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Journal <strong>of</strong> Membrane Science 320 (2008) 259–267<br />

Contents lists available at ScienceDirect<br />

Journal <strong>of</strong> Membrane Science<br />

journal homepage: www.elsevier.com/locate/memsci<br />

<strong>Surface</strong> <strong>modification</strong> <strong>and</strong> <strong>antibacterial</strong> <strong>activity</strong> <strong>of</strong> <strong>electrospun</strong> polyurethane<br />

fibrous membranes with quaternary ammonium moieties<br />

Chen Yao a,b , Xinsong Li a,∗ , K.G. Neoh b,∗ , Zhilong Shi b , E.T. Kang b<br />

a School <strong>of</strong> Chemistry <strong>and</strong> Chemical Engineering, Southeast University, Sipailou 2, Nanjing 210018, PR China<br />

b Department <strong>of</strong> Chemical <strong>and</strong> Biomolecular Engineering, National University <strong>of</strong> Singapore, Kent Ridge, 119260 Singapore, Singapore<br />

article<br />

info<br />

abstract<br />

Article history:<br />

Received 26 February 2008<br />

Received in revised form 3 April 2008<br />

Accepted 5 April 2008<br />

Available online 15 April 2008<br />

Keywords:<br />

Electrospinning<br />

Antibacterial<br />

<strong>Surface</strong> <strong>modification</strong><br />

Polyurethane<br />

A novel <strong>antibacterial</strong> material was developed by surface <strong>modification</strong> <strong>of</strong> <strong>electrospun</strong> polyurethane (PU)<br />

fibrous membranes, using a process which involved plasma pretreatment, UV-induced graft copolymerization<br />

<strong>of</strong> 4-vinylpyridine (4VP), <strong>and</strong> quaternization <strong>of</strong> the grafted pyridine groups with hexylbromide.<br />

The success <strong>of</strong> <strong>modification</strong> with poly(4-vinyl-N-hexyl pyridinium bromide) groups on these was ascertained<br />

by X-ray photoelectron spectroscopy (XPS). The morphologies <strong>and</strong> mechanical properties were<br />

investigated by scanning electron microscopy (SEM) <strong>and</strong> tensile test, respectively. The results showed<br />

that the morphologies <strong>of</strong> PU fibrous membranes changed slightly during the <strong>modification</strong> process <strong>and</strong><br />

the fiber structures were maintained. The tensile strength <strong>of</strong> PU fibrous membranes decreased after surface<br />

<strong>modification</strong>, with the smallest decrease (90%) [19]. Electrospun cellulose acetate fibers containing<br />

silver nanoparticles showed strong antimicrobial <strong>activity</strong> against<br />

both Gram-negative <strong>and</strong> Gram-positive bacteria [20,21]. Kenawy et<br />

al. modified the poly(vinyl phenol) either by sulphonation or by formation<br />

<strong>of</strong> lithium salt <strong>of</strong> the sulphonated species, <strong>and</strong> investigated<br />

the <strong>antibacterial</strong> activities <strong>of</strong> the modified poly(vinyl phenol) <strong>electrospun</strong><br />

mats [22]. Polyurethane cationomers polymerized from<br />

base PU with chain extenders having a quaternary ammonium<br />

0376-7388/$ – see front matter © 2008 Elsevier B.V. All rights reserved.<br />

doi:10.1016/j.memsci.2008.04.012


260 C. Yao et al. / Journal <strong>of</strong> Membrane Science 320 (2008) 259–267<br />

group were <strong>electrospun</strong> into non-woven nan<strong>of</strong>iber mats for antimicrobial<br />

nan<strong>of</strong>ilter applications [23].<br />

A more straightforward way is to modify the surface <strong>of</strong> polymer<br />

nan<strong>of</strong>ibers without affecting bulk properties <strong>of</strong> the treated<br />

nan<strong>of</strong>ibers. The methods used to impart surface <strong>modification</strong> usually<br />

depend strongly on the nature <strong>of</strong> the fiber-forming polymer<br />

<strong>and</strong> include, but are not limited to, covalent polymer grafting<br />

[24], plasma treatment [25], physisorption (e.g., hydrogen-bonding<br />

interactions) [26], chemisorption [27], <strong>and</strong> chemical derivatization<br />

[28]. Among the various methods, plasma treatment provides a<br />

clean <strong>and</strong> environmentally friendly way for surface <strong>modification</strong><br />

[29]. The free radicals <strong>and</strong> electrons created in the plasma treatment<br />

could be used to modify the polymer nan<strong>of</strong>ibers chemically. Covalent<br />

attachment <strong>of</strong> functional compounds to polymer fiber surfaces<br />

is the preferred approach to introduce functionalities permanently<br />

<strong>and</strong> at reasonably high efficiency.<br />

There are numerous antimicrobials suitable for immobilization<br />

on polymer surfaces. Quaternary ammonium compounds seem<br />

attractive because their target is primarily the microbial membrane<br />

<strong>and</strong> they accumulate in the cell driven by the membrane potential<br />

