Attenuation of allergic airway inflammation and hyperresponsiveness in a murine model of asthma by silver nanoparticles.

Abstract

The use of silver in the past demonstrated the certain antimicrobial activity, though this has been replaced by other treatments. However, nanotechnology has provided a way of producing pure silver nanoparticles, and it shows cytoprotective activities and possible pro-healing properties. But, the mechanism of silver nanoparticles remains unknown. This study was aimed to investigate the effects of silver nanoparticles on bronchial inflammation and hyperresponsiveness. We used ovalbumin (OVA)-inhaled female C57BL/6 mice to evaluate the roles of silver nanoparticles and the related molecular mechanisms in allergic airway disease. In this study with an OVA-induced murine model of allergic airway disease, we found that the increased inflammatory cells, airway hyperresponsiveness, increased levels of IL-4, IL-5, and IL-13, and the increased NF-κB levels in lungs after OVA inhalation were significantly reduced by the administration of silver nanoparticles. In addition, we have also found that the increased intracellular reactive oxygen species (ROS) levels in bronchoalveolar lavage fluid after OVA inhalation were decreased by the administration of silver nanoparticles. These results indicate that silver nanoparticles may attenuate antigen-induced airway inflammation and hyperresponsiveness. And antioxidant effect of silver nanoparticles could be one of the molecular bases in the murine model of asthma. These findings may provide a potential molecular mechanism of silver nanoparticles in preventing or treating asthma.

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Open Access Full Text Article
Attenuation of allergic airway inflammation
and hyperresponsiveness in a murine model
of asthma by silver nanoparticles
11664
hee sun Park1,*
Keun hwa Kim1,*
sunhyae Jang1
Ji Won Park1
hye rim cha1
Jeong eun Lee1
Ju Ock Kim1
sun Young Kim1
choong sik Lee2
Joo Pyung Kim3
sung soo Jung1
1Division of Pulmonology, Allergy and
critical care Medicine, Department
of Internal Medicine, chungnam
National University Medical school,
Daejeon, Korea; 2Department of
Pathology, chungnam National
University Medical school, Daejeon,
Korea; 3Nanochemical Incorporation,
*contributed equally to this work
correspondence: sung soo Jung
Department of Internal Medicine,
chungnam National University
hospital and cancer research
Institute, Daesadong, Daejeon
301-721, south Korea
Tel +82-42-280-8446
Fax +82-42-257-5753
email drpark@cnu.ac.kr
Abstract: The use of silver in the past demonstrated the certain antimicrobial activity, though
this has been replaced by other treatments. However, nanotechnology has provided a way
of producing pure silver nanoparticles, and it shows cytoprotective activities and possible
pro-healing properties. But, the mechanism of silver nanoparticles remains unknown. This
study was aimed to investigate the effects of silver nanoparticles on bronchial inflammation and
hyperresponsiveness. We used ovalbumin (OVA)-inhaled female C57BL/6 mice to evaluate the
roles of silver nanoparticles and the related molecular mechanisms in allergic airway disease.
In this study with an OVA-induced murine model of allergic airway disease, we found that
the increased inflammatory cells, airway hyperresponsiveness, increased levels of IL-4, IL-5,
and IL-13, and the increased NF-κB levels in lungs after OVA inhalation were significantly
reduced by the administration of silver nanoparticles. In addition, we have also found that the
increased intracellular reactive oxygen species (ROS) levels in bronchoalveolar lavage fluid
after OVA inhalation were decreased by the administration of silver nanoparticles. These results
indicate that silver nanoparticles may attenuate antigen-induced airway inflammation and hyper-
responsiveness. And antioxidant effect of silver nanoparticles could be one of the molecular bases
in the murine model of asthma. These findings may provide a potential molecular mechanism
of silver nanoparticles in preventing or treating asthma.
