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Ag nanoparticle based anti-bacterial mats for biomedical applications
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JIEC-2533; No. of Pages 7 Journal of Industrial and Engineering Chemistry xxx (2015) xxx–xxx
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Electrospun badger (Meles meles) oil/Ag nanoparticle based anti-bacterial mats for biomedical applications Jun Hee Kim a, Afeesh Rajan Unnithan a,b, Han Joo Kim a, Arjun Prasad Tiwari a, Chan Hee Park a,b,*, Cheol Sang Kim a,b,c,* a b c
Department of Bio-nano System Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea Division of Mechanical Design Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea Eco-friendly Machine Parts Design Research Center, Chonbuk National University, Jeonju 561-756, Republic of Korea
A R T I C L E I N F O
Article history: Received 4 May 2015 Received in revised form 22 May 2015 Accepted 22 May 2015 Available online xxx Keywords: Nanofibers Nano-structures Particle-reinforcement Electrospinning Wound dressing
A B S T R A C T
In this study, silver nanoparticles and badger oil were embedded into nanofibrous polyurethane (PU) mats via electrospinning. Badger oil is mainly composed of fatty acids and is used as a traditional medicine to heal wounds and the silver nanoparticles contribute to the wound healing process by reducing wound contamination. The composite mats exhibited good bactericidal activity against both of Gram positive and Gram negative bacteria, with better cell attachment and proliferation. The results of this study indicate that the proposed composite mats can be used for various biomedical purposes, including as dressings for burn wounds or to treat skin disease. ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Introduction Recently, tissue engineering has been extensively studied for its potential use in wound care management [1]. In the case of serious injuries, a considerable amount of skin must be removed from the site of the injury in order to protect the wound. The main purpose of wound dressings is to remain close to native skin, inhibit the loss of blood, assist in removing exudates, block external microorganisms from creating an infection, and advancing the appearance of the wound site [2,3]. Previously, wound dressings were intended only to wrap around the wound site to protect the skin from infection. However, at the present time, wound dressings are being developed to provide a diversity of advanced functions to improve the wound healing process. To this end, multifunctional wound dressing materials and methods have been investigated, and biological wound dressing materials have been produced from natural, biodegradable, and biocompatible polymers containing extracts from natural substances [4].
* Corresponding authors at: Department of Bionanosystem Engineering, Graduate school, Chonbuk National University, Jeonju 561-756, Republic of Korea. Tel.: +82 63 270 4284; fax: +82 63 270 2460. E-mail addresses: [email protected] (C.H. Park), [email protected] (C.S. Kim).
The use of natural extracts in wound dressings has gained much attention due to the biocompatible nature of such materials since doing so can avoid inducing side effects in surrounding tissues [5,6]. Traditional, alternative medicines have been used for a long time to assist in wound healing and as additives for burn dressings [7,8]. Natural extract based materials have been developed and used as traditional wound healing agents for hundreds of years [9–11]. The badger (Meles meles) in the family Mustelidae is indigenous to Asia and Europe, and it is omnivorous and has a fat body. In Ancient China, badger fat was used to treat wounds [11], and in Korea it was used as a burn-healing agent. Badger oil is largely composed of fatty acid (FA) from lipids isolated from depots of adipose tissues. The composition of the FAs exhibits monounsaturated fatty acids (41.25% of the total FAs on average), saturated fatty acids (33.53%), and polyunsaturated fatty acids (17.15%). The major component of the FAs is oleic acid (30.43%) with palmitic acid (20.51%), linoleic acid (10.62%), stearic acid (8.82%), and palmitoleic acid (7.04%) also present [12]. Badger oil naturally contains high levels of oleic acid, which has antibacterial properties [13]. Ag nanoparticles (AgNPs) are well-known antibiotic agents with strong antibacterial properties. The membrane structure of bacteria can be damaged by free radicals on the surface of the AgNPs [14]. In addition, AgNPs effect no harm on viable cells and do not easily raise microbial resistance [15]. Therefore, AgNPs can be
http://dx.doi.org/10.1016/j.jiec.2015.05.030 1226-086X/ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
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used in various biomedical products to improve antibacterial activity [16–19]. Electrospinning is a simple and versatile technique to produce nanofibrous mats with individual fiber diameters ranging from a few nanometers to hundreds of nanometers [20]. Nanofibers produced via electrospinning possess elevated porosity, length-todiameter ratio, and specific surface area [21]. These properties improve cell respiration, skin regeneration, hemostasis and also prevent infections [22]. In addition, therapeutic agents can be incorporated and functionalized electrospun mats can be used as effective, stable wound dressings. In this study, we demonstrate the fabrication of wound dressings that contain badger oil as a healing agent, AgNPs as an antibacterial agent, and polyurethane as the foundation polymer. The composite mats were produced via electrospinning, and we demonstrate the process, stability, characterization and the biological properties of the nanofibrous mats loaded badger oil/ AgNPs.