[30]. To maximize efficiency, quaternary ammonium compound<br />

is used as monomeric link in the polymeric leash <strong>and</strong> poly(4-<br />

vinylpyridine) (PVP) is usually selected as the carrying polymer.<br />

Tiller et al. showed that the surfaces <strong>of</strong> commercial polymers<br />

treated with N-alkylated PVP groups were lethal on contact to both<br />

Gram-positive <strong>and</strong> Gram-negative bacteria, <strong>and</strong> it was also shown<br />

that N-alkyl chain <strong>of</strong> six carbon units in length was the most effective<br />

[31].<br />

The purpose <strong>of</strong> this paper was to develop novel <strong>antibacterial</strong><br />

PU fibrous membranes by electrospinning the polymer followed<br />

by plasma pretreatment, UV-induced graft copolymerization <strong>and</strong><br />

quaternization reaction. Electrospun PU fibrous membranes were<br />

modified with poly(4-vinyl-N-hexyl pyridinium bromide) on the<br />

surfaces to achieve <strong>antibacterial</strong> activities. The modified PU fibrous<br />

membranes were subsequently characterized in terms <strong>of</strong> their<br />

morphologies, surface chemical compositions <strong>and</strong> mechanical<br />

properties. The <strong>antibacterial</strong> activities <strong>of</strong> the fibrous membranes<br />

were assessed against both Gram-positive Staphylococcus aureus (S.<br />

aureus) <strong>and</strong> Gram-negative Escherichia coli (E. coli).<br />

2. Experimental<br />

2.1. Materials<br />

Polyurethane elastomer, Elastollan ® 1180A10, was received from<br />

BASF. 4-Vinylpyridine (4VP) monomer was obtained from Aldrich<br />

Chemical Co. <strong>and</strong> freshly distilled under reduced pressure before<br />

use. Hexylbromide <strong>and</strong> solvents, such as tetrahydr<strong>of</strong>uran (THF),<br />

N,N-dimethyl formamide (DMF), heptane, 2-propanol, were <strong>of</strong><br />

reagent grade <strong>and</strong> used as received from Aldrich Chemical Co. Peptone,<br />

yeast extract, agar <strong>and</strong> beef extract were purchased from<br />

Oxoid. S. aureus (ATCC 25923) <strong>and</strong> E. coli (ATCC DH5) were<br />

obtained from American Type Culture Collection.<br />

2.2. Electrospinning <strong>of</strong> PU<br />

PU was dissolved in a mixed solvent <strong>of</strong> THF <strong>and</strong> DMF (1:1, v/v) to<br />

produce various spinning solutions with PU concentrations ranging<br />

from 5% to 11% (w/v, g/mL). Viscosity, conductivity <strong>and</strong> surface<br />

tension <strong>of</strong> the prepared solutions were measured with a viscometer<br />

(NDJ-9S, ShangPing), a conductivity meter (DDB-303A, Rex) <strong>and</strong><br />

a surface tension meter (BZY-1, HengPing) at 20 ◦ C, respectively. A<br />

syringe pump was used to feed the polymer solution through a 20-<br />

mL plastic syringe fitted with a needle <strong>of</strong> tip diameter <strong>of</strong> 0.6 mm<br />

at a delivery rate <strong>of</strong> 6 mL/h. After high voltage ranging from 10<br />

to 12 kV was applied to the needle, a positive charged jet <strong>of</strong> PU<br />

solution formed from the Taylor cone <strong>and</strong> sprayed to a grounded<br />

drum, approximately 10 cm from the needle tip. With the evaporation<br />

<strong>of</strong> solvent, PU fibers were deposited on the drum to form<br />

fibrous membrane. The as-spun fibrous membrane was then dried<br />

under vacuum <strong>and</strong> annealed at 80 ◦ C for 6 h. All electrospinning<br />

experiments were carried out at 20 ◦ C <strong>and</strong> relative humidity <strong>of</strong> 50%.<br />

2.3. <strong>Surface</strong> <strong>modification</strong> <strong>of</strong> PU fibrous membranes<br />

PU fibrous membranes were subjected to argon plasma pretreatment<br />

in an Anatech SP100 plasma system, equipped with a<br />

cylindrical quartz reactor chamber. The glow discharge was produced<br />

at a plasma power <strong>of</strong> 35 W, an applied oscillator frequency<br />

