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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 nano-

particles (AgNPs) were successfully prepared by electro-

spinning 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 activ-

ities 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 biodegrad-

ability, 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 andTechnology (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: [email protected]

Colloid Polym Sci (2009) 287:14251434

DOI 10.1007/s00396-009-2108-y

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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 mem-

branes were recognized as safe materials with strong

inhibitory and bactericidal effects, in comparison with

those containing ionic Ag which may cause the discolor-ation of ultrafine fibers and the skin [20]. A few AgNP-

containing electrospun membranes have been reported in

the literatures. Jin et al. prepared Ag/Poly(vinyl pyrroli-

done) (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 electrospin-

ning 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 prod-

ucts can be used in fabricating optical and electronic

devices [23].

Because of the unique cationic-polyelectrolyte char-

acter and gel-forming ability, CS could bind metal ions

by electrostatic attractio n [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 mem-

branes of Ag/CS/PEO were obtained by electrospinning

o f the C S/PE O solutio ns in aqu eo us acetic acid

containing Ag/CS colloids which was synthesized by

in situ chemical reduction with AgNO3. The conductiv-

ity, surface tension, and viscosity of the Ag/CS/PEO

dispersion were measured. Effects of a variety of param-

eters 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.

Experimental methods

Materials

A sample of chitosan CS (deacetylation degree 80%, Mw =

2.0105

) was purchased from Yu Huan Ocean Biochemical

Co. Ltd., China. PEO (Mw =1.5106) was kindly donated

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.

Esche richia 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 AgNO3solution 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 NaBH4solution 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. Theresultant 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

C S t o P EO w as s el ec te d a s 1 :1 , 1 .5 :1 , a nd 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 high-

voltage power supply. The flow rate of the polymer

solution was controlled by a syringe pump (KI-75439-

0005, 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 solu-

tion at 37 C for 6 h, the electrospun nanofibrous

membranes were kept in a desiccator characterization.

1426 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 C6hydroxyl groups of CS and carbonyl groups, which is

insignificant.

Characterizations

For microstructure studies of the electrospun nano-

fibrous 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 micros-copy (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 diffrac-

tion angle range 2= 1090. XPS analysis was carried out

in a Perkin-Elmer PHI-1600 spectrometer using Mg Kradiation (1,253.6 eV, 250 W), and the vacuum inside the

analysis chamber was 1.1108Pa.

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 37.00.1 C for

24 h. The wet mass of the samples after incubation

was determined by weighing immediately after remov-

ing from PBS and absorbing water on the sample

surface with filter paper. The water uptake of electro-

spun 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 mem-

branes 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 107CFU. For the Microbial test, 0.1-mg electrospun

fibrous membranes were set in each sterilized test tube and

inoculated with an equivalent volume ofE. colisuspension.

In each 50-L tube, the mixed suspension was removed asa 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

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.1 0.2 0.3 0.4 0.525

30

35

40

45

0

20

40

60

80

100Viscosity

Viscosity

(Pa

s)

Conductivity

Conductivity(mS/cm)

Ag+(mol/L)

Surface tension

Surfacetension(mN/m)

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

Colloid Polym Sci (2009) 287:14251434 1427

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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 AgNO 3concentrations.a 0 mol/L; b 0.1 mol/L; c 0.2 mol/L; d 0.3 mol/L; e 0.4 mol/L


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containing AgNPs were smooth and the diameters de-

creased 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 substan-

tial 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 97 13, 10116, and 117 18 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].

Figure4 shows TEM images of CS/PEO ultrafine fibers

electrospun from 5 wt.% CS/PEO solutions with or without

the present of AgNPs. Figure4b

drevealed that the AgNPswere 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

70 80 90 100 110 120 130 1400

5

10

15

20dave=97nm

sd=13.1

FrequencyDistribu

tion(%)

Fiber Diameter (nm)

(a)

60 80 100 120 1400

2

4

6

8

10

12

14

16dave=101nm

sd=15.8

FrequencyDistribution(%)

Fiber Diameter (nm)

(b)

80 90 100 110 120 130 140 150 1600

2

4

6

8

10

1214

16

FrequencyDistribution

(%)

Fiber Diameter (nm)

dave=117nm

sd=13.1

(c)

Fig. 3 SEM micrographs and

diameter distribution of electro-

spun Ag/CS/PEO fibers. AgNO3concentration= 0.2 mol/L; CS/

PEO mass ratio=a 2:1, b 1.5:1,

and c 1:1, respectively


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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 nano-

crystals, indicating that the electrospinning of Ag/CS/PEO

ultrafine fibers does not change the crystalline structure of

original AgNPs.

