Antimicrobial Electrospun Silk Fibroin Mats with Silver ... - Springer Link

Fibers and Polymers 2012, Vol.13, No.8, 999-1006

DOI 10.1007/s12221-012-0999-6

Antimicrobial Electrospun Silk Fibroin Mats with Silver Nanoparticles for Wound Dressing Application Pimpon Uttayarat*, Suwimol Jetawattana, Phiriyatorn Suwanmala, Jarurattana Eamsiri, Theeranan Tangthong, and Suchada Pongpat Research and Development Division, Thailand Institute of Nuclear Technology (Public Organization), Chatuchak, Bangkok 10900, Thailand (Received December 25, 2011; Revised March 12, 2012; Accepted March 20, 2012) Abstract: In this report, silk fibroin (SF) mats coated with silver nanoparticles (AgNPs) were manufactured as a prototypic wound dressing and evaluated for antimicrobial properties. SF was extracted from cocoons of Thai silkworms Bombyx mori (variant Nangnoi Si Sa Ket) and fabricated into nonwoven mats by electrospinning. In a one-step synthesis method, colloidal AgNPs were prepared from silver nitrate by gamma irradiation and inspected by transmission electron microscopy. Using the in vitro disc diffusion and growth-inhibition assays, AgNP-coated SF mats effectively inhibited the growth of Staphyllococus aureus and Pseudomonas aeruginosa when the coating solution containing colloidal AgNPs was 4 mM, or equivalent to 50.4 ng/cm2 of adsorbed AgNPs. Based on these results, the AgNP-coated SF mats can potentially be used as antimicrobial wound dressings. Keywords: Biofibers, Electrospinning, Silver nanoparticles, Silk fibroin, Wound dressings

or topical burn cream [21,22]. More recently silver nanoparticles (AgNPs), with diameters on the scale of tens of nanometers [10,18,20,23-26], have attracted attention as an alternative antibacterial agent. There are many chemical and physical methods used to synthesize AgNPs [19,24-26]. Among these, UV and gamma irradiation offer a simple, one-step process to generate AgNPs though the reduction of Ag+ to Ag0 during the irradiation process [10,24-26]. When incorporated into dressing materials, these AgNPs serve to inhibit the growth of bacteria. Previous studies have shown that the addition of AgNPs into electrospun poly(vinyl alcohol) (PVA) membranes [24] and chitosan films [10] can effectively confer both substrates with antibacterial property against Escherichia coli. Thus, the use of AgNPs in wound dressings provides a promising approach to minimize infections during wound healing. The aim of this study was to develop antibacterial wound dressing using regenerated SF as the dressing material and colloidal AgNPs as the antiseptic agent. We utilized the novel electrospinning method [2,6,27,28] to generate twodimensional (2-D) mats of SF derived from cocoons of Thai silkworms B. mori (variant Nangnoi Si Sa Ket). Using gamma irradiation, colloidal AgNPs were prepared in a onestep method in aqueous solution. The AgNP-coated SF mats were then evaluated for their antibacterial properties in comparison to commercial wound dressings embedded with ionic silver.

Introduction The loss of skin tissue from burn injuries leaves the wounded area susceptible to dehydration and infection. In severe burn injuries that cover a large body surface area, about 70 % of patients die from secondary infections [1]. To protect the body from microorganism invasion at the wound site, skin grafts or wound dressings are required to cover the wound while skin is regenerated. Due to limited availability of skin substitutes in patients with severe burns [2,3], wound dressings in the form of hydrogels, foams or textiles can provide an alternative treatment for those patients during wound healing [2,4,5]. Much of the current research in burn treatment has focused on the development of wound dressings made of naturally-derived and biocompatible materials such as silk [2,4-8] and chitosan [9-11]. In particular, silk from silkworms Bombyx mori has a long track record as textile-grade fibers [12] and surgical sutures in biomedical applications [13,14]. This protein-based biopolymer consists of a hydrophobic structural protein called fibroin encased by a glue-like coating protein called sericin [13,15,16]. In vivo studies in animal models have shown that silk fibroin (SF)-based dressings promoted the healing of full-thickness wounds with minimal inflammatory response [2,7]. To minimize wound infections wound dressings often use materials that have been coated in antiseptic agents. Silver in the form of Ag+ or Ag0 [17-23] has long been recognized for its potent antimicrobial properties against a broad spectrum of bacteria. Silver compounds including silver nitrate, silver sulfate, and silver sulphadiazine are conventionally used as a source of free silver cations in commercial wound dressings