<strong>of</strong> 40 kHz <strong>and</strong> an argon pressure <strong>of</strong> approximately 80 Pa. After<br />

subjected to glow discharge for 60 s on both surfaces, the fibrous<br />

membrane was exposed to air for 5 min to facilitate formation <strong>of</strong><br />

surface oxide <strong>and</strong> peroxide groups.<br />

The plasma pretreated fibrous membranes were immersed<br />

in 2-propanol solution <strong>of</strong> 20 vol.% 4VP in a Pyrex glass tube.<br />

Degassing <strong>of</strong> the solution was achieved by bubbling nitrogen vigorously<br />

for 30 min <strong>and</strong> sealing the tube with silicon rubber stopper.<br />

The tube was then exposed to UV irradiation in a Riko rotary<br />

photochemical reactor (RH400-10W) for 60 min on each surface.<br />

The graft-copolymerized fibrous membranes were subjected to<br />

washing with copious amounts <strong>of</strong> 2-propanol to remove residual<br />

monomer <strong>and</strong> physically adsorbed homopolymer.<br />

The fibrous membranes with graft-copolymerized 4VP were<br />

then placed in heptane solutions containing 20 vol.% hexylbromide<br />

<strong>and</strong> the reaction mixture was stirred for 48 h at 60 ◦ C. The fibrous<br />

membranes were then thoroughly rinsed with heptane <strong>and</strong> dried<br />

under vacuum.<br />

2.4. <strong>Surface</strong> characterization<br />

The morphology <strong>of</strong> PU fibrous membranes was observed with a<br />

scanning electron microscope (JEOL JSM 5600LV) after gold sputtercoating.<br />

Diameters <strong>of</strong> the <strong>electrospun</strong> fibers were measured directly<br />

from SEM images, with an average value being calculated from at<br />

least 100 measurements.<br />

<strong>Surface</strong> compositions <strong>of</strong> PU fibrous membranes were analyzed<br />

by XPS on an AXIS HSi spectrometer (Kratos Analytical Ltd.) using<br />

the monochromatized Al K X-ray source (1486.6 eV photons) at<br />

a constant dwell time <strong>of</strong> 100 ms <strong>and</strong> a pass energy <strong>of</strong> 40 eV. The<br />

anode voltage was 15 kV, <strong>and</strong> the anode current was 10 mA. The<br />

core-level signals were obtained at a photoelectron take<strong>of</strong>f angle<br />

<strong>of</strong> 90 ◦ (with respect to the membrane surface). To compensate for<br />

surface charging effect, all binding energies (BEs) were referenced<br />

to the C 1s hydrocarbon peak at 284.6 eV. In the peak synthesis,<br />

the line width (full width at half-maximum) <strong>of</strong> the Gaussian<br />

peaks was maintained constant for all components in a particular<br />

spectrum. The peak area ratios for the various elements were corrected<br />

using experimentally determined instrumental sensitivity<br />

factors.<br />

2.5. Mechanical property<br />

Tensile tests <strong>of</strong> PU fibrous membranes were performed using<br />

an Instron universal materials testing machine (Model 5544) with<br />

a 10 N load cell in a constant relative humidity (50%) room at<br />

25 ◦ C. “Dog-bone” shaped samples were cut from the fibrous membranes<br />

(5-mm wide at the narrowest point with a gage length <strong>of</strong><br />

15 mm). The thickness <strong>of</strong> these samples was measured with a digital<br />

micrometer having a precision <strong>of</strong> 1 m. A cross-head speed <strong>of</strong>


C. Yao et al. / Journal <strong>of</strong> Membrane Science 320 (2008) 259–267 261<br />

Fig. 1. Electrospinning solution parameters <strong>and</strong> average diameter <strong>of</strong> the as-spun fibers with different PU concentrations.<br />

10 mm/min was used <strong>and</strong> at least five samples were tested for each<br />

type <strong>of</strong> the fibrous membranes.<br />

2.6. Determination <strong>of</strong> <strong>antibacterial</strong> <strong>activity</strong><br />

S. aureus was incubated in 10 mL <strong>of</strong> a 3.1% yeast–dextrose broth<br />

(containing 10 g/L peptone, 8 g/L beef extract, 5 g/L sodium chloride,<br />

5 g/L glucose, <strong>and</strong> 3 g/L yeast extract at a pH 6.8) for 6–8 h at 37 ◦ C<br />

until the exponential growth phase was reached [32].<br />

The bacteria-containing broth was centrifuged at 3000 rpm for<br />

10 min, <strong>and</strong> after removal <strong>of</strong> supernatant, the cells were washed<br />

twice with sterile phosphate-buffered solution (PBS). The bacteria<br />

cells were resuspended to provide a final density <strong>of</strong> 10 6 cells/mL<br />

in PBS (based on st<strong>and</strong>ard calibration with the assumption that<br />

the optical density <strong>of</strong> 1.0 at 540 nm is equivalent to approximately<br />

