688
Macromolecular Research, Vol. 17, No. 9, pp 688-696 (2009)
Electrospun Antimicrobial Polyurethane Nanofibers Containing Silver
Nanoparticles for Biotechnological Applications
Faheem A. Sheikh
Department of Bionano System Engineering, Chonbuk National University, Jeonju 561-756, Korea
Nasser A. M. Barakat
*
Chemical Engineering Department, Faculty of Engineering, El-Minia University, El-Minia, Egypt
Center for Healthcare Technology Development, Chonbuk National University, Jeonju 561-756, Korea
Muzafar A. Kanjwal
Department of Polymer Nano Science and Technology, Chonbuk National University, Jeonju 561-756, Korea
Atul A. Chaudhari, In-Hee Jung, and John Hwa Lee
College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea
Hak Yong Kim*
Department of Textile Engineering, Chonbuk National University, Jeonju 561-756, Korea
Received November 27, 2008; Revised February 7, 2009; Accepted February 8, 2009
Abstract: In this study, a new class of polyurethane (PU) nanofibers containing silver (Ag) nanoparticles (NPs)
was synthesized by electrospinning. A simple method that did not depending on additional foreign chemicals was
used to self synthesize the silver NPs in/on PU nanofibers. The synthesis of silver NPs was carried out by exploit-
ing the reduction ability of N,N-dimethylformamide (DMF), which is used mainly to decompose silver nitrate to
silver NPs. Typically, a sol-gel consisting of AgNO
3
/PU was electrospun and aged for one week. Silver NPs were
created in/on PU nanofibers. SEM confirmed the well oriented nanofibers and good dispersion of pure silver NPs.
TEM indicated that the Ag NPs were 5 to 20 nm in diameter. XRD demonstrated the good crystalline features of
silver metal. The mechanical properties of the nanofiber mats showed improvement with increasing silver NPs
content. The fixedness of the silver NPs obtained on PU nanofibers was examined by harsh successive washing
of the as-prepared mats using a large amount of water. The results confirmed the good stability of the synthesized
nanofiber mats. Two model organisms, E. coli and S. typhimurium, were used to check the antimicrobial influence
of these nanofiber mats. Subsequently, antimicrobial tests indicated that the prepared nanofibers have a high
bactericidal effect. Accordingly, these results highlight the potential use of these nanofiber mats as antimicrobial
agents.
Keywords: electrospinning, nanofibers, silver nanoparticles, antimicrobial, zones of inhibition, morphological changes.
Introduction
Formation of super-bugs has become a main problem due
to frequent use of the antibiotics. Generally, due to genetic
transformation of microbial strains had created resistance
against the present antibiotics.
1
In order to have complete
removal of pathogenic strains during wound healing pro-
cess, there is a need to have an alternate strategy. Silver (Ag)
is a powerful natural antibiotic being used since ancient times
for the purpose of wound healing. When silver comes in
contact with microorganisms, it leads to sudden distortion
of cell wall which later on causes death of these organisms,
therefore, progressive steps are made for future use of the
silver based materials.
2
Silver is considered to be active
against multiple drug resistant microbial strains. Surpris-
ingly, there are minimal chances for development of bacte-
rial resistance due to immediate death of microorganisms
upon contact with silver ions. The mechanism for develop-
ment of minimal resistance against silver has
been well doc-
*Corresponding Authors. E-mails: khy@chonbuk.ac.kr or
nasbarakat@yahoo.com
Electrospun Antimicrobial Polyurethane Nanofibers Containing Silver Nanoparticles for Biotechnological Applications
Macromol. Res., Vol. 17, No. 9, 2009 689
umented.
3
This ability to create minimal or no resistance in
microorganisms can lead them to replace impotent antibiot-
ics.
4
Beside its antimicrobial activity, it has been found that
the use of silver based dressing materials enhances epithe-
lialization in clean wounds of animal model which indicates
synergistic effect of silver ions as antimicrobials and as well
as wound healing agents.
5
Modification of silver and its
compounds is important to be applicable in various medical
and paramedical fields which is quite challenging for scien-
tists till now. It is well known that the surface area of the
inorganic antibiotics is a main parameter, consequently, for-
mulation of Ag in nanostructural form is expected to have
interesting behavior. However, utilizing these nanostruc-
tural forms is not an easy task. Supporting of Ag nanoparti-
cles (NPs) on polymeric matrix is a unique and effective
methodology being exploited by many researchers. There-
fore, many studies had been carried out in this concern to
produce Ag based materials applicable in various applica-
tions such as an antimicrobial filters,
6
wound dressing mate-
rials,
7
water disinfectants,
8
chemical sensors,
9
and protective
cloths.
