. NIH 3T3 cells were grown on the surface of the film and incubated up to confluent growth. After incubation, by using a 200 μL pipet tip, the newly formed cell layer was unidirectionally scratched. The cell debris produced during scratching was removed by washing with 1 mL of growth medium. Five milliliters of DMEM was added, and the plates were incubated for the in vitro wound-healing assay. The cells were grown up to 24 h, and the images of the cells were taken at different time intervals, such as 0, 6, 12, and 18 h. A similar procedure of wound healing was also adopted for the A549 human lung epithelial cell line.

analysis. For the carboxylated bacterial cellulose, the characteristic resonance for the methylene proton coming from the chloroacetic acid moiety appeared at δ = 4.7 ppm, as shown in Figure 1a. The other resonances for the protons in the sugar backbone appeared at around δ = 3−4.2 ppm.51 After the modification with dopamine, new characteristic resonances appeared at δ = 8.32 ppm for the NH proton of the amide linkage, which delineates the successful modification of the carboxymethylated bacterial cellulose with the amine group of dopamine. The resonance near δ = 7.8 ppm indicates the presence of the free hydroxyl group in the dopamine moeity (Figure 1b).52 All the synthesized components were characterized with FTIR analysis to have an idea about the presence of functionality and the change of their transmittance due to the chemical modifications. Pristine BC showed characteristic vibrations at 2918, 1414, and 1375 cm−1 due to the presence of CH2 scissoring and stretching vibrations and C−H bending vibration, respectively (Figure 2). A broad peak appeared at 3250 cm−1 due to the presence of a significant number of hydroxyl groups (−OH) in the backbone of BC.22 It was observed that after the modification with chloroacetic acid, the intense peak at 3250 cm−1 broadened, indicating the replacement of the −OH groups of the BC with the

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RESULTS AND DISCUSSION In this investigation, we have prepared a mussel mimetic wound-healable transdermal patch system via adopting simple and well-known amidation chemistry. The schematic of the adopted pathway is represented in Scheme 1. Herein, the isolated BC from G. xylinus (MTCC7795) was chemically modified with dopamine, a catechol-containing compound, via succesive carboxylation and amidation between the carboxylic group of BC and the amine group of DOPA. The syntheized compounds were characterized with 1H NMR and FTIR 12087


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Figure 2. (a) FTIR analysis of the prepared components, (b) close up image of the FTIR analysis, and (c) macrophotograph of the synthesized composite film.

Figure 3. FESEM analysis of (a) bacterial cellulose, (b) BC-DOPA/rGO, (c) BC-DOPA/Ag NPs, and (d) BC-DOPA/rGO/Ag NPs.

CH2COONa. The appearance of the new band at 1621 cm−1 indicates the presence of a carboxylate group (−COO−). After the modification of CMBC with the dopamine, the trans-

mittance intensity increased significantly at 3253 cm−1, indicating the insertion of more free −OH group from the dopamine moiety. The presence of the intramolecular 12088


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Figure 4. HRTEM analysis of (a) graphene oxide (GO) and (b) BC-DOPA/rGO/Ag NPs and (c) EDAX of the BC-DOPA/rGO/Ag NPs film.

Figure 5. (a) UV−vis spectra of the GO, BC-DOPA/rGO, and BC-DOPA/rGO/Ag NPs. (b) Raman spectra of the respective components.

hydrogen bonding between −OH groups of dopamine further intensified the stretching peak of the −OH group. In addition, the appearance of the absorption peaks at 1648, 1489, and 1255 cm−1 corresponded to N−H bending vibrations, aromatic C−C stretching vibrations, and aromatic amine C−N stretching vibrations, respectively. This clearly indicates the presence of the aromatic and the amido groups coming from dopamine moiety.53 After the formation of the BC-DOPA/ rGO/Ag NPs, a decrease in the intensity of the −OH stretching band was observed, indicating the involvement of the free −OH groups of dopamine in the reduction of the Ag+ ions to form Ag NPs and GO to form rGO. The macrophotograph of the composite film is shown in Figure 2c. Morphological Analysis of the Prepared Material. All the prepared components, like BC, BC-DOPA/rGO, BCDOPA/Ag NPs, and BC-DOPA/rGO/Ag NPs, were characterized by FESEM analysis. For the pristine BC-DOPA, fibrilartype morphology was observed (Figure 3a), whereas after formation of the rGO/BC-DOPA composite film (Figure 3b),