Experimental Materials Cocoons of Thai silkworms B. mori (variant Nangnoi Si Sa Ket) were obtained from the Chiang Mai Sericulture Center (Chiang Mai, Thailand). Silver nitrate (AgNO3) and

*Corresponding author: [email protected] 999

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Fibers and Polymers 2012, Vol.13, No.8

PVA with Mw~72,000 were purchased from Merck (Darmstadt, Germany). All other chemicals used in the experiments were obtained from Sigma Aldrich (St. Louise, USA). Dialysis membrane, CelluSep, was purchased from Membrane Filtration Products (Texas, USA). Protein molecular weight standards, HiMark and Dual Color, were purchased from Invitrogen (California, USA) and Biorad (California, USA), respectively. SF Protein Extraction Before the extraction of SF protein, the cocoons were first degummed by boiling in 0.5 % (w/v) sodium carbonate (Na2CO3) solution twice, 30 min each, to remove the sericin coating. The remaining Na2CO3 on the cocoons was washed by warm double-distilled water. To extract SF protein, the degummed cocoons were dissolved in a ternary solvent system containing calcium chloride, ethanol and doubledistilled water (1:2:8 by mole) to yield a 10 % (w/v) solution and heated at 70 ºC for 2 h. Extracted SF solution was filtered through Whatman filter paper to remove debris and then dialyzed in a CelluSep tubular membrane (cut-off MW 12,000-14,000) against double-distilled water for 96 h at 4 ºC. The dialysate was then lyophilized and stored at −20 ºC prior to electrospinning. Figure 1 shows an overview of SF transformation from yellow cocoons to white, electrospun mats. Characterization of SF Molecular Weight The molecular weight of SF was estimated by SDSPAGE. After dialysis, a sample of fresh SF solution was vacuum dried to determine its dry weight. Then the fresh SF solution was analyzed by SDS-PAGE on 8 % or 6 % acrylamide gels in comparison to HiMark (31-460 kDa) and Dual Color (10-250 kDa). After electrophoresis, the gels were stained with silver in a chilled 0.1 % AgNO3 solution and then developed in 3 % (w/v) Na2CO3 solution to visualize the protein bands. Determination of Amino Acid Profile in SF Protein To determine the amino acid profile of SF by high performance liquid chromatography (HPLC), samples of lyophilized SF were prepared by acid hydrolysis. Approximately 0.1-0.2 g of lyophilized SF was suspended in 20 ml of 6 N

Pimpon Uttayarat et al.

hydrochloric acid in a Pyrex glass tube and heated at 110 oC for 24 h. The SF solution was then diluted in double-distilled water and derivatized with phenylisothiocyanate (Sigma). Five µl of the derivatized sample was dissolved in 5 ml of disodium hydrogen phosphate containing 5 % (v/v) acetonitrile and 20 µl of this solution was injected into an HPLC (Agilent 1100, Germany) equipped with a reverse-phase C18 column. The solvent system consisted of 2 eluents: (A) 940 ml of 0.14 M AR-grade sodium acetate buffer containing 0.75 ml triethylamine adjusted to pH 6.2 and 60 ml of HPLC-grade acetonitrile, and (B) 55 % HPLC-grade acetonitrile in deionized water. Eluent A was pumped into the column at a flow rate of 1 ml/min for 3 min then a gradient was run from 0-68 % B in 17 min at a flow rate of 1 ml/min. Detection was performed by diode array detector with a wavelength set at 254 nm. The minimum detection limit was 500 ppm. Amino acid kit (AAS18) was used as a standard. Preparation and Characterization of Colloidal AgNPs Gamma irradiation was used to convert Ag+ to Ag0, which was in the form of AgNPs using a previously described method [26]. Briefly, an aqueous solution containing 40 mM AgNO3, 2 % (w/v) PVA and 1 M ethanol was mixed in a glass vial. The sample was flushed under nitrogen gas for 10 min before gamma irradiation by a Co-60 source (gamma cell 220, Canada) with the total dose of 40 kGy. Colloidal AgNPs dispersed in double-distilled water were analyzed by UV-Visible spectrometer (Cintra 10e, GBC Scientific Equipment Ltd., Australia) at wavelength from 190 to 600 nm. The measurements were made at room temperature under absorbance mode. In addition, the morphology of colloidal AgNPs was also observed by transmission electron microscopy (TEM). The sample was diluted in double-distilled water at 1:1000 ratio. A drop of solution was placed on Cu grid and let dry under laminar flow hood. Images of AgNPs were visualized by TEM (JOEL JEM2100) operated at 100 kV. Fabrication of SF Mats by Electrospinning In the electrospinning process, we prepared SF solutions in two different organic solvents, formic acid (Sigma) and hexafluoroisopropanol (HFP, Sigma), to test their effect on

