Silver-Incorporated Nanocellulose Fibers for Antibacterial Hydrogels

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Article Cite This: ACS Omega 2018, 3, 16150−16157

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Silver-Incorporated Nanocellulose Fibers for Antibacterial Hydrogels Ji Un Shin,† Jaegyoung Gwon,§ Sun-Young Lee,§ and Hyuk Sang Yoo*,†,‡ †

Department of Biomedical Materials Engineering and ‡Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 24341, Republic of Korea § Department of Forest Products, National Institute of Forest Science, Seoul 02455, Republic of Korea

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ABSTRACT: A free-standing, antibacterial hydrogel was fabricated using silver-nanoparticle-immobilized cellulose nanofibers (CNFs) and alginate. Surface hydroxyl groups of CNFs were oxidized to carboxylate groups using (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (TCNF), followed by the treatment with silver nitrate solution for surface adsorption of silver ions. In situ reduction of silver ions to produce silver nanoparticles was performed for the silveradsorbed CNFs. Electron microscopy, X-ray diffraction, and spectroscopic analysis revealed that higher amounts of silver nanoparticles were immobilized on the surface of TCNF than on the surface of native CNF. Silver-nanoparticleimmobilized TCNF was embedded in alginate gels and silver ions from the matrix were slowly released for 7 days. Silver-nanoparticle-loaded alginate gels showed comparable antibacterial activity to silver-ions-loaded alginate gels, although the former showed a significantly lower cytotoxicity against animal cells. Thus, the antibacterial gels can potentially be applied to various skin surfaces to prevent bacterial infection while minimizing skin damage.

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INTRODUCTION Skin wounds are often prone to infection and difficult to heal, demanding appropriate treatment strategies to facilitate the process of wound healing.1 Conventional wound dressings include natural or synthetic bandages, cotton, and gauzes, all of which may need long-term treatment and potentially adhere to dry-wound surfaces.2 Hence, many studies have been directed to identify materials that are ideal for wound dressings. Hydrogel-based wound-dressing materials offer several advantages such as cool sensation and moist environment, allowing gaseous exchange, and absorption of wound exudate.3−6 The major cause of wound contamination is infection from bacterial pathogens that may easily enter the body via the wound and reach host tissues, leading to sepsis and death.7 The most widely used method for the treatment of bacterial infection is antibiotic therapies. To minimize the abuse of broad-spectrum antibiotics, silver nanoparticles (AgNPs) with lower bacterial resistance and fewer side effects have been widely used as topical antibacterial agents.8,9 The mechanism of action of these nanoparticles involves damage to the bacterial cell membrane upon conversion of Ag0 to Ag+ that occurs via binding to thiol residues present on membrane proteins and enzymes involved in reactive oxygen species formation,10 as well as the interruption of the intercellular metabolic pathways following Ag+ penetration into the cell.11 The toxicity of AgNPs has been recognized as a limitation of this approach, as these particles are dispersed into the cell upon Ag+ release. This issue may be solved with the stabilization of AgNP to fabricate composites with various materials.12,13 Bacterial cellulose was surface© 2018 American Chemical Society

decorated with self-assembled AgNPs to exert antibacterial activity.14 The slow release of silver ions effectively attenuated bacterial growth while minimizing cytotoxicity against epidermal cells. In another study, chitosan scaffolds were loaded with AgNPs prepared in situ to minimize host tissue damage. The released silver ions, however, were shown to exhibit considerable cytotoxicity against fibroblasts at higher concentrations.15 Chitosan scaffolds were also loaded with AgNPs to reduce bacterial infection during bone transplant operation. AgNPs effectively suppressed the growth of Staphylococcus aureus while maintaining osteogenic proliferation.16 Cellulose nanofibers (CNFs) have been widely studied in fields such as electron devices,17,18 cosmetics,19 paper films,20 biomedical devices,21 and polymer nanocomposites22,23 owing to their nontoxicity, biodegradability, biocompatibility, and economic sustainability.24−26 The CNFs have a higher aspect ratio and are more ductile, thereby serving as a useful template for integrating functionality in wound healing.27 Electrospun CNFs have been chemically immobilized with lytic enzymes to provide anti-staphylococcal activity.28 Bandages composed of surface-decorated nanofibers showed higher antibacterial activity against S. aureus but exhibited limited cytotoxicity against keratinocytes in a skin model. Bacterial cellulose-based hydrogels have been fabricated with acrylic acid polymers for full-thickness wound healing.29 These hydrogels demonstrated Received: August 27, 2018 Accepted: November 14, 2018 Published: November 28, 2018 16150