a wavy structure was observed that clearly delineates the presence of rGO. When the BC-DOPA was utilized to produce the Ag NPs, a rough surface morphology of the DOPAmodified BC was observed, where the spherically shaped Ag NPs were arranged uniformly over the BC cellulose fiber, as observed in Figure 3c. After formation of the BC-DOPA/ rGO/Ag NPs composite film, a nice architecture was observed, where Ag NPs and rGO were uniformly distributed in the composite film (Figure 3d). To investigate the bulk morphology of BC-DOPA/rGO and BC-DOPA/rGO/Ag NPs composite film, the film was cryomicrotomed and the morphology was observed via HRTEM analysis. From Figure 4a, it was observed that rGO was dispersed inside the film in a scrolled and folded-edge structure. It is reported that this type of corrugated morphology is the thermodynamically favorable, stable structure.54 A nice intercalating structure was observed for BC-DOPA and rGO, leading to an increase in the mechanical property of the film (explained later). As explained earlier, the 12089


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Figure 6. (a) XRD analysis of the prepared components and (b) mechanical property analysis of the prepared polymeric films.

free hydroxyl group of the BC-DOPA was utilized to reduce both the GO and Ag+ ions to form an organic−inorganic hybrid composite film, BC-DOPA/rGO/Ag NPs. A beautiful distribution of Ag NPs, rGO, and BC-DOPA was observed from HRTEM analysis (Figure 4b). To confirm the presence of Ag NPs in the composite film, EDAX was conducted, and it was found that a reasonable amount of Ag was present in the film (Figure 4c). The presence of both rGO and Ag NPs in the composite film increased its thermal stability as well as mechanical integrity, which is explained later. AFM was deployed to study the surface morphology of the composite film. From Figure S1a of the Supporting Information (SI) it was observed that the Ag NPs were nicely distributed over the film, having a height of 15 nm obtained from the height image data (Figure S1b, SI). When both rGO and Ag NPs were present in the system, a nice distribution of rGO containing Ag NPs was observed (Figure S1c, SI). The height profile of rGO showed an average rGO sheet thickness of 1.2 nm present in the composite film (Figure S1d, SI). UV−Vis Analysis. UV−visible spectroscopy of GO, polymer reduced GO (rGO), and the BC-DOPA/rGO/Ag NPs was carried out to verify the successful formation of the desired materials (Figure 5a). As observed from Figure 5a, for GO, the characteristic absorbance appeared at 239 and 304 nm, due to the presence of the π → π* transition of aromatic C−C bonds and the n → π* transitions of CO bonds, respectively. After the reduction reaction with dopaminemodified bacterial cellulose, a red-shift of the 239 nm peak was

observed at 263 nm, and successive elimination of the 304 nm peak delineates the maximum removal of the oxygencontaining functional group of GO upon the treatment with polymer. Elimination of the peak at 304 nm also indicates the generation of the graphene structure via reduction with the polymer.55,56 After the formation of the Ag NPs, a new absorption band was formed near 415 nm, along with the presence of the 304 nm peak; this indicates the successful formation of the Ag NPs. Surface plasmon resonance (SPR) of the Ag NPs showed the appearance of an intense peak near 415 nm.57,58 Raman Spectra. In our study, we have prepared GO from graphite flakes via a modified Hummers method, and after that the prepared GO was employed in the formation of BCDOPA/rGO/Ag NPs composite film. We conducted the Raman spectroscopy analysis (a nondestructive analytical method to characterize the carbonaceous material) of the synthesized material to get an insight into the generation of the defects in the GO layer after the modification with the polymer. It is a well-known fact that the G-band delineates the graphitic characteristic (sp2 carbon) which corresponds to the tangential vibration of the carbon atoms. As observed from Figure 5b, for the untreated graphite, a sharp vibrational peak was observed at 1591 cm−1, but there was no evidence of a peak at 1354 cm−1, generated due to the imperfection in the graphitic structure. The D/G ratio is an important parameter that determines the extent of defects induced in the graphitic layer via chemical or physical modifications.59 The defect 12090


