Colloid Polym Sci (2009) 287:14251434 DOI 10.

1007/s00396-009-2108-y

ORIGINAL CONTRIBUTION

Preparation and antibacterial activity of electrospun chitosan/poly(ethylene oxide) membranes containing silver nanoparticles
Jing An & Hong Zhang & Jitu Zhang & Yunhui Zhao & Xiaoyan Yuan

Received: 19 May 2009 / Revised: 2 August 2009 / Accepted: 28 August 2009 / Published online: 16 September 2009 # Springer-Verlag 2009

Abstract Fairly uniform chitosan (CS)/poly(ethylene oxide) (PEO) ultrafine fibers containing silver nanoparticles (AgNPs) were successfully prepared by electrospinning of CS/PEO solutions containing Ag/CS colloids by means of in situ chemical reduction of Ag ions. The presence of AgNPs in the electrospun ultrafine fibers was confirmed by X-ray diffraction patterns. The AgNPs were evenly distributed in CS/PEO ultrafine fibers with the size less than 5 nm observed under a transmission electron microscope. X-ray photoelectron spectroscopy suggested that the existence of AgO bond in the composite ultrafine fibers led to the tight combination between Ag and CS. Evaluation of antimicrobial activities of the electrospun Ag/CS/PEO fibrous membranes against Escherichia coli showed that the AgNPs in the ultrafine fibers significantly enhanced the inactivation of bacteria.

Keywords Silver nanoparticles . Chitosan . Poly(ethylene oxide) . Electrospinning . Antibacterial activity

Introduction In recent years, electrospinning, as a simple technique to produce ultrafine fibers with diameters ranging from several microns down to tens of nanometers, has attracted great attention. This promising technique can consistently generate nonwoven fibrous membranes by imposing an external electric field on a polymer solution or melt [1, 2]. The fibrous mats have shown several distinctive properties, such as high-specific surface and porosity [3, 4], which can mimic the nanosized features of natural extracellular matrix (ECM) and prove to be excellent candidates for various biomedical applications such as wound dressings [5, 6], biosensor [79], and scaffolds for tissue engineering [10, 11]. Because of intrinsic biocompatibility and biodegradability, fibrous mats of chitosan (CS), the N-deacetylated derivative of chitin, were often utilized for wound-healing [12, 13]. Electrospun CS membranes could enhance wound healing by mimicking the ECM and promote normal tissue regeneration by achieving homeostasis [14, 15]. As electrospinning of pure CS was hindered by its limited solubility and interaction of inter- and intra-chain hydrogen bonding [16], synthetic polymers such as poly (ethylene oxide) (PEO) [17, 18] and poly(vinyl alcohol) (PVA) [19] were often introduced the formation of the hybrid ultrafine fibers.

Supported by the foundation of Hebei University of Science and Technology (No. XL200819) J. An College of Sciences, Hebei University of Science and Technology, Hebei( Shijiazhuang 050018, China J. An : H. Zhang : J. Zhang : Y. Zhao : X. Yuan (*) School of Materials Science and Engineering, and Tianjin Key Laboratory of Composite and Functional Materials, Tianjin University, Tianjin 300072, China e-mail: yuanxy@tju.edu.cn

