In Situ Synthesis of Antimicrobial Silver Nanoparticles within

Feb 18, 2016 - †Department of Bionanosystem Engineering Graduate School and ‡Division ..... (TEM) was used to get more information about the size ...
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In Situ Synthesis of Antimicrobial Silver Nanoparticles within Antifouling Zwitterionic Hydrogels by Catecholic Redox Chemistry for Wound Healing Application Amin GhavamiNejad,† Chan Hee Park,*,‡ and Cheol Sang Kim*,†,‡ †

Department of Bionanosystem Engineering Graduate School and ‡Division of Mechanical Design Engineering, Chonbuk National University, Jeonju City, Republic of Korea S Supporting Information *

ABSTRACT: A multifunctional hydrogel that combines the dual functionality of both antifouling and antimicrobial capacities holds great potential for many bioapplications. Many approaches and different materials have been employed to synthesize such a material. However, a systematic study, including in vitro and in vivo evaluation, on such a material as wound dressings is highly scarce at present. Herein, we report on a new strategy that uses catecholic chemistry to synthesize antimicrobial silver nanoparticles impregnated into antifouling zwitterionic hydrogels. For this purpose, hydrophobic dopamine methacrylamide monomer (DMA) was mixed in an aqueous solution of sodium tetraborate decahydrate and DMA monomer became soluble after increasing pH to 9 due to the complexation between catechol groups and boron. Then, cross-linking polymerization of zwitterionic monomer was carried out with the solution of the protected dopamine monomer to produce a new hydrogel. When this new hydrogel comes in contact with a silver nitrate solution, silver nanoparticles (AgNPs) are formed in its structure as a result of the redox property of the catechol groups and in the absence of any other external reducing agent. The results obtained from TEM and XRD measurements indicate that AgNPs with diameters of around 20 nm had formed within the networks. FESEM images confirmed that the silver nanoparticles were homogeneously incorporated throughout the hydrogel network, and FTIR spectroscopy demonstrated that the catechol moiety in the polymeric backbone of the hydrogel is responsible for the reduction of silver ions into the AgNPs. Finally, the in vitro and in vivo experiments suggest that these mussel-inspired, antifouling, antibacterial hydrogels have great potential for use in wound healing applications.

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INTRODUCTION

completely prevent bacterial colonization and subsequent wound infection.8,9 Over the past decade, research into mussel-inspired catecholcontaining hydrogels has greatly expanded due to their fascinating chemical properties and their practical applications.10,11 Interestingly, catechol-containing hydrogels have been found to be nontoxic in many in vitro and in vivo experiments.12 One of the unique properties of catechol groups is their strong reductive property. The catechol group has the capability to reduce metal ions, including silver and gold ions, into metallic nanoparticles.13 Messersmith et al.14 took advantage of this property to form AgNPs simultaneously with hydrogel formation and obtained a silver-releasing catecholic hydrogel by using an end-functionalized polyethylene glycol (PEG). However, this method could only form and release a small amount of silver nanoparticles.15 Furthermore, PEG-based materials are insufficient for applications in which maximal biological stability and nonfouling are required.16

To date, extensive research efforts have been directed toward developing wound-dressing materials that promote the wound healing process.1 Many clinical studies have shown there are benefits to using hydrogel dressings since these can maintain a moist environment around the wound site to improve the healing process by protecting damaged skin from cellular dehydration and angiogenesis.2,3 Zwitterionic hydrogels are considered one of the most attractive types of hydrogels for use in superabsorbent and nonadherent wound dressings since these exhibit a superhydrophilic property and a strong resistance to cell attachment, protein adsorption, and bacterial adhesion.4,5 For example, it is important for cells to not adhere to wound dressing during wound healing, especially when frequent changes in dressings are needed during clinical practices since this will cause no pain to the patients.6 Although the nonfouling properties of zwitterionic hydrogels can prevent the colonization of microbes on their surfaces, they cannot kill bacteria transmitted from patients or the environment when not in contact with them.7 Therefore, it is very desirable to incorporate a releasable antibacterial agent such as silver nanoparticle (AgNPs) into the nonfouling hydrogels to © 2016 American Chemical Society