Keywords: allergic airway disease, NF-κB, oxidative stress, silver nanoparticles
Introduction
Nanomaterials are a diverse class of small-scale (,100 nm) substances, formed
by molecular-level engineering and designed to offer unique mechanical, optical,
electrical, and magnetic properties.1 Recently, much effort has been devoted to the
development of biomedical applications for nanoparticles (NPs). NPs are also being
examined for potential medical applications, such as drug delivery, because they are
able to pass readily through cell membranes.2 Metal NPs have been attracting increasing
attention due to their important applications in a number of subject areas, such as
catalysis and nanoscale electronics.3–6 While significant advances in biological label-
ing have been made, few therapeutic applications for metal NPs have been reported
in the literature.7,8 Some noble metal NPs are catalysts for reduction reactions; thus,
they may be usable as antioxidants, reducing reactive oxygen species (ROS) in the
living body.9–11 Another candidate material for NPs is silver, which has long been
known as a strong antimicrobial agent and disinfectant, which may be due to the
blockage of the respiratory enzyme pathways, alterations in microbial DNA, and cell
wall modification.12 Silver has been used for centuries to prevent and treat a variety
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of diseases, including its use in pleurodesis, cauterization,
and healing of skin wounds.13,14 The use of silver in the past
has been restrained by difficulties in the production of pure
silver compounds, thereby increasing potential side effects.
Nanotechnology has provided a way of producing pure silver
NPs. Some studies of silver NPs have demonstrated that they
exhibit cytoprotective activities toward HIV-1-infected cells
and anti-inflammatory effects, through cytokine modulation
upon topical application.15–17 However, the exact mechanism
of their anti-inflammatory effects is not fully understood.
Widespread application of nanomaterials holds enormous
potential for safe human exposure and environmental release.
Elemental silver occurs naturally. It is considered non-toxic,
non-allergic, is not cumulative, and is not known to harm
either wildlife or the environment. Products made with silver
nanoparticles have been approved by a range of authorities,
including the US FDA, US EPA, the SIAA of Japan, Korea’s
Testing and Research Institute for Chemical Industry and the
FITI Testing and Research Institute. Many consumer goods
(eg, toys, baby pacifiers, clothing, food storage containers, face
masks, HEPA filters, laundry detergent) already incorporate
silver NPs for their antimicrobial properties. While inges-
tion and skin penetration are potential exposure routes for
engineered nanomaterials, the inhalation route for airborne
nanomaterials has perhaps received the most attention.18,19
In the present study, we used a mouse model of allergic
airway disease to evaluate the effect of silver NP inhalation
on airway hyperresponsiveness and inflammation and to
investigate the related molecular mechanisms.
Material and methods
Transmission electron microscope (TeM)
imaging of silver NPs
For morphological studies of silver NPs, we had assistance
from the National Nanofab Center, Korea Advanced
Institute of Science and Technology in Daejeon. To enable
the characterization of the samples using TEM imaging with
a reasonable resolution, a silver NP solution was diluted in
alcohol and was dispersed evenly for prevention of aggre-
gating particles and elimination of water. It was coated with
mesh grid during imaging. The fine structure and particle
size images of the silver NPs powder were obtained using a
TEM (JEM-3020, 300 kV, JEOL, Japan).
Animals and experimental protocol
Female C57BL/6 mice, 8–10 weeks of age and free of murine
specific pathogens, were obtained from Damul Science Inc.
(Daejeon, South Korea) and were maintained under standard
laboratory conditions in a pathogen-free cage, with ad libitum
access to food and water. All animal experiments in this
study were conducted in accordance with the guidelines of
the Institutional Animal Care and Use Committee of the
Chungnam National University Medical School.
Mice were sensitized on days 1 and 14 via an intraperitoneal
(ip) injection of 20 µg of ovalbumin (OVA) (Sigma-Aldrich,
St. Louis, MO, USA) emulsified in 1 mg of aluminum
hydroxide (Pierce Chemical Co., Rockford, IL, USA) in
a total volume of 100 µL. On days 21–23 after the initial
sensitization, the mice were challenged for 30 min with an
aerosol of 3% (weight/volume) OVA in saline (or saline alone
as a control) using an ultrasonic nebulizer (NE-U12, Omron,
Japan). Bronchoalveolar lavage (BAL) was performed
72 hours after the final challenge. At the time of lavage, the
mice (8 per group) were sacrificed with an overdose of pento-
barbital sodium (100 mg/kg body weight, ip). The chest cavity
was exposed to allow for expansion, after which the trachea
was carefully intubated and the catheter was secured with
ligatures. Pre-warmed 0.9% NaCl solution was slowly infused
into the lungs and withdrawn. The aliquots were pooled and
kept at 4°C. A part of each pool was centrifuged, and the
supernatant was kept at −70°C until use. Total cell numbers
were counted using a hemocytometer. Smears of BAL cells
were prepared using a cytospin ( Cellspin; Hanil Science
Industrial Co, Ltd, Inchon, South Korea). To examine cell
differentials, the smears were stained with Diff-Quik solution
(Dade Diagnostics of PR Inc, Aguada, Puerto Rico, USA).
