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Surface modification and antibacterial activity of electrospun polyurethane fibrous membranes with quaternary ammonium moieties
Journal of Membrane Science 320 (2008) 259–267
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Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci
Surface modification and antibacterial activity of electrospun polyurethane fibrous membranes with quaternary ammonium moieties Chen Yao a,b , Xinsong Li a,∗ , K.G. Neoh b,∗ , Zhilong Shi b , E.T. Kang b a b
School of Chemistry and Chemical Engineering, Southeast University, Sipailou 2, Nanjing 210018, PR China Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, 119260 Singapore, Singapore
a r t i c l e
i n f o
Article history: Received 26 February 2008 Received in revised form 3 April 2008 Accepted 5 April 2008 Available online 15 April 2008 Keywords: Electrospinning Antibacterial Surface modification Polyurethane
a b s t r a c t A novel antibacterial material was developed by surface modification of electrospun polyurethane (PU) fibrous membranes, using a process which involved plasma pretreatment, UV-induced graft copolymerization of 4-vinylpyridine (4VP), and quaternization of the grafted pyridine groups with hexylbromide. The success of modification with poly(4-vinyl-N-hexyl pyridinium bromide) groups on these was ascertained by X-ray photoelectron spectroscopy (XPS). The morphologies and mechanical properties were investigated by scanning electron microscopy (SEM) and tensile test, respectively. The results showed that the morphologies of PU fibrous membranes changed slightly during the modification process and the fiber structures were maintained. The tensile strength of PU fibrous membranes decreased after surface modification, with the smallest decrease (<20%) observed in the membrane with largest diameter. The antibacterial activities of the modified PU fibrous membranes were assessed against Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli). The modified PU fibrous membranes possessed highly effective antibacterial activities and may have a wide variety of potential applications in high-performance filters, protective textiles, and biomedical devices. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In recent years, considerable attention has been paid to electrospinning as a versatile technique for producing fibers with diameters ranging from submicron to a few microns. In the typical electrospinning process, nanofibers are produced from an electrostatically driven jet of polymer solution (or melt). The discharged polymer solution jet undergoes a whipping process wherein the solvent evaporates and the highly stretched polymer fiber deposits on a grounded collector. The non-woven mats of electrospun fibers have shown unique properties, such as dramatically increased surface/volume ratios, excellent mechanical strength, and highly open porous structures. In the past few years, a wide range of synthetic and natural polymers in pure or blend solutions, as well as melts, have been electrospun to form fibers [1–4]. Thermoplastic polyurethane (PU) is a resilient elastomer of significant industrial importance which possesses a range of desirable properties such as elastomeric, resistant to abrasion, and excellent hydrolytic stability [5,6]. Numerous studies on the electrospinning of PU have been conducted [7–9]. Electrospun PU nanofiber mats exhibiting good mechanical properties may have a wide variety of potential appli-
∗ Corresponding authors. Tel.: +86 25 8379 3456; fax: +86 25 8379 3456. E-mail addresses: [email protected] (X. Li), [email protected] (K.G. Neoh). 0376-7388/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2008.04.012
cations in high-performance air filters, protective textiles, wound dressing materials, sensors, etc. [10–13]. With the growing public health awareness of the pathogenic effects, malodors and stain formations caused by microorganisms, there is an increasing need for antibacterial materials in many application areas like medical devices, health care, hygienic application, water purification systems, hospital, dental surgery equipment, textiles, food packaging, and storage [14,15]. A number of functional nanofibers and composites have been developed through electrospinning by incorporating well-selected functional agents to achieve antibacterial properties. Two common methods are incorporation of antibacterial agents in the electrospinning solutions [16,17], and electrospinning of polymers with intrinsic antibacterial properties such as quaternized chitosan [18]. Kim et al. reported that the sustained release of hydrophilic antibiotic drug (Mefoxin) from electrospun poly(lactide-co-glycolide)-based nanofibrous scaffolds was effective to inhibit Staphylococcus aureus growth (>90%) [19]. Electrospun cellulose acetate fibers containing silver nanoparticles showed strong antimicrobial activity against both Gram-negative and Gram-positive bacteria [20,21]. Kenawy et al. modified the poly(vinyl phenol) either by sulphonation or by formation of lithium salt of the sulphonated species, and investigated the antibacterial activities of the modified poly(vinyl phenol) electrospun mats [22]. Polyurethane cationomers polymerized from base PU with chain extenders having a quaternary ammonium
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group were electrospun into non-woven nanofiber mats for antimicrobial nanofilter applications [23]. A more straightforward way is to modify the surface of polymer nanofibers without affecting bulk properties of the treated nanofibers. The methods used to impart surface modification usually depend strongly on the nature of the fiber-forming polymer and include, but are not limited to, covalent polymer grafting [24], plasma treatment [25], physisorption (e.g., hydrogen-bonding interactions) [26], chemisorption [27], and chemical derivatization [28]. Among the various methods, plasma treatment provides a clean and environmentally friendly way for surface modification [29]. The free radicals and electrons created in the plasma treatment could be used to modify the polymer nanofibers chemically. Covalent attachment of functional compounds to polymer fiber surfaces is the preferred approach to introduce functionalities permanently and at reasonably high efficiency. There are numerous antimicrobials suitable for immobilization on polymer surfaces. Quaternary ammonium compounds seem attractive because their target is primarily the microbial membrane and they accumulate in the cell driven by the membrane potential [30]. To maximize efficiency, quaternary ammonium compound is used as monomeric link in the polymeric leash and poly(4vinylpyridine) (PVP) is usually selected as the carrying polymer. Tiller et al. showed that the surfaces of commercial polymers treated with N-alkylated PVP groups were lethal on contact to both Gram-positive and Gram-negative bacteria, and it was also shown that N-alkyl chain of six carbon units in length was the most effective [31]. The purpose of this paper was to develop novel antibacterial PU fibrous membranes by electrospinning the polymer followed by plasma pretreatment, UV-induced graft copolymerization and quaternization reaction. Electrospun PU fibrous membranes were modified with poly(4-vinyl-N-hexyl pyridinium bromide) on the surfaces to achieve antibacterial activities. The modified PU fibrous membranes were subsequently characterized in terms of their morphologies, surface chemical compositions and mechanical properties. The antibacterial activities of the fibrous membranes were assessed against both Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli). 2. Experimental 2.1. Materials Polyurethane elastomer, Elastollan® 1180A10, was received from BASF. 4-Vinylpyridine (4VP) monomer was obtained from Aldrich Chemical Co. and freshly distilled under reduced pressure before use. Hexylbromide and solvents, such as tetrahydrofuran (THF), N,N-dimethyl formamide (DMF), heptane, 2-propanol, were of reagent grade and used as received from Aldrich Chemical Co. Peptone, yeast extract, agar and beef extract were purchased from Oxoid. S. aureus (ATCC 25923) and E. coli (ATCC DH5␣) were obtained from American Type Culture Collection.