Received: January 12, 2016 Published: February 18, 2016 1213

DOI: 10.1021/acs.biomac.6b00039 Biomacromolecules 2016, 17, 1213−1223

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water. In the next step, the resulting solid in the solution was filtered, and the obtained colorless aqueous solution is referred to as a protected dopamine monomer solution. The amount of protected dopamine monomer inside of the solution was determined via UV− vis, confirming the total amount of DMA that has been protected through this simple procedure. Furthermore, the resulting 1H NMR of the white solid filtered compound does not show any sign of the dopamine monomer, indicating the total amount of DMA that exists in the colorless aqueous solution. It should also be mentioned that since the boronic compound that was used in this procedure is a micronutrient with an extremely low toxicity,19 the hydrogels synthesized in the rest of this work were used as-prepared from the protected dopamine monomer solution without any further purification. Synthesis of ZWDO Hydrogels. Synthesized sulfobetaine zwitterionic monomer (1 gr) and MBA (0.01 gr), as a cross-linker, were added to a vial containing 8 mL of protected dopamine monomer solution. The mixtures were stirred for 5 min, and dissolved oxygen was removed by bubbling nitrogen through the solution for 10 min. After the solution was homogenized, 2,2-azobis(2-methylpropionamidine) dihydrochloride was added into the monomer solution, and the solution was then injected into a glass reaction mold. The polymerization was initialized by heating to up to 60 °C for 2 h. After the polymerization, the prepared hydrogel (pH = 9) was removed from the mold and was immersed in double-distilled water with a low pH (pH = 3) at room temperature for at least 24 h. During this time, the water was regularly refreshed to remove the protection groups and unreacted compounds. The resulting gels are referred to as ZWDO hydrogels. The deprotection of the catechol group at this state can also be tested by adding an oxidizing agent (NaOH) to the surface, which leads to a reddening of the color of the hydrogel due to the dissociation of hydroxyl groups in the hydrogel.20 Preparation of Silver Nanoparticles within ZWDO Hydrogels (ZWDO-AgNPs). The incorporation of the silver nanoparticles into the hydrogel structure was performed by contacting hydrogel (pH = 3) with 2 mL of 2 mM AgNO3 in deionized water solution. During this stage, the silver solution was quickly absorbed and silver nanoparticles were progressively formed (due to the redox property of the catecholic moiety of dopamine methacrylamide in the polymeric backbone), and the color of the hydrogel evolved from light yellow to black. The samples were then rinsed with an excess amount of deionized water, and the resulting gels were denoted as ZWDO-AgNPs. Preparation of Poly-NIPAM/AAm (N-Isopropylacrylamideco-acrylamide) Hydrogel as Control Sample for the Cell Attachment Experiment. Nipam (7.5 mmol) and AAm (0.75 mmol) as monomers and MBA (0.15 mmol) as a cross-linker were added to a reaction system containing 10 mL of double-distilled water under nitrogen bubbling at room temperature. After the solution was homogenized, an adequate amount of APS (0.05 mmol) as initiator was added to the vial. Then TMEDA (20 μL) was added as an accelerator into the monomer solution to initiate the radical polymerization. After 6 h, the hydrogel was removed from the mold and washed with double-distilled water several times. Characterization of Hydrogels. Field Emission Scanning Electron Microscopy (FESEM). The surface structure and the morphology of the freeze-dried ZWDO and ZWDO-AgNPs hydrogel were studied via field emission scanning electron microscopy (FESEM; Zeiss Supra 40VP). To prepare freeze-dried samples, the hydrogels were transferred into liquid nitrogen for 15 min after they had been maintained in water at 15 °C for a week, and they were then freeze-dried at −43 °C under a vacuum of 0.1 Pa for 48 h to thoroughly remove the water. The cross sections were observed by placing the freeze-dried hydrogel samples into liquid nitrogen for a sufficient length of time, and they were then mechanically fractured and stuck to a sample holder. All of the samples were sputter-coated with gold for 40 s before observation. X-ray Powder Diffraction (XRD). The crystalline structure of the materials was investigated by conducting X-ray powder diffraction (XRD) analyses of the ZWDO and ZWDO-AgNPs hydrogels using a Rigaku X-ray diffractometer (Cu Kα, λ = 1.54059 Å) over Bragg angles

Here we report on a new approach to synthesize antibacterial silver nanoparticles impregnated into antifouling zwitterionic hydrogels. We take advantage of the catechol redox chemistry to carry out a typical procedure where chemical cross-linking of zwitterionic poly(sulfobetaine methacrylate) monomer and protected dopamine methacrylamide monomers (DMA) forms a hydrogel network (ZWDO) with multiple catecholic groups along the polymeric chains. The resulting hydrogel absorbed the AgNO3 solution, and the reductive catechol groups of the DMA in the cross-linked hydrogels convert the silver ions into solid silver nanoparticles in the hydrogel networks without the need for additional chemical reductants or toxic solvents. The as-prepared hybrid hydrogel contained well-dispersed and unaggregated nanoparticles with the size of less than 20 nm throughout the network. The prepared composite hydrogel exhibited good antimicrobial activity against both Gram-negative and Gram-positive bacteria, with a significant improvement in its physical properties when compared to native hydrogel. As a result of the excellent antimicrobial activity of the silver nanoparticles and the antifouling behavior of the zwitterionic polymers, the proposed composite hydrogels are considered to have great potential for use as wound dressings, as confirmed via in vivo experiments.