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Figure 1. (A) In situ reduction of silver-adsorbed TEMPO-oxidized cellulose nanofibers (TCNFs) for the fabrication of silver nanoparticle (AgNP) immobilized TCNFs. TCNFs were immersed in 10 mM AgNO3 solution (Ag+@TCNF) and subsequently reduced to form AgNP-immobilized TCNFs (AgNP@TCNFs) in a 0.1% (v/v) formaldehyde solution. (B) Schematic diagram of the preparation of AgNP@TCNF-embedded alginate hydrogels (AL/AgNP@TCNF). A solution of 2% (w/v) alginate was mixed with AgNP@TCNF, presolidified in 0.5% (w/v) CaSO4; the mixture was injected in a customized shape. A solution of 1% (w/v) CaCl2 was employed to completely solidify the TCNF hydrogel patch.

controlling the gelation times and the mechanical properties of the cross-linked hydrogels. Thus, we speculate that these antibacterial hydrogels may adopt various shapes to cover the affected skin areas and can be further solidified via secondary cross-linking to improve the mechanical properties. To fabricate the AgNP-adsorbed CNF or TCNF, we reduced the silver-ions-adsorbed nanofiber and performed characterization studies, as shown in Figure 2. We first oxidized CNF to TCNF with TEMPO treatment to convert the hydroxyl groups of carboxylic acids. Solid-state 13C NMR indicated that both TCNF and CNF exhibited peaks of C1 (104 ppm), C2, C3, C5 (72 and 74 ppm), C4 (83 and 88 ppm), and C6 (64 ppm), whereas TCNF showed a distinctive peak of carboxyl group (104 ppm), suggesting that the carboxyl groups were newly formed on TCNF (Figure S1). Although bare CNF and TCNF showed similar morphologies in the scanning electron microscopy (SEM) images, these nanofibers displayed different surface morphologies after in situ reduction; in comparison to CNF, TCNF showed a rough morphology, which was associated with the higher number of nanoparticles adsorbed on the surface. The size of the adsorbed silver nanoparticle was 12.578 ± 3.34 nm, which is based on the image analysis of the SEM images. In addition, we obtained energy-dispersive surface spectra (EDS) and confirmed that these particles were AgNP, as distinct silver peaks were observed for both nanofiber types. However, the comparison of the relative peak intensity of silver atoms revealed that the silver peak of AgNP@TCNF was higher than that of AgNP@CNF. As the carbon and oxygen peaks in both spectra showed the same intensity, the higher intensity of silver indicated that the surface of AgNP@TCNF was covered with higher amounts of silver than the surface of AgNP@CNF. This observation may be attributed to the fact that the anionic surface of TCNF electrostatically adsorbed the negativelycharged silver ions and the higher density of silver ions on the surface of TCNF led to the efficient reduction of silver ions to AgNP, whereas the low density of silver ions on CNF

excellent properties and facilitated cell attachment, migration, and proliferation. Regenerated bacterial cellulose loaded with antibiotics was also used as antibacterial sponges.30 The sponges effectively minimized the rate of wound infections and accelerated the wound-healing process. In this study, CNF was surface-immobilized with AgNPs by in situ reduction of silver ions and subsequently embedded in alginate hydrogels to promote antibacterial activity. Sustained release of silver ions was investigated from AgNP-decorated CNF. Furthermore, the antibacterial activity of the hydrogel was investigated and compared with that of CNF incorporated with silver ions. Cytotoxicity against fibroblasts was evaluated to assess the potential damage to animal cells.