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Ag nanoparticles (AgNPs) have long been a broad spectrum and highly effective antimicrobial agent in biomedical applications for treating wounds and burns on the benefit of its surface plasma resonance. Since AgNPs are relatively stable, electrospun AgNP-containing membranes were recognized as safe materials with strong inhibitory and bactericidal effects, in comparison with those containing ionic Ag which may cause the discoloration of ultrafine fibers and the skin [20]. A few AgNPcontaining electrospun membranes have been reported in the literatures. Jin et al. prepared Ag/Poly(vinyl pyrrolidone) (PVP) ultrafine fibers electrospun from the PVP solutions containing AgNPs directly or a reducing agent for the Ag ions [21]. Hong et al. reported that PVA ultrafine fibers containing AgNPs were prepared by electrospinning of PVA/silver nitrate (AgNO3) aqueous solutions, followed by short-time heat treatment [22]. Dong et al. had demonstrated in situ electrospinning method to fabricate semiconductor (Ag2S) nanostructure on the outer surfaces of PAN nanofibers. For the remarkable optical (electronic) properties of Ag2S nanostructures, the products can be used in fabricating optical and electronic devices [23]. Because of the unique cationic-polyelectrolyte character and gel-forming ability, CS could bind metal ions by electrostatic attraction [24]. It has been widely accepted that protonated amine and hydroxide sites are the main reactive sites for interaction with metal ions. AgNPs in a good dispersion state could be synthesized with the aid of CS [25]. The introduction of AgNPs in the CS ultrafine fibers by electrospinning was few reported. In the present work, ultrafine fibrous membranes of Ag/CS/PEO were obtained by electrospinning of the CS/PEO solutions in aqueous acetic acid containing Ag/CS colloids which was synthesized by in situ chemical reduction with AgNO3. The conductivity, surface tension, and viscosity of the Ag/CS/PEO dispersion were measured. Effects of a variety of parameters on the morphology of ultrafine fibers were studied and the fibrous membranes were analyzed by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). Moreover, their antimicrobial activities were investigated for wound-dressing applications.

by Sigma Co. Ltd., USA. AgNO3 and NaBH4 were obtained from Shanghai Sanpu Chemical Co., Ltd., China. Other chemicals were all analytical grade and used as received without further purification. Acetic acid was diluted to a 2% (w/v) aqueous solution before use. Escherichia coli (ATCC 44752) was purchased from Beijing Center for Disease Prevention and Control. Preparation of Ag/CS colloids Ag/CS colloid was prepared by chemical reduction with AgNO3 as precursor. Firstly, CS was dissolved in a 2% (w/v) acetic acid solution to form a clear solution with CS concentration of 5% (w/v). Secondly, 1 mL of AgNO3 solution with the concentration range of 0.10.4 mol/L was added into the above 20 mL CS solution under stirring at 30 C. Finally, the freshly prepared NaBH4 solution was quickly added into the above mixture. In order to complete the chemical reduction, the amount of NaBH4 was used in three times of that of AgNO3. The resultant Ag/CS colloids were kept at room temperature for further uses. Properties of Ag/CS/PEO solutions for electrospinning Surface tension of Ag/CS/PEO solution was determined in the Wilhelmy plate method with a tensionmeter (Model DCAT 21, Dataphysics Co., Ltd., Germany), while the clear platinum plate was used. The viscosity of the solutions was measured in a rotating rheometer (Stress Tech, Stress Tech Fluids Rheometer Co., Ltd., Sweden). Electric conductivity of the solutions was tested in a conductivity instrument (Model DDS-11A, Shanghai, China). Electrospinning of Ag/CS/PEO ultrafine fibers Ag/CS/PEO solutions in 5 wt.% polymer concentration were prepared by dissolving Ag/CS colloid and PEO in the aqueous 2 wt.% acetic acid solution. The mass ratio of CS to PEO was selected as 1:1, 1.5:1, and 2:1, respectively. The plain CS/PEO solution in the same concentration was prepared as a control. The prepared solution was driven out from a syringe with a metal capillary (ID=0.8 mm), which was connected to a highvoltage power supply. The flow rate of the polymer solution was controlled by a syringe pump (KI-754390005, Cole-Parmer Instrument). The electrospun ultrafine fibers were typically obtained at 10-kV voltage, 15-cm capillary-collector distance, and 0.1-mL/h flow rate. After cross-linking by 25% (w/v) glutaraldehyde aqueous solution at 37 C for 6 h, the electrospun nanofibrous membranes were kept in a desiccator characterization.

