Bactericidal effect of iron oxide nanoparticles on Staphylococcus aureus.

Abstract

In order to study the effects of iron oxide (IO) nanoparticles on Staphylococcus aureus, IO nanoparticles were synthesized via a novel matrix-mediated method using polyvinyl alcohol (PVA). The IO nanoparticles were characterized by transmission electron microscopy and dynamic light scattering. Further, S. aureus were grown in the presence of three different IO nanoparticle concentrations for four, 12, and 24 hours. Live/dead assays were performed and the results provide evidence that IO/PVA nanoparticles inhibited S. aureus growth at the highest concentration (3 mg/mL) at all time points.

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International Journal of Nanomedicine 2010:5 277–283
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Bactericidal effect of iron oxide
nanoparticles on Staphylococcus aureus
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Nhiem Tran1
Aparna Mir2
Dhriti Mallik2
Arvind Sinha2
Suprabha Nayar2
Thomas J Webster3
1Department of Physics, Brown
University, Providence, Rhode
Island, USA; 2Department of
Materials Science and Technology,
National Metallurgical Laboratory,
Burmamnines 831007, Jamshedpur,
India; 3Division of Engineering and
Department of Orthopedics, Brown
University, Providence,
Rhode Island, USA
Correspondence: Thomas J Webster
Division of Engineering and Department
of Orthopedics, Brown University,
Providence, RI 02912, USA
Tel +1 401 863 2318
Fax +1 401 863 9107
Email thomas_webster@brown.edu
Abstract: In order to study the effects of iron oxide (IO) nanoparticles on Staphylococcus
aureus, IO nanoparticles were synthesized via a novel matrix-mediated method using polyvinyl
alcohol (PVA). The IO nanoparticles were characterized by transmission electron microscopy
and dynamic light scattering. Further, S. aureus were grown in the presence of three different
IO nanoparticle concentrations for four, 12, and 24 hours. Live/dead assays were performed
and the results provide evidence that IO/PVA nanoparticles inhibited S. aureus growth at the
highest concentration (3 mg/mL) at all time points.
Keywords: nanotechnology, drug delivery, bactericide, magnetic nanoparticles
Introduction
Iron oxide (IO) has been widely used in biomedical research because of its
biocompatibility and magnetic properties.1,2 Nanoparticles of IO, with sizes less than
100 nm, have been developed as contrast agents for magnetic resonance imaging
(MRI),3,4 as hyperthermia agents,5,6 and as carriers for targeted drug delivery to treat
several types of cancer.7,8 It is further believed that through the use of magnetic
nanoparticles, an optimal drug delivery system can be developed by using an external
magnetic field to direct such nanoparticles to desirable sites (such as implant infection)
for immediate treatment9 (Figure 1).
Staphylococcus aureus is one of the most common human pathogens, and leads to
many types of infection.10 This bacterium is responsible not only for local infections, such
as wounds or postoperative infection, but also for prosthetic infection (such as through the
use of catheters, endotracheal tubes, and other biomaterials).11–13 S. aureus is also known
to possess an increasing ability to resist antibiotics14–16 (such as penicillin, methicillin,
tetracycline, erythromycin, and vancomycin). Thus, it is necessary to find an alternative
treatment (perhaps without the use of antibiotics) for S. aureus infection that is directed
to the site of infection, localized, and difficult for bacteria to formulate resistance.
Along this line, some have hypothesized that reactive oxygen species (ROS)
generated by Fe3O4 nanoparticles could kill bacteria without harming non-bacterial
cells.17 Specifically, Pareta et al cultured osteoblasts (bone-forming cells) with IO
nanoparticles (at a concentration of 4.25 mg/mL) and found that cell density was
greatly enhanced in the presence of IO nanoparticles compared with cells cultured
without nanoparticles.9
Polyvinyl alcohol (PVA) is a polymer commonly used in biomedical applications
because of its biocompatibility.18 IO nanoparticles stabilized by PVA were developed
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in several studies focused on targeted drug delivery.19–21
In the present study, PVA was used as a matrix to deliver
nonagglomerated nanoparticles, as suggested by previous