Two independent, blinded investigators counted the cells
under a microscope. Approximately 400 cells were counted in
each of four random locations. The inter- investigator variation
was ,5%, and the mean number of cells from both counts
was used to estimate cell differentials.
Administration of silver NPs
Silver NPs were obtained from Nano Chemical Inc.
( Daejeon, South Korea). Silver NPs were dissolved in PBS
and administered by nebulizer (20 ppm, 40 mg/kg body
weight/day) five times to each animal at 24-hour intervals for
5 days prior to each challenge day (days 19–24), beginning
1 hour before the first challenge.
Determination of airway responsiveness
Airway responsiveness was evaluated 48 hours after the final
challenge, with the mice in an unrestrained conscious state.
The mice were placed in a barometric plethysmographic
chamber (All Medicus Co., Seoul, Korea), and baseline
readings were taken and averaged for 3 min. Increasing

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silver nanoparticles in a murine asthma model
concentrations (2.5–50 mg/mL) of aerosolized methacholine
were nebulized through an inlet into the main chamber for
3 min. Readings were taken and averaged for 2 min after
each nebulization. Enhanced pause (Penh) was used as a
measure of airway responsiveness to methacholine. Penh is
a dimensionless value that represents a function of the pro-
portion of maximal expiratory to maximal inspiratory box
pressure signals and a function of the timing of expiration.
It was calculated according to the manufacturer’s protocol
as: Penh = (expiratory time/relaxation time − 1) × (peak
expiratory flow/peak inspiratory flow). The results
are expressed as the increase in Penh following challenge
with each concentration of methacholine, where the baseline
Penh (after saline challenge) is defined as 1.
hematology and blood chemistry
Blood samples were obtained within 24 hours after
the administration of silver NPs by nebulizer (20 ppm,
40 mg/kg body weight/day) five times to each animal at
24 hour- intervals for 5 days. Mouse blood or serum was
obtained from female, BALB/c inbred mice. Hematologic
parameters were analyzed in EDTA blood samples with
XE-2100 (Sysmex, Kobe, Japan), hematology analyzer.
Blood chemistry was determined in serum samples from
the control and silver NPs inhaled group 24 hours after silver
NPs administration (by nebulizer, 20 ppm, 40 mg/kg body
weight/day).
Measurement of intracellular rOs
ROS were measured using a previously described method.20
BAL cells (1 × 104 cells) were washed in PBS, incubated for
10 min at room temperature in PBS containing 3.3 µmol/L
dichlorofluorescein diacetate (DCF-DA) (Molecular Probes,
Eugene, OR, USA), transferred to polystyrene tubes with
cell-strainer caps, and analyzed using a Cytomics FC
500 CXP flow cytometry system (Beckman Coulter Inc.,
Fullerton, CA, USA). We then measured the average percent-
age of cells that were either DCF-DA-unstained or -stained.
Fluorescence images of intracellular ROS were acquired and
analyzed using a fluorescence microscope (IX-71; Olympus
Optical Co., Tokyo, Japan).
cytosolic or nuclear protein extraction
for analysis of NF-κB p65
The lungs were removed and homogenized in 2 volume of
buffer A (50 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA, 10%
glycerol, 0.5 mmol/L dithiothreitol [DTT], 5 mmol/L MgCl2,
and 1 mmol/L phenylmethanesulfonyl fluoride [PMSF])
containing protease inhibitor cocktail. The homogenates were
centrifuged at 1,000 × g for 15 min at 4°C. The supernatants
from the homogenates were incubated on ice for 10 min and
centrifuged at 100,000 × g for 1 hour at 4°C, to obtain cytoso-
lic proteins for the analysis of NF-κB p65 levels. To prepare
soluble nuclear proteins, the pellets from the homogenate
were washed twice in buffer A, re-suspended in buffer B
(1.3 mmol/L sucrose, 1.0 mmol/L MgCl2, and 10 mmol/L
potassium phosphate buffer, pH 6.8), and centrifuged at
1,000 × g for 15 min. The pellets were re-suspended in buffer
B with a final sucrose concentration of 2.2 mol/L and centri-
fuged at 100,000 × g for 1 hour. The resulting pellets were
washed once with a solution containing 0.25 mol/L sucrose,
0.5 mmol/L MgCl2, and 20 mmol/L Tris-HCl, pH 7.2, and
centrifuged at 1,000 × g for 10 min. These pellets were
solubilized with a solution containing 50 mmol/L Tris-HCl
(pH 7.2), 0.3 mol/L sucrose, 150 mmol/L NaCl, 2 mmol/L
EDTA, 20% glycerol, 2% Triton X-100, 2 mmol/L PMSF,
and protease inhibitor cocktail. The mixture was kept on
ice for 1 hour with gentle stirring and then centrifuged at
12,000 × g for 30 min. The resulting supernatants were used
as soluble nuclear proteins for the analysis of NF-κB p65.