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RESULTS AND DISCUSSION Figure 1 shows the schematic presentation of the development of antibacterial hydrogels containing AgNP-incorporated nanofibers (AL/AgNP@TCNP). The hydroxyl groups of CNFs were oxidized to carboxylate groups by (2,2,6,6tetramethylpiperidin-1-yl)oxidanyl (TEMPO) to facilitate the electrostatic adsorption of silver ions on TCNF. The carboxyl group contents in TCNF was confirmed to be 1.3−1.6 mmol/g in the previous study.26 The incorporated silver ions were then reduced to metallic silver in situ to form AgNP-incorporated TCNF (AgNP@TCNF) (Figure 1A). To fabricate nanofiberembedded hydrogels, AgNP@TCNF was mixed with alginate solution and the mixture was cross-linked in calcium solution to form the AL/AgNP@TCNF hydrogels. We sequentially employed CaSO4 and CaCl2 to control gelation rates, wherein the first gelation step produced injectable gels and the secondary gelation step increased the mechanical properties via complete cross-linking of alginate. This strategy allows the formation of a custom-made hydrogel capable of fitting into any surface. Previous studies have shown that CaSO4-based pregelation is needed to form an injectable gel for applications in three-dimensional bioprinting.33 These studies highlight that the two-step strategies of cross-linking are advantageous for 16151

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We analyzed AgNP@TCNF and AgNP@CNF with X-ray diffraction (XRD) and the amount of incorporated AgNP was quantified (Figure 3). AgNP@TCNF showed distinctive peaks of silver at degrees of 37.84, 64.09, and 77.22 in the region from 5 to 89° (Figure 3A). However, AgNP@CNF and other nanofibers failed to show any significant peaks of silver at the designated degrees. Although we observed much smaller and less-frequent AgNPs with AgNP@CNF than with AgNP@ TCNF, the amount of AgNPs on AgNP@CNF was insufficient to affect the XRD pattern. To determine the effects of silver adsorption period on the incorporation of AgNP, we dipped TCNF or CNF in HNO3 for 24 h and monitored the amount of AgNP on both nanofiber types (Figure 3B). The measurement of AgNP adsorption level after 24 h by inductively coupled plasma-optical emission spectrometry (ICP-OES) revealed that AgNP@TCNF and AgNP@CNF showed 32.66 ± 2.10 and 3.60 ± 0.64 μg/mg of AgNP adsorption, respectively. After confirming that the silver ions were not further reduced to metallic silver after a 30 min treatment with formaldehyde, we speculated that the formation of AgNP was mostly affected by the amount of silver adsorbed on nanofibrils. The initial burst of silver adsorption primarily occurred within 3 h and no significant increase in silver adsorption was observed thereafter, which is an adsorption pattern typical of electrostatic interactions. These results clearly suggest that there was extensive silver coating on the surface of TCNF in comparison to CNF, subsequently inducing the efficient formation of AgNPs on the surface of nanofibers upon in situ reduction. To compare the antibacterial properties of hydrogels with AgNP@TCNF and Ag+@TCNF, we first determined the release profiles of silver from each hydrogel (Figure 4A). After 1 week, the cumulative release of silver from AL/AgNP@ TCNF and AL/Ag+@TCNF was 15.45 ± 0.4 and 11.56 ± 0.04 μg, respectively. As the same amount of silver was incorporated in these hydrogels, the release of silver was slightly higher in AL/AgNP@TCNF and the overall levels of the released silver were considerably lower than the initial levels of silver. We speculated that alginate hydrogel significantly attenuates silver release from the matrix. To quantify the antibacterial efficacy of AL/AgNP@TCNF and AL/Ag+@TCNF, a live/dead assay for

Figure 2. Analysis of AgNP@CNF and AgNP@TCNF morphologies. (A) Scanning electron microscopy (SEM) of CNF, AgNP@CNF, TCNF, and AgNP@TCNF. Energy-dispersive spectrometry (EDS) spectra of AgNP@TCNF and AgNP@CNF were obtained for elemental detection of C, O, and Ag (inset image) (scale bar = 500 nm) (B) photos of CNF, TCNF, AgNP@CNF, and AgNP@TCNF dispersed in water.

produced very few AgNPs. As shown in Figure 2B, we also observed that the intense dark brown color indicative of reduced silver was more evident in AgNP@TCNF than in AgNP@CNF. Therefore, this result clearly indicates that TCNF is superior to CNF in the process of in situ AgNP formation.