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MATERIALS AND METHODS

Materials. 3,4-Dihydroxyphenethylamine hydrochloride, dibutyltin oxide, 3-(dimethylamino)-1-propanol, N,N-methylene bis(acrylamide) (MBA), N-isopropylacrylamide (NIPAM), acrylamide (AAm), sodium tetraborate decahydrate, 2,2-azobis(2-methylpropionamidine) dihydrochloride, methacryloyl chloride, sodium borate, sodium bicarbonate, ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TMEDA), and 1,3-propane sultone were purchased from Sigma-Aldrich, South Korea. All aqueous solutions were prepared with ultrapure water purified with a Milli-Q UV-Plus water purification system (Millipore, Bedford, MA). The water had a resistivity of >10 Ω· cm−1. Synthesis of Dopamine Methacrylamide (DMA). The dopamine methacrylamide monomer (DMA) was prepared and characterized in a manner similar to that in our previous work.17 A gray powder was produced with a yield of 80%. The structure of the monomer was confirmed via nuclear magnetic resonance spectroscopy. 1 H NMR (400 MHz, DMSO, 273 K): 6.4−6.6 (3H, m, Ph), 5.5 (1H, d, CH2C−), 5.25 (1H, d, CH2C−), 3.3 (2H, q, CH2−NH−), 2.5 (2H, tr, CH2−Ph), 1.8 (3H, s, CH3−C−). Synthesis of Zwitterionic Monomer (ZW). Zwitterionic monomer, N-(methacryloxypropyl)-N,N-dimethyl-N-(3-sulfopropyl)ammonium betaine was synthesized according to the method that was previously reported.18 The reaction yield was 78%. The structure of the monomer was confirmed via nuclear magnetic resonance spectroscopy. 1H NMR (400 MHz, D2O, 273 K): 5.986 (1H, s, H− C), 5.59 (1H, S, H−C), 4.12 (2H, t, O−CH), 3.28 (2H, t, N− CH2), 3.22 (2H, t, N−CH2), 2.94 (6H, s, N−CH3), 2.82 (2H, t, S− CH2), 2.07 (2H, m, CH2), 1.78 (3H, s, C−CH3), 1.65 (4H, m, −CH2−). Protection of DMA. The reaction media was prepared in 20 mL of double-distilled water by adding 1 g of sodium tetraborate decahydrate. The aqueous solution was degassed with bubbling nitrogen for 20 min. The DMA monomer (0.5 gr) was added to the mixture, and the pH of the mixture was adjusted to 9 by adding sodium carbonate to start the temporary protection of the catechol group through the complexation of boronate and the catechol group. When the pH increases to 9, sodium carbonate must be gradually added to the solution with vigorous mixing under a nitrogen blanket so as to avoid oxidation of the dopamine groups. The reaction mixture was stirred for 1 h at room temperature with nitrogen bubbling. At this time, the gray DMA monomers disappeared, and a white slurry-like solution formed due to the low solubility of sodium bicarbonate in 1214