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Figure 7. (i) Antimicrobial activity study of (a) CM-BC, (b) BC-DOPA, and (c) BC-DOPA/rGO/Ag NPs films against Gram-negative (E. coli and P. aeruginosa) and Gram-positive (S. aureus and L. fusiformis) bacteria. (ii) Morphological analysis of the E. coli and S. aureus bacteria (a, c) before and (b, d) after treatment with the BC-DOPA/rGO/Ag NPs composite film patch. (iii) Live−dead (DAPI and PI) assay of S. aureus and E. coli bacteria.

hydrogen bonding was the main reason behind the crystalline nature of BC. After the modification with DOPA, the crystalline nature of the BC was demolished and a broad peak was obtained near 30°, which indicates the transformation of the crystalline structure of BC to an amorphous one. The BC-DOPA-based composite films were also analyzed with the XRD. The prepared GO showed a characteristic peak at around 2θ = 11°, which corresponds to a d-spacing of 10.03 Å for the (001) plane. This d-spacing value is higher than the reported d-spacing value of graphite (approximately 3.36 Å) for the (002) plane. This indicates the successful oxidation of graphite. The BC-DOPA-stabilized Ag NPs showed characteristic diffraction peaks at 2θ = 27°, 32°, and 47°, corresponding to the (220), (311) and (420) planes of Ag (JCPDS No. 040783). The obtained results confirmed the formation of Ag NPs via a green reduction technique. The BC-DOPA/rGO/Ag NPs composite film was also analyzed by XRD, and a broad

might be generated due to the formation of ripples, charge puddles, different types of edges, etc. As observed from Figure 5b, for the pure graphite powder, a strong peak was observed at 1591 cm−1, which is due to the presence of the G-band. There was no trace of the D-band near 1354 cm−1. Upon modification of the graphite via oxidation with KMnO4, as the GO was formed, an intense peak was observed at 1354 cm−1, indication of the successful oxidation of the graphite and successive formation of GO. In case of GO, the ID/IG ratio was 0.89, but after the formation of the BC-DOPA/rGO/Ag NPs, the ID/IG ratio increased to 0.97, indicating the effective generation of defects, which might be due to the engagement of the hydroxyl group of GO in the formation of Ag NPs.60,61 Wide-Angle XRD Analysis. X-ray diffraction analyses of pristine BC showed a group of diffraction peaks at 14.59°, 16.6° ,and 22.57°, corresponding to the 11̅0, 110, and 200 planes of BC (Figure 6a).62 The formation of the interlayer 12091


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ACS Sustainable Chemistry & Engineering Table 1. Summary of the Antibacterial Activity zone of inhibition (mm) Gram negative