researchers.22,23 The objectives of this study were to design,
synthesize, and characterize PVA/IO nanoparticles and
investigate the bactericidal activity of such nanoparticles
when cultured with S. aureus.
Materials and methods
Iron oxide nanoparticle synthesis
IO nanoparticles were synthesized in situ via a matrix-
mediated method using PVA similar to previously suggested
processes.24,25 Briefly, an aqueous solution of PVA (Acros
Organics, Geel, Belgium) was mixed with equal volumes of
ferrous/ferric aqueous solutions under ambient conditions.
The iron-loaded PVA gels were soaked in stoichiometric
amounts of warm aqueous solutions of NaOH. The resulting
ferrosoferric hydroxide dehydrates gave brownish precipita-
tions in the polymer solution.
Characterization of iron oxide/PVA
A droplet of IO nanoparticles was placed on a transmission
electron microscopy (TEM) copper grid and allowed to dry. The
imaging was carried out at 100 kV on a Philips EM420 TEM
(Philips, Amsterdam, The Netherlands) and size calculations were
carried out with ImageJ (version 1.42q; NIH, Bethseda, MD).
To measure the hydrodynamic size of the nanoparticles,
100 µL of the particle solution was diluted with 1.5 mL of
water and placed into a cuvette of a Zetasizer-nano instrument
(Malvern, Philadelphia, PA). Experiments were conducted in
triplicate to obtain an average number-size distribution. The
same apparatus was used to measure zeta potentials.
X-ray diffraction (XRD) on dried IO nanoparticle powders
was performed on a Siemens D500 (Siemens, Berlin, Germany)
within a 2θ range of 20–80 degrees using Cu Kα radiation.
The magnetic properties of the dried nanoparticles
were obtained using vibrating sample magnometry (VSM,
LakeShore 7040) at room temperature.
S. aureus culture
S. aureus were obtained in frozen form from the American
Type Culture Collection (ATCC; ATCC 25923). The bacteria
were thawed on ice for 20 minutes before being plated on an
agar plate. The plate was dried before incubation for 16 hours
in a standard cell culture environment (37°C, 5% CO2, and
95% air). A single colony of S. aureus was selected using
a 10 µL loop (Sigma, St. Louis, MO) and inoculated into
centrifuge tubes containing 5 mL of tryptic soy broth. Bac-
teria in centrifuge tubes were then incubated at 37°C under
agitation at 200 rpm for another 16 hours. At that point, the
bacteria solution was diluted in tryptic soy broth to an optical
density of 0.52 at 562 nm using a microplate reader (Spectra-
Max300; Molecular Devices, Sunnyvale, CA). According to
the standard curve correlating bacteria number with optical
density, this value was equivalent to 5 × 106 cells/mL. The cells
were further diluted in tryptic soy broth to 5 × 104 cells/mL
before being added to a new centrifuge tube at 3 mL/tubes.
Concentrated IO nanoparticles in solution were added
to bacteria tubes at different doses [30 µg/mL (low dose),
300 µg/mL (medium dose), and 3 mg/mL (high dose)]. A tube
of bacteria without nanoparticles served as a control. The IO
solution was also added to tubes containing only tryptic soy at
the same concentration as above and this served as a particle
control. Bacteria were then incubated under agitation for
four hours, 12 hours, and 24 hours before a 200 µL bacteria
solution was transferred to a 96-well plate for optical density
readings at 562 nm using a microplate reader.
S. aureus live/dead assay
After four, 12, and 24 hours of incubation, 100 µL of the
bacteria suspension was transferred into a 96-well plate.
A live/dead assay was performed according to manufacturer’s
instructions (Live/Dead BacLight, L7007; Invitrogen, Carls-
bad, CA). Briefly, two solutions containing SYTO 9 dye
and propidium iodide were mixed and diluted with double
distilled water before being added to a bacteria solution at
100 µL/well. The plate was incubated at room temperature
in the dark for 15 minutes. Fluorescence intensities for live
cells (excitation: 485 nm, emission: 530 nm) and dead cells
(excitation: 485 nm, emission: 630 nm) were measured using
a fluorescence microplate reader (SpectraMax M5; Molecular
Devices). The two intensities were divided and reported as
the ratio of live/dead bacteria.
Magnetic field line
Magnetic
nanoparticles
Magnetic force
Infections
B
Figure 1 A simplified design for a magnetic drug delivery system in which magnetic
nanoparticles coated with drugs are directed to infection sites by an external
magnetic field.

