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DOI: 10.1002/adem.201180085

Safe and Effective Ag Nanoparticles ImmobilizedAntimicrobial NanoNonwovens**

By Jie Song, Menglin Chen,* Viduthalai R. Regina, Chenxuan Wang,Rikke L. Meyer, Erqing Xie, Chen Wang, Flemming Besenbacher andMingdong Dong*

Silver nanoparticles (AgNPs) with large surface-to-volume ratio have been widely studied as a valuablematerial for their strong antimicrobial effect. However, the practical applications of AgNPs in healthcare and water purification are often hampered by the concern of their toxicity and possibility ofintroduction of secondary pollution. Here, we present a novel strategy to produce a safe and effectiveantimicrobial nanononwoven material by immobilizing AgNPs on a rigid polymer nanofibrous matrixthrough simple co-electrospinning of pre-prepaired AgNPs and polystyrene (PS). Distribution of theAgNPs on the surface of PS fibers was achieved by tuning fiber diameters during electrospinning.Atomic force microscopy (AFM) analysis revealed that the AgNPs distributed at the fiber surface werestill covered by a layer of polymer, which inhibited their antimicrobial activity. UV/ozone treatmentwas thus employed to degrade the polymer coating without loosening the AgNPs, resulting in an activeantimicrobial nonwoven against Gram-positive Staphylococcus xylosus. The mechanism based oncellular uptake of silver ions via close contact to the surface of AgNPs is proposed. The novelnanononwoven retains the enhanced antimicrobial activities from nanofeatured AgNPs withoutdetectable AgNPs leaching, which holds great potential for safe and recyclable use.

The broad-spectrum antimicrobial properties of silver

encourage its use in biomedical applications, water and air

purification, foodproduction, cosmetics, clothing, andnumerous

[*] J. Song, Dr. M. Chen, Prof. F. Besenbacher, Dr. M. DongInterdisciplinary Nanoscience Center (iNANO), AarhusUniversity, Aarhus C DK-8000, DenmarkE-mail: Menglin@inano.au.dk; Dong@inano.au.dk

J. Song, Prof. E. XieSchool of Physical Science and Technology, Lanzhou University,Lanzhou 730000, People’s Republic of China

V. R. Regina, Dr. R. L. MeyerDepartment of Biological Sciences, Aarhus University, AarhusC DK-8000, Denmark

C. Wang, Prof. C. WangNational Center for Nanoscience and Technology (NCNST),Beijing 100190, People’s Republic of China

[**] We gratefully acknowledge the Danish Advanced TechnologyFoundation for funding the project NanoNonwovens, collabor-ation with Fibertex A/S, and the Danish Research Agency forfunding for the iNANO center. M. D. acknowledges a STENOgrant from the Danish Research Council. J. S. acknowledgesPhD Scholarship from China Scholarship Council of theMinistry of Education of China.

B240 wileyonlinelibrary.com � 2012 WILEY-VCH Verlag GmbH & Co

household products.[1] Emerged from rapid developments in

nanoscience and nanotechnology, silver nanomaterials exert

enhanced antimicrobial activity due to their extremely large

surface-to-volume ratio offering better contact with the micro-

organism.[2–5] Nevertheless, the practical implementation of

silver nanoparticles (AgNPs) in antimicrobial materials is often

hampered by their tendency to oxidize or aggregate, the

difficulty of recycling, or the risk of pollution from AgNPs

leaching from the material.[1,4,6,7]

Electrospinning is a remarkably simple and versatile

technique capable of generating continuous fibers directly

from a variety of polymers and composite materials.[8–11]

Electrospun nanofibers offer a range of attractive features,

such as large surface areas, high porosities, and ease

alignment, making them ideal candidates for versatile

applications such as biotechnology, textiles, membranes/

filters, composites and sensors, and so forth. Inspired by the

unique properties of electrospun fibers, introducing AgNPs

into polymer fibers via electrospinning is an attractive

approach to develop new antimicrobial materials. Previous

studies have synthesized metal nanoparticles within the

polymer by adding a metal salt to an electrospinning solution,

and upon electrospinning reducing the metal ions to

nanoparticles by a thermal, UV, or chemical treatment.[7,12–14]

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A few studies have reported the direct dosage of pre-prepared

AgNPs into the electrospinning polymer solutions.[15]

Although, the antimicrobial activities were achieved in these

studies, no investigations concerning AgNPs toxicity, how-

ever, have been conducted for electrospun nanononwovens.