Figure 1. An overview shows the transformation of SF from (A) the unprocessed cocoons of Thai silkworms B. mori (variant Nangnoi Si Sa Ket) to (B) the extracted, lyophilized SF and (C) the final, electrospun SF mat.

AgNP-coated SF Mats as Antimicrobial Dressings

the formation of SF fibers prior to the electrospinning of SF mats. The former was a typical solvent for SF [6,29,30], whereas the latter was normally used to dissolve various proteins and polymers [28,31,32]. Lyophilized SF was dissolved in formic acid and HFP to prepare solutions at concentrations in the range of 5-25 % (w/v). All SF solutions were stirred at room temperature for at least 4 h to ensure complete dissolution. A horizontal electrospinning station including an infusion pump (Scientific Infusion Pump, Fisher), a movable platform (Bangkok Cryptography, Bangkok, Thailand) and an aluminum (Al) plate was set up inside a chemical hood (Figure 1(C)). A high voltage power supply (Bangkok Cryptography, Bangkok, Thailand) was used to create an electric field between the infusion pump and the Al plate. During electrospinning, the high voltage of 12 kV was discharged over the distance of 10 cm while the infusion pump fed fibroin solution at a constant rate of 0.8 ml/h through a blunt 22-gauge stainless steel needle towards the Al target. To visualize the effect of SF concentration on the formation of electrospun fibers by light microscopy, coverslips were placed on the surface of the Al plate to collect the fibers. The fiber dimension was examined by scanning electron microscopy (SEM, JOEL JSM 6380 LV). For the fabrication of SF mats, the infusion pump was placed on a movable platform that allowed the pump to slide back and forth in a continuous cycling while injecting SF solution towards the Al target to ensure the uniform thickness of SF mats. Chemical Treatment of SF Mats The SF mats were chemically treated by immersion in methanol for stabilization in aqueous environment. To ensure the complete conversion from random coil to β-sheet, the mats were immersed in 90 % methanol for 2 h. The change in fibroin structure was detected by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy (Tensor 27, Brucker Optics, Germany). Sixteen scans were collected under absorbance mode from 4000-400 cm-1 at 4 cm-1 scan resolution. Evaluation of Antibacterial Property The antibacterial property of AgNP-coated SF mats was determined by disc diffusion assay. For the purpose of AgNP adsorption on the surface, we prepared SF mats into thin films with thickness of 20-30 µm supported by Al discs. SF fibers were electrospun onto pre-cut, 8-mm diameter Al discs. The samples were immersed in 90 % methanol for 2 h, rinsed with 70 % ethanol and left to dry inside a laminarflow hood. The 40 mM AgNP stock solution was diluted in sterile distilled water to prepare coating solutions at concentrations 0.1, 0.4, 1, 2, and 4 mM. A 10-µl drop of each coating solution was then placed on the SF discs and incubated for 2 h at room temperature. Each disc was then rehydrated with an 8-µl drop of normal saline before being


