Antibacterial Efficiency of Nanofiber Membranes with Biologically

Transcription

International Conference on Agriculture, Biology and Environmental Sciences (ICABES'14) Dec. 8-9, 2014 Bali (Indonesia)
Antibacterial Efficiency of Nanofiber
Membranes with Biologically Active
Nanoparticles
Daniela Lubasova, and Smykalova Barbora

properties such as high surface area, high porosity, and
diameters of fibers in the nanoscale. [11-13]
Herein, the incorporation of titanium dioxide (TiO2), zinc
oxide (ZnO), zirconium dioxide (ZrO2) and tin dioxide (SnO2)
nanoparticles into nanofiber membranes made of polyvinylalcohol (PVA) was studied. Morphology of nanofiber
membranes and distribution of incorporated nanoparticles
were characterized by scanning electron microscopy (SEM)
and
energy-dispersive
X-ray
spectroscopy
(EDX).
Antimicrobial activity of individual nanofiber membranes
were tested as well. Different inhibition effects against grampositive Staphylococcus aureus (S. Aureus) and gram-negative
Escherichia Coli (E. Coli) bacteria were observed.
The nanofiber membranes with incorporated antibacterial
nanoparticles can be potentially used in several fields such as
bacterial air filtration, sanitary means and cosmetics,
antibacterial textiles, and so forth.
Abstract—In this study, the antibacterial properties of nanofiber
membranes prepared using an electrospinning technique from poly(vinyl alcohol) with incorporated zinc oxide, zirconium dioxide, tin
dioxide and titanium dioxide nanoparticles were compared. Scanning
electron microscopy and X-ray maps confirmed that all tested
nanoparticles were dispersed homogenously whereas energydispersive X-ray spectra confirmed a semi-quantitative estimation of
the nanoparticles amount presented in the nanofiber membranes.
Antibacterial properties of prepared nanofiber membranes were
evaluated by a modified AATCC test method 100-2004 using both
gram-negative Escherichia coli and gram-positive Staphylococcus
aureus bacteria. All results showed that the nanofiber membranes
with zinc oxide and zirconium dioxide nanoparticles exhibit
antibacterial properties against both tested microorganisms.
Keywords—Nanofibers, nanoparticles, antibacterial efficiency,
electrospinning.
I. INTRODUCTION
II. MATERIALS AND METHODS
The human society co-exists and constantly interacts with
the world of microorganism. Unfortunately, the modern life is
characterized by objective increase of infections. Many factors
and indicators of infection activation are subject to revision
because of changing relations between the agent and its host
organism. Moreover, there are microorganisms resistant to the
majority of antibiotics and antiseptics [1-3]. Therefore the
necessity of new effective bactericidal products is obvious.
Processing and use of biocompatible nanomaterials with
antimicrobial properties are believed to be perspective from a
long-term point of view. On that account, the application of
advanced nano-compositions based on water soluble polymers
and the use of biologically active metals (copper, zinc, zircon
etc.) seems to be desirable. [4-7]
Several papers have reported that various types of
nanoparticles or additives can be entrapped in the nanofiber
membranes or encapsulated within the nanofibers. In addition,
by varying of additives and their chemical base it is possible
to obtain different characteristics of nanofiber membranes.
The incorporation of antimicrobial nanoparticles into
nanofibers prepared using the electrospinning process has
attracted a great deal of attention because of almost unlimited
variability of nanofiber membranes properties. [8-10] An
electrospinning process is a well-known and simple but
efficient technique to produce nanofibers with unique
A. Materials
PVA (Sloviol R16) was obtained from Chemical Company
Novaky-SK and used as received. All nanoparticles (TiO2,
ZnO, ZrO2 and SnO2) with average diameter under 100 nm
were purchased from Sigma Aldrich. For consistent
distribution of nanoparticles in PVA solution was used
surfactant Triton X-100 obtained from Sigma Aldrich. For the
bacterial test, E. Coli (ATCC 8739) and S. Aureus (ATCC
6538) were purchased from Fisher Company.
B. Electrospinning of PVA nanofiber membranes with
incorporated nanoparticles
10wt.% PVA solution was prepared by dissolving PVA in
DI water under magnetic stirring at room temperature. For
consistent distribution of nanoparticles, 1wt.% of surfactant
Triton X-100 was added into PVA solution. Nanoparticles
(TiO2, ZnO, ZrO2 and SnO2) were mixed individually in PVA
solution in different concentrations: 1, 5 and 10wt.%.
The polymer solutions were placed to a plastic syringe and
then charged with a high voltage of 20 kV through a metal
syringe needle (21 gauges). Fibers were electrospun at the
needle tip and collected on a spun-bond mounted onto
collector. The collector was electrically ground and placed 10
cm away from the tip of the needle. The flow rate of the
polymer solution was controlled at 0.8 ml/hr by a syringe
pump.
