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    Materials science communication

    One-step fabrication of antibacterial (silver nanoparticles/poly(ethylene

    oxide)) e Polyurethane bicomponent hybrid nanofibrous mat by

    dual-spinneret electrospinning

    Leonard D. Tijing a,*, Michael Tom G. Ruelo a, Altangerel Amarjargal b,c, Hem Raj Pant b,d, Chan-Hee Park b,Cheol Sang Kim a,b,*

    a Division of Mechanical Design Engineering, Chonbuk National University, Jeonju, Jeonbuk 561-756, Republic of Koreab Department of Bionanosystem Engineering, Chonbuk National University, Jeonju, Jeonbuk 561-756, Republic of Koreac Power Engineering School, Mongolian University of Science and Technology, Ulaanbaatar, Mongolia

    d Department of Engineering Science and Humanities, Institute of Engineering, Pulchowk Campus, Tribhuvan University, Kathmandu, Nepal

    a r t i c l e i n f o

    Article history:

    Received 26 January 2012

    Received in revised form

    9 March 2012

    Accepted 22 March 2012

    Keywords:

    Composite materials

    Electrospinning

    Nanostructures

    Polymers

    a b s t r a c t

    The one-step electrospinning fabrication of novel materials with added functionalities is being widely

    studied because of their wide array of applications. Here, the fabrication of a hybrid, bimodal nanofibrous

    mat made of two polymeric nanofibers: polyurethane (PU) and silver (Ag) nanoparticle (NP) e in situ e

    decorated poly(ethylene oxide) (PEO) utilizing an angled dual-spinneret electrospinning system is

    reported. Silver nitrate (AgNO3) is in-situ reduced in high-molecular weight PEO, and Ag NPs with sizes

    from 6 to 90 nm as checked by scanning electron microscoy and transmission electron microscopy, are

    subsequently formed on the surface of PEO nanofibers depending on the reduction time. Successful

    fabrication of bicomponent polymer matrices (PU and PEO) in the hybrid mat is confirmed by Fourier

    transform infrared spectroscopy. Metallic Ag NPs are verified to be present in the hybrid mats by energy

    dispersive X-ray spectroscopy and ultraviolet-vis spectroscopy, showing plasmon resonance band peaks

    at 415 and 425 nm. The hybrid nanofibrous mat containing Ag NPs with an average size of 8 nm (i.e.,reduction time of 3 h) exhibits strong antibacterial activity.

    2012 Elsevier B.V. All rights reserved.

    1. Introduction

    The unique properties of non-woven polymer nanofibers fabri-

    cated by electrospinning have evoked significant interests in

    differentfields [1,2]. Among the current studies, the electrospinning

    fabrication of hybrid materialsby blending twoor more polymers or

    the incorporation of metal nanoparticles (NPs) in/on polymer

    nanofibers has gained vast attention [3e6]. Recently, multi-

    spinneret electrospinning presents itself as an excellent method tofabricate hybrid materials with improved properties by utilizing the

    different properties of each component polymer [7,8]. Many of the

    studies on multi-spinneret electrospinning focused on the effect of

    blended polymers on the improvement of hybrid materials overall

    properties [9e11]. To the authors best knowledge, no one has

    reported yet on the fabrication of hybrid nanofiber mat by dual-

    spinneret electrospinning composed of mixed matrices of silver

    (Ag) NP e decorated poly(ethylene oxide) (PEO) and polyurethane

    (PU) nanofibers.