Protein levels were analyzed on Western blots, using an
antibody against NF-κB p65 (Cell Signaling Technology,
Beverly, MA, USA) as described above.
rNA isolation and rT-Pcr
Total RNA from lung tissues was isolated using a rapid extrac-
tion method (TRI-Reagent; Montgomery Rd, OH, USA)
as previously described.21 NA was quantified by measuring
absorption at 260 nm and was stored at −80°C before use.
Total RNA (4 µg) was reverse-transcribed to cDNA in a buf-
fer containing 50 mM Tris-HCl (pH 9.0), 16 mM (NH4)2SO4,
1.75 mM MgCl2, 0.1 M DTT; 1 µL oligo(dT) (500 µg/mL),
10 mM dNTP, 40 units RNase inhibitor, and 1 µL SuperScript II
RT (200 units/µL, Life Technologies, Grand Island, NY, USA),
in a final vol of 20 µL. This mixture was incubated for 50 min at
42°C and was heated for 15 min at 70°C for enzyme inactiva-
tion. The first-strand cDNAs were used for PCR amplification
of IL-4, IL-5, IL-13, or the housekeeping gene, GAPDH. PCR
amplification was performed by mixing 3 µL of the RT reaction
mixture with 47 µL of buffer containing 2.5 U of Taq DNA
polymerase (Promega, Madison, WI, USA) and 10 pmol of
specific primer pairs for mouse cDNA of IL-4, IL-5, IL-13, or
GAPDH, designed from published mouse gene sequences. The
primers used were as follows: IL-4 (predicted length 400 bp)
sense: 5′-CATCCTGCTCTTCTTTCTCGT-3′, antisense:
5′-GTACTACGTGTAATCCATTTG-3′; IL-5 (predicted length

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201 bp) sense: 5′-GAGCACAGTGGTGAAAGAGAC-3′, anti-
sense: 5′-ATGACAGGTTTTGGAATAGCATTT-3′; IL-13 (pre-
dicted length 182 bp) sense: 5′-GCCAGCCCACAGTTCTA
CAGC-3′, antisense: 5′- GTGATGTTGCTCAGCTCCTCA-3′
and GAPDH (predicted length 609 bp) sense: 5′-GCCA
TCAACGACCCCTTCATTGAC-3′, antisense: 5′-ACG
GAAGGCCATGCCAGTGAGCTT-3′. PCR reactions were
performed in a thermocycler (GeneAmp® PCR System 2400,
Foster City, CA, USA) using the following reaction conditions;
after an initial incubation for 5 min at 95°C, samples were sub-
jected to 27 cycles (GAPDH) or 35 cycles (IL-4, IL-5, IL-13)
of 5 min at 94°C, 30 sec at 60°C (GAPDH), 60°C (IL-4), 58°C
(IL-5), 60°C (IL-13) and 1 min at 72°C. A final extension step
at 72°C for 10 min was performed. The RT-PCR products were
electrophoretically fractioned on 2% agarose gels stained with
ethidium bromide. DNA bands were visualized after exposure
to UV light.