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placed face down on Mueller-Hinton agar plates inoculated with 106 colony forming unit (CFU)/ml of either Staphyllococus aureus or Pseudomonas aeruginosa. After 18 h incubation at 37 ºC, the inhibited areas of bacterial growth surrounding each sample was recorded by digital camera and measured. For comparison, the same assay was also performed on commercial, ionic silver-embeded wound dressings, Tegaderm Ag and Aquacel Ag. Additionally, the antibacterial activity of AgNP-coated SF mats was quantified using a growth-inhibition assay. AgNPcoated SF mats and commercial wound dressings were cut into 8-mm diameter discs under aseptic condition and immersed in 500 ul of sterile deionized water in test tubes for 10 min. Then 900 ul of nutrient broth and 103 CFU of S. aureus or P. aeruginosa were added to the test tubes to bring the final volume to 1.5 ml. The test tubes were incubated at 37 ºC with gentle agitation. Aliquots of 100 ul of bacterial broth were withdrawn from each test tube at 1, 2 and 4 h and transferred into new test tubes containing 1.4 ml of fresh nutrient broth and incubated with gentle agitation at 37 ºC. After an additional 16 h of incubation time, bacterial growth was assessed by optical density under absorbance mode at wavelength 600 nm. Absorbance readings from bacterial broth of the starting test tubes with immersed samples were also assessed at 24 h. Fresh broth alone was used as a blank for background subtraction. Data Analysis The amino acid profile of SF proteins was collected from 3 independent HPLC runs with fresh samples for each run. Images of AgNPs and electrospun SF fibers were analyzed by SemAfore software (JOEL, Sweden). About 100-150 AgNPs prepared from 3 different batches and 200-300 fibers from 3-5 different samples were measured from TEM and SEM images, respectively. For antibacterial evaluation, data for the disc diffusion assay were collected from 3 independent experiments and the growth-inhibition assay was performed in 3 replicates. The diameters of zones of inhibition were measured from the inhibited growth area surrounding discs of electrospun SF or commercial wound dressings in two orthogonal directions. Inhibition ratios were then calculated as the average diameters of zones of inhibition normalized to the disc diameters. Data were expressed as mean ± standard error of the mean unless stated otherwise.

Results and Discussion SF Protein Derived from Cocoons of the Thai Silkworms The amino acid profile and molecular weight of protein extracted from degummed cocoons of Thai silkworms B. mori (variant Nangnoi Si Sa Ket) was characterized for the first time in this study. Using HPLC, 14 amino acids (Table 1) were identified in the protein extracts with glycine (39.33± 1.51 %), alanine (29.26±2.78 %) and serine (13.06±0.88 %)

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Table 1. Amino acid profile of SF protein identified by HPLC. Data were collected from 3 independent experiments and presented as mean±standard deviation Amino acid Glycine Alanine Serine Tyrosine Valine Aspartic acid Phenylalanine Lysine Histidine Tryptophan Threonine Leucine Isoleucine Proline

Content (%) 39.33±1.51 29.26±2.78 13.06±0.88 9.45±0.20 1.92±0.32 1.45±0.46 1.02±0.09 0.76±0.32 0.66±0.24 0.64±0.13 0.48±0.14 0.46±0.07 0.45±0.12 0.40±0.05

Figure 2. Characterization of SF protein by gel electrophoresis. (A) A 6 % SDS-PAGE in Tris-HCL buffer shows the high molecular weight component of SF protein located above 238 kDa. Protein bands (left-right) correspond to 200, 150 and 100 µg SF, respectively. (B) A 8 % SDS-PAGE in Tris-HCL buffer shows the low molecular weight component of SF protein located between 20-25 kDa. Protein bands (left-right) correspond to 100, 75 and 50 µg SF, respectively. The protein molecular weight standards from the same gels are shown on the leftmost column in (A) and (B).