10 9 cells/mL) [33].<br />

PU fibrous membranes <strong>electrospun</strong> from 10% (w/v) solutions in<br />

THF <strong>and</strong> DMF (1:1, v/v) (either pristine or modified, 8 mm × 8 mm)<br />

were sterilized with ultraviolet irradiation for 30 min, <strong>and</strong> then<br />

immersed in 10 mL <strong>of</strong> the bacterial suspension in an Erlenmeyer<br />

flask <strong>and</strong> shaken at 200 rpm at 37 ◦ C. The viable cell counts <strong>of</strong><br />

bacteria were measured by surface spread plate method. At the<br />

predetermined time, 1 mL <strong>of</strong> bacteria culture was taken from the<br />

flask <strong>and</strong> decimal serial dilutions with PBS were repeated with each<br />

Fig. 2. XPS wide scans <strong>of</strong> (a) pristine PU fibrous membranes, (d) PU membranes grafted with PVP, <strong>and</strong> (g) PU membranes modified with poly(4-vinyl-N-hexylpyridinium<br />

bromide); C 1s core-level spectra <strong>of</strong> (b) pristine PU fibrous membranes, (e) PU membranes grafted with PVP, <strong>and</strong> (h) PU membranes modified with poly(4-vinyl-Nhexylpyridinium<br />

bromide); N 1s core-level spectra <strong>of</strong> (c) pristine PU fibrous membranes, (f) PU membranes grafted with PVP, <strong>and</strong> (i) PU membranes modified with<br />

poly(4-vinyl-N-hexylpyridinium bromide). PU fibrous membranes were <strong>electrospun</strong> from 10% (w/v) PU solutions in THF <strong>and</strong> DMF (1:1, v/v).


262 C. Yao et al. / Journal <strong>of</strong> Membrane Science 320 (2008) 259–267<br />

initial sample. A 0.1-mL drop <strong>of</strong> the diluted sample was then spread<br />

onto solid growth agar plates. After incubation <strong>of</strong> the plates at 37 ◦ C<br />

for 24 h, the number <strong>of</strong> viable cells (colonies) was counted manually,<br />

<strong>and</strong> expressed as mean colony-forming units per milliliter<br />

(CFU/mL) after multiplication with the dilution factor. After 4 h, the<br />

PU fibrous membranes were removed from the bacterial suspension<br />

with sterile forceps <strong>and</strong> gently washed with PBS. The bacteria<br />

retained on membranes were dislodged by mild ultrasonication (for<br />

10 min) in a 100 W ultrasonic bath. Serial 10-fold dilutions were performed<br />

<strong>and</strong> viable counts estimated following the surface spread<br />

plate method. The number <strong>of</strong> colony-forming units on each membrane<br />

surface was computed <strong>and</strong> expressed relative to the apparent<br />

surface area <strong>of</strong> the membrane (CFU/cm 2 ). All experiments were performed<br />

in triplicate <strong>and</strong> the quantitative value was expressed as the<br />

average ± st<strong>and</strong>ard deviation.<br />

The extent <strong>of</strong> bacterial adhesion on PU fibrous membranes<br />

(both pristine <strong>and</strong> modified) were also assessed by examining<br />

these membranes after 4 h immersion in the PBS suspension <strong>of</strong><br />

Fig. 3. SEM images <strong>of</strong> (a), (c) <strong>and</strong> (e) pristine, (b), (d) <strong>and</strong> (f) modified PU fibrous membranes with poly(4-vinyl-N-hexylpyridinium bromide). PU fibrous membranes were<br />

<strong>electrospun</strong> from (a) <strong>and</strong> (b) 6%, (c) <strong>and</strong> (d) 8%, (e) <strong>and</strong> (f) 10% (w/v) solutions in THF <strong>and</strong> DMF (1:1, v/v).


C. Yao et al. / Journal <strong>of</strong> Membrane Science 320 (2008) 259–267 263<br />

10 7 cells/mL S. aureus. Control experiment was carried out with the<br />

filter paper. The membranes were then examined under SEM (JEOL<br />

JSM 5600LV) to assess the adhesion <strong>and</strong> viability <strong>of</strong> the bacteria.<br />

The membrane fixation <strong>and</strong> preparation for SEM were as follows:<br />

the membranes were first washed with PBS <strong>and</strong> 3 vol.% glutaraldehyde<br />

in PBS was added for 5 h <strong>and</strong> stored at 4 ◦ C. The glutaraldehyde<br />

solution was then removed <strong>and</strong> the membranes were washed with<br />

PBS, followed by step dehydration with 25%, 50%, 70%, 95%, <strong>and</strong><br />