Western blot analysis
Lung tissues were homogenized in the presence of protease
inhibitors, and protein concentrations were determined
using the Bradford assay (Bio-Rad Laboratories Inc.,
Hercules, CA, USA). Samples were loaded onto a gel and
subjected to SDS-PAGE at 120 V for 90 min. The separated
proteins were transferred to nitrocellulose membranes (GE
Healthcare Bio-Sciences, Piscataway, NJ, USA) via the wet
transfer method (250 mA, 90 min). Nonspecific sites on the
membranes were blocked by incubation for 1 hour in 5%
non-fat dry milk in Tris-buffered saline containing Tween 20
(TBST; 25 mmol/L Tris, pH7.5, 150 mmol/L NaCl, 0.1%
Tween 20), and the blots were then incubated overnight
at 4°C with an anti-IL-4 antibody (Serotec Ltd, Oxford,
UK), anti-IL-5 antibody (Santa Cruz Biotechnology, Santa
Cruz, CA, USA), or anti-IL-13 antibody (R&D Systems,
Inc., Minneapolis, MN, USA). Anti-rat or anti-mouse horse-
radish peroxidase-conjugated IgG was used to detect antibody
binding. The membranes were stripped and re-probed with an
anti-actin antibody (Sigma-Aldrich) to verify equal loading of
proteins in each lane. Specific antibody binding was visual-
ized by exposure of the membranes to photographic film, after
treatment with enhanced chemiluminescence system reagents
(GE Healthcare Bio-Sciences, Piscataway, NJ, USA).
histological image analysis
For histological analysis, the mice were sacrificed 72 hours
after the final challenge, and the lungs and trachea were filled
with fixative (0.8% formalin, 4% acetic acid). The trachea
was ligated, and the lungs and trachea were dissected out.
The lung tissues were fixed with 10% (volume/volume)
neutral-buffered formalin. Specimens were dehydrated and
embedded in paraffin, and 4-µm-thick sections were cut using
a Leica model 2165 rotary microtome (Leica Microsystems
Nussloch GmbH, Nussloch, Germany). The sections were
placed on slides, deparaffinized, and stained sequentially
with hematoxylin 2 and eosin-Y (Richard-Allan Scientific,
Kalamazoo, MI, USA). All stained slides were evalu-
ated via light microscopy under identical conditions with
respect to magnification (20×), gain, camera position, and
background illumination.22 The degrees of peribronchial and
perivascular inflammation were scored by three independent,
blinded investigators using a subjective scale of 0–3 (0, no
detectable inflammation; 1, occasional cuffing with inflam-
matory cells; 2, most bronchi or vessels surrounded by a thin
layer of between one and five inflammatory cells; 3, most
bronchi or vessels surrounded by a thick layer of more than
five inflammatory cells), as described elsewhere.23 Total lung
inflammation was defined as the average of the peribronchial
and perivascular inflammation scores.
Densitometric analysis and statistics
All immunoreactive signals were scanned and densitometric
analyses were performed using a Gel-Pro Analyzer (Media
Cybernetics, Silver Spring, MD, USA). Data are expressed
as the mean ± SEM. Statistical comparisons were made
using one-way analysis of variance (ANOVA) followed by
Scheffe’s test. Significant differences between two groups
were determined using an unpaired Student’s t-test. Statistical
significance was set at P , 0.05.
Results
Morphological characterization
of silver NPs
TEM was used to study the morphology and particle size
of the silver NPs (Figure 1). The particles have a spherical
shape. Mean particle size is 6.0 ± 0.29 nm.
Figure 1 A) Photomicrographs of silver nanoparticles (NPs). B) Transmission
electron microscopic image of bulk of silver NPs. Bars in A) and B) indicate 20 nm
and 5 nm, respectively.

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silver nanoparticles in a murine asthma model
effect of silver NPs on hematologic
and serum chemistry profiles
To determine systemic effects of silver NPs, we introduced
hematologic and serum chemistry profiles. For example,
C-reactive protein, an acute-phase protein, is used as a bio-
marker of inflammation and lactate dehydrogenase is used as
a marker of cytotoxicity. No significant changes in any of the
hematological or serum chemistry parameters measured were
observed in the inhalation exposure to silver NPs compared
with the control (Tables 1 and 2). Only minor differences on
serum chemistry levels were observed (Table 2).
effect of silver NPs on intracellular rOs
levels in BAL cells from OVA-sensitized
and OVA-challenged mice
Intracellular ROS generation was examined under a fluo-
rescence microscope, and ROS levels were quantified via
FACScan analysis. ROS generation in BAL cells was sig-
nificantly higher at 72 hours after OVA inhalation compared
with ROS generation after saline inhalation (31.3 ± 1.1 vs
24.5 ± 1.4%, respectively). This increase in ROS generation
was substantially attenuated by the administration of silver
NPs (28.0 ± 1.2%) (Figure 2).