Significant evidence has been reported in relation to the

toxicity of AgNPs to higher organisms, including fish, fruit fly,

and different mammalian cell lines of mice, rats, and also

humans.[16] Therefore, safety has been an arising issue for

AgNPs-based antimicrobial materials.

Here, we present a novel strategy for preparation of a safe

and effective antimicrobial AgNPs immobilized nonwoven

materials applying simple co-electrospinning of AgNPs with

polystyrene (PS). Delicate control of nanofiber morphology

assures the surface distribution of AgNPs; while UV–ozone

treatment activates their antimicrobial effects. The mechanism

based on cellular uptake of silver ions by close contact to the

surface of AgNPs is proposed.

1. Experimental

1.1. Preparation of AgNPs

The AgNPs were synthesized by the polyol method

implementing some modifications.[17] A solution of 0.792 g

polyvinylpyrrolidone (PVP, Mw� 1 300 000; Sigma) in 32mL

ethylene glycol (�99%, Sigma–Aldrich) was heated and

thermally stabilized at 150 8C. Then 0.204 g AgNO3 (silver

nitrate, 99%, Sigma–Aldrich) dissolved in 12mL ethylene

glycol was injected into the above refluxed mixture at a rate of

�2mL �min�1. After injection, the reaction mixture under

vigorous magnetic stirring was further refluxed for an

additional 30min. After growth, the AgNPs were transferred

into methanol by two steps of centrifugation at 10 000 rpm for

5min each.

1.2. Electrospinning of AgNPs-Immobilized Nanononwoven

Homogeneous polymer solutions were prepared by dissol-

ving PS in DMF at room temperature after stirring for 5h. The

polymer concentrations varied from 10 up to 25%w/v, which

were next dispersed with 0, 0.175, 0.700, and 2.800%w/v

AgNPs at room temperature and stirred for 12h. These

homogeneous solutions were then placed in a 5mL syringe

fitted with a metallic needle of 0.4mm inner diameter.

The syringe was fixed horizontally on a syringe pump (Model:

KDS 101, KD Scientific) and an electrode of high voltage power

supply (Spellman High Voltage Electronics Corporation, MP

Series) was clamped to the metal needle tip. The flow rate of

solutionwas 1mL �h�1, and the applied voltagewas 15kV. The

tip-to-collector distance was set to 20 cm, and both aluminum

foil and glass slidewere used for the fiber collection in the favor

of different measurements.

1.3. Measurements and Characterization

The morphology of the composite nanofibers was exam-

ined by high-resolution scanning electron microscopy (SEM;

FEI, Nova 600 NanoSEM). The SEM was operated in

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high-voltage (10 kV) mode to apply the charge contrast

imaging data. Transmission electron microscopy (TEM;

Tecnai G2 F20 U-TWIN, 200 kV) was applied to illustrate

the nanostructures of sample. The AFM experiments were

carried out on a Multimode VIII system (Bruker Corporation,

Santa, Barbara, CA) in peak force tapping (PFT) mode. Si3N4

cantilevers with the spring constants of 0.4N �m�1 were used,

and measurements were performed under ambient condi-

tions. For adhesion force measurements, quantitative forces

were obtained by control peak force (PF). Surface chemistry

was probed by X-ray photoelectron spectroscopy (XPS) using

a Kratos Axis UltraDLD instrument equipped with a

monochromatedAl KaX-ray source (hn¼ 1486.6eV) operating

at 10 kV and 15mA (15W). UV/ozone treatment of the

nanononwoven was performed in UV/ozone chamber, in

which the UV emission is generated from low-pressure quartz

mercury vapor lamps and the UV intensity at the wavelength

of 254 nm is �28mW � cm�2 at a distance of 6mm from the

lamps. UV–Vismeasurements were conducted on a Shimadzu

UV-3600 UV–Vis–NIR spectrophotometer.