10,11
Actually, not only the antimicrobial agents (i.e. silver) do
have solitary importance but also the polymer matrix also
plays a vital role, so, many polymers have been exploited.
Generally, for the purpose of using a polymer in antimicro-
bial or wound healing materials, it should have the follow-
ing characters: water insolubility, appropriate pore size, non
invasive to human cells so as to support the epithelializa-
tion. Recently, many polymers have been utilized; like cel-
lulose acetate,
12
poly(acrylonitrile),
13
poly(caprolactone),
14
poly(methyl methacrylate),
15
poly(vinyl alcohol)
16
and polyim-
ide fibers.
17
Unfortunately, all the reports in such a field have
focused only on the synthesizing procedure and ignored some
important parameters affecting utilizing the final products in
nano-biotechnological applications, for instance fixedness
of the obtained silver nanoparticles and analyses of these
nanofibers for antimicrobial assay. Therefore, preparing of
silver NPs/polymer nanofiber matrix with taken in consider-
ation all the aforementioned parameters was the aim of the
present study.
During the past few decades, electrospinning technique
has been paid a considerable attention due to production of
fibers having diameter in the range of few microns to nanome-
ter scale by applying high electric fields.
18,19
In a typical
electrospinning process, electrostatically driven polymer jet
is ejected from polymer solution which undergoes bending
instability wherein the solvent evaporates and ultra fine stretched
fibers are deposited on the grounded collector.
20
Nanofibers
received from this technique have been drawn attention due
to their web like nature which exactly mimic the topology
of extracellular matrix present in human body therefore, are
used as scaffolds in tissue engineering.
21
Nanostructured mate-
rials especially, nanofibers prepared via electrospinning of
biocompatible polymer materials have tremendous abilities
such as a high surface to volume ratio and high porosity
which can be used as supporting aid for filter membranes
for water purifying systems.
22
Polyurethane (PU) is thermoplastic polymer having excel-
lent mechanical properties
23
and water insolubility.
24
More-
over, it can be used as biomaterials. Recently, it has been
explored that PU nanofibers have tremendous applications
in various fields; biosensors,
25
protective cloths,
26
and enhanc-
ing epithelial growth.
27
There are various articles regarding
electrospinning of modified PU to be used as antimicrobial
fibers.
28,29
However, according to best of our knowledge,
there is no report dealing with producing PU nanofibers/sil-
ver NPs to exploit their prompt features.
In the present study PU nanofibers containing Ag NPs
polymeric matrix has been successfully produced by using
the electrospinning technique without adding any foreign
reducing agent. Moreover, evaluation of the obtained nanofi-
ber matrices for morphological properties and crystalline
structure were investigated. Stability of the produced silver
NPs/PU nanofiber mats has been studied by several succes-
sive washing processes. Antimicrobial activities for two gram
negative test microorganisms have been evaluated. Accord-
ing to the obtained results, one can say that the prepared sil-
ver NPs/PU nanofibrous matrix could be properly employed
as recommended candidate for many biological applications
such as internal aid for water and air filters membranes and
for prolonged antimicrobial wound dressing agents.
Experimental
Materials. Polyurethane (PU, MW=110,000, medical grade)
and silver nitrate (AgNO
3
) were purchased from Cardio Tech.
Intern., Japan, and Junsei Chemical Ltd., Japan, respectively.
Tetrahydrofuran (THF) and N,N-dimethylformamide (DMF)
(analytical grade, Showa Chemicals Ltd., Japan) were used
as solvents without further purification. Difco Mueller Hin-
ton Agar and Standard Plate Count Agar were purchased
from Becton, Dickinson & Co., (Spark, MD 221 Ltd., USA)
and Hampshire, (Oxoid Ltd. England). Phosphate buffer saline
(PBS) 0.1 M, pH 7.4 was purchased from (Aldrich Co., USA).
For checking antimicrobial activity two microbial strains as
E. coli (ATCC 43890) and S. typhimurium obtained from
(National Veterinary Quarantine service Korea) were used
as a model organisms to check antimicrobial activity.
Characterization. The morphology of the nanofiber mats
had been analyzed by JEOL JSM-5900 scanning electron
microscope, JEOL Ltd., Japan. Information about the phase
and crystallinity was obtained by using Rigaku X-ray dif-
fractometer (XRD, Rigaku Co., Japan) with Cu Kα (λ=
1.540 Å) radiation over Bragg angle ranging from 30
o
to 80
o
.