100% ethanol for 10 min each. The membranes were dried under<br />

vacuum <strong>and</strong> gold sputter-coated before SEM observation.<br />

For E. coli, the same assay procedures were used as those<br />

described above for S. aureus.<br />

3. Results <strong>and</strong> discussion<br />

3.1. Electrospinning <strong>of</strong> PU<br />

While electrospinning has proven to be a versatile <strong>and</strong> powerful<br />

means <strong>of</strong> fabricating polymer micro/nan<strong>of</strong>ibers, its applicability<br />

to obtain smooth, uniform fibrous structure is not straightforward.<br />

Among various parameters <strong>of</strong> electrospinning process, such<br />

as applied voltage, needle tip-to-receiver distance <strong>and</strong> solution<br />

delivery rate, concentration or corresponding viscosity <strong>of</strong> spinning<br />

solution is one <strong>of</strong> the most effective variables for controlling fiber<br />

morphology <strong>and</strong> diameter. Results obtained from our study showed<br />

that solution concentration was found to be the major factor controlling<br />

the morphology <strong>of</strong> the fibers in the electrospinning <strong>of</strong> PU.<br />

Various PU solutions with concentration in the range <strong>of</strong> 5–11%<br />

(w/v, g/mL) in THF <strong>and</strong> DMF (1:1, v/v) were <strong>electrospun</strong>. A beadon-string<br />

morphology with several big beads was obtained at<br />

PU concentration <strong>of</strong> 5% (w/v). The distribution density <strong>of</strong> beads<br />

decreased when PU concentration increased to 6% (w/v). The shape<br />

<strong>of</strong> the beads became spindle-like. Smooth <strong>and</strong> homogeneous fibers<br />

without beads were produced when PU concentration reached 8%<br />

(w/v). As PU concentration increased from 9% to 11% (w/v), the<br />

<strong>electrospun</strong> fibers became thicker <strong>and</strong> more adhesive at various<br />

bonding sites, which led to a film-like character <strong>and</strong> structural<br />

integrity <strong>of</strong> the fibrous membranes.<br />

The effects <strong>of</strong> electrospinning solution parameters on the average<br />

diameter <strong>of</strong> PU nan<strong>of</strong>ibers were investigated. As shown in<br />

Fig. 1a, viscosity <strong>of</strong> PU solutions increased with increasing PU<br />

concentration. However, both conductivity <strong>and</strong> surface tension <strong>of</strong><br />

PU solutions with different concentrations did not differ much<br />

(Fig. 1b). The viscosity <strong>of</strong> a polymer solution reflects the intermolecular<br />

interactions between polymer chains. Polymer solutions<br />

with higher viscosity usually exhibit longer stress relaxation times,<br />

which may facilitate the formation <strong>of</strong> fibers with large diameters<br />

during electrospinning [34]. When the viscosity <strong>of</strong> PU solutions<br />

increased greatly from 0.186 Pa s up to 0.459 Pa s with increasing<br />

PU concentration from 9% to 11% (w/v), the average fiber diameter<br />

increased dramatically from 820 nm to 1.95 m(Fig. 1a). Therefore,<br />

Fig. 4. (a) Tensile strength, (b) elongation at break <strong>and</strong> (c) Young’s modulus <strong>of</strong> PU fibrous membranes <strong>electrospun</strong> from solutions with different concentrations (both pristine<br />

membranes <strong>and</strong> those modified with poly(4-vinyl-N-hexylpyridinium bromide)).


264 C. Yao et al. / Journal <strong>of</strong> Membrane Science 320 (2008) 259–267<br />

it was considered that the viscosity <strong>of</strong> solution was the major factor<br />

affecting the average diameter <strong>of</strong> <strong>electrospun</strong> PU nan<strong>of</strong>ibers.<br />

3.2. <strong>Surface</strong> <strong>modification</strong> <strong>of</strong> PU fibrous membranes<br />

The success <strong>of</strong> <strong>modification</strong> with poly(4-vinyl-N-hexyl pyridinium<br />

bromide) on <strong>electrospun</strong> PU fibrous membranes can be<br />

ascertained by comparing the XPS spectra <strong>of</strong> the pristine <strong>and</strong> modified<br />

membranes as shown in Fig. 2.<br />

The spectra <strong>of</strong> pristine PU fibrous membranes showed three<br />

main signals corresponding to C 1s (284.6 eV), O 1s (532 eV) <strong>and</strong><br />

N 1s (400 eV) (Fig. 2a). The XPS C 1s core-level spectrum <strong>of</strong> pristine<br />