Gram positive

composition

E. coli

P. aeruginosa

S. aureus

L. fusiformis

CM-BC BC-DOPA BC-DOPA/rGO/Ag NPs ciprofloxacin-doped BC-DOPA

15 ± 1.0 24 ± 0.5

11 ± 0.5

13 ± 0.5 23 ± 0.5

14 ± 0.75

electrodes. So this primary data supports our assumptions on the conductivity of the composite film (BC-DOPA/rGO/Ag NPs). Antimicrobial Assay. The antimicrobial activity of CMBC, BC-DOPA, and BC-DOPA/rGO/Ag NPs was determined by using E. coli, P. aeruginosa, S. aureus, and L. fusiformis (Figure 7) as model bacteria. It was noticed that CM-BC (a) and BC-DOPA (b) bore no antimicrobial activity against any of the bacterial isolates, whereas BC-DOPA/rGO/Ag NPs (c) had significant bactericidal activity and produced a clear inhibition zone. BC-DOPA/rGO/Ag NPs composite film inhibits the growth of E. coli, P. aeruginosa, S. aureus, and L. fusiformis, producing a zone of inhibition with a diameter of 15 ± 1.0, 11 ± 0.5, 13 ± 0.5, and 14 ± 0.75 mm, respectively (Figure 7i). The variation of the inhibition zone may be due to the diversity of the cell surface components of all the isolates. The effect of BC-DOPA/rGO/Ag NPs was further confirmed by analyzing the cell surface morphology of E. coli and S. aureus assessed under a scanning electron microscope. Figure 7ii represents the morphological alteration of the cell wall of both the bacterial isolates with the exposure of BC-DOPA/rGO/Ag NPs. In this figure, the cell surface of the Gram-negative bacteria E. coli and the Gram-positive bacteria S. aureus was altered drastically and the cell wall was squeezed in the presence of BC-DOPA/rGO/Ag NPs, indicating the significant antimicrobial activity of BC-DOPA/rGO/Ag NPs. For the control study, the antibacterial activity of the antibiotic (ciprofloxacin)-doped BC-DOPA film was also assessed. The plate is shown in Figure S3 (SI). The obtained ZOI of all the samples is summarized in Table 1. The release kinetics of the Ag+ ions from the composite film (BC-DOPA/rGO/Ag NPs) is shown in Figure S4 (SI). Live−Dead Assay. DAPI and PI are the two DNA-binding fluorescent dyes utilized to check the viability status of the cells in the presence of BC-DOPA/rGO/Ag NPs. DAPI can stain both the live and dead cells by binding with the AT-rich minor groove of the DNA, but PI is a DNA- or RNA-intercalating die and unable to pass through the cell membrane of a metabolically active and healthy cell. As a result, it can only enter into dead cells and stain the nucleic acids of dead cells and cells with damaged or compromised membranes. It was observed from Figure 7iii that approximately all of the E. coli and S. aureus cells retain the color of DAPI, demonstrating that many fewer dead cells were present in the control setup (BCDOPA). Approximately all of the S. aureus and E. coli cells in the presence of BC-DOPA/rGO/Ag NPs took the color of PI, representing massive cell death or damaged cell membrane and walls with the exposure of BC-DOPA/rGO/Ag NPs. The results of fluorescent staining demonstrated that BC-DOPA/ rGO/AgNPs showed a significantly high level of antibacterial properties against both S. aureus and E. coli. Cell Cytotoxicity and Wound-Healing Assay. The cytotoxicity of the synthesized BC-DOPA (control), BC-

peak appeared at 2θ = 25° via significant reduction of the intense peak at 2θ = 10.03°, an indication of the successful reduction of GO by the BC-DOPA moiety. The characteristic peaks for Ag NPs were also present in the XRD diffractogram, indicating the presence of Ag NPs in the composite film.63 Mechanical Property Analysis. To repair a wound located at a motional part of the human body requires a high-stretch material. Here we have prepared a composite transdermal patch system based on BC-DOPA/rGO/Ag NPs with significantly high tensile stretch and toughness (Figure 6b). It was observed that a dramatic increase in the mechanical property occurred due to the incorporation of rGO in the composite film. The tensile strength observed in the case of BC-DOPA was 1 ± 0.05 MPa, whereas after the incorporation of the rGO in the composite formulation via the green reduction of GO with BC-DOPA, a significant increase of 5.52 ± 0.07 MPa was observed. The strong increment in the tensile value was due to the high energy absorption property of rGO, leading to the higher mechanical stability of the patch system. Upon further incorporation of the metallic nanoparticles in the composite film, a strong improvement in the elastic modulus occurred, but the tensile strength reduced a bit (5.21 ± 0.03 MPa). It might be due to the resistive force given by the BCDOPA/rGO-coordinated Ag NPs, as shown in the Scheme 1. The mechanical interlocking by the Ag NPs in between the rGO and BC-DOPA films introduces the higher modulus to the system. TGA Analysis. The thermal property of the composite hydrogel was measured using thermogravimetric analysis. From Figure S2 (SI), it was observed that DOPA-modified BC showed a two-step degradation process, where at 250 °C an initial degradation starts due to the thermal decomposition of the DOPA moiety along with the elimination of bound water.64 After that, a final degradation starts at 330 °C, due to the pyrolytic degradation of cellulose subunits.65 For the BCDOPA/rGO complex, a similar kind of trend was observed, but the initial degradation starts a bit earlier as compared to the pristine BC-DOPA. It might be due to the relative hydrophilicity of the rGO group.66 Significant ash content was observed in the case of the BC-DOPA/rGO complex, which clearly indicates the presence of rGO. Upon incorporation of the Ag NPs, a further increase in the ash content was observed. This is due to the presence of metals in the ash. From the obtained ash content, the amount of Ag NPs present in the composite film was 34 μg/g of composite film. Study of the Conductivity. As mentioned in the Experimental Section, the current vs voltage measurement was done using a Keithley 4200 SCS semiconductor parameter analyzer keeping 1 cm distance between the two crocodile clip electrodes. A step size of 0.1 V was applied and the respective current was measured. From Figure S3 (SI), a linear ohmic response was observed and the average resistance was 84 kΩ, which is pretty high considering the distance between the 12092