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Bacterial Suspension Assays. First, 50 μL of bacterial stock was inoculated in 5 mL of LB medium for 24 h with constant shaking at 300 rpm and 37 °C. Then the bacteria suspension was diluted to obtain an optical density (OD) of 0.07 at 600 nm. This solution was used as the starting bacterial solution (control), and hydrogel disks with and without silver nanoparticle were used as test samples. The samples were placed into a 48-well plate, and 400 μL of the bacterial solution was transferred into each well. The plates were wrapped in aluminum foil and placed in a shaker incubator for 24 h. For the OD readings, 100 μL of the bacterial solution was transferred into a transparent 96 well and growth was monitored by measuring the absorbance at 600 nm. The assay was performed in triplicate, with three replicates for each independent experiment. In Vitro Biocompatibility. The viability of the cultured cell was monitored on the first, third, and seventh days of culture using a CCK (Dojindo’s cell counting kit-8) assay. Samples of the same size were sterilized under UV light and thoroughly rinsed in phosphate buffer saline (pH 7.4). Later, samples were transferred to a 48-well plate and rinsed with medium prior to cell seeding. Cell (MC3T3-E1) suspension of 500 μL was seeded at a density of 1 × 104 cells/well and was incubated at 37 °C in a 5% CO2 atmosphere. Culture medium was changed every 2 days. Following the manufacturer’s instructions, 200 μL of cultured medium was transferred to a 96-well plate, and 20 μL of a CCK-8 solution was added to each well and incubated for 3 h. After 3 h of incubation, the absorbance was measured at a wavelength of 450 nm using a microplate reader (Sunrise Tecan, Austria). A standard curve was established by measuring the known number of cells prior to the experiment, and cell viability was determined from the standard curves. In order to examine the cell attachment and spreading, Human HS68/F3T3 Fibroblasts were maintained in continuous growth in Dulbecco’s modified Eagle medium (DMEM, Gaithersburg, MD) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C under a humidified atmosphere containing 5% CO2. Hydrogel disks (ZW, ZWDO, and ZWDO-AgNPs) were used as test samples, and poly-NIPAM/AAm hydrogels were used as control. The samples were placed into a 24-well plate, were incubated with 75% ethanol for 1 h at room temperature, and were washed three times with PBS (pH 7.4) prior to cell seeding. Then, 1 mL of cell suspension (204 cells/mL) was added to each well and was incubated for 24 h at 37 °C. The cells were then chemically fixed with 4% paraformaldehyde for 10 min, followed by staining with ActinGreen 488 (cytoskeleton) and DAPI (nucleus) according to the manufacturer’s protocol. Finally, the stained samples, including control, were examined using confocal laser scanning microscopy (Carl Zeiss, Japan). Wound Healing Test and Histological Analysis. The in vivo animal study was approved by the Institutional Animal Ethical Committee, IACUC at Chonbuk National University, Jeonju, South Korea (certification number CBU 2014−00062). For these experiments, the hydrogel pads were synthesized in a glass reaction mold consisting of two glass plates separated by a 0.5 mm thick silicon spacer final composite hydrogel before application on the wound with a thicknesses of around 0.8 mm and a length and width of 1.5 cm (see Figure S1). The wound healing characteristics were evaluated in rats weighing approximately 250 g. The animals were fed with commercial rodent chow diet and tap water ad libitum and were housed in a room with a constant temperature (22 ± 1 °C) and humidity (60 ± 3%) following a 12 h light/12 h dark cycle. On the day of the wound creation, the rats were anaesthetized with 80 mg/kg ketamine and 8 mg/kg xylazine by intraperitoneal injection. The dorsal area of the rats was depilated, and the operative area of the skin was cleaned with alcohol. One full thickness skin wound of 1.5 cm2 area was prepared by excising the dorsum of the animals. The wounds were then covered with the ZWDO-AgNPs and ZWDO hydrogels, and rats with bare wounds were covered with cotton gauze. After the dressing materials had been applied, the rats were housed individually in cages under normal room temperature. The dressing materials were changed at days 5, 10, and 15. When the dressings had been changed, the animals’ hairs were cropped to take photographs. Upon completion of the wound-healing experiments after 15 days, the

ranging from 10° to 90°. In addition, the exact sizes of the resulting nanoparticles were calculated using the Scherrer formula in the High Score Plus software. Transmission Electron Microscopy (TEM). Transmission electron microscopy (TEM, JEOL JEM, Japan) was used to characterize the size of the silver nanoparticles. Briefly, the freeze-dried samples were carefully scratched and washed several times with water to detach the nanoparticles from the samples. Then a drop of suspended nanoparticle was placed on a copper grid and excess solution on the grid was removed using filter paper. Fourier Transform Infrared Spectroscopy (FTIR). The bonding configurations of the samples were characterized by means of their FTIR spectra using a Paragon 1000 spectrometer (PerkinElmer). X-ray Photoelectron Spectroscopy (XPS) and Energy Dispersive X-ray Spectroscopy (EDS). The elemental composition and surface state of the samples was verified using X-ray photoelectron spectroscopy (XPS, AXIS-NOVA, Kratos, Inc.) with an Al Kα irradiation source. Energy dispersive X-ray spectroscopy (EDS) was also used to confirm the formation of silver nanoparticles. Thermal Analysis. Thermogravimetric analysis (TGA) was conducted using a SDT Q600 TGA/DSC system under N2 purge from room temperature to 800 °C at a heating rate of 10 °C min. Ultraviolet−Visible Spectroscopy (UV−vis). UV−vis analyses were carried out with a PerkinElmer Lambda 45 UV−vis spectrometer using quartz cuvettes (1 cm path length). pH Measurement. The pH of the gels was measured using a pH meter (IQ 150 pH Meter) with an electrode designed for solids, semisolids, and liquids. The sensor was calibrated with standard solutions prior to the measurements. Rheology. The mechanical/rheological properties of the hydrogels were evaluated using a rheometer (Malvern Kinexus Pro) with a cone and plate geometry (20 mm diameter rotating top plate). All of the rheological measurements were then carried out under a regimen of linear viscoelasticity, that is, the material parameters were independent of the applied stress. In Vitro Silver Release. The amount of silver released from the composite hydrogel was analyzed via inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7500 ICP-MS). The hydrogels were synthesized in a Mini-PROTEAN 3 casting mold (Bio-Rad) consisting of two glass plates separated by a 1 mm thickness (see Figure S1). After the silver loading and washing process, the samples were cut into 5 × 5 cm2 pieces. Then the specimens were weighed and placed into centrifuge tubes with 10 mL of phosphate-buffered saline for 1 week at 37 °C. A total of 2.5 mL of the released solutions was extracted at different time intervals and then added back 2.5 mL of fresh PBS to keep the total amount. In the next step, the samples were diluted with 2.5 mL of ultrapure water and were digested with nitric acid (65%) at a 1:1 ratio by volume and transferred to the heating block (60 °C, 5 h), followed by keeping at room temperature overnight. Finally, the solutions were diluted to 10 mL with 2% nitric acid. The calibration between 1 and 200 ppb was made using a 1000 ppm standard Ag(I) solution. All experiments were repeated three times in triplicate, and the data are reported in μg/L (ppb) in the form of average ± SD. Bacterial Inhibition Test. The differences in the ZW and the ZWDO-AgNPs were measured in terms of the reduction in the bacterial activity. We tested the samples by conducting a zone of inhibition test in independent triplicates with Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) as model organisms. The spread plate method was used with nutrient agar plates incubated with 1 mL of bacterial suspension containing around 106 colony-forming units (CFU)/mL. The samples were then placed with ample distance from each other, and the agar plates were then placed in an incubating oven at 37 °C and were left for 3 days to accurately assess the zone of inhibition for each of the samples. The percentage of the inhibition for each sample was estimated through the following equation.