at a delivery rate of 6 mL/h. After high voltage ranging from 10 to 12 kV was applied to the needle, a positive charged jet of PU solution formed from the Taylor cone and sprayed to a grounded drum, approximately 10 cm from the needle tip. With the evaporation of solvent, PU fibers were deposited on the drum to form fibrous membrane. The as-spun fibrous membrane was then dried under vacuum and annealed at 80 ◦ C for 6 h. All electrospinning experiments were carried out at 20 ◦ C and relative humidity of 50%. 2.3. Surface modification of PU fibrous membranes PU fibrous membranes were subjected to argon plasma pretreatment in an Anatech SP100 plasma system, equipped with a cylindrical quartz reactor chamber. The glow discharge was produced at a plasma power of 35 W, an applied oscillator frequency of 40 kHz and an argon pressure of approximately 80 Pa. After subjected to glow discharge for 60 s on both surfaces, the fibrous membrane was exposed to air for 5 min to facilitate formation of surface oxide and peroxide groups. The plasma pretreated fibrous membranes were immersed in 2-propanol solution of 20 vol.% 4VP in a Pyrex glass tube. Degassing of the solution was achieved by bubbling nitrogen vigorously for 30 min and sealing the tube with silicon rubber stopper. The tube was then exposed to UV irradiation in a Riko rotary photochemical reactor (RH400-10W) for 60 min on each surface. The graft-copolymerized fibrous membranes were subjected to washing with copious amounts of 2-propanol to remove residual monomer and physically adsorbed homopolymer. The fibrous membranes with graft-copolymerized 4VP were then placed in heptane solutions containing 20 vol.% hexylbromide and the reaction mixture was stirred for 48 h at 60 ◦ C. The fibrous membranes were then thoroughly rinsed with heptane and dried under vacuum. 2.4. Surface characterization The morphology of PU fibrous membranes was observed with a scanning electron microscope (JEOL JSM 5600LV) after gold sputtercoating. Diameters of the electrospun fibers were measured directly from SEM images, with an average value being calculated from at least 100 measurements. Surface compositions of PU fibrous membranes were analyzed by XPS on an AXIS HSi spectrometer (Kratos Analytical Ltd.) using the monochromatized Al K␣ X-ray source (1486.6 eV photons) at a constant dwell time of 100 ms and a pass energy of 40 eV. The anode voltage was 15 kV, and the anode current was 10 mA. The core-level signals were obtained at a photoelectron takeoff angle of 90◦ (with respect to the membrane surface). To compensate for surface charging effect, all binding energies (BEs) were referenced to the C 1s hydrocarbon peak at 284.6 eV. In the peak synthesis, the line width (full width at half-maximum) of the Gaussian peaks was maintained constant for all components in a particular spectrum. The peak area ratios for the various elements were corrected using experimentally determined instrumental sensitivity factors.
2.2. Electrospinning of PU 2.5. Mechanical property PU was dissolved in a mixed solvent of THF and DMF (1:1, v/v) to produce various spinning solutions with PU concentrations ranging from 5% to 11% (w/v, g/mL). Viscosity, conductivity and surface tension of the prepared solutions were measured with a viscometer (NDJ-9S, ShangPing), a conductivity meter (DDB-303A, Rex) and a surface tension meter (BZY-1, HengPing) at 20 ◦ C, respectively. A syringe pump was used to feed the polymer solution through a 20mL plastic syringe fitted with a needle of tip diameter of 0.6 mm
Tensile tests of PU fibrous membranes were performed using an Instron universal materials testing machine (Model 5544) with a 10 N load cell in a constant relative humidity (50%) room at 25 ◦ C. “Dog-bone” shaped samples were cut from the fibrous membranes (5-mm wide at the narrowest point with a gage length of 15 mm). The thickness of these samples was measured with a digital micrometer having a precision of 1 m. A cross-head speed of
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Fig. 1. Electrospinning solution parameters and average diameter of the as-spun fibers with different PU concentrations.
10 mm/min was used and at least five samples were tested for each type of the fibrous membranes. 2.6. Determination of antibacterial activity S. aureus was incubated in 10 mL of a 3.1% yeast–dextrose broth (containing 10 g/L peptone, 8 g/L beef extract, 5 g/L sodium chloride, 5 g/L glucose, and 3 g/L yeast extract at a pH 6.8) for 6–8 h at 37 ◦ C until the exponential growth phase was reached [32]. The bacteria-containing broth was centrifuged at 3000 rpm for 10 min, and after removal of supernatant, the cells were washed twice with sterile phosphate-buffered solution (PBS). The bacteria
cells were resuspended to provide a final density of 106 cells/mL in PBS (based on standard calibration with the assumption that the optical density of 1.0 at 540 nm is equivalent to approximately 109 cells/mL) [33]. PU fibrous membranes electrospun from 10% (w/v) solutions in THF and DMF (1:1, v/v) (either pristine or modified, 8 mm × 8 mm) were sterilized with ultraviolet irradiation for 30 min, and then immersed in 10 mL of the bacterial suspension in an Erlenmeyer flask and shaken at 200 rpm at 37 ◦ C. The viable cell counts of bacteria were measured by surface spread plate method. At the predetermined time, 1 mL of bacteria culture was taken from the flask and decimal serial dilutions with PBS were repeated with each
Fig. 2. XPS wide scans of (a) pristine PU fibrous membranes, (d) PU membranes grafted with PVP, and (g) PU membranes modified with poly(4-vinyl-N-hexylpyridinium bromide); C 1s core-level spectra of (b) pristine PU fibrous membranes, (e) PU membranes grafted with PVP, and (h) PU membranes modified with poly(4-vinyl-Nhexylpyridinium bromide); N 1s core-level spectra of (c) pristine PU fibrous membranes, (f) PU membranes grafted with PVP, and (i) PU membranes modified with poly(4-vinyl-N-hexylpyridinium bromide). PU fibrous membranes were electrospun from 10% (w/v) PU solutions in THF and DMF (1:1, v/v).