being the three major constituents. The relatively high amounts of glycine, alanine and serine are in agreement with previous reports [13,33,34] that analyzed the amino acid components of SF protein extracted from other variants of B. mori cocoons. These amino acids form the core repetitive sequences such as GAGAGA, GAGAGS and GAGAGY that are responsible for the formation of the β-sheets that make up the crystalline regions of heavy-chain fibroin [15,34]. Heavy-chain fibroin, light-chain fibroin and glycoprotein p25 [16] have been identified as the major components of the core SF protein. In this study, the molecular weight of the extracted proteins was estimated by SDS-PAGE. Using 6 % and 8 % acrylamide gels we identified two major bands of protein at above 238 kDa (Figure 2(A)) and between 20-25 kDa (Figure 2(B)), respectively. The molecular weights of


these bands are in accordance with previous observations of heavy-chain fibroin (ca. 220-500 kDa) [16] and light-chain fibroin (ca. 25 kDa) [15,16]. Thus, the protein extracted from cocoons of Thai silkworms B. mori (variant Nangnoi Si Sa Ket) in our study contains SF as its major component. Colloidal AgNPs Prepared by Gamma Irradiation A simple, one-step method to prepare AgNPs can be achieved by gamma [25,26] or UV [10,24] irradiation of silver compounds dissolved in aqueous solutions. For the former, the high energy gamma ray produces strongly reductive electrons from water molecules in the solution that reduce silver ions from their high valence state to the atomic metal [35]. Thus, nano-scale metallic silver in its Ag0 state can be obtained as the final product from the starting silver compound which is presented in its Ag+ state. In our study, colloidal AgNPs were prepared from AgNO3 by exposure to gamma ray irradiated from the Co-60 source. Prior to the irradiation procedure, PVA was added into AgNO3 solution as a stabilizer to prevent agglomeration of AgNPs and to disperse these particles as colloids. After irradiation for a total dose of 40 kGy, the UV absorbance peak showed a shift from 180-200 nm to 400-420 nm corresponding to the higher wavelength absorbance region of colloidal AgNPs [10,24-26]. In addition to characteristic UV absorbance, TEM images showed that colloidal AgNPs were also uniformly dispersed (Figure 3) as individual particles with round shape (Figure 3(B)) and an average size of 23.82±0.73 nm (Figure 3(B), inset). By contrast, the nonirradiated AgNO3 (Figure 3(A)) remained in aggregates. Therefore, gamma irradiation can be used as an effective synthesis method to generate dispersed, colloidal AgNPs from the starting silver compounds. Electrospun SF Mats For the fabrication of nonwoven SF mats, we first tested the effect of two common solvents, formic acid and HFP, as well as the concentration of SF on the formation of electrospun fibers (Figure 4). For formic acid-based solutions, irregularities such as beads and varicosities were observed

Figure 3. TEM images show (A) aggregates of silver in the starting AgNO3 solution and (B) dispersed, colloidal AgNPs after gamma irradiation. Inset in (B) shows individual AgNPs at 300,000X magnification.



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Figure 4. SEM images of fibers electrospun from SF solutions prepared in formic acid at (A) 15 %, (B) 20 % and (C) 25 % (w/v) and in HFP at (D) 5 %, (E) 10 % and (F) 18 % (w/v). Histogram in (F) shows the normal distribution of fiber diameter. Arrows in (B) and (E) indicate varicosities on the fibers. Scale bars in (C) and (F) are for all images on top and bottom panel, respectively.

on the electrospun SF fibers at the lower SF concentrations of 15 % and 20 % (w/v) (Figure 4(A) and 4(B)). By contrast, when the SF concentration was raised to 25 % (w/v) (Figure 4(C)), the fibers became smooth and continuous. Similar results were obtained when HFP was used as the solvent. Fibers electrospun from 5 % and 10 % (w/v) SF solutions contained beads and varicosities (Figure 4(D) and 4(E)), whereas these irregularities disappeared and the fibers became smooth and continuous when SF concentration increased to 18 % (w/v) (Figure 4(F)). Compared to formic acid, lower SF concentration in HFP solution was used to prepare smooth and continuous electrospun fibers. Thus, the SF concentration of 18 % (w/v) prepared in HFP solvent was selected to fabricate SF mats. The diameter of fibers electrospun from this 18 % (w/v) SF in HFP ranged from 100-1600 nm with peak frequencies occurring between 500800 nm (Figure 4(F)). The average diameter of these fibers was 670±12 nm. In conclusion, HFP was the optimal electrospinning solvent to produce SF nanofibers. A total volume of 2.5 ml fibroin solution resulted in electrospun SF mats with thickness ~200 µm. These thick SF mats were used previously in our laboratory in the study of biocompatibility in rat model. Methanol-Treated SF Mats To maintain the structural stability of electrospun SF mats in aqueous environment, the SF mats were treated in methanol to induce a conversion within SF’s secondary structure from random coils to β-sheets. The methanoltreated mats were shown to be stable in water whereas nontreated mats rapidly degraded upon contact with water. After treatment in 90 % methanol for 2 h, FTIR spectra (Figure 5) showed shifts in N-H bending vibration intensity from 1652 to 1628 cm-1 (amide I), 1543 to 1530 cm-1 (amide II) and additional shoulder at 1266 cm-1 (amide III). These shifts in