effect of silver NPs on NF-κB p65
protein levels in lung tissues from
OVA-sensitized and OVA-challenged mice
Western blot analysis showed a significant increase in
NF-κB p65 in the nuclear protein extracts from lung tissues
at 72 hours after OVA inhalation, compared with the level
in control mice (Figure 3). This increase in NF-κB p65
after OVA inhalation was decreased significantly with the
administration of silver NPs. In contrast, NF-κB p65 in the
cytosolic protein fractions from lung tissues was decreased
after OVA inhalation, compared with the level in control
mice, and this decrease in cytosolic NF-κB p65 was also
attenuated by the administration of silver NPs.
effect of silver NPs on IL-4, IL-5,
and IL-13 expression in lung tissues of
OVA-sensitized and OVA-challenged mice
RT-PCR revealed that IL-4, IL-5, and IL-13 mRNA levels
were increased substantially in lung tissues at 72 hours
after OVA inhalation, compared with the levels after saline
inhalation (Figure 4A). These increases were inhibited by the
administration of silver NPs. Consistent with these results,
Western blot analysis also demonstrated that cytokine levels
in lung tissues were increased significantly at 72 hours after
OVA inhalation, compared with the levels in control mice
(Figures 4B and 4C) and that these increases were inhibited
by the administration of silver NPs.
effect of silver NPs on cellular changes
in BAL fluids and lung inflammation of
OVA-sensitized and OVA-challenged mice
The numbers of total cells, eosinophils, lymphocytes, and
neutrophils in BAL fluids were increased significantly at
72 hours after OVA inhalation, compared with the cell counts
after saline inhalation (Figure 5F), and the administration
of silver NPs blocked these increases in cell counts after
OVA inhalation. Similarly, the scores for total lung inflam-
mation were significantly increased at 72 hours after OVA
inhalation, compared with the scores after saline inhalation
(Figure 5E). The total lung inflammation scores after OVA
inhalation were significantly lower with the administration
of silver NPs.
effect of silver NPs on airway
hyperresponsiveness
Airway responsiveness was assessed based on the increase
in Penh in response to increasing doses of methacholine.
Table 1 effect of inhalation exposure to silver NPs on hematologic
profiles
SALAgP
WBc
rBc
hb
hct
PLT (×106)
3,530 ± 460.32
8.60 ± 0.32
14.33 ± 0.16
41.96 ± 1.39
4.13 ± 2.33
3,550 ± 625.539
8.96 ± 0.47
13.66 ± 0.23
42.83 ± 0.81
3.59 ± 5.34
0.98
0.55
0.08
0.62
0.42
Abbreviations: sAL, saline inhaled mice; Ag, silver NPs inhaled mice; WBc, white
blood cell; rBc, red blood cell; hb, hemoglobin; hct, hematocrit; PLT, platelet.
Table 2 effect of inhalation exposure to silver NPs on serum
chemistry profiles
SALAgP
TP
ALB
AsT
ALT
ALP
LDh
crP
BUN
creA
4.4 ± 0.72
1.6 ± 0.03
111.14 ± 14.18
30.85 ± 1.1
159.71 ± 5.56
742.66 ± 82.39
0.1 ± 0.00
18.18 ± 1.7
0.11 ± 0.01
4.44 ± 0.06
1.62 ± 0.01
153.16 ± 18.15
34.33 ± 1.2
139.14 ± 4.76
841 ± 145.51
0.1 ± 0.00
23.58 ± 2.44
0.13 ± 0.01
0.65
0.51
0.91
0.56
0.16
0.60
NA
0.14
0.2
Abbreviations: sAL, saline inhaled mice; Ag, silver NPs inhaled mice; TP, total
protein; ALB, albumin; AsT, asparte aminotransferase; ALT, alanine aminotransferase;
ALP, alkaline phosphatase; LDh, lactate dehydrogenase; crP, c-reactive protein;
BUN, blood urea nitrogen; creA, creatinine.

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In OVA-sensitized and OVA-challenged mice, the Penh
dose-response curve was shifted to the left compared with the
control curve (Figure 6). In addition, Penh produced by the
administration of 50 mg/mL methacholine was significantly
greater in OVA-inhaled mice than in control mice. OVA-
sensitized and OVA-challenged mice treated with silver
NPs showed a substantial reduction in Penh at 50 mg/mL
methacholine compared with untreated mice after OVA
inhalation. These results indicate that silver NP treatment
reduces OVA-induced airway hyperresponsiveness.