%inhibition =

diameter of zone of inhibition(mm) × 100 diameter of hydrogel(mm) 1215

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Biomacromolecules animals were sacrificed by excess diethyl ether. The skin wound tissue of the rats was excised, fixed with 10% formalin, and stained with hematoxylin−eosin (H&E) for further histological observations. Statistical Analysis. Data were compared using one-way, in GraphPad Prism 6. Data are expressed as means ± SD of measurements (*p < 0.05 and **p < 0.01).

After protecting the catechol group, the zwitterionicdopamine methacrylamide hydrogels (ZWDO) were synthesized by adding sulfobetaine zwitterionic (ZW) as the main monomer and other reagents to a vial containing a fresh solution of protected DMA monomer (Figure 2a). The synthesis procedure and the composition of the polymerization mixture are discussed in the experimental parts. After synthesizing the ZWDO hydrogel, the hydrogel was treated with double-distilled water with a low pH (pH = 3) to obtain the catechol group in a deprotonated state (Figure 2b). The deprotection of the catechol group in this state can also be tested by increasing pH to 12, which leads to a reddening in color for the hydrogel due to the oxidation of the catechol group and the dissociation of hydroxyl groups in the hydrogel (Figure 2c).20 The silver nanoparticles are then incorporated into the ZWDO hydrogel structure by contacting the ZWDO hydrogel (pH = 3) with 2 mM AgNO3 in deionized water. Silver nanoparticles progressively formed through catechol redox chemistry and the color of the hydrogel evolved from yellow to black (Figure 2d). The schematic of this reaction is shown in Figure 2e. The inner morphologies of freeze-dried ZWDO and ZWDOAgNPs hydrogels were evaluated using FESEM. The ZWDO hydrogels show macroporous structures, and their pores are interconnected to form an “open-cell” structure (Figure 3a).26 After treating them with the silver nitrate solution, many nanoparticles formed on the pore walls of the hydrogels (Figure 3b). The uniform distribution of the nanoparticles inside of the hydrogel suggests that the use of DMA monomer in the ZWDO hydrogel provides the synthesis of monodispersed AgNPs with a minimum degree of aggregation, which is expected to produce a strong antimicrobial effect. Furthermore, the pore structures of the nanocomposite hydrogels do not change remarkably after the reduction process, resulting in the presence of channels that allow for the efficient, facile exchange of oxygen, which could also benefit the wound healing process (Figure 3c−d). In addition to the FESEM, transmission electron microscopy (TEM) was used to get more information about the size of AgNPs. The result illustrate that the diameters of the silver nanoparticles were less than 20 nm (Figure 3d, inset). Fourier-transform infrared (FTIR) measurements were carried out to characterize the chemical composition of the hydrogels, and the typical spectrum is shown in Figure 4. The existence of sulfobetaine methacrylate segments in pure zwitterionic hydrogel, as well as in other hydrogels, could be ascertained from the presence of the ester carbonyl groups and sulfonate groups observed from the bands corresponding to −SO3 stretching at 1033 cm−1 and O−CO stretching at 1727 cm−1, respectively. Furthermore, the FTIR-spectrum band at 1183 cm−1 could be attributed to the SO group, and the absorption band 1487 cm−1 is mainly due to the C−H stretching of −N + (CH3)2−. After introducing the DMA monomer in the hydrogel structure, the IR spectrum shows the phenolic C−O−H stretching vibration (1290 cm−1) of the catechol groups in the ZWDO hydrogels, which significantly decreases after the immobilization of the silver nanoparticles, indicating that the catechol group is responsible for the reduction process that takes place. A similar finding was observed by our group for the formation of silver nanoparticles on catecholic nanofibers.27 X- ray diffraction was carried out to confirm the formation of silver nanoparticles in the composite hydrogel. The freeze-dried