    There are basically two ways to prepare metal NP-decorated

    polymer nanofibers: (a) by synthesis of NPs and then dispersion

    on nanofibers [12]; and (b) by in-situ reduction of metal NPs within

    or at the interface of nanofibers [1]. The former is a two-step

    process, and presents difficulty in achieving well-dispersed NPs.In this study, we present a one-step fabrication of hybrid nanofiber

    mat composed of two different polymeric nanofibers (PEO and PU),

    wherein only PEO is decorated with Ag NPs without using any

    chemicals, i.e. PEO is used as both the reducing agent of silver

    nitrate (AgNO3) and the templateof the formedAg NPs.PU isoneof

    the most used polymers in biomedical applications due to its good

    biocompatibility and excellent mechanical properties [13,14]. PEO

    is a non-toxic and biocompatible polymer that is widely used for

    tissue engineering, scaffolds, and wound dressing [15]. Silver NPs

    possess an inherent antibacterial activity against a number of pa-

    thogenic bacteria, have good biocompatibility [13], and are widely

    * Corresponding authors. Division of Mechanical Design Engineering, Chonbuk

    National University, Jeonju, Jeonbuk 561-756, Republic of Korea. Fax: 82

    632702460.

    E-mail addresses: [email protected] (L.D. Tijing), [email protected] (C.S. Kim).

    Contents lists available at SciVerse ScienceDirect

    Materials Chemistry and Physics

    j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m a t c h e m p h y s

    0254-0584/$ e see front matter 2012 Elsevier B.V. All rights reserved.

    doi:10.1016/j.matchemphys.2012.03.037

    Materials Chemistry and Physics 134 (2012) 557e561

    mailto:[email protected]:[email protected]://www.sciencedirect.com/science/journal/02540584http://www.elsevier.com/locate/matchemphyshttp://dx.doi.org/10.1016/j.matchemphys.2012.03.037http://dx.doi.org/10.1016/j.matchemphys.2012.03.037http://dx.doi.org/10.1016/j.matchemphys.2012.03.037http://dx.doi.org/10.1016/j.matchemphys.2012.03.037http://dx.doi.org/10.1016/j.matchemphys.2012.03.037http://dx.doi.org/10.1016/j.matchemphys.2012.03.037http://www.elsevier.com/locate/matchemphyshttp://www.sciencedirect.com/science/journal/02540584mailto:[email protected]:[email protected]
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    used as catalysts, and photosensitive components [16]. A recent

    review by Dallas et al. [17] discussed about the use of silver poly-

    meric nanocomposites as advanced antimicrobial agents.

    Here, the combination of PU and Ag/PEO nanofibers to make

    into a bicomponent and bimodal hybrid mat is potentially useful as

    antibacterial tissue scaffolds or for wound-healing applications.

    The bigger PU nanofibers give structural stability and prolonged use

    of the hybrid materials [18], while the presence of thinner PEO

    nanofibers provide more reactive surface area, and the decoration

    of Ag NPs on PEO surfaces without the use of chemicals gives

    antibacterial functionality to the hybrid mat.

    2. Experimental section

    In a typical experiment, two separate solutions were prepared

    for dual-spinneret electrospinning. In solution A, 3.3 wt% poly(-

    ethylene oxide) (PEO, molecular weight (MW) 60 kDa, Acros

    Organics) and 5 wt% silver nitrate (AgNO3, 99.8%, Showa) relative to

    the weight of PEO, were dissolved in distilled water (DW) in two

    separate glass containers. In one container, 2 g PEOwas dissolved in

    48 g DW, and in another container,100 mg AgNO3 was dissolved in

    10 g DW. After complete dissolution (around 4 h of magnetic stir-

    ring for PEO/DW solution), the two solutions were combined bymagnetic stirring in covered containers and kept away from light

    for the reduction of AgNO3 into Ag NPs.The ratio of AgNO3 toPEO in

    the mixed solution was 1:20. In Solution B, 10 wt% of polyurethane

    (PU, Estane Skythane X595A-11, Lubrizol) was dissolved over-

    night in solvent mixture of N,N dimethylformamide (DMF, Showa)

    and methyl ethyl ketone/2-butanone (MEK, extra pure, Junsei) (50/

    50, wt:wt%).