Experimental methods Materials A sample of chitosan CS (deacetylation degree 80%, M w = 2.0105) was purchased from Yu Huan Ocean Biochemical Co. Ltd., China. PEO (M w =1.5106) was kindly donated

Colloid Polym Sci (2009) 287:14251434

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Glutaraldehyde is prone to be contacted by nucleophile reagents, so the amine and hydroxyl groups, which are nucleophilic, on CS glucosamine rings can react with glutaraldehyde to form cross-linking structure. There are mainly two kinds of cross-linking reaction: one is Schiff base reaction between C2 amine groups of CS and carbonyl groups on glutaraldehyde, which is predominant during cross-linking, the other is acetalization reaction between C6 hydroxyl groups of CS and carbonyl groups, which is insignificant. Characterizations For microstructure studies of the electrospun nanofibrous membranes, samples were sputter-coated with gold and observed using a scanning electron microscope (SEM, Philips XL-30). The average diameter of the electrospun fibers was measured from SEM micrographs in the original magnification of 10,000 using Adobe Photoshop 7.0 software. Transmission electron microscopy (TEM) micrographs were obtained by using Tecnai G2 F20 with samples deposited on carbon-coated copper grids. XRD patterns were recorded at 40 kV and 100 mA on Rigaku D/MAX-2500 diffractometer using Cu Ka radiation ( =0.15406 nm) with the diffraction angle range 2 =1090. XPS analysis was carried out in a Perkin-Elmer PHI-1600 spectrometer using Mg K radiation (1,253.6 eV, 250 W), and the vacuum inside the analysis chamber was 1.1108 Pa. To determine the water uptake of Ag/CS/PEO and CS/ PEO membranes, square samples (2020 mm2) were weighed using an electronic balance, placed in closed bottles containing 20 mL of phosphate buffered saline (PBS, pH = 7.4), and incubated at 37.0 0.1 C for 24 h. The wet mass of the samples after incubation was determined by weighing immediately after removing from PBS and absorbing water on the sample surface with filter paper. The water uptake of electrospun membranes were calculated using the following equation: Water uptake % m1 m0 =m0 100% where m0 and m1 are the mass of the membranes before and after immersion in PBS, respectively. Mechanical properties of electrospun nanofibrous membranes were determined using a universal testing machine (Testmetric M350-20KN, UK) equipped with a 100 N load-cell at a cross-head speed of 5 mm/min in the ambient environment. The gauge length of tensile samples was 40 mm. The samples were prepared in the rectangular shape with dimensions of 6010 mm2 from dry electrospun nanofibrous membranes. For obtaining

mechanical properties of membranes in the wet condition, tensile samples were placed in closed bottles containing 10 mL of PBS (pH=7.4) and incubated at 37.00.1 C for 24 h. The samples were then taken out of the bottles and tested under tension. The tensile modulus, tensile strength, and elongation at break were presented averaged results of five tests. Microbial experiments The antimicrobial activities of electrospun fibrous membranes containing AgNPs were tested against the bacteria of E. coli by the nonwoven fabric attachment method [26]. E. coli was cultivated in sterilized LuriaBertani (LB) broth and then incubated overnight at 37 C with shaking. The bacterial suspensions employed for the tests contained from 106 to 107 CFU. For the Microbial test, 0.1-mg electrospun fibrous membranes were set in each sterilized test tube and inoculated with an equivalent volume of E. coli suspension. In each 50-L tube, the mixed suspension was removed as a function of contact time (h) and cultured in LB agar plates. The LB agar plates containing test samples and the control were kept at 37 C, and the number of survival colonies was counted every hour.