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RESULTS AND DISCUSSION Dopamine methacrylamide (DMA) monomer has been selected as the functional unit to bind the catechol moiety along the polymer backbone. However, it is impossible to use it directly for hydrogel preparation due to its hydrophobicity and polymerization inhibitory characteristic.21 In a recent paper, Marcelo et al.22 used a binary mixture of water and dimethylformamide (DMF) to address the solubility issue. However, the addition of a toxic solvent, such as DMF, limits the biocompatibility of the final hydrogel. In another study, Matsumoto et al.23,24 suggested that complexation between hydrophobic boronic monomers and hydrophilic compounds has a significant effect on the solubility of the boronic monomers. In the present study, we hypothesize that catechol groups of DMA may also coordinate and bind with watersoluble boronic compounds, and this complexation may affect the solubility of this monomer. For this reason, DMA monomer was mixed in sodium tetraborate decahydrate solution, and surprisingly, the DMA monomer became soluble as the pH increased to 9 through the addition of sodium carbonate, which also provides a temporary protection of the catechol group through the reversible covalent bonding of the catechol groups of the DMA monomer and the boron in sodium tetraborate decahydrate.19 In Figure 1, the UV−vis spectra of the protected

Figure 1. UV−vis spectra obtained for the solution of protected dopamine monomer at pH = 9.0 and 3.0.

dopamine solution clearly shows that catechol−boronate bonds form at a basic pH, as shown in the UV−vis spectra at 490 nm.20 This peak disappears when the pH changes to an acidic value, indicating that the protected groups of the catechols could be conveniently removed by lowering the pH (in a pH 3 buffer). This could be an advantage of using this method since deprotection does not require the use of hazardous materials. It should also be mentioned that the pH sensitivity of catechol− boron interaction at the molecular level is well characterized in the previous works.19,20,25 1216

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Figure 2. Photographs showing the changes in color of hydrogels by changing the pH: (a) ZWDO pH = 9, (b) ZWDO pH = 3, (c) ZWDO pH = 12, (d) ZWDO after being put in contact with the AgNO3 solution (ZWDO-AgNPs), and (e) schematic of the reaction that converts the silver ions into solid silver nanoparticles.

Figure 3. FESEM images of (a) ZWDO hydrogel and (b−d) ZWDO-AgNPs hydrogel with different magnification; (d) inset shows a TEM image of AgNPs detached from the ZWDO-AgNPs.

ZWDO hydrogel shows a diffraction peak at 2θ = 19.70° that corresponds to amorphous polymers.28 However, the diffrac-

tion peaks observed at 38.1°, 44.2°, 64.5°, and 77.5° correspond to the (111), (200), (220), and (311) diffractions 1217

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Figure 4. FTIR spectra of (a) ZWDO and (b) ZWDO-AgNPs hydrogels. The phenolic vibration of the catechol groups is within the rectangular marked.

Figure 6. XPS scans of (a) ZWDO and (b) ZWDO-AgNPs composite hydrogels (Inset: Ag 3d core−level spectrum of the ZWDO-AgNPs composite hydrogel).

of metallic Ag (JCPDS No. 04−0783), confirming the presence of silver nanoparticles in the hydrogel (Figure 5). 29

silver nanoparticles in the hydrogel networks. (Figures S2 and S3). Zwitterionic hydrogel is well-known to have poor mechanical properties, which is a major disadvantage. The mechanical properties of our hydrogels changed significantly after the formation of the silver nanoparticles, so we used oscillatory rheometry to quantify the changes in the mechanical properties of the swollen hydrogels with and without AgNPs. Figure 7

Figure 5. X-ray diffraction patterns of the ZWDO and ZWDO-AgNPs hydrogels.

Furthermore, the average size of the silver nanoparticles was estimated using the Scherrer formula. The results indicate that the size of the silver nanoparticles contained in the AgNPsZWDO hydrogel was around 18 ± 2 nm. The formation of silver nanoparticles could also be evidenced via XPS spectroscopy. The wide-scan XPS data for ZWDO hydrogels only shows peaks for S 2p (167.4 eV), S 1s (230.9 eV), C 1s (284.7 eV), N 1s (399 eV), and O 1s (531 eV). In contrast, the XPS spectrum of the ZWDO-AgNPs exhibits the presence of S 2p, S 1s, C 1s, N 1s, O 1s, Ag 3d, and Ag 3p (Ag 3p5/2, Ag 3p3/2), where a strong Ag signal peaks at a binding energy of about 370.0 eV, indicating the presence of silver.30 As shown in the spectrum in the inset of Figure 6, there is a 6.0 eV split at the 3d doublet of Ag, implying that the synthesized silver nanoparticles were zerovalent.31 To further determine the identity of the silver metal and its corresponding weight percentage, EDX analysis and TGAs experiments were carried out as Supporting Information, confirming the presence of