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initial sample. A 0.1-mL drop of the diluted sample was then spread onto solid growth agar plates. After incubation of the plates at 37 ◦ C for 24 h, the number of viable cells (colonies) was counted manually, and expressed as mean colony-forming units per milliliter (CFU/mL) after multiplication with the dilution factor. After 4 h, the PU fibrous membranes were removed from the bacterial suspension with sterile forceps and gently washed with PBS. The bacteria retained on membranes were dislodged by mild ultrasonication (for 10 min) in a 100 W ultrasonic bath. Serial 10-fold dilutions were per-
formed and viable counts estimated following the surface spread plate method. The number of colony-forming units on each membrane surface was computed and expressed relative to the apparent surface area of the membrane (CFU/cm2 ). All experiments were performed in triplicate and the quantitative value was expressed as the average ± standard deviation. The extent of bacterial adhesion on PU fibrous membranes (both pristine and modified) were also assessed by examining these membranes after 4 h immersion in the PBS suspension of
Fig. 3. SEM images of (a), (c) and (e) pristine, (b), (d) and (f) modified PU fibrous membranes with poly(4-vinyl-N-hexylpyridinium bromide). PU fibrous membranes were electrospun from (a) and (b) 6%, (c) and (d) 8%, (e) and (f) 10% (w/v) solutions in THF and DMF (1:1, v/v).
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107 cells/mL S. aureus. Control experiment was carried out with the filter paper. The membranes were then examined under SEM (JEOL JSM 5600LV) to assess the adhesion and viability of the bacteria. The membrane fixation and preparation for SEM were as follows: the membranes were first washed with PBS and 3 vol.% glutaraldehyde in PBS was added for 5 h and stored at 4 ◦ C. The glutaraldehyde solution was then removed and the membranes were washed with PBS, followed by step dehydration with 25%, 50%, 70%, 95%, and 100% ethanol for 10 min each. The membranes were dried under vacuum and gold sputter-coated before SEM observation. For E. coli, the same assay procedures were used as those described above for S. aureus.
3. Results and discussion 3.1. Electrospinning of PU While electrospinning has proven to be a versatile and powerful means of fabricating polymer micro/nanofibers, its applicability to obtain smooth, uniform fibrous structure is not straightforward. Among various parameters of electrospinning process, such as applied voltage, needle tip-to-receiver distance and solution delivery rate, concentration or corresponding viscosity of spinning solution is one of the most effective variables for controlling fiber morphology and diameter. Results obtained from our study showed
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that solution concentration was found to be the major factor controlling the morphology of the fibers in the electrospinning of PU. Various PU solutions with concentration in the range of 5–11% (w/v, g/mL) in THF and DMF (1:1, v/v) were electrospun. A beadon-string morphology with several big beads was obtained at PU concentration of 5% (w/v). The distribution density of beads decreased when PU concentration increased to 6% (w/v). The shape of the beads became spindle-like. Smooth and homogeneous fibers without beads were produced when PU concentration reached 8% (w/v). As PU concentration increased from 9% to 11% (w/v), the electrospun fibers became thicker and more adhesive at various bonding sites, which led to a film-like character and structural integrity of the fibrous membranes. The effects of electrospinning solution parameters on the average diameter of PU nanofibers were investigated. As shown in Fig. 1a, viscosity of PU solutions increased with increasing PU concentration. However, both conductivity and surface tension of PU solutions with different concentrations did not differ much (Fig. 1b). The viscosity of a polymer solution reflects the intermolecular interactions between polymer chains. Polymer solutions with higher viscosity usually exhibit longer stress relaxation times, which may facilitate the formation of fibers with large diameters during electrospinning [34]. When the viscosity of PU solutions increased greatly from 0.186 Pa s up to 0.459 Pa s with increasing PU concentration from 9% to 11% (w/v), the average fiber diameter increased dramatically from 820 nm to 1.95 m (Fig. 1a). Therefore,
Fig. 4. (a) Tensile strength, (b) elongation at break and (c) Young’s modulus of PU fibrous membranes electrospun from solutions with different concentrations (both pristine membranes and those modified with poly(4-vinyl-N-hexylpyridinium bromide)).
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it was considered that the viscosity of solution was the major factor affecting the average diameter of electrospun PU nanofibers. 3.2. Surface modification of PU fibrous membranes The success of modification with poly(4-vinyl-N-hexyl pyridinium bromide) on electrospun PU fibrous membranes can be ascertained by comparing the XPS spectra of the pristine and modified membranes as shown in Fig. 2. The spectra of pristine PU fibrous membranes showed three main signals corresponding to C 1s (284.6 eV), O 1s (532 eV) and N 1s (400 eV) (Fig. 2a). The XPS C 1s core-level spectrum of pristine PU fibrous membrane (Fig. 2b) was resolved into three component peaks: a hydrocarbon environment (C–H, C–C, 284.6 eV), a carbon singly bound to oxygen environment (C–O, 286.0 eV), and a carbon in a carbamate environment (–CO–, 289.0 eV) [35]. The corresponding N 1s spectrum (Fig. 2c) showed an intense peak at the binding energy (BE) of 399.5 eV attributable to the nitrogen (–NH–) in carbamate of PU. After UV-induced graft copolymerization of PVP onto the PU fibrous membranes, the C 1s core-level spectrum (Fig. 2e) showed an additional peak at 285.5 eV attributable to C–N species. The intensity of the peak assigned to C–O species became weaker and that of –CO– species was barely discernible compared with Fig. 2b. Similarly, the corresponding N 1s spectrum (Fig. 2f) showed an additional peak at the BE of 398.5 eV attributable to imine moiety (–N ) of the pyridine rings [36] and a decrease in the intensity of –NH– peak component compared with Fig. 2c. The extent of surface grafting of PVP can be estimated from the sensitivity factor corrected ratio of the total N 1s peak over the total C 1s peak, expressed as [N]/[C]. Previous work of surface graft copolymerization showed that graft concentration of PVP was affected by the monomer concentration [37]. In this experiment, 4VP concentration of 20% was chosen for effective surface graft copolymerization. As determined by XPS, the surface [N]/[C] ratio of the 4VP grafted onto PU fibrous membrane was 0.12, close to the value of 0.14, expected for the 4VP monomeric unit (C7 H7 N1 ). It indicated that the surface was almost completely covered by 4VP copolymers, which could provide abundant reactive sites for the subsequent quaternization. Fig. 2g–i shows the XPS spectra of the graft-copolymerized PU fibrous membrane N-alkylated with hexylbromide. No significant difference was observed in C 1s core-level spectrum before (Fig. 2e) and after (Fig. 2h) N-alkylation. The corresponding N 1s core-level
spectrum in Fig. 2i showed an additional peak at BE above 400 eV, attributable to the N+ groups of the pyridinium ions [38], which confirmed the derivatization of the –N groups by hexylbromide. On the basis of the [N+ ]/[N] ratio, the degree of alkylation of the pyridine rings on the PU fibrous membrane was around 50–60%. Fig. 3 shows the typical SEM images of PU fibrous membranes before and after surface modification. The morphologies of the surface modified PU fibrous membranes electrospun from 6%, 8% and 10% (w/v) solution changed slightly compared with those of pristine PU fibrous membranes. The result indicated that the fiber structures were maintained during the modification process, and no significant change in fiber diameter was observed. 3.3. Mechanical property PU fibrous membranes both pristine and modified with poly(4-vinyl-N-hexylpyridinium bromide) were tested for their mechanical integrity in terms of tensile strength, elongation at break and Young’s modulus, as indicated in Fig. 4. It can be seen that the pristine electrospun PU fibrous membranes had tensile strength of 3.27–11.8 MPa, elongation at break of 159.2–349.7%, and Young’s modulus of 1.78–3.73 MPa, depending on the concentration of polymer solution used for electrospinning. PU fibrous membranes electrospun from 7% (w/v) solution had the lowest tensile strength of 3.27 MPa (Fig. 4a). As the concentration increased to 8% (w/v), the tensile strength increased to 3.88 MPa. The Young’s modulus and elongation at break showed an increasing trend with concentration as well (Fig. 4b and c). This is mainly due to more interstices between the fibers with smaller diameters, resulting in a lower number of closely adjacent binding in the fibrous membranes. Since strong “sticky” binding interactions make a major contribution to the strength of the membranes, increasing the fiber diameters can lead to increases of tensile strength [39]. When PU concentration increased from 9% to 11% (w/v), the tensile strength of the pristine as-spun fibrous membranes increased significantly from 4.32 to 11.80 MPa, in agreement with the increase in fiber diameters as shown in Fig. 1a. On the other hand, the tensile strength and elongation at break of PU fibrous membranes decreased after surface modification, whereas the Young’s moduli did not show much change. As shown in Fig. 4a, the tensile strength of PU fibrous membranes electrospun from 7% (w/v) solution decreased from 3.27 to 1.99 MPa after modification, losing almost 40% of tensile strength. As the solution
Fig. 5. Viable cell numbers of (a) Staphylococcus aureus, and (b) Escherichia coli as a function time in contact with: () control, (䊉) pristine and () modified PU fibrous membranes. The cell number was determined by surface spread plate method.