Results and discussion Morphology of electrospun ultrafine fibers The morphology and diameter of electrospun fibers were dependent on a number of parameters including properties and composition of the polymer solution such

1.0

45 Viscosity Surface tension Conductivity Conductivity (mS/cm) 40

100

0.8 s)

80 Surface tension (mN/m)

Viscosity (Pa

0.6

60 35 40 30 20

0.4

0.2

0.0

25 0.0

0.1

0.2 0.3 Ag+(mol/L)

0.4

0 0.5

Fig. 1 Changes in viscosity, surface tension, and conductivity of 5 wt.% Ag/CS/PEO solutions (CS/PEO mass ratio=1:1) with increasing the AgNO3 amount

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as viscosity, concentration, conductivity, and surface tension [27]. Figure 1 shows variations of Ag/CS/PEO solution properties with different amount of AgNO3 added. The conductivities of Ag/CS/PEO solutions were linearly

increased, while the viscosities and surface tensions were not changed with the increase content of AgNO3. SEM images of Ag/CS/PEO electrospun ultrafine fibers with different concentrations of AgNO3 aqueous solutions are shown in Fig. 2. It could be found that the fibers

(a)

(b)

(c)

(d)

(e)
Fig. 2 SEM micrographs of electrospun ultrafine fibers from 5 wt.% Ag/CS/PEO solutions (CS/PEO mass ratio=1:1) with different AgNO3 concentrations. a 0 mol/L; b 0.1 mol/L; c 0.2 mol/L; d 0.3 mol/L; e 0.4 mol/L

Colloid Polym Sci (2009) 287:14251434

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containing AgNPs were smooth and the diameters decreased when the addition of AgNO3 increased, while the solution at the same CS/PEO concentration without AgNPs, beads, and bead fibers were generated (Fig. 2a). Since AgNO3 increased the charge density in Ag/CS/PEO solutions, stronger stretch forces were imposed on the ejected jets under the electrical field, resulting in substantial formation of finer ultrafine fibers [28]. Electrospun Ag/CS/PEO ultrafine fibers with different average diameters at varied CS/PEO mass ratios are shown in Fig. 3. The average diameters of the Ag/CS/PEO ultrafine fibers electrospun with CS/PEO mass ratios of

2:1, 1.5:1, and 1:1, were 9713, 10116, and 11718 nm, respectively. The fiber diameters tended to decrease slightly when the CS amount increased. The result was similar to that of CS/PEO fibers prepared by Duan et al. with diameter ranging from 120 to 140 nm [29]. Figure 4 shows TEM images of CS/PEO ultrafine fibers electrospun from 5 wt.% CS/PEO solutions with or without the present of AgNPs. Figure 4bd revealed that the AgNPs were evenly distributed in the CS/PEO ultrafine fibers with an average size less than 5 nm. This suggested that the AgNPs were well stabilized by CS during the synthesis of Ag/CS colloids and the electrospinning process. Sufficient

Frequency Distribution (%)

Fig. 3 SEM micrographs and diameter distribution of electrospun Ag/CS/PEO fibers. AgNO3 concentration=0.2 mol/L; CS/ PEO mass ratio= a 2:1, b 1.5:1, and c 1:1, respectively

20 15 10 5 0 70

dave=97nm sd=13.1

80

90 100 110 120 Fiber Diameter (nm)

130

140

(a)
16 Frequency Distribution (%) 14 12 10 8 6 4 2 0 60 80 100 120 140 dave=101nm sd=15.8

Fiber Diameter (nm)

(b)
Frequency Distribution (%) 16 14 12 10 8 6 4 2 0 80 90 100 110 120 130 140 150 160 Fiber Diameter (nm) dave=117nm sd=13.1

(c)

1430 Fig. 4 TEM micrographs of CS/PEO ultrafine fibers (a) and Ag/CS/PEO ultrafine fibers prepared from 0.2 mol/L AgNO3 solution (b, c, d) in different magnifications. CS/PEO mass ratio=1:1

Colloid Polym Sci (2009) 287:14251434

(a)

(b)

(c)

(d)

stability of AgNP dispersions prevented aggregating, which could lead to enhancing the antibacterial activity [30]. XRD analysis XRD was used to confirm the formation of AgNPs and examine the crystal structure of the nanoparticles. Figure 5 shows the XRD patterns of Ag/CS/PEO ultrafine fibers electrospun from the solutions with different concentrations of AgNO3. The strong reflection at 21.3 and 26.5 was attributed to the crystalloid of original CS, while those at 19.2 and 23.6 were assigned to the crystalline PEO. The characteristic five peaks at 2 of 38.1, 44.3, 64.4, 77.3, and 81.6 were corresponding to (111), (200), (220), (311), and (222) planes of the face-centered cubic structure of the metallic AgNPs encapsulated in the electrospun ultrafine fibers [31], respectively. The XRD patterns of AgNPs in the ultrafine fibers were similar to that of original Ag nanocrystals, indicating that the electrospinning of Ag/CS/PEO ultrafine fibers does not change the crystalline structure of original AgNPs.