Transmission electron microscopy (TEM) was done by
JEOL JEM 2010 (TEM) operating at 200 kV, JEOL Ltd.,
Japan. The mechanical properties of the nanofiber mats
were investigated by universal testing machine (UTM, AG-
F. A. Sheikh et al.
690 Macromol. Res., Vol. 17, No. 9, 2009
5000G, Shimadzu Co., Japan) under a crosshead speed of 5
mm/min. Samples were prepared in the form of standard
dumbbell shaped according to guidelines of ASTM stand-
ard via die cutting from nonwoven mats and tested in the
machine direction at least five specimens were tested for
each sample. UV-Visible measurements were performed by
using HP 8453 UV-Visible spectroscopy system (Germany),
the absorbance was measured from 400 to 1,100 nm; wave-
length. The spectra obtained were analysed by HP ChemiS-
tation software 5890 series. Average pore diameter, pore
volume, pore area, and total porosity of the electrospun
membrane were measured by the Auto Pore IV 9500 V1.05
mercury porosimeter (Micrometritis Instrument, Co., Ger-
many). The pre-weighed rectangular shaped electrospun
membranes of average 0.25 mm thickness were cut into 1×3
cm
2
as sample for analyses. Sample was placed in the cup of
the penetrometer (s/n-(10) 5 Bulb, 1.131 stem power), which
was closed by tightening the cap. Mercury filling pressure
was 3.4 kPa (0.42 psia) with evacuation time of 5 min. To
find out the effect of silver NPs, on bacterial morphology;
nanofibrous membranes were examined by Bio-SEM (Hita-
chi Co., Japan).
Procedure.
Fabrication of Nanofibers by Electrospinning: Pure PU
10 wt% was prepared by stepwise dissolving in THF and
DMF. Initially PU pellets were overnight dissolved in THF
after that DMF was added to produce final sol−gel contain-
ing 10 wt% of PU in THF/DMF (1:1, w/w). AgNO
3
/DMF
solutions were prepared and added to the PU sol−gel to
have final mixtures containing silver nitrate of 2, 5, 7, 10 wt%;
with respect to the polymer concentration. A care was taken
to protect the samples from light while working with AgNO
3
.
A high voltage power supply (CPS-60 K02V1, Chungpa
EMT Co., Republic of Korea), capable of generating volt-
ages up to 60 kV, was used as a source of electric field for
spinning of nanofibers. Polymer solution to be electrospun
was supplied through a glass syringe attached to a capillary
tip. The copper wire originating from positive electrode
(anode) connected with graphite pin was inserted into the
polymer solution and a negative electrode (cathode) was
attached to a metallic collector. Briefly, the solutions were
electrospun at 20 kV voltage and 15 cm working distance
(the distance between the needle tip and the collector). The
as-spun fibers were stored for 1 week then vacuously dried
for 24 h to remove the residual solvents.
Zone of Inhibition: Analysis of zones of inhibition (ZoI)
was carried out by making samples from silver-free PU
nanofiber mats and silver containing ones, the samples were
cut into round disk shape by cork borer having diameter of
13 mm. Before using, the samples were sterilized by ethyl-
ene oxide (EO). The microbial population were efficiently
raised and spectroscopically checked to reach density of
1×10
8
cells/mL by McFarland method. The test has been
carried out as follow, a cell suspension of 1×10
8
cells/mL
was inoculated over a plate containing solidified nutrient agar
medium onto disposable sterilized petriplates by spread plate
method. Later on, the sterilized nanofiber samples were
gently placed over the solidified agar gel under aseptic con-
ditions. Plates were incubated for 12 h at 37
o
C, and then
observation of the zones of inhibition was investigated.
Cell Morphology Changes: Cell suspensions of E. coli
and S. typhimurium having density of 1×10
8
cells/mL in PBS
were prepared. Pre-weighed electrospun membranes of 0.25
mm thickness were cut into 1×3 cm
2
of rectangular shapes
were immersed in 1×10
8
cells/mL in PBS solutions in shak-
ing incubator at 37
o
C for a period of 4 h. After such time
period; samples were taken out form the microbial popula-
tion. The bacterial fixation on nanofiber mats was con-
ducted as follow: Nanofiber disks obtained after immersion
in 1×10
8
cells/mL in PBS solutions were washed with fresh
PBS in a six well plate to remove the excess of bacteria,
PBS drain was removed. The remaining cells on the nanofi-
bers were well fixed on the nanofiber mats by adding a
proper amount of PBS containing 3% glutaraldehyde, and
then the six well plate containing the samples immersed in
PBS/glutaraldehyde solution were stored at 4
o
C for 5 h.