Figure 5. FTIR spectra of SF mats (a) before and (b) after methanol treatment. Methanol-treated SF mats show shifts in N-H bending vibration at 1628 and 1530 cm-1, which correspond to regions of amide I and amide II, respectively. Additional shoulder is observed at 1266 cm-1 at the spectral region of amide III.

N-H bending vibration correspond to the presence of βsheets in the methanol-treated SF mats [6,36,37]. The methanol-treated SF mats can be stored in phosphate buffer saline or 70 % ethanol for at least one month without being degraded. AgNP-Coated SF Mats As a prototypic antibacterial SF wound dressing, colloidal AgNPs prepared by gamma irradiation were subsequently used to coat SF mats. The presence of AgNPs on SF fibers was confirmed by SEM. Figure 6(A)-6(C) shows representative SEM images of AgNPs that were uniformly distributed on individual electrospun fibers after the SF mats were coated with AgNP solution at concentrations 1, 2 and 4 mM. Arrows indicate individual AgNPs, which appeared as dots, on the fibers. By way of comparison, the fibrous matrices of

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Figure 6. SEM images show the distribution of AgNPs on SF mats at AgNP coating concentrations (A) 1 mM, (B) 2 mM and (C) 4 mM and ionic silver in commercial wound dressings (D) Tegaderm Ag and (E) Aquacel Ag. Arrows indicate AgNPs and ionic silver on the fibers.

Figure 7. Antibacterial property of AgNP-coated SF mats against S. aureus (A-D) and P. aeruginosa (E-H). AgNP concentrations were 0 mM (A and E), 1 mM (B and F), 2 mM (C and G) and 4 mM (D and H). Lower panels show growth curves for both strains of bacteria following exposure to AgNP-coated SF mats and commercial wound dressings Tegaderm Ag and Aquacel Ag. Data are means ± standard error of the mean from 3 replicates.

two commercial wound dressings, Tegaderm Ag and Aquacel Ag, were also shown to be uniformly coated with ionic silver (Figure 6(D) and 6(E)). Therefore, the coating procedure used in this study provides an even distribution of antibacterial agents on SF mats that is comparable to commercial wound dressings.

Antibacterial Properties of AgNP-coated SF Mats AgNPs bind to bacterial cell walls and also penetrate inside the bacterial cells, which disrupts the functions of AgNP-bound organelles and proteins [20,23,38]. The binding of AgNPs to bacterial cell walls forces the membrane morphology to become irregular, disrupting membrane