Experimental details Materials Polyurethane (PU, Estane1Skythane1 X595A-11, Lubrizol Advanced Materials, Inc, USA) and badger oil (Yeongcheon Badger Farms, Korea) were mixed into a solution, and the AgNPs were produced from silver nitrate (AgNO3, Showa, Japan). N,N dimethyl formamide (DMF, Showa, Japan), methyl ethyl ketone(2-buthanone) (MEK, Junsei, Japan), and a mixed solvent were used to create the composite polymer solution. PU (10 wt%) was dissolved in DMF/MEK (50:50 by weight), and a 10 wt% PU solution was prepared with a magnetic stirrer with badger oil added at a 5 wt% concentration. The PU/badger oil solution was stirred for 4 h with a magnetic stirrer. The AgNPs were produced from silver nitrate (3 wt%) and were added to the badger oil/PU solution by stirring for 12 h with a magnetic stirrer before electrospinning. The solution was placed in a syringe tube and was injected into a nozzle with a 1/4-in. diameter. A high-voltage power supply (CPS-60 K02V1, Chungpa EMT, Korea) was used to supply 20 kV of power to the nozzle tip. The working distance between the tip of the nozzle and the collector was of 15 cm. After electrospinning, the nanofibrous mats were vacuum dried for 24 h to remove the solvent, and the samples were used for further analyses. Characterization The surface morphology of the electrospun mats was observed via scanning electron microscopy (SEM, S-7400, Hitach, Japan), field emission spectroscopy (FE-SEM, S-7400, Hitach, Japan), and Bio-transmission microscopy (Bio-TEM, Hitachi, Japan). The distribution of the fiber diameters was investigated using the Image J software (NIH, USA). An average of 100 nanofiber diameters was measured from the SEM images. Dark-field TEM images and TEM-EDX (JEM-2200, JEOL, Japan) were used to observe the particle deposition. The interaction between the PU and the badger oil was analyzed via Fourier-transform infrared (FT-IR) spectroscopy, and the mechanical properties were measured with a universal testing machine (AG-5000G, Shimadzu, Japan) under a crosshead speed of 10 mm/min producing a dumbbell shape according to the ASTM 638 standard by die cutting the mat in the direction of the machine. A contact angle meter (GBX, Digidrop, France) was used to measure the contact angle of deionized water with the mats in order to determine the wettability.
Antibacterial assessment Microbial strains and culturing conditions With respect to bacterial inactivation, Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) were precultured in Luria-Bertani (LB) containing tryptone, yeast extract, sodium chloride, and distilled water at an exact ratio, and before placing the bacteria, the LB was sterilized with an autoclave for 20 min at 121 8C with 15 psi. One colony of each bacterium was taken out from the original stock in an agar plate and was cultured in LB at 500 ml. The bacterial solution was incubated in a shaking incubator at 37 8C with a setting of 200 rpm for 24 h. Antibacterial activity measurement The bacterial solution was prepared by adding 100 ml of inoculated LB to 50 ml of sterilized distilled water in a beaker. The electrospun mats were cut to a size of 4 cm 4 cm and were placed in a Petri dish. A 10-ml E. coli solution (initial concentration = 3 106 CFU/ml; CFU: colony forming unit) and a 10-ml S. aureus solution (initial concentration = 3 106 CFU/ml) were placed over each mat. At given time intervals until 180 min, 100 ml suspensions were collected and diluted by several decimal dilutions in distilled water. Sampling was performed in triplicate and the average was calculated together with the standard deviation. The bacterial concentration was counted with readyto-use petrifilm (3 M Petrifilm, USA) and prepared agar plates. After 24 h of incubation, the number of bacteria was manually counted with a colony counter, and the bacterial zone of inhibition was measured using the disc diffusion method. Cell attachment assay The electrospun scaffolds on the cover slips were sterilized overnight under ultraviolet light. Then, the scaffolds were rinsed with phosphate buffer saline (pH 7.4), placed in a 48-well plate containing culture medium, and incubated overnight in a humidified atmosphere at 37 8C prior to cell seeding. The cell attachment was observed on days 1, 3 and 6. To this end, 200 ml of NIH-3T3 (fibroblasts) cell suspension (10,000 cells/well, DMEM/ high glucose supplemented with 10% FBS and 1% penicillinstreptomycin) were dispensed in a pre-incubated 48-well plate containing scaffolds and were allowed to incubate for an appropriate length of time at 37 8C under a 5% CO2 atmosphere. The medium was changed every 48 h. In order to examine the manner of the cell attachment on the composite nanofibers, chemical fixation was carried out for each sample. At days 1, 3 and 6 of incubation, the scaffolds were first rinsed twice with phosphate buffer saline (pH 7.4) and were subsequently fixed with 2.5% glutaraldehyde for 1 h. Then, the scaffolds were washed with 25%, 50%, 75%, and 100% ethanol for 10 min each. Finally, the samples were dried in a laminar flow hood overnight and were then sputter-coated with gold to investigate cell attachment via SEM. Cell proliferation assay The electrospun scaffolds were sterilized overnight on cover slips under ultraviolet light. Then, the scaffolds were rinsed with phosphate buffer saline (pH 7.4), placed in a 48-well plate containing culture medium, and incubated overnight in a humidified atmosphere at 37 8C prior to cell seeding. The cell proliferation was investigated at 24, 72, and 144 h using Dojindo’s cell counting kit-8. To this end, 200 ml of NIH-3T3 (fibrioblasts) cell suspension (10,000 cells/well, DMEM/high glucose supplemented with 10% FBS and 1% penicillin-streptomycin) were dispensed in a