Figure 7. Frequency sweeps of the composite hydrogel compared to a gel without silver nanoparticles in a swollen state.

shows a significant increase in the storage modulus for the composite hydrogel compared to the native hydrogel, indicating that the incorporation of silver nanoparticles has a strong effect on the mechanical properties of the hydrogels. Figure 8 shows the release rate of silver from the composite hydrogel in phosphate buffered saline (PBS). Silver was released with a concentration of 46 μg/L for a 5 × 5 cm2 sample during the first day and gradually increased thereafter. This release behavior might be due to the faster release from the nanoparticles that formed near the network surface rather than internal to the 3D network. Silvercontaining hydrogels 1218

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Figure 8. (a) In vitro release profile of Ag from the ZWDO-AgNPs hydrogel as a function of time. (b) Graph showing the MC3T3-E1 cell viability indices for different samples on 1, 3, and 5 days. The viability of the control cell was set at 100%, and viability relative to control was expressed; * indicates statistical significance (p < 0.05; one way ANOVA post hoc Tukey test).

Figure 9. Results of the antibacterial activity of ZWDO-AgNPs hydrogel against P. aeruginosa, S. aureus, and E. coli; * indicates statistical significance (p < 0.05), ** indicates statistical significance (p < 0.01; one way ANOVA post hoc Tukey test).

in Figure 7b. The CCK-8 assay showed that due to the low concentrations of silver released, Mammalian cell viability was not greatly affected during 5 days of incubation. The finding is also in agreement with a previous study by Travan et al.34 who proved that silver release at a concentration of less than 58 μg/ L did not have a significant lethal effect on cell growth. Though,

(SilvaSorb) currently used in clinical wound dressing have been reported to release up to 55.5 μg for the same sample size within 48 h.32,33 Therefore, the silver amounts released from ZWDO-AgNPs are likely to be within the clinically acceptable range. To provide further evidence we also examined the cytotoxicity of the samples, and the results of this test are given 1219

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Figure 10. Confocal microscopy images of HT-1080 cell attachment on the surfaces of (a) poly-NIPAM/AAm hydrogel, (b) ZW, (c) ZWDO, and (d) ZWDO-AgNP hydrogel.

the silver nanoparticles that are released and diffuse into the test media, acting as biocidal agents that deactivate microorganism.8 Figure 10 shows the adhesion of fibroblasts cells on the surface of poly NIPAM/AAm hydrogel as control as well as for ZW, ZWDO and ZWDO-AgNPs hydrogels. As expected, cell adherence and cell spreading was observed for control, but no cells were found on the pure zwitterionic hydrogel surfaces due to the repulsion between the oppositely charged ions of the polymer.4 However, this nonadhesion property was not observed in the case of the ZWDO hydrogel, and few cells adhered to the surfaces of the hydrogel as a result of the interaction between the catechol groups and various molecules on the cell membrane.36 Interestingly, after the silver nanoparticles were immobilized in the hydrogel, no cells were found once again on the surfaces of the ZWDO-AgNPs gel, which supports the hypothesis that after the reduction of silver by the catechol groups, the silver nanoparticle may have occupied the catechol groups through a complexation that does not let the catechol group interact with cells. Therefore, the ZWDO-AgNPs sample can be used as an effective barrier against cell adhesion and can lead to multiple benefits for wound care.6 The possibility of an interaction between the catechol and silver nanoparticles is also in agreement with the results obtained via ICP-Mass measurements where most of the silver nanoparticles where not observed to have been released to the test media during the silver release measurements. The final objective of our study was to demonstrate the efficiency of the samples in healing a real wound by conducting an in vivo experiment. For this experiment, wounds were created on the back of Wistar rats, and the rats were then divided into three groups. The control group had wounds that remained without dressings, one test group had wounds treated with ZWDO hydrogel, and the other test group had wounds treated with ZWDO-AgNPs hydrogel. The decrease in the wound area was assessed by taking digital images of the wounds