XPS analysis Figure 6 shows X-ray photoelectron spectra of Ag/CS/PEO ultrafine fibers with CS/PEO mass ratio of 1:1. The XPS analysis in Fig. 6a shows the presence of carbon, oxygen, nitrogen, and silver, with the atomicity percentage of 75.3%, 23.1%, 1.4%, and 0.2% in the samples, respectively. In Fig. 6b, the Ag3d peaks at 367.1 eV (3d5/2) and 373.5 eV (3d3/2) are characteristic of metallic Ag and Ag ion [32]. The presence of Ag+ could result from the combining of AgNPs with the oxygen in the air or the solution. In Fig. 6c, the O1s photoemission spectra were shifted to a higher energy and resolved into two peaks being fitted by multiple Gaussians. The lower peak at 532.0 eV was similar to that of original CS while the higher one at 533.3 eV was attributed to the interaction between carbonyl oxygen atoms and AgNPs. This result indicated that the carbonyl oxygen atoms have a lower electronic density than those in original CS [33]. The N1s photoemission of the ultrafine fibers at 399.3 eV was not changed by the addition of AgNO3 shown in Fig. 6d, implying that nitrogen atoms did not interact directly with AgNPs. It can

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AgNPs in the electrospun membrane, enhancement of the water uptake was very limited. Mechanical properties
Ag (111) Ag (200)

Ag (220)

Ag Ag (311) (222)

Intensity (a.u.)

(e)

(d)

(c)

(b)

(a)
0 10 20 30 40 50 2 (degree) 60 70 80 90

Fig. 5 XRD patterns of Ag/CS/PEO ultrafine fibers (CS/PEO mass ratio=1:1) prepared from different AgNO3 concentrations: a 0 mol/L; b 0.1 mol/L; c 0.2 mol/L; d 0.3 mol/L; e 0.4 mol/L

be drawn a conclusion that the bond of AgO could exist in ultrafine fibers and lead to the tight combination between AgNPs and CS molecules. Water uptake The water uptake of the electrospun Ag/CS/PEO and CS/PEO membranes with the CS/PEO mass ratio of 1:1 is summarized in Table 1. The water uptake of CS/PEO nanofibrous membranes was about 3915% after 24 h incubation in PBS, which was due to the polymer nanofiber morphology that had extremely high specific surface area. With the introduction of AgNPs into the membrane, it could absorb as much as 42715% of water, which was very similar to the water absorption of electrospun CS membranes (49917%) [31]. Therefore, the introduction of AgNPs could enhance the water uptake of the electrospun CS/PEO membranes. The phenomenon could be attributed to the high superficial energy of AgNPs; it made AgNPs combine with the oxygen in the aqueous solution and then the united oxygen could integrated with hydrogen to form hydrogen bond, which resulted in enhancing the hydrophilicity of CS/PEO ultrafine fibers. Since there was only 2.2 wt.% content of