1.4. Antimicrobial Activity Tests

Antimicrobial activity was assayed against a biofilm

forming Gram-positive bacterium: Staphylococcus xylosus

(DSM 20266, DSMZ, Braunschweig, Germany). The pure

nanofiber (without AgNPs) was used as control and

nanofibers with 2.8%w/v AgNPs before and after UV/ozone

treatment were used as test samples. For the zone of inhibition

test, tryptic soy broth (TSB) agar was filled onto disposable

sterilized petri dishes, and then 100mL of S. xylosus was

spread uniformly after the agar solidified. Circular pieces

(1 cm2) of nanofibers were cut and gently placed over the

solidified agar gel. Petri dishes were incubated for 24 h at 37 8Cto visualize the zone of inhibition. For the antibacterial test in

solution, the S. xylosus starter culture was grown overnight in

3mL 1% TSB medium in a 50mL conical bottom flask tube by

incubating overnight with shaking at 25 8C. One milliliter

from this culture was then used to inoculate 100mL of 1% TSB

medium, which was incubated under the same conditions

until the optical density measured at 600 nm (OD600) was

0.8–1.0. Cells were harvested by centrifugation (10min, at

3000 g), washed twice, resuspended in phosphate buffered

saline (PBS, pH 7.0), and diluted to obtain OD600 of 0.1. Three

tubes each with 1mL of the above medium were then

introduced with the nanofibers before and after UV/ozone

treatment. The mixtures were cultured at 37 8C in a shaking

incubator for overnight. Then the number of colony forming

units (CFU)was counted to determine the antimicrobial effect.

2. Results and Discussion

2.1. Fabrication and Characterization of AgNPs

The polyol method involving using polyol, such as ethylene

glycol, was used to reduce silver salts and obtainAgNPs.[17] The

as-grown AgNPs obtained were spherical in shape had a broad

size distribution with an average diameter of approximately

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Fig. 1. Representative (a) SEM and (b) TEM images of the as-grown AgNPs; (c) HRTEM image of a selectedAgNP with the diameter about 25 nm; (d) XPS spectrum of the as-grown AgNPs.

80nm (Figure 1a and 1b). High resolution TEM analysis

of a selected AgNP (approx. 25nm in diameter) illustrated

the typical single crystalline structure of Ag (Figure 1c),

and also demonstrated the presence of an amorphous

layer (�2nm marked in white) around the Ag particle. XPS

analysis, furthermore, revealed that the AgNPs not only

contained Ag but also C, N, and O elements from PVP

(Figure 1d). As previously reported, PVP is a polymeric

surfactant that interacts with silver particles through carbonyl

groups.[18] It is, therefore, concluded that the as-grown AgNPs

Fig. 2. Illustration of electrospinning PS fibers with immobilized AgNPs, as well as pure PS fibers.

B242 http://www.aem-journal.com � 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

were coated by a thin PVP layer, which is

approximate 2nm thick.

2.2. Electrospinning and Distribution ofAgNPs in PS Nanofibers

PS, with a glass transition temperature (Tg)

of around 100 8C, was chosen as a rigid

matrix polymer for immobilize AgNPs.

Considering the limited polymer chain

movement in its glassy state at biological

temperature, using PS could assure no

leach-out of AgNPs. Further, since there is

no known microorganism to be shown

to biodegrade PS, it is thus ideal for

developing safe and effective antimicrobial

nonwovens.

AgNPs-immobilized PS fibers, as well as

pure PS fibers were successfully electrospun

and their SEM images are shown in Figure 2.

Due to the difference in electron charge

transport properties between the conductive

AgNPs and the insulating PS polymer

matrix, the secondary electron yield is

enriched at the location of the AgNPs, in

particular for higher electron beam ener-

gies.[9] Therefore, the brightness variations

seen in the SEM charge contrast images can

be related to the position of AgNPs in the

sample, and SEM images thus confirm that

AgNPs were successfully immobilized into the PS polymer

fibers.

SEM images of fibers produced fromPS solutions (15%w/v)

containing three different concentrations of AgNPs (0.175,

0.70, and 2.8%w/v) are shown in Figure 3a–c, respectively.