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pre-incubated 48-well plate containing scaffolds and were allowed to incubate for an appropriate length of time at 37 8C under a 5% CO2 atmosphere. The medium was changed every 48 h. The cell proliferation on the scaffolds was checked at specific times (24, 72 and 144 h) with 20 ml of CCK-8 solution added to each well and incubated for a further 3 h, as mentioned above for the cell culture condition. In the next step, 100 ml of medium from each well was transferred to a 96-well plate and the absorbance at 450 nm was measured using an iMarkTM Microplate reader. The cell proliferation was expressed as a percentage of that of control (untreated) cells. Results and discussion Morphological structure studies The morphology of the nanofiber membranes is shown in Fig. 1. SEM images of pure PU nanofibers, badger oil/PU, and badger oil/Ag–PU nanocomposites are shown in Fig. 1a, c and f, respectively. A smooth nanofiber surface and randomly oriented nanofibers with almost uniform diameters can be observed in Fig. 1. The distribution of the nanofiber diameters is shown in Fig. 1b, d and g, respectively, through a histogram. The average fiber diameter of pristine PU
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(Fig. 1b) is of about 518.64 253.43 nm. Fig. 1d shows PU with 5% badger oil, which reduces the average diameter of the composite mats to 427.38 204.47 nm. The average fiber diameter of badger oil/Ag/PU mats is of about 375.29 140.21 nm, as shown in Fig. 1g. The pristine PU electrospun mats (Fig. 1a) had fibers that didn’t show any kind of interconnection among the fibers. However, the PU mats with badger oil showed some changes in the fibrous morphology (Fig. 1c and f). Here the addition of badger oil resulted in a number of connections formed between the fibers. We observed that a point-bonded fiber structure was generated by the badger oil during electrospinning. Fig. 1a clearly shows that pristine PU mats have non-bonded fibers whereas an increasing amount of badger oil leads to a transformation in the non-bonded fibers to point-bonded fibers. Fig. 2 shows FE-SEM images of badger oil/Ag/PU mats more clearly. We find that some tiny spider net-like nano-structures formed between the fibers. These nano-nets could not be seen in pure PU or in badger oil-loaded PU nanofibers. Therefore, the inclusion of AgNPs led to the formation of spider net-like structures in the badger oil/Ag/PU mats. The point-bonded structure of the fibers can be explained to be a result of the badger oil components. The majority of components in badger oil are fatty acids (FA). FAs are made of chains of carbon atoms connected to hydrogen atoms (Fig. 3a). The number of carbon atoms varies in each molecule, and at one end of the carbon
Fig. 1. SEM images of electrospun (a) PU, (c) badger oil/PU, (f) badger oil/Ag/PU nanofibrous mats. Histogram of diameter distribution for (b) PU, (d) badger oil/PU, (g) badger oil/Ag/PU nanofibrous mat.
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Fig. 2. FE-SEM images showing nano-nets of badger oil/Ag/PU (a and b) nanofibrous mats. Diameter distributions of fibers in the synthesized nano-nets in badger oil/Ag/PU nanofibrous mats are also shown.
Fig. 3. Schematic of (a) hydrogen bond formation between PU and FA and (b) the corresponding behavior of silver and nitrate ions in solution during electrospinning.
chain is a carboxyl group (-COOH) and, a methyl group (–CH3; Fig. 3a) is at the other end. Saturated FAs are all connected with single C–C bonds, but unsaturated FAs have one or more C5 5C double bond in the carbon chain (Fig. 3a) [13]. The point-bonded structure was a result of hydrogen bonding between the PU and the FAs (Fig. 3a). The spider net-like structures can be explained to be a result of the inclusion of AgNPs. When making AgNPs, we added silver nitrate to the PU solution. The silver nitrate dissolved into silver ions (Ag+) and nitrate ions (NO3 ). And Ag+ reacts with DMF
as a solvent in the PU solution to form Ag NPs through a reduction reaction [23,24]. However, unreacted Ag+ and NO3 randomly disperse in the solution and are electrospun with the polymer solution. Therefore, the differently-charged electric poles in the nanofibers led to the formation of a joint bond between each of two differently-charged poles [25], as shown in Fig. 3b. Fig. 4 shows the Ag NPs in the badger oil/Ag–PU nanofibrous mat. The Ag NPs had an average diameter of almost 15 nm and were found both in and outside the nanofibers. This indicated that
Fig. 4. Bio-TEM (a), dark-field TEM image and TEM-EDX of badger oil/Ag/PU mats.
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Fig. 5. FT-IR spectra of electrospun (a) PU nanofibers, (b) badger oil/PU, (c) badger oil/Ag/PU mats.
sufficient Ag NPs had formed and were well distributed in the fiber. Fig. 4a and b clearly show tiny Ag NPs that are well-dispersed and spherical NPs that are effectively encapsulated in the nanofiber. We used TEM-EDX to confirm the presence of the Ag particles in the fibers. Fig. 4c shows the elemental composition of the composite fibers, which confirms Ag NPs were incorporated throughout.
Fig. 6. Mechanical strength of (a) PU nanofibers, (b) badger oil/PU, (c) badger oil/Ag/ PU nanofibrous mats.
The interaction of the badger oil and the PU was investigated via FT-IR spectroscopy. Fig. 5 shows the specific transmittance peaks of pure PU, badger oil/PU, and badger oil/Ag/PU composites. PU has a specific absorption bands at 3320 cm 1(N–H), 2960 cm 1(C–H),
Fig. 7. Antibacterial efficiency of different mats in (a) E. coli and (c) S. aureus, and zone of inhibition tests for (b) E. coli and (d) S. aureus. Pure PU, badger oil/PU, and badger oil/ Ag/PU discs are denoted as A, B and C, respectively, in the petri plates (arrow denotes the zone of inhibition).