Ing. Daniela Lubasova, Ph.D., Technical University of Liberec, Czech
Republic. (Email id: daniela.lubasova@tul.cz)
http://dx.doi.org/10.17758/IAAST.A1214054
55
The nanofiber membranes used for further analysis were
about 300 m in thickness. For comparison, pure PVA
nanofiber membrane without any antibacterial nanoparticles
was also fabricated using the same procedure. After
electrospinning, the samples were stored in a desiccator for
further characterization.
In case of 1wt.% of nanoparticles incorporated into PVA
polymer solution, most of nanoparticles were not observed on
the surface of nanofibers and therefore it was assumed that
most of nanoparticles were encapsulated within the
nanofibers. In contrast, SEM with nanofiber membranes
containing 10wt.% of nanoparticles indicates that
nanoparticles were encapsulated within the nanofiber and
furthermore they created bigger clusters on the top of
nanofibers. The biggest clusters created nanoparticles ZnO in
comparison to the other tested.
C. SEM, EDS and EDX mapping of nanofiber membranes
with incorporated nanoparticles
Fiber formation and morphology of the electrospun PVA
nanofiber membranes with nanoparticles were determined
using a scanning electron microscope (SEM) ULTRA PLUS
(Carl Zeiss STM AG). Distribution of nanoparticles and their
concentration in nanofiber membrane was detected by EDS
detector (X-Max 20) with software Smart SEM Version 5.05,
AZtec 2.1. SEM and EDX maps of nanofiber membranes
were taken under nitrogen atmosphere, without any previous
coating..
D.Antibacterial test of nanofiber membranes with
Coli and S. Aureus were cultivated in sterilized LB broth
and then incubated overnight at 37 °C within a shaking
incubator. The bacterial suspensions employed for the tests
contained from 102 to 103 colony forming units (CFU). For
the antibacterial test, nanofiber membranes with nanoparticles
were cut into circular swatches 4.8 cm in diameter and
sterilized in an oven for 60 min at 80°C. A nanofiber
membrane without any antimicrobial nanoparticles was used
as a blank specimen. Sterilized nanofiber membranes were
individually placed into a sterilized test tube and inoculated
with 30ml of E. coli or S. Aureus bacterial suspension. In “0”
contact time and after 24hours, 600 L of bacterial suspension
was extracted and quickly spread on Tryptic Soy agar plates.
The number of viable E. coli or S. Aureus was determined by
plating the extracted solution onto the Tryptic Soy agar plates
and counting colonies after 24 hrs of incubation at 37 °C.
The percentage reduction of test microorganisms in test
tubes with nanofiber membranes after 24 hours was calculated
using the Eqn. 1.
 B  A
(1)
R%  100 
B
where R is the reduction of test microorganism in
percentage; A is the number of bacteria recovered from the
inoculated nanofiber membrane with the nanoparticles in the
test tube after 24hours, and B is the number of bacteria
recovered from the inoculated nanofiber membrane with the
nanoparticles in the test tube at “0” contact time.
III. RESULTS AND DISCUSSION
A. Morphology of nanofiber membranes and homogeneity
of incorporated nanoparticles
Fig. 1 shows the SEM and EDX (1st and 2nd line,
respectively) maps of electrospun nanofiber membranes PVA
with 1 and 10wt.% of SnO2,TiO2, ZnO, ZrO2 nanoparticles. In
all cases, nanofibers were elongated and straight with
relatively homogenous diameters ranging from 150 to 350nm.
Fig. 1 SEM and EDX maps of PVA nanofiber membranes with
incorporated nanoparticles: (a) SnO2, (b) TiO2, (c) ZnO and (d) ZrO2
For better understanding of the homogeneity of
nanoparticles inside nanofibers, EDX maps were taken, see
Fig. 1 (the 1st line images). EDX mapping (also called element
56
TiO2, ZnO, ZrO2 and SnO2 nanoparticles were analyzed by a
modified AATCC method 100-2004 (Antibacterial finishes on
textile materials). Two tested microorganisms representing
both gram positive (S. Aureus) and gram negative (E. Coli)
were chosen. Fig. 3 shows the reduction of test
microorganisms R caused by increasing concentration of
TiO2, ZnO, ZrO2 and SnO2 nanoparticles entrapped in PVA
nanofiber membranes.
In both cases of tested microorganisms, number of bacteria
recovered from the inoculated nanofiber membrane with the
nanoparticles in the test tube after 24hours was reduced with
increasing concentration of nanoparticles. However, the
reductions of tested microorganisms R were different and
depended on the type of nanoparticles in nanofiber
membranes. The reduction of E. coli in bacterial suspension
reached 100% when PVA nanofiber membrane with 10 wt.%
of ZnO nanoparticles was in contact with tested suspension
for 24 hrs. In addition, nanofiber membrane with 10 wt.% of
ZrO2 was able to reduce about 90% of bacteria E. Coli in
bacterial suspension, whereas SnO2 had a minimal impact on
the reduction of bacteria E. Coli.
In contrast, nanofiber membrane with 10wt.% of SnO2
reduced about 81% of bacteria S. aureus in bacterial
suspension. The best antimicrobial activity against S. Aureus
showed nanofiber membrane with 10wt.% of ZrO2, where the
reduction of bacteria in bacterial suspension reached almost
99% after 24hrs. Blank control, pure PVA nanofiber
membrane without any nanoparticles did not evince any
antimicrobial activity.