Figure 3. Qualitative and quantitative analyses of AgNP on CNF and TCNF. (A) X-ray diffraction patterns of CNF, TCNF, AgNP@CNF, and AgNP@TCNF. XRD patterns of the samples were measured using an X-ray beam with a Cu radiation of 1.54 Å at 40 kV and 30 mA. The patterns were collected in the region of 2θ from 5 to 89° at a scanning rate of 0.08°/min. (B) Quantification of adsorbed AgNP on the surface of CNF and TCNF by ICP-OES. The samples dissolved in 10% HNO3 were subjected to quantification with ICP-OES. 16152

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Figure 4. Silver release and antibacterial behavior of AL/AgNP@TCNF. (A) Ag+ release profiles from AL/AgNP@TCNF and AL/Ag+@TCNF hydrogels. The amount of Ag+ in the supernatants was quantified by ICP-OES at 37 °C for 7 days. (B) The fluorescence ratio of live E. coli with negative control (without AgNO3), AL/AgNP@TCNF, AL/Ag+@TCNF, and positive control (AgNO3 25 μg/mL). Hydrogels containing TCNF with AgNP and Ag+ were used as a hydrogel-released fractions. Live bacterium ratio was calculated by dividing the red fluorescence intensity by the green fluorescence intensity. (C) Fluorescence imaging of live/dead staining of E. coli co-cultured with control (without AgNO3), AgNP@TCNF, and Ag+@TCNF after 0, 3, and 15 h. Green fluorescence and red fluorescence of live bacterial cell and dead bacterial cell, respectively. (D) The zones of E. coli growth inhibition in the presence of AL/AgNP@TCNF, AL/Ag+@TCNF, and AL/TCNF for 24 h (scale bar = 2 mm).

bacterial cells and a disk diffusion test with E. coli were performed (Figure 4B−D). On day 7, all the hydrogels containing TCNF with silver ions (AgNP@TCNF and Ag+@ TCNF) showed similar levels of antibacterial activity, whereas the G/R ratio decreased by 3- to 4-fold as compared to the untreated control group. We observed that the G/R ratio of bacteria within these hydrogels was similar to that observed following incubation in a minimum bacterial concentration (MBC) of silver solution (25 μg/mL). The MBC concentration was determined by incubating bacterial cells in AgNO3 solutions of different concentrations (0−50 μg/mL) (Figure S2). The antibacterial activities were also assessed by confocal microscopy (Figure 4C) and the bacterial cells treated with the released fractions of hydrogels containing AgNP@TCNF or Ag+@TCNF showed higher intensities of red fluorescence than those without treatment. Thus, both hydrogels significantly suppressed the bacterial growth for 24 h because the released amount of silver was between 11.4 and 15.4 μg.

To assess the antibacterial properties of these hydrogels, we placed the hydrogel patches containing AgNP@TCNF or Ag+@TCNF on bacteria-containing agar plates and measured the zones of growth inhibition after 24 h (Figure 4D). An AL/ TCNF hydrogel patch was employed as a negative control. All the TNCF-embedded hydrogel showed a homogeneous mixing in the hydrogels, where brownish color of AgNP@TCNF was distributed throughout the matrix (Figure 4D, AL/AgNP@ TCNF). The diameters of the inhibition zones were 15, 17, and 19 mm for AL/TCNF, AL/AgNP@TCNF, and AL/Ag+@ TCNF, respectively. A slightly larger zone was observed with the hydrogel patch containing Ag+@TCNF than with the patch containing AgNP@TCNF, which may be attributed to the rapid diffusion of silver ions. The hydrogel patch was in place for only 24 h, which seems too short a period for AgNP@TCNF to release enough silver ions to generate antibacterial activity. Although we could not employ an incubation period longer than 24 h because of the limitation of the test, we speculate higher antibacterial activities owing to 16153