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1710 cm 1(C–O), 1530 cm 1(C–C), 1220 cm 1(C–C), 1110 cm 1(C– O) and 777 cm 1(C–H) on substituted benzene [26]. The spectrum for badger oil/PU and badger oil/Ag/PU composites indicated additional absorption bands at around 2800 to 3000 cm 1 due to the FA from the badger oil. Since FAs are the main component of badger oil, which mainly consists of C–H and C–C segments [13] (Fig. 3a), bands at 2960 cm 1 and 1530 cm 1 are visible. We measured the mechanical strength of each mat, and Fig. 6 clearly shows that the badger oil/Ag/PU mat has the highest mechanical strength of all samples. The high mechanical strength of the badger oil/PU and badger oil/Ag–PU nanofibrous mats is associated with the interconnection due to point-bonding formed by hydrogen bonds. In addition, the spider web-like structures in the badger oil/Ag/PU samples contributes to these showing the highest mechanical strength. Antibacterial test Bacterial inactivation was evaluated for the nanofibrous mats. A decrease in the numbers of bacterial (E. coli and S. aureus) colonies indicates good antibacterial activity. Fig. 7a shows the results from antibacterial tests for E. coli (Gram-negative) at an initial concentration of 3 106 CFU/ml. The control test using only bacterial solution without nanocomposite mats shows an almost constant concentration of E. coli after 180 min. Meanwhile, pure PU and badger oil/PU nanofibrous mats also exhibited a similar concentration as control, signifying that pure PU mats could not inactivate E. coli. The results for the badger oil/Ag/PU mats show a much higher antibacterial efficiency since the AgNPs play an important role as antibacterial agents. Fig. 7c shows the test results for S. aureus (Gram-positive) at an initial concentration of 3 106 CFU/ml. The control remained with the same condition, as mentioned above, and the concentration of the bacterial colonies is quite similar to that for E. coli, indicating that badger oil/Ag/PU mats had good antibacterial properties. We also measured the zone of inhibition around the nanofibers in the case where a clear area of inhibition had developed. The diameter of the inhibition zone depends on the antibacterial efficiency.
Circular nanofiber mats (d = 5 mm) were placed in an agar plate inoculated with bacteria and were incubated for 24 h. Fig. 7b and d clearly shows that pure PU and badger oil/PU mats have no zone of inhibition. On the other hand, the badger oil/Ag/PU mats showed a clear zone of inhibition. The zones of inhibition had diameters of 11 mm for E. coli and 12 mm for S. aureus, respectively. It has been suggested that Ag ions inhibit DNA replication and obstruct the expression of ribosomal proteins and enzymes for ATP hydrolysis [27]. These results indicate that badger oil/Ag/PU mats have good antibacterial properties, so these materials can be applied in cosmetics and pharmaceutical products. Cell study The cell viability was proven by observing the morphological appearance of cells on the hybrid mats after 3 and 6 days. Fig. 8 shows SEM images of cell attachment on PU, badger oil/PU and badger oil/Ag/PU mats. After 3 days of incubation, cells cultured on badger oil and Ag NPs contained nanofibers and had already spread (Fig. 8d and g) while pristine PU nanofibers exhibited little cell spreading (Fig. 8a). After 6 days, the badger oil/PU and badger oil/Ag/PU nanofiber matrix were completely covered with cells (Fig. 8e and h), and the badger oil/Ag/PU nanofibers showed the highest cell attachment and infiltration. Hydrophilicity is an important property for wound dressing applications since it can seriously influence biocompatibility in terms of cell adhesion and proliferation. The contact angle measurements are shown in Fig. 8c, f, and i. Pure PU nanofiber mats were hydrophobic, with a contact angle of around 129.83 0.658. The addition of badger oil to PU resulted in reduction in the contact angle to around 120.0 0.148, and badger oil/Ag/PU mats became hydrophilic, with a contact angle around 93.3 0.128. This result confirmed the hydrophilic behaviour of badger oil/Ag–PU mats, which can enhance cell attachment behavior during wound healing. Fibroblast proliferation on pure PU, badger oil/PU, and badger oil/Ag–PU matrices was evaluated via CCK assay on days 1, 3 and 6. Fig. 9 shows that fibroblasts cultured with badger oil/PU and
Fig. 8. SEM images showing cell attachment (3T3-L1 fibroblasts) on PU (a and b), badger oil/PU (d and e), badger oil/Ag/PU (g and h) after day 3 and day 6, respectively, and water contact angles for electrospun nanofibers from (c) PU, (f) badger oil/PU and (i) badger oil/Ag/PU mats.
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Fig. 9. CCK cell growth measurement assay.
badger oil/Ag/PU composites exhibited more cell growth than control and PU. These results indicate that the prepared nanofibrous mats were non-toxic to the cells. Moreover, the addition of badger oil and Ag NPs improves cell growth, which can be useful for wound dressing applications. Conclusions In summary, we have successfully obtained badger oil/Ag/PU blended composite nanofibers via electrospinning. Pure badger oil cannot yield continuous and uniform nanofibers, and thus, a synthetic polymer, such as PU, has to be blended with the badger oil solution to improve spinnability. Ag NPs can be introduced in the solution to improve the antibacterial properties of the composite mats, and the interaction between PU and badger oil through hydrogen bonds with added silver ions produced spider web-like nano-structures. Furthermore, the badger oil/Ag/PU nanofibers exhibited significantly improved antibacterial properties. The improvement in the cytocompatibility indicates that badger oil/Ag/ PU nanofibers are preferable for use in tissue engineering and in other cosmetic and therapeutic application, such as for wound dressings. Acknowledgments This study was supported by 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 no. 2013-012911 and 2013R1A2A2A04015484). References [1] R.A. Kamel, J.F. Ong, E. Eriksson, J.P.E. Junker, E.J. Caterson, J. Am. Coll. Surg. 217 (2013) 533.