These results confirmed that nanofiber membranes with
incorporated nanoparticles enhanced antimicrobial activity
against bacteria E. Coli and S. Aureus compared to the pure
PVA nanofibers without any nanoparticles. Both ZnO and
ZrO2 nanoparticles exhibit antibacterial properties against both
tested microorganism.
maps) provides, in addition to the conventional SEM image, a
meaningful picture of the elements distribution in the
nanofibers which are shown as colored maps representing
elements distribution. All EDX maps confirmed that
nanoparticles
inside
nanofibers
were
distributed
homogenously. A higher color intensity of the EDX maps
corresponds to the higher concentration of elements in the
nanofiber membrane. From the maps it is clearly visible that
the higher amount of nanoparticles (elements) incorporated
into PVA solution before electrospinning corresponds to the
higher amount of nanoparticles (elements) found in the
nanofibers after electrospinning. Unfortunately, EDX
mapping is entirely a qualitative method, therefore
quantitative confirmation of nanoparticles amount in
nanofiber membranes was analyzed by EDS.
B. EDS analysis of nanofiber membranes with
The EDS analysis of nanofiber membranes with a different
concentration of incorporated nanoparticles (1, 5 and 10wt.%)
was carried out, see Fig. 2.
The presence of Ti, Zn, Zr and Sn peaks in EDS spectra
indicates the successful incorporation of nanoparticles in PVA
nanofiber membrane through electrospinning technique.
Additionally, a higher intensity of peaks corresponds very
well with increasing concentration of incorporated
nanoparticles
Subtracting the background, the X-ray counts (y-axe in
EDS spectrum, Fig. 2) of the elements Ti, Sn, Zn or Zr in
PVA nanofiber membranes are proportional to the amount of
TiO2, ZnO, ZrO2 and SnO2 nanoparticles dispersed in the
PVA solution before electrospinning process. Instance, the
incorporation of 1, 5 and 10wt.% of SnO2 into PVA polymer
solution, the X-ray counts in EDS spectrum of nanofiber
membrane reached for element Sn values 0.42, 2.4 and 4.8
cps/eV, respectively. Similar results were observed in case of
nanofiber membranes with 1, 5 and 10wt.% of TiO2, ZrO,
ZnO2 nanoparticles. All EDS spectra confirmed a semiquantitative estimation of the amount of nanoparticles in the
nanofiber membranes after electrospinning.
Fig. 2 EDS spectra of PVA nanofiber membranes with incorporated
nanoparticles: (a) SnO2, (b) TiO2, (c) ZnO and (d) ZrO2
C.Antibacterial properties of nanofiber membranes with
Antibacterial properties of PVA nanofiber membranes with
57
[4]
[5]
[6]
Fig. 3 The reduction of test microorganisms (a) E. Coli and (b) S.
Aureus caused by increasing concentration of TiO2, ZnO, ZrO2 and
SnO2 nanoparticles entrapped in PVA nanofiber membranes
[7]
IV. CONCLUSION
[8]
PVA nanofiber membranes with incorporated nanoparticles
were prepared using electrospinning technique. The
homogenous distribution of nanoparticles TiO2, ZnO, ZrO2,
SnO2 entrapped by nanofiber membranes was confirmed by
EDX mapping along with the increasing concentration of
incorporated nanoparticles. The PVA nanofiber membranes
with all tested nanoparticles enhanced antibacterial activity
against both gram-negative E. Coli and gram-positive
S. Aureus bacteria based on the bactericidal properties of
nanoparticles. It was found out that nanofiber membrane with
SnO2 exhibited antimicrobial properties only against S. aureus
whereas nanofiber membranes with ZnO or ZrO2, exhibited
antimicrobial properties against both bacteria, particularly E.
Coli and S. aureus. These results suggest that PVA nanofiber
membranes with incorporated TiO2, ZnO, ZrO2 or SnO2
nanoparticles have a potential to be used for many different
environments where bacteria are harmful such as an airconditioning system in hospitals, variable antibacterial
products for medicine, sanitary and antibacterial textiles etc.”
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ACKNOWLEDGMENT
Daniela Lubasova, PhD., Post-doctoral
researcher at the Institute for Nanomaterials,
Advanced Technology and Innovation
The paper was supported in part by the project of the
Ministry of Education, Youth and Sports in the framework of
the targeted support of the “National Programme for
Sustainability I” LO1201, the project of Nanofibrous textile
composites for special filtration applications FR-TI3/621, the
OPR&DI project of Centre for Nanomaterials, Advanced
Technologies and Innovation CZ.1.05/2.1.00/01.0005,
CZ.1.05/3.1.00/14.0295 and the project development of
research teams of R&D Projects at the Technical university of
Liberec CZ.1.07/2.3.00/30.0024
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Antibacterial Efficiency of Nanofiber Membranes with Biologically

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