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comparable antibacterial activities between AL/AgNP@TCNF and AL/Ag+@TCNF in the released fractions for 7 days. Furthermore, as silver ions are considered cytotoxic to animal cells at high concentrations, the sustained, slow release of silver would be optimal at concentrations that are low enough to minimize cytotoxicity but sufficient enough for exert antibacterial activity. Several studies have indicated that silver ions are released from AgNP upon the gradual oxidation of the atomic silver in the air over a period of approximately 3−7 days. AgNP-embedded bacterial cellulose showed a gradual release profile for 6 days, and 20−28% release of silver was observed according to NaBH4/AgNO3 molar ratios.34 In another study, in situ formation of AgNP on the surface of bacterial cellulose was shown to attenuate the release of Ag cations because of the strong adhesion between AgNP and the cellulose surface, wherein the release profile was dictated by Fickian diffusion model in response to the ionization of atomic silver.14 Most published reports have employed AgNP or AgNPimmobilized matrices to facilitate optimum silver release. However, we embedded AgNP-adsorbed nanofibers to ensure maximum AgNP immobilization, owing to the high surface-tovolume ratio of the nanostructures. Furthermore, we speculate that the burst release of silver could be attenuated because the diffusion of positively charged silver ions out of the negatively charged alginate cage was controlled both by physical and electrostatic interactions. Thus, AL/AgNP@TCNF could release silver ions at levels similar to those observed with hydrogels (AL/Ag+@TCNF) incorporated with silver ions, and the released silver from AL/AgNP@TCNF showed antibacterial activity that was comparable to that of the silver-ion-incorporated hydrogel matrices. Additionally, we did not examine the mechanical properties of AgNP@TCNFembedded alginate hydrogels because the alginate hydrogel play a role of gluing silver-incorporated nanofibrils in the matrix. However, we speculate that mechanical properties should be further examined for industrial application of our hydrogel system. To determine the cytotoxicity of silver-incorporated nanofibers, NIH3T3 cells were incubated with various concentrations of AgNP@TCNF and Ag+@TCNF, and the viability was monitored for 48 h (Figure 5). AgNP@TCNF showed cytotoxicity similar to that observed for the untreated group (black bar) in response to various silver concentrations (1−10 μg/mL) for 48 h, indicating that AgNP@TCNF displays minimum cytotoxicity against eukaryotic cells. Ag+@TCNF showed major cytotoxicity at concentrations of 5−10 μg/mL, but no cytotoxicity was observed at the lowest concentration (1 μg/mL); the viability was 74−78 and 76−78% at silver concentrations of 5 and 10 μg/mL, respectively. Previous studies indicated that higher concentrations of silver cations are cytotoxic to eukaryotic cells. AgNP-incorporated chitosan scaffolds showed extreme cytotoxicity against fibroblasts in the presence of silver from 3.7 to 177.9 mg/device and a 9−18% inhibition of cell proliferation.15 In another study, however, AgNP-incorporated bacterial cellulose showed low toxicity and supported the proliferation and differentiation of epidermal cells for longer than 3 days.14 We speculate that additional animal study can be required to claim the potential advantages of our hydrogels; however, animal model simulating a bacterial infection condition is required to monitor the dramatic difference among the groups because the antibacterial effect of silver is supportive of the wound-healing process.35

Figure 5. Cytotoxicity assay of AgNP@TCNF and Ag+@TCNF in NIH3T3 cells. WST-1-based cytotoxicity assay was performed for AgNP@TCNF and Ag+@TCNF with different concentrations of silver equivalents ranging from 1 to 10 μg/mL. Absorbance at 450 nm was recorded at 0, 12, and 48 h (n = 3). NIH3T3 cells (5 × 103 cells/ well) were seeded in each well (96-well plate). Asterisk (*) indicates statistically significant level at p < 0.05 by one-way analysis of variance (ANOVA).

Additionally, we did not employ alginate for cytotoxicity assay for animal cells because antiproliferation effect was further attenuated when AgNP@TCNF was encapsulated in the alginate gel. Thus, we speculate that AL/AgNP@TCNF and AL/Ag+@TCNF should be further noncytotoxic. Thus, these findings highlight that the sustained release of silver cations from AgNP@TCNF is highly advantageous for minimizing the cytotoxicity to eukaryotic cells. We did not test the cytotoxicity of AL/AgNP@TCNF because of limitations of the assay; however, we expect little or no cytotoxicity of AL/ AgNP@TCNF, as the same AgNP@TCNF was embedded in the hydrogel.

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CONCLUSIONS Hydroxyl groups of CNFs were converted to carboxyl groups to ensure efficient adsorption of silver ions on the surfaces of CNFs, wherein AgNPs were formed in situ in formaldehyde solution. The AgNP@TCNF-embedded alginate hydrogel showed sustained release of Ag+ for extended period of time and the released media from the hydrogel significantly attenuated the bacterial growth without any cytotoxicity toward animal cells. Thus, the current hybrid hydrogel is expected to be a superior candidate for wound dressing with prolonged antibacterial activity.