    Electrospinning was carried out using a multinozzle electro-

    spinning system with robot-controlled, movable and tunable spin-

    nerets made by ourlaboratory (Fig.1a). Solutions A (atreductiontime

    of 3 or 5.5 h) and B were each collected separately in 10 ml plastic

    syringe connected to two spinneret holders with 21G needle tip

    (inner diameter510 nm). The two spinnerets were oriented hori-

    zontally on one side of thecollectorat an angle of 80 between them.The two syringes werefixed in a syringe pump set at a constant feed

    rate of 1.0 mlh1. Each needle tip was connected to two separate

    power supplies with identical voltages of 15 kV. The grounded

    collector was made of aluminium plate covered with Teflon sheet,

    and the tip-to-collector distance was kept at 20 cm for all tests. For

    comparison, neat PU nanofiber was also prepared by conventional

    single nozzle electrospinning,using the sameconditionsabove.After

    electrospinning, the nanofibrous membrane was dried in a dry oven

    at 60 C for 48 h.

    The morphological structure of the samples was character-

    ized by field emission scanning electron microscopy (FESEM,

    Hitachi S-4800), and transmission electron microscopy (TEM, H-

    7650, Hitachi). The samples for FESEM were coated with a very

    thin layer of platinum before characterization. Samples for TEM

    were prepared by electrospinning directly on a copper grid mesh

    with carbon coating for 20e30 s. Elemental analysis was carried

    out by energy dispersive X-ray spectroscopy (EDS). The fiber

    diameter and nanoparticle size distribution were determined

    using ImageJ (NIH, USA) software, getting the average of 100

    measurements based from the FESEM images. Fourier transform

    infrared (FTIR) spectra of the samples were obtained using

    a Paragon 1000 Spectrometer (Perkin Elmer) at a signal resolu-

    tion of 1 cm1 within the range of 400e4000 cm1. The ultra-

    violet (UV)evisible spectra of the samples were obtained with

    a Lambda 900 UVevis spectrometer (PerkineElmer) over the

    range of 200e800 nm.

    The antibacterial activity of composite nanofibers against

    Escherichia coli, a Gram-negative bacteria, was investigated by

    a zone inhibition method. The nanofiber mat samples were formed

    into circular discs with a diameter of 6 mm. Using a spread platemethod, nutrient agar plates were inoculated with 1 ml of bacterial

    suspension containing around 105 colony forming units for each

    bacteria. The same amounts of samples were gently placed on the

    inoculated plates, and were then incubated at 37 C for 24 h. Zones

    of inhibition were determined by measuring the clear area formed

    around each sample.

    3. Results and discussion

    Electrospinning of hybrid mats was carried out at two different

    reduction times of AgNO3 in PEO: 3 h and 5.5 h (referred herein as

    Hybrid 1 and Hybrid 2, respectively). A study by Saquing et al. [19]

    showed that high-molecular weight PEO has the capability of

    reducing AgNO3 into Ag NPs at ambient conditions. The reductionof AgNO3 to Ag NPs could be observed in the color change of the

    AgNO3/PEO/DW solution from milky white color at t 0 to dark

    green color at 3 h reduction time (Fig. 2d). The reduction of AgNO3into Ag NPs in high MW PEO is attributed to the formation of

    pseudo-crown ethers that can bind silver ions within the coiled

    polyether helix, and these bound silver ions are reduced via

    oxidation of PEO fragments by the metal center [19,20]. The reason

    Fig.1. (a) Schematic of the present angled two-spinneret electrospinning set-up; photographic images of (b) Hybrid 2 mat and; (c) neat PU nanofi

    brous mat.