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Figure 8. (i) Graphical representation of the XTT assay analysis carried out over the NIH 3T3 fibroblast cell line. (ii) Inverted phase-contrast microscopic image of the NIH 3T3 cell proliferation over the BC-DOPA (control), BC-DOPA/rGO, and BC-DOPA/rGO/Ag NPs films. (iii) Images of the wound-healing activity of the synthesized film carried out over the NIH 3T3 cell line.

DOPA/rGO, and BC-DOPA/rGO/Ag NPs films on the NIH 3T3 fibroblast cell line was checked by an in vitro XTT assay. The XTT assay is a more sensitive, newer, and modified approach over the most commonly used MTT assay. The higher sensitivity, the formation of the highly soluble formazan

salt, and the opportunity to measure OD immediately after a short period of incubation during the XTT assay make it a superior candidate in comparison with the MTT assay. To investigate the cell cytotoxicity assay, the NIH 3T3 fibroblast cell line was incubated with BC-DOPA, BC-DOPA/rGO, and 12093


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Figure 9. Images of the wound-healing activity of the synthesized film carried out over the A549 human lung epithelial cell line.

from the fibriblast. Myofibroblast secrets elastin and actin fibers that help to close the wound.69 The wound-healing efficiency of the composite film was also assessed over the A549 human lung epithelial cell line. Like over the NIH 3T3 fibroblast cell line, in this case, the composite film showed efficient wound-healing activity, as observed from Figure 9. The wound-healing efficiency of the rGO/Ag NPs-doped film was higher than that of the rGO-doped BC film.

BC-DOPA/rGO/Ag NPs. From the acquired OD values (Figure 8i), it was observed that all of the BC-DOPA, BCDOPA/rGO, and BC-DOPA/rGO/Ag NPs showed much less cytotoxicity over the NIH 3T3 fibroblast cell line. The surface morphology and the adhesion property of the cell line was observed using a phase-contrast microscope (Figure 8ii). From the acquired OD values and the phase-contrast microscopic images, it can be observe that the presence of BC-DOPA/rGO and BC-DOPA/rGO/Ag NPs stimulated the proliferation of the NIH 3T3 cells, which naturally supports the wound-healing process. The release of the nanoparticles from BC-DOPA/ rGO/Ag NPs remarkably influences the proliferation of cells and supports the wound-healing process.67 The in vitro wound-healing assay proved our hypothesis (Figure 8iii). From the figure it is observed that BC-DOPA/ rGO/Ag NPs composite film efficiently influences the healing of wounds compared to BC-DOPA/rGO and BC-DOPA (control) films. The healing with BC-DOPA/rGO was comparatively higher than with BC-DOPA (control), as the presence of reduced graphene can accelerate the cell proliferation via preventing microbial growth.68 Impressively, it was noticed that the efficiency of wound healing for the BCbased film is significantly enhanced in the presence of Ag NPs. The healing efficiency of BC-DOPA/rGO/Ag NPs was comparatively higher than that of BC-DOPA/rGO, whereas BC-DOPA (control sample) can induce the healing process up to 45%. The presence of Ag NPs stimulated the migration and the proliferation of the cells, and from the figure, it is observed that the BC-DOPA/rGO/Ag NPs composite film was able to heal the generated wound over the NIH 3T3 fibroblast cells within 18 h. It is reported that, in the presence of Ag NPs, the migration and the proliferation of the keratinocytes increased to a significant extent, helping in the wound closure. The fibroblast cells also took part in the wound closure. The presence of Ag NPs accelerated the formation of myofibroblast