further study is required to identify the critical factors that influence the release of silver from the ZWDO-AgNPs hydrogel, this initial fast release rate and the subsequent sustained release rate of Ag could be helpful in reducing the risk of colonization and preventing reinfection at the wound site. It should also be mentioned that the small release amount of the silver with respect to the total amount of silver element (characterized by TGA and EDX) in our composite hydrogel indicates that most of the silver nanoparticles were tightly bound to the catechol groups.27 The Disc diffusion method was used to test the antibacterial properties of ZWDO and ZWDO-AgNPs against both Gramnegative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus and Pseudomonas aeruginosa). As expected, no significant zones of inhibition were observed around the ZWDO hydrogel, indicating that the antibacterial activity was due to the presence of silver nanoparticles in the ZWDO-AgNPs. The percentage increases of the diameters for ZWDO-AgNPs hydrogels were measured after 72 h and the results indicated values of 157, 148 and 129% for E. coli, S. aureus, and P. auregenosa, respectively (Figure 9). It can be seen that the ZWDO-AgNPs have a less significant effect on the growth of Gram-positive than on Gram-negative bacteria, which might be due to the fact that the Gram-positive bacteria is encased in a plasma membrane covered with a thick layer of peptidoglycan (about 20−80 nm) that limits the penetration of the silver nanoparticle.35 To further assess the bacterial killing capability of the hydrogels, suspension assays were also carried out by measuring optical density (OD) of bacteria at 600 nm. These results are shown in Figure 9b, c, and d for E. coli, S. aureus, and P. auregenosa, respectively. For all three bacterial strains, the OD values decreased significantly for ZWDOAgNPs hydrogels, in comparison to other samples. Indicating that the AgNPs containing hydrogels are not only able to inhibit bacterial growth but also are able to kill them. The antimicrobial activity of ZWDO-AgNPs is mainly imputed to 1220

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Figure 11. (a) Wound closure in untreated animals and animals treated with ZWDO and ZWDO-AgNP hydrogels after 15 days; * indicates statistical significance (p < 0.05), ** indicates statistical significance (p < 0.01; one way ANOVA post hoc Tukey test). (b) H&E stained sections at 15 days: (a) control, (b) ZWDO, (c, d) ZWDO-AgNPs (the triangle, arrow, two-sided arrow, star, quadrangle, and circle indicate epidermis, bacterial colonies, keratinocytes, rete pegs, granular tissue, glands, and blood vessels, respectively).

an aqueous mixture of sodium tetraborate decahydrate and it became protected and soluble upon an increase in the pH. The cross-linking polymerization of the zwitterionic monomer was then carried out with a solution of protected dopamine monomer to obtain a new hydrogel, which formed silver nanoparticle in their structure when in contact with AgNO3 in the absence of any other external reducing agent or toxic solvent. The excellent antimicrobial activity of the silver nanoparticles and the antifouling behavior of the zwitterionic polymers introduced into the hydrogels are expected to provide suitable conditions for wound healing applications. Furthermore, a simple procedure is proposed to introduce the dopamine moiety along the polymer chain during hydrogel preparation can be used to fabricate various kinds of dopaminecontaining hydrogels or nanogels just by using other monomer materials instead of the zwitterionic monomer used in this work, and this may be a productive area of research for future work.

(Figure S4) and is presented in Figure 11a as the percentage of the original wound size at certain times. After 15 days, the defect area in different groups became small and was filled with fibro-proliferative tissue. The percentage of the wound size reduction was 59, 80, and 98% for control, ZWDO, and ZWDO-AgNPs hydrogel groups, respectively. These findings indicate that the ZWDO-AgNPs hydrogel exhibits excellent wound healing efficiency when compared to the other groups. Table S11 compares the wound closer ratio of our ZWDOAgNPs with some of the most effective wound dressing materials reported so far.26,37−41The results show the materials used in this research are very competitive when compared with them. It should also be mentioned that most reported in vivo studies used hydrogels with strong cell attachment properties. However, the nonsticking properties of our ZWDO-AgNPs can provide multiple benefits for the wound healing process, such a non-cell-adherent wound dressing hydrogel can be applied as easily as removable, painless bandages,42 and the newly formed skin layer would not be damaged during the frequent removal and replacement of wound dressings.43,44 The histopathological evaluation of the wounds on day 15 indicated the presence of densely packed keratinocytes in the epidermis of the healed wounds with ZWDO-AgNP hydrogels without sign of inflammation. Moreover, the results clearly indicate that the quality of the granulation tissue, vascularization, big rete pegs, and keratinocyte restoration in wounds treated with ZWDO-AgNPs significantly improved when compared to other groups. The excellent wound healing efficiency of the ZWDO-AgNPs could be attributed to all of the unique features of this gel, including (a) the well-known noncytotoxicity and biocompatibility of biomimetic polymers used in its structure; (b) the strong antibiofouling property of the SBMA segments; (c) the antibacterial performance of the releasable silver nanoparticles in a sufficient range; and (d) the nature of the hydrogel in maintaining a moist environment at the wound interface while also allowing for a gaseous exchange.

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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/acs.biomac.6b00039. Dimensions of hydrogel for the wound healing experiments, EDX and TGA results (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported with grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (Project Nos. 2015-020449 and 2013R1A2A2A04015484). The authors would also like to thank the staff of the CBNU central lab for their help with the

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CONCLUSION We have demonstrated the simple and controllable synthesis of silver nanoparticles embedded in antifouling zwitterionic hydrogels. For this purpose, a DMA monomer was mixed in 1221

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ICP-MS. The authors also thank Prof. Dongmei Wu for the in vivo animal study.

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