PU fibrous membrane (Fig. 2b) was resolved into three component<br />

peaks: a hydrocarbon environment (C–H, C–C, 284.6 eV), a carbon<br />

singly bound to oxygen environment (C–O, 286.0 eV), <strong>and</strong> a carbon<br />

in a carbamate environment (–CO–, 289.0 eV) [35]. The corresponding<br />

N 1s spectrum (Fig. 2c) showed an intense peak at the binding<br />

energy (BE) <strong>of</strong> 399.5 eV attributable to the nitrogen (–NH–) in carbamate<br />

<strong>of</strong> PU.<br />

After UV-induced graft copolymerization <strong>of</strong> PVP onto the PU<br />

fibrous membranes, the C 1s core-level spectrum (Fig. 2e) showed<br />

an additional peak at 285.5 eV attributable to C–N species. The<br />

intensity <strong>of</strong> the peak assigned to C–O species became weaker <strong>and</strong><br />

that <strong>of</strong> –CO– species was barely discernible compared with Fig. 2b.<br />

Similarly, the corresponding N 1s spectrum (Fig. 2f) showed an<br />

additional peak at the BE <strong>of</strong> 398.5 eV attributable to imine moiety<br />

(–N ) <strong>of</strong> the pyridine rings [36] <strong>and</strong> a decrease in the intensity <strong>of</strong><br />

–NH– peak component compared with Fig. 2c. The extent <strong>of</strong> surface<br />

grafting <strong>of</strong> PVP can be estimated from the sensitivity factor corrected<br />

ratio <strong>of</strong> the total N 1s peak over the total C 1s peak, expressed<br />

as [N]/[C]. Previous work <strong>of</strong> surface graft copolymerization showed<br />

that graft concentration <strong>of</strong> PVP was affected by the monomer concentration<br />

[37]. In this experiment, 4VP concentration <strong>of</strong> 20% was<br />

chosen for effective surface graft copolymerization. As determined<br />

by XPS, the surface [N]/[C] ratio <strong>of</strong> the 4VP grafted onto PU fibrous<br />

membrane was 0.12, close to the value <strong>of</strong> 0.14, expected for the 4VP<br />

monomeric unit (C 7 H 7 N 1 ). It indicated that the surface was almost<br />

completely covered by 4VP copolymers, which could provide abundant<br />

reactive sites for the subsequent quaternization.<br />

Fig. 2g–i shows the XPS spectra <strong>of</strong> the graft-copolymerized PU<br />

fibrous membrane N-alkylated with hexylbromide. No significant<br />

difference was observed in C 1s core-level spectrum before (Fig. 2e)<br />

<strong>and</strong> after (Fig. 2h) N-alkylation. The corresponding N 1s core-level<br />

spectrum in Fig. 2i showed an additional peak at BE above 400 eV,<br />

attributable to the N + groups <strong>of</strong> the pyridinium ions [38], which<br />

confirmed the derivatization <strong>of</strong> the –N groups by hexylbromide.<br />

On the basis <strong>of</strong> the [N + ]/[N] ratio, the degree <strong>of</strong> alkylation <strong>of</strong> the<br />

pyridine rings on the PU fibrous membrane was around 50–60%.<br />

Fig. 3 shows the typical SEM images <strong>of</strong> PU fibrous membranes<br />

before <strong>and</strong> after surface <strong>modification</strong>. The morphologies <strong>of</strong> the surface<br />

modified PU fibrous membranes <strong>electrospun</strong> from 6%, 8% <strong>and</strong><br />

10% (w/v) solution changed slightly compared with those <strong>of</strong> pristine<br />

PU fibrous membranes. The result indicated that the fiber structures<br />

were maintained during the <strong>modification</strong> process, <strong>and</strong> no<br />

significant change in fiber diameter was observed.<br />

3.3. Mechanical property<br />

PU fibrous membranes both pristine <strong>and</strong> modified with<br />

poly(4-vinyl-N-hexylpyridinium bromide) were tested for their<br />

mechanical integrity in terms <strong>of</strong> tensile strength, elongation at<br />

break <strong>and</strong> Young’s modulus, as indicated in Fig. 4. It can be seen<br />

that the pristine <strong>electrospun</strong> PU fibrous membranes had tensile<br />

strength <strong>of</strong> 3.27–11.8 MPa, elongation at break <strong>of</strong> 159.2–349.7%,<br />

<strong>and</strong> Young’s modulus <strong>of</strong> 1.78–3.73 MPa, depending on the concentration<br />

<strong>of</strong> polymer solution used for electrospinning. PU fibrous<br />

membranes <strong>electrospun</strong> from 7% (w/v) solution had the lowest tensile<br />

strength <strong>of</strong> 3.27 MPa (Fig. 4a). As the concentration increased<br />

to 8% (w/v), the tensile strength increased to 3.88 MPa. The Young’s<br />

modulus <strong>and</strong> elongation at break showed an increasing trend with<br />

concentration as well (Fig. 4b <strong>and</strong> c). This is mainly due to more<br />

interstices between the fibers with smaller diameters, resulting in<br />

a lower number <strong>of</strong> closely adjacent binding in the fibrous membranes.<br />