Discussion
Bronchial asthma is a chronic inflammatory disease of the
airways characterized by airway eosinophilia, goblet cell
hyperplasia with mucus hypersecretion, and hyperrespon-
siveness to inhaled allergens and nonspecific stimuli.24,25
There is increasing evidence that inflammation, which is
characteristic of asthma, results in increased oxidative stress
in the airways.26 Eosinophils, alveolar macrophages, and neu-
trophils from asthmatic patients produce more ROS than do
those from normal subjects.27–30 ROS are involved in airway
smooth muscle contraction, impairment of β-adrenergic
receptors, decreased number and function of epithelial cilia,
increased mucus production, altered release of inflamma-
tory mediators, influx of inflammatory cells, and increased
vascular permeability.31
The overproduction of ROS or depression of protective
mechanisms also results in bronchial hyperreactivity, which
is characteristic of asthma.32–34 ROS directly cause contrac-
tion of airway smooth muscle preparations, and this effect
is enhanced when the epithelium is injured or removed.32
Studies in animal models have indicated that ROS contribute
to airway hyperresponsiveness by increasing vagal tone due
to damage of oxidant-sensitive β-adrenergic receptors as well
as decreased mucociliary clearance.35,36
ROS appear to directly stimulate the release of histamine
from mast cells and mucus secretion from airway epithelial
cells.37 Increased ROS release may directly result in oxida-
tive damage to epithelial cells and cell shedding.29 Previous
studies have suggested that ROS may lead to endothelial
barrier dysfunction, with subsequent increased permeability
to fluid, macromolecules, and inflammatory cells.31
The present study using an OVA-induced model of
allergic airway disease demonstrated that ROS production
Figure 2 effect of silver nanoparticles (NPs) on intracellular reactive oxygen species (rOs) levels in bronchoalveolar lavage (BAL) cells from ovalbumin (OVA)-sensitized and
OVA-challenged mice. Sampling was performed 72 hours after the final challenge. A–D) Representative microphotographs show the dichlorofluorescein (DCF) fluorescence
intensity of cells from saline-inhaled mice administered saline A), OVA-inhaled mice administered saline B), saline-inhaled mice administered 40 mg/kg of silver NPs (C), and
OVA-inhaled mice administered 40 mg/kg of silver NPs D). E–H) corresponding transmission light microphotographs are shown. I–L) A representative frequency histogram
of the fluorescence intensity of cells from saline-inhaled mice administered saline (SAL), OVA-inhaled mice administered saline (OVA), saline-inhaled mice administered
40 mg/kg of silver NPs (Ag 40), and OVA-inhaled mice administered 40 mg/kg of silver NPs (OVA + Ag 40).
SALOVAAg 40OVA + Ag 40
A
BCD
E
FG
H
L
K
JI
Counts
FL-1
103
102
101
103
102
101
FL-1
103
102
101
FL-1
103
102
101
FL-1
103
102
101
0
0
24.5%
31.3%20.4%
28.0%

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silver nanoparticles in a murine asthma model
was increased in cells from BAL fluids and that the
administration of silver NPs significantly attenuated ROS
generation, Th2 cytokine expression, bronchial inflammation,
and airway hyperresponsiveness in OVA-challenged mice.
We also noted that the increased levels of nuclear NF-κB
induced by OVA inhalation were decreased with the admin-
istration of silver NPs. NF-κB is a multiprotein complex
that is known to activate a large number of genes involved
in early cellular defense reactions in higher organisms. In
non-stimulated cells, inactive NF-κB is found in the cytosol.38
NF-κB plays an important role in immune and inflammatory
responses and is present in most cell types.39–43 The role of
ROS in the activation of NF-κB signal transduction was
initially observed in cells treated with H2O2.44 A recent study
reported that the development of an oxidant/ antioxidant
imbalance in asthma leads to the activation of the redox-
sensitive transcription factor NF-κB.45 ROS have been
directly implicated as second messengers in the activation of
NF-κB, based upon the ability of oxidants to activate NF-κB
via oxidation of its cysteine-SH group or via ubiquitination
and proteolysis of IκB.46–48
NF-κB activation induces the transcription of a variety of
inflammatory genes that are abnormally expressed in asthma,
including cytokines (eg, IL-4, IL-5, IL-9, IL-15, and tumor
necrosis factor [TNF]-α), chemokines (eg, regulated upon
activation, normal T-cell expressed and secreted; eotaxin; and
monocyte chemotactic protein-3), and adhesion molecules
Figure 3 effect of silver nanoparticles (NPs) on the protein expression of NF-κB
p65 in lung tissues collected from ovalbumin (OVA)-sensitized and OVA-challenged
mice. NF-κB p65 were measured 72 hours after the final challenge in saline-inhaled
mice administered saline (sAL), OVA-inhaled mice administered saline (OVA), saline-
inhaled mice administered 40 mg/kg of silver NPs (Ag 40), and OVA-inhaled mice
administered 40 mg/kg of silver NPs (OVA + Ag 40). A) Western blot analyses of
NF-κB p65 in nuclear (Nuc) and cytosolic (cyt) protein extracts from lung tissues.