transport and subsequently causing cell death [23]. As silver tends to have high affinity for phosphorous and sulfur compounds [20], internalized AgNPs can bind to and interrupt the functions of proteins and DNA, which contain significant amounts of sulfur and phosphorous, respectively. The antibacterial properties of AgNP-coated SF mats were evaluated by disc diffusion assay, which is a conventional method to test the inhibitory effect of antibacterial agents [39-41]. In our study we used this assay to evaluate the antibacterial activity of SF mats coated with AgNPs at several of different concentrations. SF mats coated with AgNP solutions ≥1 mM produced clear zones of inhibition on agar plates inoculated with either Gram-positive (S. aureus, Figure 7(B)-7(D)) or Gram-negative (P. aeruginosa, Figure 7(F)-7(H)) bacteria. This result was due to AgNPs because the uncoated SF mats (Figure 7(A) and 7(E)) or mats coated with AgNP solution at concentrations less than 1 mM did not produce any observable inhibition of bacterial growth. To determine the effect of AgNP release, we assessed the growth of bacteria in suspension culture following exposure to AgNP-coated SF mats. We found that at 4 mM AgNP concentration, the bacterial growth decreased with increasing exposure time. Similar results were also obtained for the commercial wound dressings. The assay, however, was not sensitive enough to detect a similar effect at lower AgNP coating concentrations. Such differences in sensitivity among various assays used to determine the antibacterial activity of silver were also reported in previous studies [42,43]. Taken together, these data show that antibacterial properties can be conferred to SF-mats by coating them with AgNPs. When incorporated into SF mats, metallic silver in the form of AgNPs exhibited significant antibacterial activity at very low concentrations relative to ionic silver used in two commercial wound dressings. To quantify this effect, we calculated the inhibition ratio (Table 2) using the disc diffusion data. On AgNP-coated SF mats, the maximum antibacterial efficacy for S. aureus was achieved at AgNP Table 2. Inhibition ratios of AgNP-coated SF mats, Tegaderm Ag and Aquacel Ag on S. aureus- and P. aeruginosa-inoculated plates. The inhibition ratio was calculated as the diameter of the zone of inhibition normalized to the mat diameter, which was measured from 3 independent disc diffusion assays, and presented as mean± standard deviation Inhibition ratio S. aureus P. aeruginosa AgNP concentration 4 mM 2 mM 1 mM 0.4 mM 0.1 mM Tegaderm Ag Aquacel Ag

1.76±0.14 1.69±0.11 1.68±0.16 N/D N/D 1.82±0.02 2.57±0.02

1.31±0.02 1.26±0.02 1.09±0.03 N/D N/D 1.46±0.02 2.05±0.02

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concentration as low as 1 mM, which corresponded to an inhibition ratio of ~1.7. For P. aeruginosa, it required the concentration of AgNPs between 2 and 4 mM (Figure 7(G) and 7(H)) to obtain the maximum inhibition ratio of ~1.3. As the antibacterial activity was observed in both disc diffusion and growth-inhibition assays at AgNP concentration of 4 mM, we estimated the amount of adsorbed AgNPs on SF mats which was found to be 50.4 ng/cm2 based on the amount of AgNPs in the 10-µl drop that was used to coat the 8-mmdiameter mats. As a comparison, the commercial wound dressings Tegaderm Ag and Aquacel Ag achieved inhibition ratios of 1.8 and 2.6, respectively, against S. aureus and 1.5 and 2.0, respectively, against P. aeruginosa. The higher inhibition ratios of the commercial wound dressings can be attributed to the much higher concentration of ionic silver contained in the wound dressing matrices. Tegaderm Ag and Aquacel Ag dressings contain silver sulfate and silver ions at 8 mg per gram of dressing and 1.2 % (data provided by manufacturers), respectively, which are equivalent to 8,000 ppm and 12,000 ppm of silver. By contrast, the effective concentration of AgNP solution at 4 mM in inhibiting bacterial growth is estimated to contain only ~432 ppm of AgNPs. Therefore, these results demonstrated that the SF mats coated with low concentrations of AgNPs could exhibit antibacterial property similar to commercial wound dressings embedded with high concentrations of ionic silver.

Conclusion The manufacture and evaluation of silk-based wound dressings in this study has shown that the incorporation of AgNPs at low concentrations on electrospun SF mats could confer significant antibacterial activity against S. aureus and P. aeruginosa. In the fabrication processes, the electrospinning parameters to generate nonwoven SF mats were established and gamma irradiation method has shown to be effective in preparing dispersed, colloidal AgNPs. All these results demonstrated that AgNP-coated SF mats can be fabricated as a prototypic wound dressing with antibacterial properties.

Acknowledgements This research was funded by the National Metal and Materials Technology Center of Thailand, the Thailand Research Fund and the Office of the National Research Council of Thailand. The authors also thanked Ms. Nipaporn Yongprapankhun at the Central Laboratory Thailand for her help on analyzing the amino acid profile of silk protein.

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