Mechanical properties of electorspun membranes depend on a number of factors including the fiber structure, properties of the constituent components, and the interaction between AgNPs and polymer chains. The average thickness of the two dry membrane samples was 47 m for Ag/CS/PEO and 56 m for CS/PEO. After incubation in PBS, the cross-linked Ag/CS/PEO and CS/PEO membranes shrank in about 1.5% and 2.0%, respectively. The typical stressstrain curves of Ag/CS/PEO and CS/PEO membranes are presented in Fig. 7, while the characteristic data including tensile strength, tensile modulus, and elongation at break for both dry and wet states are shown in Table 1. For both dry and wet membranes, it was observed that electrospun Ag/CS/PEO membrane exhibited higher tensile strength and tensile modulus, but lower elongation than those of CS/PEO membranes. The Ag component, whose concentration was only 2.2 wt.% in nanofibrous membranes, had more significant effect on tensile modulus than on the tensile strength and elongation. In the dry condition, there was a large increase in tensile modulus from 59.2 to 322.4 MPa, which was accompanied by a little increase in tensile strength from 4.6 to 6.5 MPa and a reduction in elongation from 4.5% to 2.5%, for membranes from CS/PEO to Ag/CS/PEO. Such a large increase in tensile modulus could be attributed to the interaction and bonding structure between Ag and CS, as well as hydrogen bonding between CS and PEO fibers. For the electrospun membranes with about 48.6% of CS and the bond of AgO leading to the tight combination between AgNPs and CS molecules, AgNPs could disperse well in the composite membranes and probably promoted the interaction between CS and PEO. In addition, due to AgNPs contained in cross-linked CS taking a negative effect on providing frictional entanglements between fibers, the elongation of Ag/CS/PEO membrane was reduced compared with the CS/PEO membrane. In wet state, ultimate tensile strength and tensile modulus of both Ag/CS/PEO and CS/PEO eletrospun membranes decreased to some extent corresponded with those in dry state, except the elongation. The result consisted with the mechanical properties of CS and CS/ PEO fibrous membranes [34, 35]. The absorption of PBS resulted in the stretch of polymer network bonds, which lowered the sliding friction between polymer chains [36]. In addition, the water molecules entering ultrafine fibers may play a role as plasticizers. Uptaken water appeared to working with the reductions of

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2.5x105

Colloid Polym Sci (2009) 287:14251434

6400

(a)
2.0x10
5

O1s

Intensity (a.u.)

Intensity (a.u.)

Atomic % C1s 75.3 O1s 23.1 C1s N1s 1.4 Ag3d 0.2

(b)
6000 5600 5200 4800 4400 0.4 mol/L 0.3 mol/L 0.2 mol/L 0.1 mol/L 0 mol/L 376 374 372 370 368 Binding Energy (ev) 373.5 ev

Ag3d 367.1 ev

1.5x105 1.0x105

N1s Ag3d
5.0x10
4

0.0 1000 800 600 400 200

C2s 0

4000 378

366

364

Binding Energy (ev)

(c)
533.3 eV

532.0 eV

O1s

5.5x103

(d)
399.3 eV

N(1s)

Intensity (a.u.)

5.0x10

0.4 mol/L

Intensity (a.u.)

0.3 mol/L

4.5x103

0.2 mol/L

4.0x103 410

405

400

395

390

Binding Energy (eV)

0.1 mol/L

0 mol/L 538 536 534 532 530 Binding Energy (eV) 528 526

Fig. 6 XPS spectra of Ag/CS/PEO ultrafine fibers (CS/PEO mass ratio=1:1). a Survey spectrum from 0.2 mol/L AgNO3 solutions; b Ag3d, c O1s XPS spectra from different AgNO3 concentrations; and d N1s XPS spectra

tensile strength and tensile modulus and increases of elongation. Antimicrobial activities The contact biocidal properties of Ag/CS/PEO and CS/PEO ultrafine fibers with the CS/PEO mass ratio

of 1:1 were investigated, and the effects of the electrospun membranes on the growth of the recombinant bacteria of E. coli are shown in Fig. 8. The Ag/CS/PEO membrane electrospun with 0.1 and 0.2 mol/L AgNO3 added (the concentration of Ag in the membrane was 1.1 and 2.2 wt.%, respectively) inactivated all the bacteria within 10 and 6 h, respectively, whereas the CS/PEO