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EXPERIMENTAL SECTION Materials. (2,2,6,6-Tetramethylpiperidin-1-yl)oxidanyl (TEMPO)-oxidized cellulose nanofibers (TCNFs) and CNFs were donated by the Korean Forest Research Institute (Seoul, Korea) and used for the study without further modifications.31−32 Silver(I) nitrate (AgNO3), calcium sulfate anhydrous (CaSO4), calcium chloride (CaCl2), and t-butanol (C4H10O) were purchased from Daejung Chemical (Seoul, Korea). Formaldehyde solution (36−38%, w/w) was obtained from Wako (Tokyo, Japan) and sodium carbonate (Na2CO3) and nitric acid (HNO3), from Junsei Chemical (Tokyo, Japan). Alginate acid sodium salt was supplied by Sigma-Aldrich (St. Louis, MO) and Difco Luria-Bertani (LB) broth (Miller), by BD Bioscience (CA). Bacterial Viability Kit was purchased 16154

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from Thermo Fisher Scientific (Waltham, MA). Escherichia coli (KCCM 11234) was donated by Dr. Juhee Ahn. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and Dulbecco’s phosphate-buffered saline (PBS) were procured from Gibco BRL (Staley Rd Grand island, New York). Murine fibroblast cell line NIH3T3 was obtained from the Korean Cell Line Bank (Seoul, South Korea), whereas WST-1 (EZ Cytox) was acquired from DoGenBio Co. Ltd. (Seoul, Korea). In Situ Reduction of Silver Ions (Ag+) on TCNF and CNF. To modify the surfaces of TCNF and CNF with AgNPs, silver ions were in situ reduced to AgNP on TCNF or CNF in an AgNO3 solution (Figure 1A). Briefly, 3 mL of CNF (10 mg/mL) or TCNF (15 mg/mL) was mixed with 30 mL of AgNO3 solution (10 mM) and the mixture was incubated at 37 °C with gentle shaking for 1 h to facilitate Ag+ adsorption. Silver-ions-adsorbed TCNF (Ag+@TCNF) and CNF (Ag+@ CNF) were thoroughly washed with distilled water (dH2O) twice to remove the unbound Ag+. Ag+@TCNF and Ag+@ CNF were subsequently reduced with 30 mL of Na2CO3 solution (3%, w/v) containing formaldehyde (0.1%, v/v) at room temperature for 20 min. After washing twice with dH2O to remove the residual chemicals, the amount of reduced AgNPs on the surface of TCNF or CNF was quantified by inductively coupled plasma optical emission spectrometry (ICP-OES) (Optima 7300 DV, Perkin Elmer, Waltham, MA). To prepare samples for ICP-OES, the AgNPs on the surface of TCNF and CNF were dissolved in HNO3 (10%, v/v), and the solutions were filtered through 0.45 μm filters. Characterization of AgNP-Modified TCNF (AgNP@ TCNF) and CNF (AgNP@CNF). We characterized TCNF and CNF with nuclear magnetic resonance spectrometry (13C NMR; Unity-Inova 600 MHz, Varian Technologies) and the degree of AgNP adsorption on the surfaces was examined by X-ray diffractometry (XRD; X’pert-pro MPD, PANalytical, Netherlands), ICP-OES, and energy-dispersive spectrometry (EDS) equipped ultrahigh-resolution scanning electron microscopy (UHR-SEM) in the Central Laboratory of Kangwon National University. For XRD spectroscopy, an X-ray beam with Cu radiation of 1.54 Å at 40 kV and 30 mA was irradiated on 100 mg of TCNF, CNF, AgNP@TCNF, and Ag@CNF. Atomic silver was employed for a reference spectrum to determine the surface deposition of AgNP. For the quantification of AgNP, AgNP@TCNF, and Ag@CNF were dipped in 10 mM AgNO3 solution for 0−24 h and then reduced to AgNP in a 0.1% (v/v) formaldehyde solution containing 3% (w/v) Na2CO3 to determine the effects of preincubation time on AgNP incorporation. The amount of silver incorporated was determined by ICP-OES, as described above. For SEM examination, the AgNP@TCNF or AgNP@ CNF solution was semidried at ambient temperature and the solvent exchanged with n-butanol for freeze-drying. The freezedried samples were coated with iridium and examined by UHR-SEM at 1 kV (S-4800, Hitachi, Japan). Chemical microanalysis was performed with EDS at an acceleration voltage of 15 kV. In Situ Formation of AL/AgNP@TCNF and AL/Ag+@ TCNF Hybrid Gel. To fabricate an injectable hybrid gel, an alginate solution (2%, w/v) was loosely cross-linked with CaSO4 solution (0.5%, w/v) and then uniformly mixed with AgNP@TCNF in a 1:1 ratio through gentle pipetting (Figure 1B). The mixture was completely cross-linked with CaCl2 (1%, w/v) for 20 min to increase the physical strength and then