Fiber diameters is summarized in the Figure 3d, which

demonstrates that along with the increase of AgNPs

concentrations, the average diameters of the composite fibers

decreased from around 210 to 180 nm. As indicated in our

previous work, the electrospinning process is involved with

the whipping instability originating from the

electrostatic interactions between the exter-

nal field and the surface charges on the jet.[9]

Therefore, the stretching force would

increase when the amount of AgNPs

increase, and consequently the diameters of

the composite fibers decrease. Further

increase of the AgNPs above 2.8%w/v

resulted in unstable electrospinning, where

unusual beads instead of fibers were fabri-

cated. Therefore, the composite fibers used

for antibacterial activity experiments were

prepared using highest dosage of AgNPs

(2.8%w/v).

The distribution of the AgNPs in PS fibers

was tuned by varying the fiber diameter,

which can be tuned by varying the concen-

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Fig. 3. SEM images of AgNP-containing PS nano-nonwoven prepared with different concentrations of AgNPsin the precursory solution: (a) 0.175%, (b) 0.700%, and (c) 2.80%. (d) The mean fiber diameter of PSnanononwoven with different PVP-AgNPs concentrations.

Fig. 4. The SEM images and TEM images of the electrospun PS fibers from solution with 20% w/v (a and b),15% w/v (c and d), and 10% w/v PS (e and f). (g) Illustrates the process about the surface distribution of theAgNPs in PS fibers by decreasing the diameters of electrospun fibers from micron to nanometer.

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tration of the matrix polymer, where gen-

erally a higher concentration of polymers

resulted in increased fiber diameter.[19]

The fibers from the solutions contain 20,

15, and 10%w/v PS were approximately

1mm, 200nm, and 100nm, respectively

(Figure 4a–f). As illustrated in Figure 4g,

most of the AgNPs were encapsulated inside

the fibers when their diameters are

large (Figure 4b, arrow). As the diameters

decreased to 200 nm, an increased number of

AgNPs appeared at the fiber surface (Figure

4d). When diameters dropped down to

100 nm, AgNPs started to leach from the

fiber matrix (Figure 4f, white circle). There-

fore, the fibers with diameter around 200 nm

were chosen for further antibacterial activity

experiments.

2.3. Antimicrobial Activity

The antimicrobial activity against S. xylo-

sus was carried out. Surprisingly, no anti-

microbial activities were seen neither in any

of the AgNPs contained PS nanofibres,

including the one with maximized surface

distribution of AgNPs (obtained from

the solution containing 15%w/v PS and

2.8%w/v AgNPs), nor in AgNPs alone (data

not shown).

Considering PVP has a reducing effect on

metal ions, such as Agþ, it is postulated that

PVP might prevent the generation of Agþ

and consequently impair the antimicrobial

activity. To clarify this, we analyzed the

surfaces of nanofibers obtained from

the solution containing 15%w/v PS and

2.8%w/v AgNPs by AFM imaging in PF

tapping mode, which allows identifying

surface properties along with its morpho-

logies. Figure 5a shows the height image,

and the corresponding height profile

(Figure 5c) demonstrates that the bead-

on-a-string morphology contained AgNPs

as beads (I–I0 in Figure 5c) and the PS

nanofibers as strings (II–II0 in Figure 5c),

and that these were around 250 and 150 nm

high, respectively, which is consistent with

the SEM results (Figure 4). Further, using the

PF tapping mode, the result from adhesion

mapping obtained by AFM analysis in the

PF tapping mode is shown in Figure 5b.

Interestingly, the adhesion force values of

the beads (III–III0 in Figure 5c) and the

strings (IV–IV0 in Figure 5c) were similar. It

is thus concluded that the AgNPs located

at the fiber surfaces were still covered by a

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Fig. 5. AFM topography images (a) and adhesion force (b) of AgNPs-PS nanofibers and (c) the profiles corresponds to labels in (a) and (b).

thin layer of polymer, which was also confirmed by XPS

analysis, showing that no Ag signals were detectable

(Figure 6b). Besides, no N1s and O1s from PVP were found

in the XPS spectrum. Considering that the XPS

depth resolution is around 10 nm, it could be concluded

that the thickness of PS on the surface was at least 10 nm.

Combining PVP’s reducing effect with the limited chain

Fig. 6. (a) The schematic of removing polymer layer with UV/ozone treatment; (b–d) the XPimmobilized PS nanononwoven after 0, 10, and 15 min of treatment, respectively.