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Antibacterial effects of iron oxide nanoparticles
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Statistical analysis
All experiments were conducted in triplicate and repeated
at least three times. Differences between means were
determined using a Student’s t-test.
Results and discussion
Nanoparticle synthesis
and characterization
TEM images of the synthesized PVA-coated IO nanoparticles
(Figure 2) showed that the size of the nanoparticles was
9 nm ± 4 nm. The nanoparticles formed necklace-like
chains with a typical length of approximately 100–200 nm.
A similar formation was reported in an earlier study, in
which IO nanoparticles were believed to precipitate along
the polymer chain of PVA.22 The nanoparticle solution was
a clear brownish color and no major types of aggregation
were visible after months from synthesis.
The hydrodynamic diameter measurement results
(Figure 3) showed that with the PVA coating, the IO
chain-like particles had an average size of 140 nm.
The measured average zeta potential was −19 mV
(Figure 4). This low value suggested that the nanoparticle
solution was stable mostly because of steric repulsion but
not electrostatic repulsion. The long hydrophilic chains of
the PVA associated with water molecules, thus, preventing
agglomeration.
The XRD pattern confirmed that the final product was a
mixture of Fe3O4 and γ-Fe2O3 (Figure 5). The existence of
γ-Fe2O3 was common and was because of the oxidation of
Fe3O4 during synthesis.26
According to the VSM results (Figure 6), the negligible
coercivity of IO nanoparticles showed properties of
superparamagnetic materials, meaning that these nanoparticles
do not retain any magnetism after removal of a magnetic field.
The high magnetization and superparamagnetic properties
are highly desirable for biomedical applications because
larger magnetic particles form aggregates after exposure to a
magnetic field.2 The saturation magnetization of the synthesized
IO nanoparticles was 15 emu/g. As expected, because of the
PVA coating saturation, magnetization of nanoparticles was
lower than that of bulk phase magnetite (∼90 emu/g).
Bactericidal activity of PVA-coated IO
After four hours of incubation, the optical density measurements
(Figure 7) showed that there was no significant difference
in bacteria numbers in the presence of IO nanoparticles.
Fe304 PVA_002.tif
Cal:3.045pix/nm
15:24 07/23/09
Microscopist: 410
20 nm
HV = 100 kV
Direct Mag: 35000x
Figure 2 Transmission electron microscope images of the synthesized IO/PVA
nanoparticles. Scale bar = 20 nm.
Abbreviations: IO, iron oxide; PVA, polyvinyl alcohol.
0
1 10 1001000 10000
10
20
30
Number (%)
Size (d.nm)
Figure 3 IO nanoparticle size distribution as measured by dynamic light scattering.
Abbrevation: IO, iron oxide.

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However, and more importantly, the results from the live/dead
assay demonstrated that after four hours, the ratio of live/dead
bacteria was significantly lower in the solution with the high-
est dose (3 mg/mL) of IO nanoparticles compared with the
control sample as well as the low and medium dose samples;
the same trend was observed after 12 hours and 24 hours
(Figure 8).
There are several factors that caused the presently studied
IO nanoparticles to be bactericidal. The main mechanism by
which antibacterial drugs and antibiotics work is via oxidative
stress generated by ROS.27 ROS, including superoxide
radicals (O2
(H2O2), and singlet oxygen (1O2), can cause damage to pro-
teins and DNA in bacteria.28 Park et al also demonstrated
an antibacterial activity from silver metals because of ROS
generation.29 In this case, metal oxide Fe3O4 could be the
source that created ROS leading to the inhibition of S. aureus.
A similar process was described by Keenan et al in which
Fe2+ reacted with oxygen to create hydrogen peroxide.30 This
H2O2 consequently reacted with ferrous irons via the Fenton
−), hydroxyl radicals (−OH), hydrogen peroxide
300000
200000
100000
0
−200−100 100−2000
Zeta potential (mV)
Intensity (kcps)
Figure 4 Zeta potential of IO nanoparticles in an aqueous solution was measured by a Malvern Zetasizer-nano instrument. Average zeta potential was −19.09 mV.
Abbreviation: IO, iron oxide.
Intensity
Degrees 2-Theta
100
80
60
40
20
20
0
30
40
50
60 7080
220
311
221
400
422
511
440
533
Fe3O4
Fe3O4
Fe3O4
Fe3O4
Fe3O4
Fe3O4
γ -Fe2O3
γ -Fe2O3
Figure 5 X-ray diffraction results of synthesized nanoparticles confirmed the existence of magnetite (Fe3O4) and maghemite γ-Fe2O3.