Since strong “sticky” binding interactions make a major<br />

contribution to the strength <strong>of</strong> the membranes, increasing the fiber<br />

diameters can lead to increases <strong>of</strong> tensile strength [39]. When PU<br />

concentration increased from 9% to 11% (w/v), the tensile strength<br />

<strong>of</strong> the pristine as-spun fibrous membranes increased significantly<br />

from 4.32 to 11.80 MPa, in agreement with the increase in fiber<br />

diameters as shown in Fig. 1a.<br />

On the other h<strong>and</strong>, the tensile strength <strong>and</strong> elongation at break<br />

<strong>of</strong> PU fibrous membranes decreased after surface <strong>modification</strong>,<br />

whereas the Young’s moduli did not show much change. As shown<br />

in Fig. 4a, the tensile strength <strong>of</strong> PU fibrous membranes <strong>electrospun</strong><br />

from 7% (w/v) solution decreased from 3.27 to 1.99 MPa after<br />

<strong>modification</strong>, losing almost 40% <strong>of</strong> tensile strength. As the solution<br />

Fig. 5. Viable cell numbers <strong>of</strong> (a) Staphylococcus aureus, <strong>and</strong> (b) Escherichia coli as a function time in contact with: () control, () pristine <strong>and</strong> () modified PU fibrous<br />

membranes. The cell number was determined by surface spread plate method.


C. Yao et al. / Journal <strong>of</strong> Membrane Science 320 (2008) 259–267 265<br />

concentration increased, smaller decreases in the tensile strength<br />

were observed. With a concentration <strong>of</strong> 11% (w/v) which resulted in<br />

the largest diameter <strong>of</strong> fibers in PU fibrous membranes, the loss <strong>of</strong><br />

tensile strength was approximately 16%. The results indicated that<br />

the surface <strong>modification</strong> may have less adverse effect on the tensile<br />

strength <strong>of</strong> fibrous membranes with larger diameters. In view<br />

<strong>of</strong> these results, the <strong>antibacterial</strong> assays were carried out with surface<br />

modified PU fibrous membranes <strong>electrospun</strong> from 10% (w/v)<br />

solutions in THF <strong>and</strong> DMF (1:1, v/v) in order to achieve a balance<br />

between mechanical properties <strong>and</strong> fibrous structures.<br />

3.4. Antibacterial <strong>activity</strong><br />

Antibacterial efficacy <strong>of</strong> surface modified PU fibrous membranes<br />

with poly(4-vinyl-N-hexylpyridinium bromide) was investigated<br />

by estimating the number <strong>of</strong> viable bacteria cells in the S. aureus<br />

<strong>and</strong> E. coli suspension after being in contact with membranes for<br />

various periods <strong>of</strong> time, <strong>and</strong> the results were shown in Fig. 5. As<br />

expected, no significant loss <strong>of</strong> viable bacteria was detected in the<br />

control experiment (i.e. without the membranes). For pristine PU<br />

fibrous membranes, the viable cell numbers in bacteria suspen-<br />

Fig. 6. SEM images <strong>of</strong> (a) <strong>and</strong> (d) filter paper (control), (b) <strong>and</strong> (e) pristine, <strong>and</strong> (c) <strong>and</strong> (f) modified PU fibrous membranes after immersed in PBS suspension <strong>of</strong> (a)–(c) S.<br />

aureus, or (d)–(f) E. coli at 10 7 cells/mL for 4 h. PU fibrous membranes were <strong>electrospun</strong> from 10% (w/v) solutions in THF <strong>and</strong> DMF (1:1, v/v).


266 C. Yao et al. / Journal <strong>of</strong> Membrane Science 320 (2008) 259–267<br />

sions were similar to the control (Fig. 5a). The surface modified PU<br />

fibrous membranes had a high <strong>antibacterial</strong> efficacy for S. aureus,<br />

reaching 99.9% <strong>and</strong> 99.999% after 1 <strong>and</strong> 4 h contact, respectively.<br />

In comparison, the <strong>antibacterial</strong> efficacy for E. coli was 99.9% after<br />