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CONCLUSION In conclusion, a mussel mimetic antimicrobial and woundhealing transdermal patch system based on a green resource, bacterial cellulose, has been prepared. The microbial cellulose was isolated from G. xylinus (MTCC7795) bacteria and chemically modified with catechol-containing compound (dopamine) to impart an adhesive nature to the synthesized transdermal patch system. To obtain the bacterial-cellulosebased composite film doped with rGO and Ag NPs (BCDOPA/rGO/Ag NPs), the GO and Ag+ ions were coreduced with the free hydroxyl group of the catechol moeity via a green reduction process. The as-prepared BC-DOPA/rGO/Ag NPs composite film was characterized with several techniques to established its prepration method and productivity. The antimicrobial activity of the composite film was examined against Gram-positive (S. aureus and L. fusiformis) and Gramnegative (E. coli and P. aeruginosa) bacteria, as this is a prime factor in the design of a wound-healing patch. The bactericidal property of the composite film was assessed through the zone inhibition method and live−dead assay. The morphological analysis of the affected bacteria was evidenced through the SEM analysis. A cytotoxicity study of the prepared films were carried out over the NIH 3T3 fibroblast cell line, and it was found that all the fillms were compatible with the cells. It is also evident that the presence of the Ag NPs in the composite 12094


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ACS Sustainable Chemistry & Engineering film accelerates the proliferation and the migration of the NIH 3T3 fibroblast cells, as well as A549 human lung epithelial cells, leading to accelerated wound healing. We believe that our formulated mussel mimetic BC-based wound-healing transdermal patch system can be a promising candidate in wound cure therapy.