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Antibacterial effects of iron oxide nanoparticles
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reaction and produced hydroxyl radicals which are known to
damage biological macromolecules.17
Other research has demonstrated that the small size of
nanoparticles can also contribute to bactericidal effects.
For example, Lee et al reported that the inactivation
of Escherichia coli by zero-valent iron nanoparticles31
could be because of the penetration of the small particles
(sizes ranging from 10–80 nm) into E. coli membranes.
Nano-Fe0 could then react with intracellular oxygen, leading
to oxidative stress and eventually causing disruption of the
cell membrane. Several other studies on ZnO and MgO
nanoparticles also concluded that antibacterial activity
increased with decreasing particle size.32–34
In this study, the concentration of nanoparticles was
a major contribution to S. aureus activity inhibition.
A similar concentration-dependent behavior was observed
by Kim et al when they investigated the antimicrobial effects
of Ag and ZnO nanoparticles on S. aureus and E. coli.32,35
In a study of bactericidal effects of IO nanoparticles on
S. epidermidis, Taylor et al also reported concentration-
dependent bacteria inhibition.36 Briefly, S. epidermidis
density progressively decreased at time points of 12, 24,
and 48 hours when incubated with 100 µg/mL, 1 mg/mL,
and 2 mg/mL IO.
It is also important to note that IO nanoparticles do not
negatively influence all cells. Specifically, osteoblast (bone-
forming cell) proliferation was enhanced in the presence
of Fe2O3 nanoparticles (at 4.25 mg/mL, the same order of
magnitude which inhibited bacteria in this study).9 Such
results showed that IO nanoparticles could have a dual
therapeutic function which can enhance bone growth and
inhibit bacteria infection. Lastly, this present study provided
evidence that with an appropriate external magnetic field, IO
magnetic particles may be directed to kill bacteria as needed
throughout the body.
Moment (emu/g)
Field (G)
15
−15
10
−10
5
−5
0
−1200012000
−80008000−400040000
Figure 6 Magnetization curves of IO/PVA nanoparticles as measured by VSM at
room temperature. The results clearly showed superparamagnetic behavior of the
nanoparticles.
Abbreviations: IO, iron oxide; PVA, polyvinyl alcohol; VSM, vibrating sample
magnometry.
Cell number
28000000
26000000
24000000
22000000
20000000
18000000
16000000
14000000
12000000
10000000
Fe3O4 low Fe3O4 high Fe3O4 medium
Control
4 hrs
12 hrs
24 hrs
*
*
*
*
Figure 7 Optical density reading of bacteria in IO/PVA solution after four hours, 12 hours, and 24 hours. The results are mean ± SEM. (n = 3). All densities are significantly
(*P  0.01) greater at 12 and 24 hours compared with the four-hour time point.
Abbreviations: IO, iron oxide; PVA, polyvinyl alcohol.

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Conclusions
Stable IO/PVA nanoparticles were successfully synthe-
sized. The particles were characterized with TEM, dynamic
light scattering, XRD and VSM. A live/dead assay showed
that at the highest dose of iron oxide (3 mg/mL), the growth
of S. aureus was inhibited significantly compared with the
control samples. Further studies should investigate the
bactericidal effect of iron oxide nanoparticles on other
types of bacteria for potentially widening such antibacte-
rial applications.
Acknowledgments
The authors thank the Hermann Foundation, Vietnam
Education Foundation, and the Indo-US Center for
Biomaterials for Healthcare for funding.
Disclosure
The authors report no conflict of interest in this work.
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0
10
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60
Live/Dead
A
*
IO highIO mediumIO lowControl
IO highIO mediumIO lowControl
IO high
IO medium
IO low
Control
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B) 12 hours, and C) 24 hours. Data = mean ± SEM; n = 3. *P  0.05 compared with control sample and samples with low (30 mg/mL) and medium doses (300 mg/mL) of IO
nanoparticles.
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