B244 http://www.aem-journal.com � 2012 WILEY-VCH Verlag GmbH & C

movement of PS in its glassy state and the low solubility of

silver ions in biological environment, the polymer layer

inhibited the release of Agþ and further impaired the

antimicrobial activities.

Ozone molecules react mostly with unsaturated double

bonds and sometimes with saturated bonds to decompose

or degrade polymers.[20] UV/ozone treatment was thus

S spectra of AgNPs

o. KGaA, Weinheim

employed to remove both PS and PVP on

the surface of the AgNPs as illustrated in

Figure 6a. Ag 3d3/2 and Ag 3d5/2 peaks

appeared in the high-resolution spectrum

upon 10min UV/ozone treatment (Figure

6c), and became sharper upon 15min UV/

ozone treatment (Figure 6d). These results

illustrate that the polymer layer was gradu-

ally degraded and Ag became exposed. On

the other hand, the appearance of O1s peak is

resulted from ozone reaction generated

carbonyl species.

The antimicrobial activity against S.

xylosus was carried out again using the

UV/ozone treated nanononwoven with

immobilized AgNPs (Figure 7). No inhibition

was seen from nonwovens that had not

received UV/ozone treatment, whereas

inhibition zones were observed at nano-

nonwoven that had received 10 or 15min

UV/ozone treatment (Figure 7a). The

antimicrobial activity was also confirmed

by immersing samples into a bacterial

suspension (Figure 7b). The safe and

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Fig. 7. The antimicrobial activity test against S. xylosus in (a) agar dish and (b) in solution.

effective antimicrobial activity of the nanononwoven could

thus be achieved by UV/ozone treatment

without loosening the AgNPs.

Further stability studies and UV–Vis measurements

showed that there was no leach-out of AgNPs (data not

shown). The safe and effective antimicrobial activity of the

nanononwoven could thus be achieved by UV/ozone

treatment without loosening the AgNPs.

2.4. Proposed Mechanism

Although, the mechanism of antimicrobial activity of

AgNPs is still not well known, it is mainly proposed to be

related to three passages (1) uptake of free Agþ followed by

disruption of ATP production and DNA replication, (2) silver

nanoparticle and silver ion generation of reactive oxygen

species, and (3) silver nanoparticle direct damage to cell

membranes.[16]

Considering possible toxicity of AgNPs, we have immo-

bilized them on the surface of nanononwovens to eliminate

the third passage, AgNPs uptake. No AgNPs leach out was

found. As antimicrobial activity from the AgNPs immobilized

nanofibers has been observed, it is believed that the activity

comes from Agþ. Furthermore, it has been found that the use

of Agþ as an antimicrobial agent is limited by the solubility

of Agþ in biological and environmental media containing

Cl�, because AgCl has a very low solubility and rapidly

precipitates out of solutions.

Therefore, we believe that the antimicrobial effect relies

on cellular uptake of Agþ by close contact to the surface

of Ag.

3. Conclusions

A safe and effective antimicrobial nanononwoven with

immobilized AgNPs was fabricated via electrospinning

followed by UV–ozone activation. PVP-coated AgNPs

were pre-prepared via polyol process and suspended in

the PS solution prior to co-electrospinning. The process

of electrospinning was optimized to achieve maximum

surface distribution of AgNPs. The optimal diameter

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of the fibers was approximately 200 nm,

obtained from a solution containing

15%w/v PS and 2.8%w/v AgNPs. Further

AFM analysis showed that AgNPs at the fiber

surface were still covered by a layer of

polymer, which inhibited the antimicrobial

activity. Time controlled UV/ozone treat-

ment was therefore, employed to degrade

the polymer layer and expose the AgNPs

without loosening them. Antimicrobial activ-

ity against S. xylosus was observed with the

UV/ozone treated nanononwovens. We

believe the antimicrobial mechanism behind

the novel antimicrobial nonwoven material

relies on cellular uptake of Agþ by close

contact to the surface of AgNPs. In conclu-

sion, the novel nanononwoven retains the enhanced anti-

microbial activities from nanofeatured AgNPs without

detectable AgNPs leaching, which holds great potential for

safe and recyclable use.

Received: September 15, 2011

Final Version: November 26, 2011

Published online: January 20, 2012

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