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concentration increased, smaller decreases in the tensile strength were observed. With a concentration of 11% (w/v) which resulted in the largest diameter of fibers in PU fibrous membranes, the loss of tensile strength was approximately 16%. The results indicated that the surface modification may have less adverse effect on the tensile strength of fibrous membranes with larger diameters. In view of these results, the antibacterial assays were carried out with surface modified PU fibrous membranes electrospun from 10% (w/v) solutions in THF and DMF (1:1, v/v) in order to achieve a balance between mechanical properties and fibrous structures.
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3.4. Antibacterial activity Antibacterial efficacy of surface modified PU fibrous membranes with poly(4-vinyl-N-hexylpyridinium bromide) was investigated by estimating the number of viable bacteria cells in the S. aureus and E. coli suspension after being in contact with membranes for various periods of time, and the results were shown in Fig. 5. As expected, no significant loss of viable bacteria was detected in the control experiment (i.e. without the membranes). For pristine PU fibrous membranes, the viable cell numbers in bacteria suspen-
Fig. 6. SEM images of (a) and (d) filter paper (control), (b) and (e) pristine, and (c) and (f) modified PU fibrous membranes after immersed in PBS suspension of (a)–(c) S. aureus, or (d)–(f) E. coli at 107 cells/mL for 4 h. PU fibrous membranes were electrospun from 10% (w/v) solutions in THF and DMF (1:1, v/v).
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sions were similar to the control (Fig. 5a). The surface modified PU fibrous membranes had a high antibacterial efficacy for S. aureus, reaching 99.9% and 99.999% after 1 and 4 h contact, respectively. In comparison, the antibacterial efficacy for E. coli was 99.9% after 4 h contact (Fig. 5b), lower than that for S. aureus. Although the mechanism of the antibacterial activity of immobilized quaternary ammonium groups is not entirely clear. It has been hypothesized that these immobilized moieties disrupt the integrity of the cytoplasmic membrane to cause cell death, similar to the mechanism of free biocides [40]. The difference in antibacterial efficacy is postulated to be the result of the different cell membrane structures between S. aureus and E. coli bacteria. The multilayered cell envelope structure of Gram-negative bacteria may be more resistant to access by the bactericidal moieties to the inner membrane of the organism [15]. Fig. 6 shows the SEM images of different substrates after immersion in bacteria suspensions. Numerous distinguishable S. aureus cells can be observed on the filter paper as shown in Fig. 6a. The number of S. aureus cells on the pristine PU fibrous membranes was significantly less (Fig. 6b). The S. aureus was distributed not only on the pristine PU fibrous membrane upper surface but also entrapped in the space between the thin fibers. In contrast, very few sparsely distributed bacteria cells could be spotted over the entire surface of the modified PU fibrous membrane (Fig. 6c). Similar results were observed for E. coli on the surface of fibrous membranes as shown in Fig. 6d–f. The results indicated that both filter paper and pristine PU fibrous membranes are good templates for the proliferation of bacteria and biofilm formation may occur readily on such surfaces in contact with bacteria. The presence of quaternary ammonium groups attached to the surface of PU fibrous membranes was thus demonstrated to be very effective in preventing biofilm formation. Since the interstitial spaces of the pristine PU fibrous membranes are large enough, it can be expected that some bacteria would have penetrated into the fibrous membranes during immersion in the bacteria suspension. Therefore, the bacteria retained in fibrous membranes were dislodged by mild ultrasonication and the number of viable cells was counted by surface spread plate method. For the pristine PU fibrous membranes, the mean number of S. aureus and E. coli cells relative to the surface area was (9.03 ± 0.63) × 104 and (1.65 ± 0.24) × 105 CFU/cm2 , respectively. For the modified PU fibrous membranes, the corresponding numbers were 735 ± 35 of S. aureus and 2555 ± 315 CFU/cm2 of E. coli cells. Since very few bacteria cells could be observed on the surface of the modified PU fibrous membrane (Fig. 6c), the bacteria may be entrapped deep in the membrane either on fibers which have lower concentration of the antibacterial N-alkyl pyridinium units or on dead bacteria deposited on the fibers which provided a shielding effect from the N-alkyl pyridinium groups. 4. Conclusions The surface of electrospun PU fibrous membranes was successfully modified with poly(4-vinyl-N-hexyl pyridinium bromide) using a method involving plasma pretreatment, UV-induced surface graft copolymerization and N-alkylation reaction. The morphologies of PU fibrous membranes changed slightly and the fiber structures were maintained after the modification process. The tensile strength and elongation at break of modified PU fibrous membranes decreased, whereas the Young’s moduli showed no significant change. Antibacterial assays showed that the modified PU fibrous membranes possessed highly effective antibacterial activities against both Gram-positive S. aureus and Gram-negative E. coli. The novel antibacterial PU fibrous membranes may have a wide variety of potential applications in high-performance filters, protective textiles, and biomedical devices.