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    for the conformation similar to that of the crown ethers is the

    ionedipole interaction between the metal ion and the electron

    pair of the ether oxygen in the PEO fragments [21]. Furthermore,

    high MW PEO has longer polymer chains, which in turn can form

    more crown ether cavities and provide more reaction sites thus

    making it faster in reduction rate, comparing to using low MW

    PEO, which require at least 20 days of reduction time as reported

    elsewhere [20]. The electrospun nanofibrous mats (Fig. 2aec) all

    showed highly-porous structure, and randomly-oriented, ultrafinenanofibers [22]. The neat PU nanofibers showed continuous and

    smooth fibrous structure with an average fiber diameter of

    950270 nm (Table 1). On the other hand, the hybrid Ag/PEO e

    PU nanofibers showed two distinct fiber sizes, i.e., larger fiber sizes

    for PU nanofibers and much smaller sizes for PEO nanofibers. In

    Hybrid 1 (reduction time of 3 h, Fig. 2b), the PU and PEO nano-

    fibers showed an average diameter of 800290 nm and

    194 40 nm, respectively. A closer look at the PEO nanofiber

    (inset of Fig. 2b) shows very small, well-dispersed Ag NPs with

    diameters ranging from 6 to 10 nm. These Ag NPs were verified by

    TEM imaging as shown in Fig. 2e, showing highly-dispersed Ag

    NPs with narrow size distribution along the nanofiber. Similar

    structures in Hybrid 1 were observed in Hybrid 2 (Fig. 2c and f)

    but the Ag NPs were larger in diameter (d9030 nm) and hadwider diameter distribution. These large Ag NP sizes are attributed

    to the aggregation of metallic silvers to form clusters, due to their

    high surface energy [23]. The increase in NP size is also supported

    by the UVevis spectroscopy results (Fig. 3b). Here, the duration of

    reduction time affected the sizes of the Ag NPs, wherein the longer

    reduction time produced bigger Ag NPs, which is consistent with

    the results of other studies [19]. A recent study reported that the

    reduction of AgNO3 into Ag NPs starts as early as 30 min and

    complete reduction is attained after 8 h, i.e., very little additional

    Ag NP formation is observed at reduction time from 8 h until 24 h.

    When the silver ions are reduced into metallic silver, the silver

    atoms aggregate into clusters which possess very high surface

    energy, thereby aggregating more into larger particles [23]. Thus,

    the longer reduction time, there is more chance of clusters to

    Fig. 2. FESEM and TEM images of nanofibrous mats of (a) neat PU; (b, e) Hybrid 1; (c, f) Hybrid 2, and; (d) photos of Ag/PEO solutions at reduction times of 0 and 3 h.

    Table 1

    Size distribution of electrospun neat PU and hybrid mats, and Ag NPs.

    Ne at P U Ag/PEO e PU

    (3 h) (Hybrid 1)

    Ag/PEO e PU

    (5.5 h) (Hybrid 2)

    Fiber diameter

    (nm)

    PU 950270 800 290 770280

    PEO e 194 40 20036

    Ag NP size

    (nm)

    e 8 2 9030

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    aggregate more and form into bigger particles as observed in thepresent study.

    To analyze the composition of electrospun nanofibers, we took

    FTIR spectra of the nanofibrous mats (Fig. 3a). The characteristic

    peaks of neat PU nanofibers can be assigned as: 2955 cm1 (CH2asymmetric vibration); 2881 cm1 (CH3 symmetric vibrations);

    1733 cm1 (free C]O); 1709 cm1 (C]O bond); 1532 cm1

    (urethane amide II); 1081 cm1 C(O)eOeC stretching of the hard

    segment; and 821 cm1 (bending vibration in benzene ring)

    [24,25]. The typical absorption bands for PEO are 2894 cm1 (CH2stretching), CeOeC stretching vibration at 1153, 1101, and

    963 cm1, and at 1343 cm1 assigned as CeH deformation mode

    [26]. All peaks of neat PU were detected in the hybrid mats, and the

    additional peaks in Fig. 3a are attributed to the peaks of PEO.

    However, the band at 1478 (eCeH bending mode) of neat PU wasobserved to have shifted to lower frequency in the hybrid mats,

    indicating the formation of stronger hydrogen bond between PU

    and PEO [27]. These FTIR spectra results confirm the successful

    fabrication of bicomponent hybrid nanofibrous mat consisting of

    PU and PEO nanofiber matrices.