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JIEC-2533; No. of Pages 7 Journal of Industrial and Engineering Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
Electrospun badger (Meles meles) oil/Ag nanoparticle based anti-bacterial mats for biomedical applications Jun Hee Kim a, Afeesh Rajan Unnithan a,b, Han Joo Kim a, Arjun Prasad Tiwari a, Chan Hee Park a,b,*, Cheol Sang Kim a,b,c,* a b c
Department of Bio-nano System Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea Division of Mechanical Design Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea Eco-friendly Machine Parts Design Research Center, Chonbuk National University, Jeonju 561-756, Republic of Korea
A R T I C L E I N F O
Article history: Received 4 May 2015 Received in revised form 22 May 2015 Accepted 22 May 2015 Available online xxx Keywords: Nanofibers Nano-structures Particle-reinforcement Electrospinning Wound dressing
A B S T R A C T
In this study, silver nanoparticles and badger oil were embedded into nanofibrous polyurethane (PU) mats via electrospinning. Badger oil is mainly composed of fatty acids and is used as a traditional medicine to heal wounds and the silver nanoparticles contribute to the wound healing process by reducing wound contamination. The composite mats exhibited good bactericidal activity against both of Gram positive and Gram negative bacteria, with better cell attachment and proliferation. The results of this study indicate that the proposed composite mats can be used for various biomedical purposes, including as dressings for burn wounds or to treat skin disease. ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Introduction Recently, tissue engineering has been extensively studied for its potential use in wound care management [1]. In the case of serious injuries, a considerable amount of skin must be removed from the site of the injury in order to protect the wound. The main purpose of wound dressings is to remain close to native skin, inhibit the loss of blood, assist in removing exudates, block external microorganisms from creating an infection, and advancing the appearance of the wound site [2,3]. Previously, wound dressings were intended only to wrap around the wound site to protect the skin from infection. However, at the present time, wound dressings are being developed to provide a diversity of advanced functions to improve the wound healing process. To this end, multifunctional wound dressing materials and methods have been investigated, and biological wound dressing materials have been produced from natural, biodegradable, and biocompatible polymers containing extracts from natural substances [4].
* Corresponding authors at: Department of Bionanosystem Engineering, Graduate school, Chonbuk National University, Jeonju 561-756, Republic of Korea. Tel.: +82 63 270 4284; fax: +82 63 270 2460. E-mail addresses: [email protected] (C.H. Park), [email protected] (C.S. Kim).
The use of natural extracts in wound dressings has gained much attention due to the biocompatible nature of such materials since doing so can avoid inducing side effects in surrounding tissues [5,6]. Traditional, alternative medicines have been used for a long time to assist in wound healing and as additives for burn dressings [7,8]. Natural extract based materials have been developed and used as traditional wound healing agents for hundreds of years [9–11]. The badger (Meles meles) in the family Mustelidae is indigenous to Asia and Europe, and it is omnivorous and has a fat body. In Ancient China, badger fat was used to treat wounds [11], and in Korea it was used as a burn-healing agent. Badger oil is largely composed of fatty acid (FA) from lipids isolated from depots of adipose tissues. The composition of the FAs exhibits monounsaturated fatty acids (41.25% of the total FAs on average), saturated fatty acids (33.53%), and polyunsaturated fatty acids (17.15%). The major component of the FAs is oleic acid (30.43%) with palmitic acid (20.51%), linoleic acid (10.62%), stearic acid (8.82%), and palmitoleic acid (7.04%) also present [12]. Badger oil naturally contains high levels of oleic acid, which has antibacterial properties [13]. Ag nanoparticles (AgNPs) are well-known antibiotic agents with strong antibacterial properties. The membrane structure of bacteria can be damaged by free radicals on the surface of the AgNPs [14]. In addition, AgNPs effect no harm on viable cells and do not easily raise microbial resistance [15]. Therefore, AgNPs can be
http://dx.doi.org/10.1016/j.jiec.2015.05.030 1226-086X/ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
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used in various biomedical products to improve antibacterial activity [16–19]. Electrospinning is a simple and versatile technique to produce nanofibrous mats with individual fiber diameters ranging from a few nanometers to hundreds of nanometers [20]. Nanofibers produced via electrospinning possess elevated porosity, length-todiameter ratio, and specific surface area [21]. These properties improve cell respiration, skin regeneration, hemostasis and also prevent infections [22]. In addition, therapeutic agents can be incorporated and functionalized electrospun mats can be used as effective, stable wound dressings. In this study, we demonstrate the fabrication of wound dressings that contain badger oil as a healing agent, AgNPs as an antibacterial agent, and polyurethane as the foundation polymer. The composite mats were produced via electrospinning, and we demonstrate the process, stability, characterization and the biological properties of the nanofibrous mats loaded badger oil/ AgNPs.