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Contents lists available at ScienceDirect
Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci
Surface modification and antibacterial activity of electrospun polyurethane fibrous membranes with quaternary ammonium moieties Chen Yao a,b , Xinsong Li a,∗ , K.G. Neoh b,∗ , Zhilong Shi b , E.T. Kang b a b
School of Chemistry and Chemical Engineering, Southeast University, Sipailou 2, Nanjing 210018, PR China Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, 119260 Singapore, Singapore
a r t i c l e
i n f o
Article history: Received 26 February 2008 Received in revised form 3 April 2008 Accepted 5 April 2008 Available online 15 April 2008 Keywords: Electrospinning Antibacterial Surface modification Polyurethane
a b s t r a c t A novel antibacterial material was developed by surface modification of electrospun polyurethane (PU) fibrous membranes, using a process which involved plasma pretreatment, UV-induced graft copolymerization of 4-vinylpyridine (4VP), and quaternization of the grafted pyridine groups with hexylbromide. The success of modification with poly(4-vinyl-N-hexyl pyridinium bromide) groups on these was ascertained by X-ray photoelectron spectroscopy (XPS). The morphologies and mechanical properties were investigated by scanning electron microscopy (SEM) and tensile test, respectively. The results showed that the morphologies of PU fibrous membranes changed slightly during the modification process and the fiber structures were maintained. The tensile strength of PU fibrous membranes decreased after surface modification, with the smallest decrease (<20%) observed in the membrane with largest diameter. The antibacterial activities of the modified PU fibrous membranes were assessed against Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli). The modified PU fibrous membranes possessed highly effective antibacterial activities and may have a wide variety of potential applications in high-performance filters, protective textiles, and biomedical devices. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In recent years, considerable attention has been paid to electrospinning as a versatile technique for producing fibers with diameters ranging from submicron to a few microns. In the typical electrospinning process, nanofibers are produced from an electrostatically driven jet of polymer solution (or melt). The discharged polymer solution jet undergoes a whipping process wherein the solvent evaporates and the highly stretched polymer fiber deposits on a grounded collector. The non-woven mats of electrospun fibers have shown unique properties, such as dramatically increased surface/volume ratios, excellent mechanical strength, and highly open porous structures. In the past few years, a wide range of synthetic and natural polymers in pure or blend solutions, as well as melts, have been electrospun to form fibers [1–4]. Thermoplastic polyurethane (PU) is a resilient elastomer of significant industrial importance which possesses a range of desirable properties such as elastomeric, resistant to abrasion, and excellent hydrolytic stability [5,6]. Numerous studies on the electrospinning of PU have been conducted [7–9]. Electrospun PU nanofiber mats exhibiting good mechanical properties may have a wide variety of potential appli-
∗ Corresponding authors. Tel.: +86 25 8379 3456; fax: +86 25 8379 3456. E-mail addresses: [email protected] (X. Li), [email protected] (K.G. Neoh). 0376-7388/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2008.04.012
cations in high-performance air filters, protective textiles, wound dressing materials, sensors, etc. [10–13]. With the growing public health awareness of the pathogenic effects, malodors and stain formations caused by microorganisms, there is an increasing need for antibacterial materials in many application areas like medical devices, health care, hygienic application, water purification systems, hospital, dental surgery equipment, textiles, food packaging, and storage [14,15]. A number of functional nanofibers and composites have been developed through electrospinning by incorporating well-selected functional agents to achieve antibacterial properties. Two common methods are incorporation of antibacterial agents in the electrospinning solutions [16,17], and electrospinning of polymers with intrinsic antibacterial properties such as quaternized chitosan [18]. Kim et al. reported that the sustained release of hydrophilic antibiotic drug (Mefoxin) from electrospun poly(lactide-co-glycolide)-based nanofibrous scaffolds was effective to inhibit Staphylococcus aureus growth (>90%) [19]. Electrospun cellulose acetate fibers containing silver nanoparticles showed strong antimicrobial activity against both Gram-negative and Gram-positive bacteria [20,21]. Kenawy et al. modified the poly(vinyl phenol) either by sulphonation or by formation of lithium salt of the sulphonated species, and investigated the antibacterial activities of the modified poly(vinyl phenol) electrospun mats [22]. Polyurethane cationomers polymerized from base PU with chain extenders having a quaternary ammonium
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group were electrospun into non-woven nanofiber mats for antimicrobial nanofilter applications [23]. A more straightforward way is to modify the surface of polymer nanofibers without affecting bulk properties of the treated nanofibers. The methods used to impart surface modification usually depend strongly on the nature of the fiber-forming polymer and include, but are not limited to, covalent polymer grafting [24], plasma treatment [25], physisorption (e.g., hydrogen-bonding interactions) [26], chemisorption [27], and chemical derivatization [28]. Among the various methods, plasma treatment provides a clean and environmentally friendly way for surface modification [29]. The free radicals and electrons created in the plasma treatment could be used to modify the polymer nanofibers chemically. Covalent attachment of functional compounds to polymer fiber surfaces is the preferred approach to introduce functionalities permanently and at reasonably high efficiency. There are numerous antimicrobials suitable for immobilization on polymer surfaces. Quaternary ammonium compounds seem attractive because their target is primarily the microbial membrane and they accumulate in the cell driven by the membrane potential [30]. To maximize efficiency, quaternary ammonium compound is used as monomeric link in the polymeric leash and poly(4vinylpyridine) (PVP) is usually selected as the carrying polymer. Tiller et al. showed that the surfaces of commercial polymers treated with N-alkylated PVP groups were lethal on contact to both Gram-positive and Gram-negative bacteria, and it was also shown that N-alkyl chain of six carbon units in length was the most effective [31]. The purpose of this paper was to develop novel antibacterial PU fibrous membranes by electrospinning the polymer followed by plasma pretreatment, UV-induced graft copolymerization and quaternization reaction. Electrospun PU fibrous membranes were modified with poly(4-vinyl-N-hexyl pyridinium bromide) on the surfaces to achieve antibacterial activities. The modified PU fibrous membranes were subsequently characterized in terms of their morphologies, surface chemical compositions and mechanical properties. The antibacterial activities of the fibrous membranes were assessed against both Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli). 2. Experimental 2.1. Materials Polyurethane elastomer, Elastollan® 1180A10, was received from BASF. 4-Vinylpyridine (4VP) monomer was obtained from Aldrich Chemical Co. and freshly distilled under reduced pressure before use. Hexylbromide and solvents, such as tetrahydrofuran (THF), N,N-dimethyl formamide (DMF), heptane, 2-propanol, were of reagent grade and used as received from Aldrich Chemical Co. Peptone, yeast extract, agar and beef extract were purchased from Oxoid. S. aureus (ATCC 25923) and E. coli (ATCC DH5␣) were obtained from American Type Culture Collection.