Table 1 Mechanical properties and water uptake of Ag/CS/PEO and CS/PEO membranes after cross-linking. CS/PEO mass ratio = 1:1 Samples Ag/CS/PEO CS/PEO State Dry Wet Dry Wet Tensile strength (MPa) 6.481.94 2.150.26 4.630.76 0.550.32 Tensile modulus (MPa) 32236.2 5.460.43 59.222.9 2.100.62 Elongation (%) 2.540.83 10.53.2 4.480.68 14.05.5 Water uptake (%) 42715 3916

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10

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8 Stress (MPa)

(a)

6 (b) 4 (c) (d) 0 0.00

impregnated into bacterial cellulose through chemical reduction by immersing bacterial cellulose in silver nitrate solution [42]. The performed research has proven that antibacterial activity of the AgNPs is dependent on not only the size but also the shape. Therefore, when AgNPs were incorporated into the ultrafine fibers by electrospinning, these fibers also could show excellent antimicrobial activities besides its unique characteristics of electrospun materials.

Conclusions
0.05 0.10 Strain (mm/mm) 0.15 0.20

Fig. 7 Typical stressstrain curves of electrospun Ag/CS/PEO (a, c) and CS/PEO (b, d) membranes after cross-linking. a, b dry state; c, d wet state. CS/PEO mass ratio 1:1. 0.2 mol/L AgNO3

membrane was bacteriostatic only at the test concentration. CS itself is known to have strong antibacterial properties by disrupting cell membranes. The proposed mechanism for its antimicrobial action is binding to the negatively charged bacterial cell wall, with consequent destabilization of the cell envelope and altered permeability attached to DNA with inhibition of its replication [37]. In addition, the nanosized composite fiber provides relatively higher surface area to contact with bacteria. The results indicated that the nanofibrous membrane containing AgNPs exhibited much more antibacterial activity than that of noncontaining AgNPs, and the treated membrane with higher content of AgNPs had a more effective contact biocidal property than the one with lower content of AgNPs. Therefore, the electrospun nanofiber containing AgNPs has superior contact antibacterial property to that of the nanofiber without containing AgNPs. In comparison with the antibiotics, the bacterial resistance against AgNPs has not been observed up to the present and AgNPs do not constitute any significant complication [38]. This fact is supposedly caused by the antibacterial mechanism of AgNPs [39]. One believed inhibition mechanism of Ag on microorganism is that AgNPs affect DNA molecules by losing their replication abilities [40]. Recently, Kim et al. confirmed that the free radicals, derived from the surface of AgNPs, could attack membrane lipids of microorganisms and then break down the membrane functions [41]. The AgNPs prepared in this study were approximately 5 nm in diameter with cubic crystal structure. Similar observations were reported by Maneerung et al., in which AgNPs

The incorporation of AgNPs into uniform CS/PEO ultrafine fibers could be achieved by electrospinning. The diameter of Ag/CS/PEO ultrafine fibers was approximately 100 nm. Since CS molecules played an important role in the preparation and stabilization of the nanoparticles, AgNPs were found evenly distributed on the surface of CS/PEO ultrafine fibers with the size less than 5 nm by TEM. XRD examination confirmed the cubic crystal structure of AgNPs, and XPS results suggested that AgO bond existing in the composite ultrafine fibers. Microbial experimentation indicated that the electrospun nanofibrous membranes containing AgNPs had much better bacteriostatic effects on the bacteria of E. coli than the casting membranes without AgNPs. It was supposed that the electrospun Ag/CS/PEO membranes with very strong antimicrobial activity could be used in various biomedical fields such as wound dressings, body wall repairs, antimicrobial filters, and tissue scaffolds.

12 10 log CFU/mL 8 6 4 2 0 0

Control CS/PEO Ag-CS/PEO (CAgNO3=0.1 mol/L) Ag-CS/PEO (CAgNO3=0.2 mol/L)

6 Time (h)

10

12

Fig. 8 Comparative effects of Ag/CS/PEO and CS/PEO membranes on recombinant E. coli viability. CS/PEO mass ratio=1:1

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