Experimental details Materials Polyurethane (PU, Estane1Skythane1 X595A-11, Lubrizol Advanced Materials, Inc, USA) and badger oil (Yeongcheon Badger Farms, Korea) were mixed into a solution, and the AgNPs were produced from silver nitrate (AgNO3, Showa, Japan). N,N dimethyl formamide (DMF, Showa, Japan), methyl ethyl ketone(2-buthanone) (MEK, Junsei, Japan), and a mixed solvent were used to create the composite polymer solution. PU (10 wt%) was dissolved in DMF/MEK (50:50 by weight), and a 10 wt% PU solution was prepared with a magnetic stirrer with badger oil added at a 5 wt% concentration. The PU/badger oil solution was stirred for 4 h with a magnetic stirrer. The AgNPs were produced from silver nitrate (3 wt%) and were added to the badger oil/PU solution by stirring for 12 h with a magnetic stirrer before electrospinning. The solution was placed in a syringe tube and was injected into a nozzle with a 1/4-in. diameter. A high-voltage power supply (CPS-60 K02V1, Chungpa EMT, Korea) was used to supply 20 kV of power to the nozzle tip. The working distance between the tip of the nozzle and the collector was of 15 cm. After electrospinning, the nanofibrous mats were vacuum dried for 24 h to remove the solvent, and the samples were used for further analyses. Characterization The surface morphology of the electrospun mats was observed via scanning electron microscopy (SEM, S-7400, Hitach, Japan), field emission spectroscopy (FE-SEM, S-7400, Hitach, Japan), and Bio-transmission microscopy (Bio-TEM, Hitachi, Japan). The distribution of the fiber diameters was investigated using the Image J software (NIH, USA). An average of 100 nanofiber diameters was measured from the SEM images. Dark-field TEM images and TEM-EDX (JEM-2200, JEOL, Japan) were used to observe the particle deposition. The interaction between the PU and the badger oil was analyzed via Fourier-transform infrared (FT-IR) spectroscopy, and the mechanical properties were measured with a universal testing machine (AG-5000G, Shimadzu, Japan) under a crosshead speed of 10 mm/min producing a dumbbell shape according to the ASTM 638 standard by die cutting the mat in the direction of the machine. A contact angle meter (GBX, Digidrop, France) was used to measure the contact angle of deionized water with the mats in order to determine the wettability.
Antibacterial assessment Microbial strains and culturing conditions With respect to bacterial inactivation, Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) were precultured in Luria-Bertani (LB) containing tryptone, yeast extract, sodium chloride, and distilled water at an exact ratio, and before placing the bacteria, the LB was sterilized with an autoclave for 20 min at 121 8C with 15 psi. One colony of each bacterium was taken out from the original stock in an agar plate and was cultured in LB at 500 ml. The bacterial solution was incubated in a shaking incubator at 37 8C with a setting of 200 rpm for 24 h. Antibacterial activity measurement The bacterial solution was prepared by adding 100 ml of inoculated LB to 50 ml of sterilized distilled water in a beaker. The electrospun mats were cut to a size of 4 cm 4 cm and were placed in a Petri dish. A 10-ml E. coli solution (initial concentration = 3 106 CFU/ml; CFU: colony forming unit) and a 10-ml S. aureus solution (initial concentration = 3 106 CFU/ml) were placed over each mat. At given time intervals until 180 min, 100 ml suspensions were collected and diluted by several decimal dilutions in distilled water. Sampling was performed in triplicate and the average was calculated together with the standard deviation. The bacterial concentration was counted with readyto-use petrifilm (3 M Petrifilm, USA) and prepared agar plates. After 24 h of incubation, the number of bacteria was manually counted with a colony counter, and the bacterial zone of inhibition was measured using the disc diffusion method. Cell attachment assay The electrospun scaffolds on the cover slips were sterilized overnight under ultraviolet light. Then, the scaffolds were rinsed with phosphate buffer saline (pH 7.4), placed in a 48-well plate containing culture medium, and incubated overnight in a humidified atmosphere at 37 8C prior to cell seeding. The cell attachment was observed on days 1, 3 and 6. To this end, 200 ml of NIH-3T3 (fibroblasts) cell suspension (10,000 cells/well, DMEM/ high glucose supplemented with 10% FBS and 1% penicillinstreptomycin) were dispensed in a pre-incubated 48-well plate containing scaffolds and were allowed to incubate for an appropriate length of time at 37 8C under a 5% CO2 atmosphere. The medium was changed every 48 h. In order to examine the manner of the cell attachment on the composite nanofibers, chemical fixation was carried out for each sample. At days 1, 3 and 6 of incubation, the scaffolds were first rinsed twice with phosphate buffer saline (pH 7.4) and were subsequently fixed with 2.5% glutaraldehyde for 1 h. Then, the scaffolds were washed with 25%, 50%, 75%, and 100% ethanol for 10 min each. Finally, the samples were dried in a laminar flow hood overnight and were then sputter-coated with gold to investigate cell attachment via SEM. Cell proliferation assay The electrospun scaffolds were sterilized overnight on cover slips under ultraviolet light. Then, the scaffolds were rinsed with phosphate buffer saline (pH 7.4), placed in a 48-well plate containing culture medium, and incubated overnight in a humidified atmosphere at 37 8C prior to cell seeding. The cell proliferation was investigated at 24, 72, and 144 h using Dojindo’s cell counting kit-8. To this end, 200 ml of NIH-3T3 (fibrioblasts) cell suspension (10,000 cells/well, DMEM/high glucose supplemented with 10% FBS and 1% penicillin-streptomycin) were dispensed in a