4 h contact (Fig. 5b), lower than that for S. aureus. Although the<br />

mechanism <strong>of</strong> the <strong>antibacterial</strong> <strong>activity</strong> <strong>of</strong> immobilized quaternary<br />

ammonium groups is not entirely clear. It has been hypothesized<br />

that these immobilized moieties disrupt the integrity <strong>of</strong> the cytoplasmic<br />

membrane to cause cell death, similar to the mechanism<br />

<strong>of</strong> free biocides [40]. The difference in <strong>antibacterial</strong> efficacy is postulated<br />

to be the result <strong>of</strong> the different cell membrane structures<br />

between S. aureus <strong>and</strong> E. coli bacteria. The multilayered cell envelope<br />

structure <strong>of</strong> Gram-negative bacteria may be more resistant to<br />

access by the bactericidal moieties to the inner membrane <strong>of</strong> the<br />

organism [15].<br />

Fig. 6 shows the SEM images <strong>of</strong> different substrates after immersion<br />

in bacteria suspensions. Numerous distinguishable S. aureus<br />

cells can be observed on the filter paper as shown in Fig. 6a. The<br />

number <strong>of</strong> S. aureus cells on the pristine PU fibrous membranes was<br />

significantly less (Fig. 6b). The S. aureus was distributed not only on<br />

the pristine PU fibrous membrane upper surface but also entrapped<br />

in the space between the thin fibers. In contrast, very few sparsely<br />

distributed bacteria cells could be spotted over the entire surface<br />

<strong>of</strong> the modified PU fibrous membrane (Fig. 6c). Similar results were<br />

observed for E. coli on the surface <strong>of</strong> fibrous membranes as shown<br />

in Fig. 6d–f. The results indicated that both filter paper <strong>and</strong> pristine<br />

PU fibrous membranes are good templates for the proliferation <strong>of</strong><br />

bacteria <strong>and</strong> bi<strong>of</strong>ilm formation may occur readily on such surfaces<br />

in contact with bacteria. The presence <strong>of</strong> quaternary ammonium<br />

groups attached to the surface <strong>of</strong> PU fibrous membranes was thus<br />

demonstrated to be very effective in preventing bi<strong>of</strong>ilm formation.<br />

Since the interstitial spaces <strong>of</strong> the pristine PU fibrous membranes<br />

are large enough, it can be expected that some bacteria<br />

would have penetrated into the fibrous membranes during immersion<br />

in the bacteria suspension. Therefore, the bacteria retained<br />

in fibrous membranes were dislodged by mild ultrasonication <strong>and</strong><br />

the number <strong>of</strong> viable cells was counted by surface spread plate<br />

method. For the pristine PU fibrous membranes, the mean number<br />

<strong>of</strong> S. aureus <strong>and</strong> E. coli cells relative to the surface area was<br />

(9.03 ± 0.63) × 10 4 <strong>and</strong> (1.65 ± 0.24) × 10 5 CFU/cm 2 , respectively.<br />

For the modified PU fibrous membranes, the corresponding numbers<br />

were 735 ± 35 <strong>of</strong> S. aureus <strong>and</strong> 2555 ± 315 CFU/cm 2 <strong>of</strong> E. coli<br />

cells. Since very few bacteria cells could be observed on the surface<br />

<strong>of</strong> the modified PU fibrous membrane (Fig. 6c), the bacteria<br />

may be entrapped deep in the membrane either on fibers which<br />

have lower concentration <strong>of</strong> the <strong>antibacterial</strong> N-alkyl pyridinium<br />

units or on dead bacteria deposited on the fibers which provided a<br />

shielding effect from the N-alkyl pyridinium groups.<br />

4. Conclusions<br />

The surface <strong>of</strong> <strong>electrospun</strong> PU fibrous membranes was successfully<br />

modified with poly(4-vinyl-N-hexyl pyridinium bromide)<br />

using a method involving plasma pretreatment, UV-induced<br />

surface graft copolymerization <strong>and</strong> N-alkylation reaction. The morphologies<br />

<strong>of</strong> PU fibrous membranes changed slightly <strong>and</strong> the fiber<br />

structures were maintained after the <strong>modification</strong> process. The<br />

tensile strength <strong>and</strong> elongation at break <strong>of</strong> modified PU fibrous<br />

membranes decreased, whereas the Young’s moduli showed no significant<br />

change. Antibacterial assays showed that the modified PU<br />

fibrous membranes possessed highly effective <strong>antibacterial</strong> activities<br />

against both Gram-positive S. aureus <strong>and</strong> Gram-negative E. coli.<br />

The novel <strong>antibacterial</strong> PU fibrous membranes may have a wide<br />

variety <strong>of</strong> potential applications in high-performance filters, protective<br />

textiles, <strong>and</strong> biomedical devices.<br />

Acknowledgements<br />

Project 50573011 <strong>and</strong> 50673019 supported by the National Natural<br />

Science Foundation <strong>of</strong> China. Grant no.: R 279000202112 from<br />

the National University <strong>of</strong> Singapore, Ministry <strong>of</strong> Education, Singapore.<br />

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