at a delivery rate of 6 mL/h. After high voltage ranging from 10 to 12 kV was applied to the needle, a positive charged jet of PU solution formed from the Taylor cone and sprayed to a grounded drum, approximately 10 cm from the needle tip. With the evaporation of solvent, PU fibers were deposited on the drum to form fibrous membrane. The as-spun fibrous membrane was then dried under vacuum and annealed at 80 ◦ C for 6 h. All electrospinning experiments were carried out at 20 ◦ C and relative humidity of 50%. 2.3. Surface modification of PU fibrous membranes PU fibrous membranes were subjected to argon plasma pretreatment in an Anatech SP100 plasma system, equipped with a cylindrical quartz reactor chamber. The glow discharge was produced at a plasma power of 35 W, an applied oscillator frequency of 40 kHz and an argon pressure of approximately 80 Pa. After subjected to glow discharge for 60 s on both surfaces, the fibrous membrane was exposed to air for 5 min to facilitate formation of surface oxide and peroxide groups. The plasma pretreated fibrous membranes were immersed in 2-propanol solution of 20 vol.% 4VP in a Pyrex glass tube. Degassing of the solution was achieved by bubbling nitrogen vigorously for 30 min and sealing the tube with silicon rubber stopper. The tube was then exposed to UV irradiation in a Riko rotary photochemical reactor (RH400-10W) for 60 min on each surface. The graft-copolymerized fibrous membranes were subjected to washing with copious amounts of 2-propanol to remove residual monomer and physically adsorbed homopolymer. The fibrous membranes with graft-copolymerized 4VP were then placed in heptane solutions containing 20 vol.% hexylbromide and the reaction mixture was stirred for 48 h at 60 ◦ C. The fibrous membranes were then thoroughly rinsed with heptane and dried under vacuum. 2.4. Surface characterization The morphology of PU fibrous membranes was observed with a scanning electron microscope (JEOL JSM 5600LV) after gold sputtercoating. Diameters of the electrospun fibers were measured directly from SEM images, with an average value being calculated from at least 100 measurements. Surface compositions of PU fibrous membranes were analyzed by XPS on an AXIS HSi spectrometer (Kratos Analytical Ltd.) using the monochromatized Al K␣ X-ray source (1486.6 eV photons) at a constant dwell time of 100 ms and a pass energy of 40 eV. The anode voltage was 15 kV, and the anode current was 10 mA. The core-level signals were obtained at a photoelectron takeoff angle of 90◦ (with respect to the membrane surface). To compensate for surface charging effect, all binding energies (BEs) were referenced to the C 1s hydrocarbon peak at 284.6 eV. In the peak synthesis, the line width (full width at half-maximum) of the Gaussian peaks was maintained constant for all components in a particular spectrum. The peak area ratios for the various elements were corrected using experimentally determined instrumental sensitivity factors.
2.2. Electrospinning of PU 2.5. Mechanical property PU was dissolved in a mixed solvent of THF and DMF (1:1, v/v) to produce various spinning solutions with PU concentrations ranging from 5% to 11% (w/v, g/mL). Viscosity, conductivity and surface tension of the prepared solutions were measured with a viscometer (NDJ-9S, ShangPing), a conductivity meter (DDB-303A, Rex) and a surface tension meter (BZY-1, HengPing) at 20 ◦ C, respectively. A syringe pump was used to feed the polymer solution through a 20mL plastic syringe fitted with a needle of tip diameter of 0.6 mm
Tensile tests of PU fibrous membranes were performed using an Instron universal materials testing machine (Model 5544) with a 10 N load cell in a constant relative humidity (50%) room at 25 ◦ C. “Dog-bone” shaped samples were cut from the fibrous membranes (5-mm wide at the narrowest point with a gage length of 15 mm). The thickness of these samples was measured with a digital micrometer having a precision of 1 m. A cross-head speed of
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Fig. 1. Electrospinning solution parameters and average diameter of the as-spun fibers with different PU concentrations.
10 mm/min was used and at least five samples were tested for each type of the fibrous membranes. 2.6. Determination of antibacterial activity S. aureus was incubated in 10 mL of a 3.1% yeast–dextrose broth (containing 10 g/L peptone, 8 g/L beef extract, 5 g/L sodium chloride, 5 g/L glucose, and 3 g/L yeast extract at a pH 6.8) for 6–8 h at 37 ◦ C until the exponential growth phase was reached [32]. The bacteria-containing broth was centrifuged at 3000 rpm for 10 min, and after removal of supernatant, the cells were washed twice with sterile phosphate-buffered solution (PBS). The bacteria
cells were resuspended to provide a final density of 106 cells/mL in PBS (based on standard calibration with the assumption that the optical density of 1.0 at 540 nm is equivalent to approximately 109 cells/mL) [33]. PU fibrous membranes electrospun from 10% (w/v) solutions in THF and DMF (1:1, v/v) (either pristine or modified, 8 mm × 8 mm) were sterilized with ultraviolet irradiation for 30 min, and then immersed in 10 mL of the bacterial suspension in an Erlenmeyer flask and shaken at 200 rpm at 37 ◦ C. The viable cell counts of bacteria were measured by surface spread plate method. At the predetermined time, 1 mL of bacteria culture was taken from the flask and decimal serial dilutions with PBS were repeated with each
Fig. 2. XPS wide scans of (a) pristine PU fibrous membranes, (d) PU membranes grafted with PVP, and (g) PU membranes modified with poly(4-vinyl-N-hexylpyridinium bromide); C 1s core-level spectra of (b) pristine PU fibrous membranes, (e) PU membranes grafted with PVP, and (h) PU membranes modified with poly(4-vinyl-Nhexylpyridinium bromide); N 1s core-level spectra of (c) pristine PU fibrous membranes, (f) PU membranes grafted with PVP, and (i) PU membranes modified with poly(4-vinyl-N-hexylpyridinium bromide). PU fibrous membranes were electrospun from 10% (w/v) PU solutions in THF and DMF (1:1, v/v).