After such time, the nanofiber mats were removed from the
solution and washed with PBS followed by dehydration
steps via 25, 50, 75, and 100% ethanol for 15 min each. Fur-
ther on, the excess of ethanol was removed from the nanofi-
ber mats by overnight vacuum drying, and then the samples
were investigated by Bio-SEM.
Colony Forming Units: Bacterial cells of E. coli and S.
typhimurium were resuspended to provide a final density of
1×10
8
CFU/mL in PBS according to 0.5 McFarland turbid-
ity standard (approximately 1 to 2×10
8
CFU/mL). In order to
obtain countable colony number, it is noteworthy mentioning
that 10-fold dilutions were performed to obtain the final
density of 1×10
7
CFU/mL in nutrient broth. Disks (13×13 mm)
from silver-free and silver containing PU nanofiber mats
were sterilized with EO gas for 30 min, and then immersed
in 5 mL of the bacterial suspension in a 50 mL conical tube,
the media were shaken at 200 rpm at 37
o
C for 30, 60, 90
and 120 min each. After shaking incubation, 100 µL of cul-
ture media was spread over entire agar surface. Thereafter
plates were incubated at 37
o
C for overnight and CFU/mL
was counted.
Results and Discussion
Previous works have indicated that DMF does have the
ability to reduce some metallic precursors to the corre-
sponding metallic NPs,
30,31
particularly; this organic solvent
has been exploited to synthesize silver NPs from silver nitrate
at room temperature without using catalyst.
31,32
Moreover,
as aforementioned, DMF being a main solvent for PU.
33
Therefore, we have exploited these observations to self-
synthesize silver NPs within PU/(THF/DMF) electrospun
Electrospun Antimicrobial Polyurethane Nanofibers Containing Silver Nanoparticles for Biotechnological Applications
Macromol. Res., Vol. 17, No. 9, 2009 691
nanofiber mats. To get full decomposition of silver nitrate
into silver NPs, after electrospinning; the nanofiber mats
were aged for 1 week, during this storing time; visible
changes in color was observed in the electrospun mats.
These mats exhibited pinkish to brown color during a period
of one week; the brightness of the original and final colors
was depending on the silver content in the electrospun mat,
later on, no visible change in color was observed which
indicated maximum decomposition of silver salt into silver
metal. Figure 1 shows SEM images for all nanofiber formu-
lations. As shown in this figure, pure PU and PU/AgNO
3
produces smooth and bead-free nanofibers. Observable NPs
can be noticed with high silver nitrate contents (Figure 1,
panel A, B, C, D and E).
The typical XRD patterns for pristine and PU composite
nanofiber mats are presented in Figure 2. The diffraction
peaks at 2θ values of 38.11, 44.27, 64.42 and 77.47
o
corre-
sponding to (111), (200), (220), (311) crystal planes implies
the persistence of crystalline silver NPs (JCDPS, card no
04-0783). As shown in Figure 2, all the standard peaks of
pure silver are observed in the case of nanofibers obtained
from PU/silver nitrate sol−gels. While as in case of pristine
no such peaks were observed as compared with other modi-
fied counterparts. This indicates that the obtained nanofiber
mats from PU/silver nitrates sol−gels do have silver nitrate
in reduced form as pure silver particles and simultaneously
confirming ability of DMF as reducing agent. Moreover, it
can also be observed from this figure that, the intensity at
(111) main plane in the standard silver crystal lattice (at 2θ
values of 38.11
o
) increases with increasing the silver nitrate
content in the original sol−gel which leads to more silver
NPs in the electrospun nanofiber mats.
Figure 3 shows high magnification TEM image of one of
the modified nanofiber it can be observed that NPs are present
in/on the nanofiber. Overall, size of the NPs is within range
5 to 20 nm. In this context, Figure 4 shows the TEM images
of the PU nanofibers containing different amounts of silver
NPs after electrospinning. It can be observed in all the com-
binations ranging from 2, 5, 7 and 10 wt%, nearly spherical
NPs can be seen. It can be also noticed when TEM images
are compared with SEM ones that the NPs density is higher
in TEM images than SEM for all silver nitrate contents.