    In order to verify the presence of Ag NPs on the hybrid nano-

    fibrous mats, EDS and UVevis spectra were obtained. In the present

    study, PEO was used as the in situ reductant of AgNO3 to form Ag

    NPs. A change of color was observed visually on the nanofiber mat

    from white (for neat PU, Fig. 1c) to yellowish mat (for the hybrid

    mats, Fig. 1b), which confirms the reduction of Ag ions and the

    formation of metallic silver nuclei [28]. The EDS spectra (inset of

    Fig. 3b) show Ag peak for the hybrid nanofibrous mats, confirming

    the presence of Ag NPs. The UVevis spectra (Fig. 3b) show peaksfrom 400 to 470 nm, which signifies the surface plasmon band

    resonance of Ag NPs [19,29], while no peak absorbance was

    obtained for neat PU in this region. Hybrid 1 showed a peak at

    415 nm, while Hybrid 2 shifted its peak to a longer wavelength (i.e.,

    425 nm) (blue shift), signifying an increase in NP sizes [30], which

    corresponds well with the sizes observed in SEM and TEM images

    (Fig. 2), and as also observed by other studies [31].

    Fig. 4 shows the antibacterial activity of the electrospun

    nanofibrous mats via a zone inhibition test. Previous studies

    observed that the antimicrobial activity of Ag NPs depended on

    their average size [32]. It was reported that Ag NPs with sizes of

    1e10 nm can disturb the permeability of bacteria and respiratory

    functions, thus can efficiently inactivate them [33,34]. In this

    study, Hybrid 1 nanofibrous mat, which contains Ag NPs withaverage diameter of 8 nm (Fig. 4c) showed much higher bacterial

    inactivation as compared to Hybrid 2 (Ag NP average diame-

    ter 90 nm) (Fig. 4d), while individual nanofibrous mats of neat

    PU (Fig. 4a) and neat PEO (Fig. 4b) alone did not show any signs of

    antibacterial activity. This suggests that without the aid of Ag NPs,

    the individual neat nanofibrous components of PU and PEO could

    not provide any antibacterial effect. The higher bacterial activity

    of Hybrid 1 could be attributed to the presence of smaller Ag NPs,

    which provide large surface area to come in contact with bacteria,

    when compared with bigger Ag NPs in Hybrid 2 [35]. Several

    other researchers reported high bacterial inactivation efficiency of

    Ag NP/polymer composites with Ag NP sizes ranging from 2 to

    21 nm [23,32,36,37].

    Fig. 3. (a) FTIR and (b) UVevis spectra of nanofibrous mats of neat PU, Hybrid 1 and Hybrid 2; and (c) EDS spectra of Hybrid 2 mat.

    Fig. 4. Zone of inhibition test against E. coli bacteria for nanofi

    brous mats of (a) neat PU, (b) neat PEO, (c) Hybrid 1, and (d) Hybrid 2 (scale bar

    5 mm).

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    4. Conclusion

    In summary, we have demonstrated a facile way to fabricate

    hybrid nanofibrous mats with antibacterial functionality by dual-

    spinneret electrospinning. Using PEO both as reducing agent of

    AgNO3 and template for the formed Ag NPs, we can utilize the dual-

    spinneret electrospinning to selectively functionalize the compo-

    nent nanofibers for making new materials. Here, the larger PU

    nanofibers give structural stability and prolonged use of the hybrid

    materials, while the thinner PEO nanofibers provide more reactive

    surface area, and the decoration of Ag NPs on PEO surfaces without

    the use of chemicals gives antibacterial functionality to the hybrid

    mat. The reduction time of AgNO3 in PEO plays an important role in

    the size control of the Ag NPs. The hybrid mat with Ag NPs of 8 nm

    average size (i.e., reduction time of 3 h) exhibited strong antibac-

    terial activity. The present hybrid mat has great potential for anti-

    bacterial tissue scaffolds or for wound-healing applications.

    Acknowledgement

    This research was supported by a Grant from the Ministry of

    Education, Science and Technology of Korea through the National

    Research Foundation (Project no. 2011-0011807).

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