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pre-incubated 48-well plate containing scaffolds and were allowed to incubate for an appropriate length of time at 37 8C under a 5% CO2 atmosphere. The medium was changed every 48 h. The cell proliferation on the scaffolds was checked at specific times (24, 72 and 144 h) with 20 ml of CCK-8 solution added to each well and incubated for a further 3 h, as mentioned above for the cell culture condition. In the next step, 100 ml of medium from each well was transferred to a 96-well plate and the absorbance at 450 nm was measured using an iMarkTM Microplate reader. The cell proliferation was expressed as a percentage of that of control (untreated) cells. Results and discussion Morphological structure studies The morphology of the nanofiber membranes is shown in Fig. 1. SEM images of pure PU nanofibers, badger oil/PU, and badger oil/Ag–PU nanocomposites are shown in Fig. 1a, c and f, respectively. A smooth nanofiber surface and randomly oriented nanofibers with almost uniform diameters can be observed in Fig. 1. The distribution of the nanofiber diameters is shown in Fig. 1b, d and g, respectively, through a histogram. The average fiber diameter of pristine PU
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(Fig. 1b) is of about 518.64 253.43 nm. Fig. 1d shows PU with 5% badger oil, which reduces the average diameter of the composite mats to 427.38 204.47 nm. The average fiber diameter of badger oil/Ag/PU mats is of about 375.29 140.21 nm, as shown in Fig. 1g. The pristine PU electrospun mats (Fig. 1a) had fibers that didn’t show any kind of interconnection among the fibers. However, the PU mats with badger oil showed some changes in the fibrous morphology (Fig. 1c and f). Here the addition of badger oil resulted in a number of connections formed between the fibers. We observed that a point-bonded fiber structure was generated by the badger oil during electrospinning. Fig. 1a clearly shows that pristine PU mats have non-bonded fibers whereas an increasing amount of badger oil leads to a transformation in the non-bonded fibers to point-bonded fibers. Fig. 2 shows FE-SEM images of badger oil/Ag/PU mats more clearly. We find that some tiny spider net-like nano-structures formed between the fibers. These nano-nets could not be seen in pure PU or in badger oil-loaded PU nanofibers. Therefore, the inclusion of AgNPs led to the formation of spider net-like structures in the badger oil/Ag/PU mats. The point-bonded structure of the fibers can be explained to be a result of the badger oil components. The majority of components in badger oil are fatty acids (FA). FAs are made of chains of carbon atoms connected to hydrogen atoms (Fig. 3a). The number of carbon atoms varies in each molecule, and at one end of the carbon
Fig. 1. SEM images of electrospun (a) PU, (c) badger oil/PU, (f) badger oil/Ag/PU nanofibrous mats. Histogram of diameter distribution for (b) PU, (d) badger oil/PU, (g) badger oil/Ag/PU nanofibrous mat.
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Fig. 2. FE-SEM images showing nano-nets of badger oil/Ag/PU (a and b) nanofibrous mats. Diameter distributions of fibers in the synthesized nano-nets in badger oil/Ag/PU nanofibrous mats are also shown.
Fig. 3. Schematic of (a) hydrogen bond formation between PU and FA and (b) the corresponding behavior of silver and nitrate ions in solution during electrospinning.
chain is a carboxyl group (-COOH) and, a methyl group (–CH3; Fig. 3a) is at the other end. Saturated FAs are all connected with single C–C bonds, but unsaturated FAs have one or more C5 5C double bond in the carbon chain (Fig. 3a) [13]. The point-bonded structure was a result of hydrogen bonding between the PU and the FAs (Fig. 3a). The spider net-like structures can be explained to be a result of the inclusion of AgNPs. When making AgNPs, we added silver nitrate to the PU solution. The silver nitrate dissolved into silver ions (Ag+) and nitrate ions (NO3 ). And Ag+ reacts with DMF
as a solvent in the PU solution to form Ag NPs through a reduction reaction [23,24]. However, unreacted Ag+ and NO3 randomly disperse in the solution and are electrospun with the polymer solution. Therefore, the differently-charged electric poles in the nanofibers led to the formation of a joint bond between each of two differently-charged poles [25], as shown in Fig. 3b. Fig. 4 shows the Ag NPs in the badger oil/Ag–PU nanofibrous mat. The Ag NPs had an average diameter of almost 15 nm and were found both in and outside the nanofibers. This indicated that
Fig. 4. Bio-TEM (a), dark-field TEM image and TEM-EDX of badger oil/Ag/PU mats.
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Fig. 5. FT-IR spectra of electrospun (a) PU nanofibers, (b) badger oil/PU, (c) badger oil/Ag/PU mats.
sufficient Ag NPs had formed and were well distributed in the fiber. Fig. 4a and b clearly show tiny Ag NPs that are well-dispersed and spherical NPs that are effectively encapsulated in the nanofiber. We used TEM-EDX to confirm the presence of the Ag particles in the fibers. Fig. 4c shows the elemental composition of the composite fibers, which confirms Ag NPs were incorporated throughout.
Fig. 6. Mechanical strength of (a) PU nanofibers, (b) badger oil/PU, (c) badger oil/Ag/ PU nanofibrous mats.
The interaction of the badger oil and the PU was investigated via FT-IR spectroscopy. Fig. 5 shows the specific transmittance peaks of pure PU, badger oil/PU, and badger oil/Ag/PU composites. PU has a specific absorption bands at 3320 cm 1(N–H), 2960 cm 1(C–H),
Fig. 7. Antibacterial efficiency of different mats in (a) E. coli and (c) S. aureus, and zone of inhibition tests for (b) E. coli and (d) S. aureus. Pure PU, badger oil/PU, and badger oil/ Ag/PU discs are denoted as A, B and C, respectively, in the petri plates (arrow denotes the zone of inhibition).