XPS analysis

Figure6 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 photoemis-

sion 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

(a) (b)

(c) (d)

Fig. 4 TEM micrographs of

CS/PEO ultrafine fibers (a) and

Ag/CS/PEO ultrafine fibers pre-

pared from 0.2 mol/L AgNO3solution (b, c, d) in different

magnifications. CS/PEO mass

ratio=1:1


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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 (499 17%) [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

AgNPs in the electrospun membrane, enhancement of the

water uptake was very limited.

Mechanical properties

Mechanical properties of electorspun membranes depend

on a number of factors including the fiber structure,

proper ties of the con stitue nt components, and theinteraction 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 tensilestrength 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 mem-

branes 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

0 10 20 30 40 50 60 70 80 90

Ag

(111) Ag

(200) Ag

(220) Ag

(311) Ag(222)

(c)

(a)

(d)

(b)

Intensity(a.u.)

2 (degree)

(e)

Fig. 5 XRD patterns of Ag/CS/PEO ultrafine fibers (CS/PEO mass

ratio= 1:1) prepared from different AgNO3concentrations: a 0 mol/L;

b 0.1 mol/L; c 0.2 mol/L; d 0.3 mol/L; e 0.4 mol/L


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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 electro-

spun 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 AgNO3added (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 State Tensile strength (MPa) Tensile modulus (MPa) Elongation (%) Water uptake (%)

Ag/CS/PEO Dry 6.481.94 32236.2 2.540.83 42715

Wet 2.150.26 5.460.43 10.53.2

CS/PEO Dry 4.630.76 59.222.9 4.480.68 3916

Wet 0.550.32 2.100.62 14.05.5

1000 800 600 400 200 00.0

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

In

tensity(a.u.)

Binding Energy (ev)

Atomic %

C1s 75.3

O1s 23.1

N1s 1.4

Ag3d 0.2

O1s

C1s

C2s

N1s Ag3d

(a)

378 376 374 372 370 368 366 3644000

4400

4800

5200

5600

6000

6400

(b)

367.1 ev373.5 ev

0 mol/L

0.1 mol/L

0.2 mol/L0.3 mol/L

0.4 mol/L

Ag3d

In

tensity(a.u.)

Binding Energy (ev)

538 536 534 532 530 528 526

532.0 eV533.3 eV

(c)O1s

0.3 mol/L

0.2 mol/L

0.1 mol/L

0.4 mol/L

Intensity(a.u.)

Binding Energy (eV)

0 mol/L

410 405 400 395 390

4.5x103

5.0x103

5.5x103

N(1s)

Inten

sity(a.u.)

Binding Energy (eV)

(d)

399.3 eV

4.0x103

Fig. 6 XPS spectra of Ag/CS/PEO ultrafine fibers (CS/PEO mass ratio=1:1). aSurvey spectrum from 0.2 mol/L AgNO3solutions;b Ag3d,

c O1s XPS spectra from different AgNO3 concentrations; and d N1s XPS spectra


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membrane was bacteriostatic only at the test concentra-

tion. 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 perme-

ability 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 anti-

bacterial 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

r ep or te d b y M an ee ru ng e t a l. , i n w hi ch A gN Ps

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 characteristicsof electrospun materials.

Conclusions

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 onthe 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.

0 8 10 120

2

4

6

8

10

12

log

CFU/mL

Time (h)

Control

CS/PEO

Ag-CS/PEO (CAgNO3

=0.1 mol/L)

Ag-CS/PEO (CAgNO3

=0.2 mol/L)

2 4 6

Fig. 8 Comparative effects of Ag/CS/PEO and CS/PEO membranes

on recombinant E. coli viability. CS/PEO mass ratio=1:1

0.00 0.05 0.10 0.15 0.200

2

4

6

8

10

Stre

ss(MPa)

Strain (mm/mm)

(a)

(b)

(c)

(d)

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


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