With taken into consideration that the electron beam in TEM
analysis passes through the nanofibers, so, it can detect the
silver NPs incorporated inside the nanofibers, however, in a
case of SEM; only the surface morphology is investigated,
consequently, we can say that the number of silver NPs
inside the obtained nanofibers are so many compared with
the NPs synthesized on the outer surface. For instance, as
shown in Figure 1(B) which reveals the nanofibers obtained
from a sol−gel containing 2% silver nitrate, no NPs can be
observed, however, in the corresponding TEM image (Fig-
ure 4(A)) some NPs are clearly visible (the dark dots). This
observation might add good feature to the prepared nanofi-
Figure 1. Scanning electron microscopy images of the PU nanofi-
bers containing different amounts of silver nitrate, 0%; (A), 2%;
(B), 5%; (C), 7%; (D) and 10%; (E) silver nitrate with respect to PU.
Figure 2. XRD results of the obtained nanofiber mats.
Figure 3. High magnification transmission electron micrograph
of individual nanofibers showing nanoparticles.
F. A. Sheikh et al.
692 Macromol. Res., Vol. 17, No. 9, 2009
ber matrix, since some authors have been proofed that PU is
biodegradable
34
so with time passing more silver NPs will
appeared which enhances the antibacterial activity for the
prepared nanofiber mats.
The obtained nanofibers were analyzed for the mechani-
cal properties by following our early designed method.
35
Figure 5 reveals the obtained stress-strain curves. As shown
in this figure, incorporating silver NPs in PU nanofibers do
have noticeable positive effect on the mechanical properties,
since the fracture tensile strength increases with increasing
the silver NPs content. Improving the mechanical properties
of the obtained silver NPs/PU nanofibers mats strongly sup-
ports utilizing of prepared mats in many versatile applications
such as filter membranes. Actually, effect of salt addition on
the mechanical properties of the electrospun mats was stud-
ied in details by other researchers whom concluded that it
has positive effect due to decrease the average fiber diame-
ter and accordingly increase the total number of fibers in the
electrospun mat.
36
Silver has an interesting feature so-called surface plasmon
resonance (SPR), this character is making the spectropho-
tometer more trustable to investigate silver content in the
solutions. SPR is a phenomenon which occurs when light is
reflected off thin metal films or NPs. A fraction of the light
energy incident at a sharply defined angle can interact with
the delocalised electrons in the metal surface (plasmon) thus
reducing the reflected light intensity.
37
In more details, when
small metallic NPs are illuminated, the oscillating electric
field causes the conduction electrons to oscillate coherently.
In particular, silver and gold metals are the most popular
materials used in this concern,
38
however, silver is most
commonly utilized,
39
because its d-s band gap is in the UV
region and does not damp out the plasmon mode as strongly
as for gold.
40
Consequently, we exploited this phenomenon
to check the stability of the synthesized silver nanoparticles
on the polymeric nanofibers. To be sure that the silver NPs
are firmly bounded with the nanofibers and will not be lost
under harsh conditions, e.g., in water purifier systems, we
performed series of wash experiments. In this regard, a pre-
weighted amount of fiber was washed with distill water at
solid to liquid ratio of 3:5,000 under vigorously stirring for
25 h. During this time period, every 2.5 h; distilled water
was replaced by fresh one. To precisely invoke spectropho-
tometer, the polymeric nanofibers (before and after wash-
ing) were dissolved in a proper solvent. In a case of silver
NPs/PU nanofibers mats, THF has been used to dissolve the
polymer and leave out silver NPs suspended in the solution.
Figure 5 shows the obtained UV-Vis absorption spectra PU
containing different amounts of silver NPs solutions (pris-
tine PU/THF solution was used as blank). It is noteworthy
mentioning that the weight of utilized samples was same in
all silver contents and for the blank solution as well. As shown
in this figure, nanofiber containing silver NPs showed
absorbance band in the range of 450 nm and this absorbance
is attributed to the characteristic surface plasmon resonance
of silver NP.
41
As can be also observed from Figure 6, the
absorbance intensity increases with increasing the silver
content in the nanofibers. This increase is observed until 7%
due to the UV instrument specification which can not detect
high silver content solution. Moreover, nearly no difference
between the spectra was observed for the washed and
unwashed mats. To practically demonstrate the results,
Table I shows the absorbance intensities at 450 nm for the
solutions of the as-prepared and after washing process; also
the relative differences were calculated. As shown in this
table, the relative difference is very small even for the high
silver content samples. According to these results, it can be
concluded that silver NPs are still remaining in the washed
Figure 4. Transmission electron micrographs of the prepared nanofi-
bers obtained from silver nitrate/PU sol−gels containing different
amounts of silver nitrate, 2%; (A), 5%; (B), 7%; (C) and 10%;
(D) silver nitrate with respect to PU.
Figure 5. Stress over strain curves of the prepared silver NPs/PU
nanofibers matrix obtained from silver nitrate/PU sol−gels con-
taining different amounts of silver nitrate.