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1710 cm 1(C–O), 1530 cm 1(C–C), 1220 cm 1(C–C), 1110 cm 1(C– O) and 777 cm 1(C–H) on substituted benzene [26]. The spectrum for badger oil/PU and badger oil/Ag/PU composites indicated additional absorption bands at around 2800 to 3000 cm 1 due to the FA from the badger oil. Since FAs are the main component of badger oil, which mainly consists of C–H and C–C segments [13] (Fig. 3a), bands at 2960 cm 1 and 1530 cm 1 are visible. We measured the mechanical strength of each mat, and Fig. 6 clearly shows that the badger oil/Ag/PU mat has the highest mechanical strength of all samples. The high mechanical strength of the badger oil/PU and badger oil/Ag–PU nanofibrous mats is associated with the interconnection due to point-bonding formed by hydrogen bonds. In addition, the spider web-like structures in the badger oil/Ag/PU samples contributes to these showing the highest mechanical strength. Antibacterial test Bacterial inactivation was evaluated for the nanofibrous mats. A decrease in the numbers of bacterial (E. coli and S. aureus) colonies indicates good antibacterial activity. Fig. 7a shows the results from antibacterial tests for E. coli (Gram-negative) at an initial concentration of 3 106 CFU/ml. The control test using only bacterial solution without nanocomposite mats shows an almost constant concentration of E. coli after 180 min. Meanwhile, pure PU and badger oil/PU nanofibrous mats also exhibited a similar concentration as control, signifying that pure PU mats could not inactivate E. coli. The results for the badger oil/Ag/PU mats show a much higher antibacterial efficiency since the AgNPs play an important role as antibacterial agents. Fig. 7c shows the test results for S. aureus (Gram-positive) at an initial concentration of 3 106 CFU/ml. The control remained with the same condition, as mentioned above, and the concentration of the bacterial colonies is quite similar to that for E. coli, indicating that badger oil/Ag/PU mats had good antibacterial properties. We also measured the zone of inhibition around the nanofibers in the case where a clear area of inhibition had developed. The diameter of the inhibition zone depends on the antibacterial efficiency.
Circular nanofiber mats (d = 5 mm) were placed in an agar plate inoculated with bacteria and were incubated for 24 h. Fig. 7b and d clearly shows that pure PU and badger oil/PU mats have no zone of inhibition. On the other hand, the badger oil/Ag/PU mats showed a clear zone of inhibition. The zones of inhibition had diameters of 11 mm for E. coli and 12 mm for S. aureus, respectively. It has been suggested that Ag ions inhibit DNA replication and obstruct the expression of ribosomal proteins and enzymes for ATP hydrolysis [27]. These results indicate that badger oil/Ag/PU mats have good antibacterial properties, so these materials can be applied in cosmetics and pharmaceutical products. Cell study The cell viability was proven by observing the morphological appearance of cells on the hybrid mats after 3 and 6 days. Fig. 8 shows SEM images of cell attachment on PU, badger oil/PU and badger oil/Ag/PU mats. After 3 days of incubation, cells cultured on badger oil and Ag NPs contained nanofibers and had already spread (Fig. 8d and g) while pristine PU nanofibers exhibited little cell spreading (Fig. 8a). After 6 days, the badger oil/PU and badger oil/Ag/PU nanofiber matrix were completely covered with cells (Fig. 8e and h), and the badger oil/Ag/PU nanofibers showed the highest cell attachment and infiltration. Hydrophilicity is an important property for wound dressing applications since it can seriously influence biocompatibility in terms of cell adhesion and proliferation. The contact angle measurements are shown in Fig. 8c, f, and i. Pure PU nanofiber mats were hydrophobic, with a contact angle of around 129.83 0.658. The addition of badger oil to PU resulted in reduction in the contact angle to around 120.0 0.148, and badger oil/Ag/PU mats became hydrophilic, with a contact angle around 93.3 0.128. This result confirmed the hydrophilic behaviour of badger oil/Ag–PU mats, which can enhance cell attachment behavior during wound healing. Fibroblast proliferation on pure PU, badger oil/PU, and badger oil/Ag–PU matrices was evaluated via CCK assay on days 1, 3 and 6. Fig. 9 shows that fibroblasts cultured with badger oil/PU and
Fig. 8. SEM images showing cell attachment (3T3-L1 fibroblasts) on PU (a and b), badger oil/PU (d and e), badger oil/Ag/PU (g and h) after day 3 and day 6, respectively, and water contact angles for electrospun nanofibers from (c) PU, (f) badger oil/PU and (i) badger oil/Ag/PU mats.
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Fig. 9. CCK cell growth measurement assay.
badger oil/Ag/PU composites exhibited more cell growth than control and PU. These results indicate that the prepared nanofibrous mats were non-toxic to the cells. Moreover, the addition of badger oil and Ag NPs improves cell growth, which can be useful for wound dressing applications. Conclusions In summary, we have successfully obtained badger oil/Ag/PU blended composite nanofibers via electrospinning. Pure badger oil cannot yield continuous and uniform nanofibers, and thus, a synthetic polymer, such as PU, has to be blended with the badger oil solution to improve spinnability. Ag NPs can be introduced in the solution to improve the antibacterial properties of the composite mats, and the interaction between PU and badger oil through hydrogen bonds with added silver ions produced spider web-like nano-structures. Furthermore, the badger oil/Ag/PU nanofibers exhibited significantly improved antibacterial properties. The improvement in the cytocompatibility indicates that badger oil/Ag/ PU nanofibers are preferable for use in tissue engineering and in other cosmetic and therapeutic application, such as for wound dressings. Acknowledgments This study was supported by 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 no. 2013-012911 and 2013R1A2A2A04015484). References [1] R.A. Kamel, J.F. Ong, E. Eriksson, J.P.E. Junker, E.J. Caterson, J. Am. Coll. Surg. 217 (2013) 533.
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