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initial sample. A 0.1-mL drop of the diluted sample was then spread onto solid growth agar plates. After incubation of the plates at 37 ◦ C for 24 h, the number of viable cells (colonies) was counted manually, and expressed as mean colony-forming units per milliliter (CFU/mL) after multiplication with the dilution factor. After 4 h, the PU fibrous membranes were removed from the bacterial suspension with sterile forceps and gently washed with PBS. The bacteria retained on membranes were dislodged by mild ultrasonication (for 10 min) in a 100 W ultrasonic bath. Serial 10-fold dilutions were per-
formed and viable counts estimated following the surface spread plate method. The number of colony-forming units on each membrane surface was computed and expressed relative to the apparent surface area of the membrane (CFU/cm2 ). All experiments were performed in triplicate and the quantitative value was expressed as the average ± standard deviation. The extent of bacterial adhesion on PU fibrous membranes (both pristine and modified) were also assessed by examining these membranes after 4 h immersion in the PBS suspension of
Fig. 3. SEM images of (a), (c) and (e) pristine, (b), (d) and (f) modified PU fibrous membranes with poly(4-vinyl-N-hexylpyridinium bromide). PU fibrous membranes were electrospun from (a) and (b) 6%, (c) and (d) 8%, (e) and (f) 10% (w/v) solutions in THF and DMF (1:1, v/v).
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107 cells/mL S. aureus. Control experiment was carried out with the filter paper. The membranes were then examined under SEM (JEOL JSM 5600LV) to assess the adhesion and viability of the bacteria. The membrane fixation and preparation for SEM were as follows: the membranes were first washed with PBS and 3 vol.% glutaraldehyde in PBS was added for 5 h and stored at 4 ◦ C. The glutaraldehyde solution was then removed and the membranes were washed with PBS, followed by step dehydration with 25%, 50%, 70%, 95%, and 100% ethanol for 10 min each. The membranes were dried under vacuum and gold sputter-coated before SEM observation. For E. coli, the same assay procedures were used as those described above for S. aureus.
3. Results and discussion 3.1. Electrospinning of PU While electrospinning has proven to be a versatile and powerful means of fabricating polymer micro/nanofibers, its applicability to obtain smooth, uniform fibrous structure is not straightforward. Among various parameters of electrospinning process, such as applied voltage, needle tip-to-receiver distance and solution delivery rate, concentration or corresponding viscosity of spinning solution is one of the most effective variables for controlling fiber morphology and diameter. Results obtained from our study showed
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that solution concentration was found to be the major factor controlling the morphology of the fibers in the electrospinning of PU. Various PU solutions with concentration in the range of 5–11% (w/v, g/mL) in THF and DMF (1:1, v/v) were electrospun. A beadon-string morphology with several big beads was obtained at PU concentration of 5% (w/v). The distribution density of beads decreased when PU concentration increased to 6% (w/v). The shape of the beads became spindle-like. Smooth and homogeneous fibers without beads were produced when PU concentration reached 8% (w/v). As PU concentration increased from 9% to 11% (w/v), the electrospun fibers became thicker and more adhesive at various bonding sites, which led to a film-like character and structural integrity of the fibrous membranes. The effects of electrospinning solution parameters on the average diameter of PU nanofibers were investigated. As shown in Fig. 1a, viscosity of PU solutions increased with increasing PU concentration. However, both conductivity and surface tension of PU solutions with different concentrations did not differ much (Fig. 1b). The viscosity of a polymer solution reflects the intermolecular interactions between polymer chains. Polymer solutions with higher viscosity usually exhibit longer stress relaxation times, which may facilitate the formation of fibers with large diameters during electrospinning [34]. When the viscosity of PU solutions increased greatly from 0.186 Pa s up to 0.459 Pa s with increasing PU concentration from 9% to 11% (w/v), the average fiber diameter increased dramatically from 820 nm to 1.95 m (Fig. 1a). Therefore,
Fig. 4. (a) Tensile strength, (b) elongation at break and (c) Young’s modulus of PU fibrous membranes electrospun from solutions with different concentrations (both pristine membranes and those modified with poly(4-vinyl-N-hexylpyridinium bromide)).
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it was considered that the viscosity of solution was the major factor affecting the average diameter of electrospun PU nanofibers. 3.2. Surface modification of PU fibrous membranes The success of modification with poly(4-vinyl-N-hexyl pyridinium bromide) on electrospun PU fibrous membranes can be ascertained by comparing the XPS spectra of the pristine and modified membranes as shown in Fig. 2. The spectra of pristine PU fibrous membranes showed three main signals corresponding to C 1s (284.6 eV), O 1s (532 eV) and N 1s (400 eV) (Fig. 2a). The XPS C 1s core-level spectrum of pristine PU fibrous membrane (Fig. 2b) was resolved into three component peaks: a hydrocarbon environment (C–H, C–C, 284.6 eV), a carbon singly bound to oxygen environment (C–O, 286.0 eV), and a carbon in a carbamate environment (–CO–, 289.0 eV) [35]. The corresponding N 1s spectrum (Fig. 2c) showed an intense peak at the binding energy (BE) of 399.5 eV attributable to the nitrogen (–NH–) in carbamate of PU. After UV-induced graft copolymerization of PVP onto the PU fibrous membranes, the C 1s core-level spectrum (Fig. 2e) showed an additional peak at 285.5 eV attributable to C–N species. The intensity of the peak assigned to C–O species became weaker and that of –CO– species was barely discernible compared with Fig. 2b. Similarly, the corresponding N 1s spectrum (Fig. 2f) showed an additional peak at the BE of 398.5 eV attributable to imine moiety (–N ) of the pyridine rings [36] and a decrease in the intensity of –NH– peak component compared with Fig. 2c. The extent of surface grafting of PVP can be estimated from the sensitivity factor corrected ratio of the total N 1s peak over the total C 1s peak, expressed as [N]/[C]. Previous work of surface graft copolymerization showed that graft concentration of PVP was affected by the monomer concentration [37]. In this experiment, 4VP concentration of 20% was chosen for effective surface graft copolymerization. As determined by XPS, the surface [N]/[C] ratio of the 4VP grafted onto PU fibrous membrane was 0.12, close to the value of 0.14, expected for the 4VP monomeric unit (C7 H7 N1 ). It indicated that the surface was almost completely covered by 4VP copolymers, which could provide abundant reactive sites for the subsequent quaternization. Fig. 2g–i shows the XPS spectra of the graft-copolymerized PU fibrous membrane N-alkylated with hexylbromide. No significant difference was observed in C 1s core-level spectrum before (Fig. 2e) and after (Fig. 2h) N-alkylation. The corresponding N 1s core-level
spectrum in Fig. 2i showed an additional peak at BE above 400 eV, attributable to the N+ groups of the pyridinium ions [38], which confirmed the derivatization of the –N groups by hexylbromide. On the basis of the [N+ ]/[N] ratio, the degree of alkylation of the pyridine rings on the PU fibrous membrane was around 50–60%. Fig. 3 shows the typical SEM images of PU fibrous membranes before and after surface modification. The morphologies of the surface modified PU fibrous membranes electrospun from 6%, 8% and 10% (w/v) solution changed slightly compared with those of pristine PU fibrous membranes. The result indicated that the fiber structures were maintained during the modification process, and no significant change in fiber diameter was observed. 3.3. Mechanical property PU fibrous membranes both pristine and modified with poly(4-vinyl-N-hexylpyridinium bromide) were tested for their mechanical integrity in terms of tensile strength, elongation at break and Young’s modulus, as indicated in Fig. 4. It can be seen that the pristine electrospun PU fibrous membranes had tensile strength of 3.27–11.8 MPa, elongation at break of 159.2–349.7%, and Young’s modulus of 1.78–3.73 MPa, depending on the concentration of polymer solution used for electrospinning. PU fibrous membranes electrospun from 7% (w/v) solution had the lowest tensile strength of 3.27 MPa (Fig. 4a). As the concentration increased to 8% (w/v), the tensile strength increased to 3.88 MPa. The Young’s modulus and elongation at break showed an increasing trend with concentration as well (Fig. 4b and c). This is mainly due to more interstices between the fibers with smaller diameters, resulting in a lower number of closely adjacent binding in the fibrous membranes. Since strong “sticky” binding interactions make a major contribution to the strength of the membranes, increasing the fiber diameters can lead to increases of tensile strength [39]. When PU concentration increased from 9% to 11% (w/v), the tensile strength of the pristine as-spun fibrous membranes increased significantly from 4.32 to 11.80 MPa, in agreement with the increase in fiber diameters as shown in Fig. 1a. On the other hand, the tensile strength and elongation at break of PU fibrous membranes decreased after surface modification, whereas the Young’s moduli did not show much change. As shown in Fig. 4a, the tensile strength of PU fibrous membranes electrospun from 7% (w/v) solution decreased from 3.27 to 1.99 MPa after modification, losing almost 40% of tensile strength. As the solution
Fig. 5. Viable cell numbers of (a) Staphylococcus aureus, and (b) Escherichia coli as a function time in contact with: () control, (䊉) pristine and () modified PU fibrous membranes. The cell number was determined by surface spread plate method.