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(9) Fang, Z.; Zhu, H.; Yuan, Y.; Ha, D.; Zhu, S.; Preston, C.; Chen, Q.; Li, Y.; Han, X.; Lee, S.; et al. Novel nanostructured paper with ultrahigh transparency and ultrahigh haze for solar cells. Nano Lett. 2014, 14 (2), 765−773. (10) Hsieh, M. C.; Kim, C.; Nogi, M.; Suganuma, K. Electrically conductive lines on cellulose nanopaper for flexible electrical devices. Nanoscale 2013, 5 (19), 9289−9295. (11) Nyholm, L.; Nyström, G.; Mihranyan, A.; Strømme, M. Toward flexible polymer and paper-based energy storage devices. Adv. Mater. 2011, 23 (33), 3751−3769. (12) Li, J.; Vadahanambi, S.; Kee, C. D.; Oh, I. K. Electrospun fullerenol-cellulose biocompatible actuators. Biomacromolecules 2011, 12 (6), 2048−2054. (13) Iwatake, A.; Nogi, M.; Yano, H. Cellulose nanofiber-reinforced polylactic acid. Compos. Sci. Technol. 2008, 68 (9), 2103−2106. (14) Iwamoto, S.; Nakagaito, A. N.; Yano, H.; Nogi, M. Optically transparent composites reinforced with plant fiber-based nanofibers. Appl. Phys. A: Mater. Sci. Process. 2005, 81 (6), 1109−1112. (15) Wan, Y.; Huang, Y.; Yuan, C.; Raman, S.; Zhu, Y.; Jiang, H.; He, F.; Gao, C. Biomimetic synthesis of hydroxyapatite/bacterial cellulose nanocomposites for biomedical applications. Mater. Sci. Eng., C 2007, 27 (4), 855−864. (16) Czaja, W. K.; Young, D. J.; Kawecki, M.; Brown, R. M. The future prospects of microbial cellulose in biomedical applications. Biomacromolecules 2007, 8 (1), 1−12. (17) Ohwoavworhua, F.; Adelakun, T. Some physical characteristics of microcrystalline cellulose obtained from raw cotton of Cochlospermum planchonii. Trop. J. Pharm. Res. 2005, 4 (2), 501− 507. (18) Abe, K.; Iwamoto, S.; Yano, H. Obtaining cellulose nanofibers with a uniform width of 15 nm from wood. Biomacromolecules 2007, 8 (10), 3276−3278. (19) Ouajai, S.; Shanks, R. Composition, structure and thermal degradation of hemp cellulose after chemical treatments. Polym. Degrad. Stab. 2005, 89 (2), 327−335. (20) Trovatti, E.; Freire, C. S.; Pinto, P. C.; Almeida, I. F.; Costa, P.; Silvestre, A. J.; Neto, C. P.; Rosado, C. Bacterial cellulose membranes applied in topical and transdermal delivery of lidocaine hydrochloride and ibuprofen: in vitro diffusion studies. Int. J. Pharm. 2012, 435 (1), 83−87. (21) Patel, D. P.; Setty, C. M.; Mistry, G. N.; Patel, S. L.; Patel, T. J.; Mistry, P. C.; Rana, A. K.; Patel, P. K.; Mishra, R. S. Development and evaluation of ethyl cellulose-based transdermal films of furosemide for improved in vitro skin permeation. AAPS PharmSciTech 2009, 10 (2), 437−442. (22) Khamrai, M.; Banerjee, S. L.; Kundu, P. P. Modified bacterial cellulose based self-healable polyeloctrolyte film for wound dressing application. Carbohydr. Polym. 2017, 174, 580−590. (23) Brown, A. J. XLIII.On an acetic ferment which forms cellulose. J. Chem. Soc., Trans. 1886, 49, 432−439. (24) Pecoraro, É .; Manzani, D.; Messaddeq, Y.; Ribeiro, S. J. Bacterial cellulose from Glucanacetobacter xylinus: preparation, properties and applications, Monomers, polymers and composites from renewable resources. Elsevier 2007, 369−383. (25) Hong, F.; Qiu, K. An alternative carbon source from konjac powder for enhancing production of bacterial cellulose in static cultures by a model strain Acetobacter aceti subsp. xylinus ATCC 23770. Carbohydr. Polym. 2008, 72 (3), 545−549. (26) Lin, Q.; Zheng, Y.; Ren, L.; Wu, J.; Wang, H.; An, J.; Fan, W. Preparation and characteristic of a sodium alginate/carboxymethylated bacterial cellulose composite with a crosslinking semi-interpenetrating network. J. Appl. Polym. Sci. 2014, 131 (3), 39848. (27) Lam, E.; Male, K. B.; Chong, J. H.; Leung, A. C.; Luong, J. H. Applications of functionalized and nanoparticle-modified nanocrystalline cellulose. Trends Biotechnol. 2012, 30 (5), 283−290. (28) Barbiroli, A.; Bonomi, F.; Capretti, G.; Iametti, S.; Manzoni, M.; Piergiovanni, L.; Rollini, M. Antimicrobial activity of lysozyme and lactoferrin incorporated in cellulose-based food packaging. Food Control 2012, 26 (2), 387−392.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.9b01163. AFM images of the BC-DOPA/Ag NPs and BC-DOPA/ rGO/Ag NPs composite films, thermogravimetric analysis, I−V characteristic plot, antimicrobial activity of the ciprofloxacin-doped samples, and release kinetics of the Ag+ ions (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. Fax and Tel: 91-33-2352-510. ORCID

Patit Paban Kundu: 0000-0001-6787-8641 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.K. acknowledges the Department of Polymer Science & Technology, University of Calcutta, and the Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, for providing the required research facilities.

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REFERENCES

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