washed twice with PBS (pH = 7.4). For antibacterial tests, AL/ AgNP@TCNF was fabricated into the shape of a disk using a poly(dimethylsiloxane) mold (ϕ = 1.5 cm). AL/Ag+@TCNF was prepared under the same conditions. In Vitro Release of Ag+ from AL/AgNP@TCNF and AL/ Ag+@TCNF. To investigate the release profile of Ag+ from AL/ AgNP@TCNF and AL/Ag+@TCNF, the samples (thickness = 2 mm, diameter = 10 mm) were incubated in 5 mL of PBS (pH = 7.4) containing 10% t-butanol at 37 °C with shaking at 300 rpm. At predetermined timepoints (1, 3, and 7 days), the samples were centrifuged, supernatants were collected for ICPOES analysis, and the same volume of fresh releasing medium was added. To prepare samples for ICP-OES, the supernatants were freeze-dried, dissolved in 10% HNO3, and the solutions filtered through 0.45 μm filters. Antibacterial Activity Test. Three different methods were used to investigate the antibacterial activities of AL/AgNP@ TCNF and AL/Ag+@TCNF against E. coli. To visualize and quantify the antibacterial effect of the hybrid hydrogel, a live/ dead assay was performed according to the manufacturer’s instructions. Briefly, the hydrogels were incubated in a releasing buffer for 7 days and the supernatants were collected, freeze-dried, and resuspended in 1 mL of dH2O. Each sample was treated with 1 mL of LB broth containing E. coli (1 × 108 cells/mL) and incubated on an orbital shaker at 37 °C for 12 h. To quantify bacterial viability, 100 μL of the cell suspension was mixed with the same volume of a dye mixture containing SYTO-9 and propidium iodide (PI) in a 96-well plate and incubated for 15 min. Fluorescence intensity was measured using a fluorescence microplate reader (Synergy H1, Biotech) with an excitation wavelength of 485 nm (SYTO-9 and PI) and emission wavelengths of 530 nm (SYTO-9) and 630 nm (PI). Cell viability was calculated by dividing the live cell fluorescence intensity at 530 nm (I530) by the dead cell fluorescence intensity at 630 nm (I630) using the following equation cell viability =

I530 I630

For visualization, bacteria in the incubation medium were fluorescently stained with SYTO-9 and PI for 30 min and observed by high-resolution confocal laser scanning microscopy (LSM880, Carl Zeiss, Germany) at the Central Laboratory of Kangwon National University. For the disk diffusion test in LB agar plate, a hybrid hydrogel (AL/AgNP@ TCNF, AL/Ag+@TCNF, and AL/TCNF) (thickness = 2 mm, diameter = 1.5 cm) was autoclaved and placed on an agar plate inoculated with 100 μL of E. coli (1 × 108 cells/mL). After incubation at 37 °C for 24 h, the diameter of the zone of inhibition was measured. Animal Cell Viability Assay. To compare the cytotoxicity of AgNP@TCNF and Ag+@TCNF against animal cells, WST1-based cytotoxicity assay was performed using the NIH3T3 cell line. The NIH3T3 cells were seeded in a 96-well plate at a cell density of 5 × 103 cells/well in 200 μL of DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/ streptomycin (P/S) and cultivated at 37 °C in the presence of 5% CO2 in a humidified atmosphere for 24 h. After removing the media, the cells were treated with different concentrations (1, 5, and 10 μg/mL) of AgNP@TCNF or Ag+@TCNF in DMEM (200 μL). After 24 and 48 h treatment, the media were replaced with 200 μL of fresh media and 20 μL of WST-1 16155

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solution, followed by incubation for 1 h at 37 °C. The optical density of the supernatant was measured at 450 nm wavelength to evaluate the cell viability. Statistical Analysis. All the data were evaluated with oneway analysis of variance (ANOVA) and presented as the mean ± standard deviation. A value of p < 0.05 was considered statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02180. Solid-state 13C NMR spectra of CNF and TCNF; growth curves of E. coli (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. website: http://nano-bio. kangwon.ac.kr. ORCID

Hyuk Sang Yoo: 0000-0002-4346-9154 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Institute of Forest Science. REFERENCES

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DOI: 10.1021/acsomega.8b02180 ACS Omega 2018, 3, 16150−16157