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concentration increased, smaller decreases in the tensile strength were observed. With a concentration of 11% (w/v) which resulted in the largest diameter of fibers in PU fibrous membranes, the loss of tensile strength was approximately 16%. The results indicated that the surface modification may have less adverse effect on the tensile strength of fibrous membranes with larger diameters. In view of these results, the antibacterial assays were carried out with surface modified PU fibrous membranes electrospun from 10% (w/v) solutions in THF and DMF (1:1, v/v) in order to achieve a balance between mechanical properties and fibrous structures.
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3.4. Antibacterial activity Antibacterial efficacy of surface modified PU fibrous membranes with poly(4-vinyl-N-hexylpyridinium bromide) was investigated by estimating the number of viable bacteria cells in the S. aureus and E. coli suspension after being in contact with membranes for various periods of time, and the results were shown in Fig. 5. As expected, no significant loss of viable bacteria was detected in the control experiment (i.e. without the membranes). For pristine PU fibrous membranes, the viable cell numbers in bacteria suspen-
Fig. 6. SEM images of (a) and (d) filter paper (control), (b) and (e) pristine, and (c) and (f) modified PU fibrous membranes after immersed in PBS suspension of (a)–(c) S. aureus, or (d)–(f) E. coli at 107 cells/mL for 4 h. PU fibrous membranes were electrospun from 10% (w/v) solutions in THF and DMF (1:1, v/v).
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sions were similar to the control (Fig. 5a). The surface modified PU fibrous membranes had a high antibacterial efficacy for S. aureus, reaching 99.9% and 99.999% after 1 and 4 h contact, respectively. In comparison, the antibacterial efficacy for E. coli was 99.9% after 4 h contact (Fig. 5b), lower than that for S. aureus. Although the mechanism of the antibacterial activity of immobilized quaternary ammonium groups is not entirely clear. It has been hypothesized that these immobilized moieties disrupt the integrity of the cytoplasmic membrane to cause cell death, similar to the mechanism of free biocides [40]. The difference in antibacterial efficacy is postulated to be the result of the different cell membrane structures between S. aureus and E. coli bacteria. The multilayered cell envelope structure of Gram-negative bacteria may be more resistant to access by the bactericidal moieties to the inner membrane of the organism [15]. Fig. 6 shows the SEM images of different substrates after immersion in bacteria suspensions. Numerous distinguishable S. aureus cells can be observed on the filter paper as shown in Fig. 6a. The number of S. aureus cells on the pristine PU fibrous membranes was significantly less (Fig. 6b). The S. aureus was distributed not only on the pristine PU fibrous membrane upper surface but also entrapped in the space between the thin fibers. In contrast, very few sparsely distributed bacteria cells could be spotted over the entire surface of the modified PU fibrous membrane (Fig. 6c). Similar results were observed for E. coli on the surface of fibrous membranes as shown in Fig. 6d–f. The results indicated that both filter paper and pristine PU fibrous membranes are good templates for the proliferation of bacteria and biofilm formation may occur readily on such surfaces in contact with bacteria. The presence of quaternary ammonium groups attached to the surface of PU fibrous membranes was thus demonstrated to be very effective in preventing biofilm formation. Since the interstitial spaces of the pristine PU fibrous membranes are large enough, it can be expected that some bacteria would have penetrated into the fibrous membranes during immersion in the bacteria suspension. Therefore, the bacteria retained in fibrous membranes were dislodged by mild ultrasonication and the number of viable cells was counted by surface spread plate method. For the pristine PU fibrous membranes, the mean number of S. aureus and E. coli cells relative to the surface area was (9.03 ± 0.63) × 104 and (1.65 ± 0.24) × 105 CFU/cm2 , respectively. For the modified PU fibrous membranes, the corresponding numbers were 735 ± 35 of S. aureus and 2555 ± 315 CFU/cm2 of E. coli cells. Since very few bacteria cells could be observed on the surface of the modified PU fibrous membrane (Fig. 6c), the bacteria may be entrapped deep in the membrane either on fibers which have lower concentration of the antibacterial N-alkyl pyridinium units or on dead bacteria deposited on the fibers which provided a shielding effect from the N-alkyl pyridinium groups. 4. Conclusions The surface of electrospun PU fibrous membranes was successfully modified with poly(4-vinyl-N-hexyl pyridinium bromide) using a method involving plasma pretreatment, UV-induced surface graft copolymerization and N-alkylation reaction. The morphologies of PU fibrous membranes changed slightly and the fiber structures were maintained after the modification process. The tensile strength and elongation at break of modified PU fibrous membranes decreased, whereas the Young’s moduli showed no significant change. Antibacterial assays showed that the modified PU fibrous membranes possessed highly effective antibacterial activities against both Gram-positive S. aureus and Gram-negative E. coli. The novel antibacterial PU fibrous membranes may have a wide variety of potential applications in high-performance filters, protective textiles, and biomedical devices.
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