B) NF-κB p65 protein levels in (A) were quantified using a Gel-Pro Analyzer (Media
cybernetics, silver spring, MD, UsA) and plotted as the integrated optical density,
using Microsoft excel. Bars indicate the mean ± seM and are representative of eight
independent experiments using different preparations of nuclear and cytosolic
extracts. *P , 0.05 versus sAL; #P , 0.05 versus OVA.
A
B
SALOVAAg 40 OVA + Ag 40
Integrated optical density
SAL
Nuc
Cyt
NF-κB (Cyt)
NF-κB (Nuc)
*
#
*
#
*
*
3500
3000
2500
2000
1500
1000
500
0
OVAAg 40 OVA + Ag 40
Figure 4 effect of silver nanoparticles (NPs) on IL-4, IL-5, and IL-13 expression
in lung tissues collected from ovalbumin (OVA)-sensitized and OVA-challenged
mice. Sampling was performed 72 hours after the final challenge in saline-inhaled
mice administered saline (sAL), OVA-inhaled mice administered saline (OVA),
saline-inhaled mice administered 40 mg/kg of silver NPs (Ag 40), and OVA-inhaled
mice administered 40 mg/kg of silver NPs (OVA + Ag 40). A) rT-Pcr results.
B) Western blot analyses of IL-4, IL-5, and IL-13 in lung tissues. C) Quantification
of the IL-4, IL-5, and IL-13 protein levels in B) using gel-Pro Analyzer (Media
cybernetics, silver spring, MD, UsA). The relative protein content was calculated as
the ratio of the integrated optical density of each protein to that of actin. The ratio is
arbitrarily presented as 100%. Bars indicate the mean ± seM and are representative
of eight independent experiments using different preparations of lung tissues.
*P , 0.05 versus sAL; #P , 0.05 versus OVA.
A
B
C
SAL
OVA
Ag 40
OVA + Ag 40
SAL
OVA
Ag 40
OVA + Ag 40
Integrated optical density
SAL
IL-4
IL-5
IL-13
IL-4
IL-5
IL-13
GAPDH
IL-4
IL-5
IL-13
Actin
#
#
#
*
*
*
*
300
250
200
150
100
50
0
OVA
Ag 40
OVA + Ag 40

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512
Park et al
(eg, intercellular adhesion molecule-1 and vascular cell
adhesion molecule-1).21,43,49,50 In a recent study, the anti-
oxidant L-2-oxothiazolidine-4-carboxylic acid (OTC) was
shown to inhibit airway inflammation in a murine model
of asthma.51 In that study, OTC administration significantly
reduced NF-κB translocation into the nucleus and the expres-
sion of adhesion molecules, chemokines, and cytokines.
These results show that OTC inhibits NF-κB activity by pre-
venting the ROS-induced translocation of this transcription
factor into nucleus. Antioxidants have also been reported to
inhibit NF-κB activation by preventing IκB degradation in
response to various stimuli.52–56 Taken together, these findings
indicate that the activation of NF-κB by ROS is a critical
signaling mechanism for evoking inflammatory responses.
Our results suggest that silver NPs attenuate airway
inflammation and hyperresponsiveness through the
modulation of ROS generation, which subsequently
attenuates the expression of inflammatory cytokines and
NF-κB activation in mice with allergic airway disease.
Thus, silver NPs appear to act as antioxidative and/or anti-
inflammatory agents, based on this study. Nanocrystalline
silver (NPI 32101) has been demonstrated to have anti-
inflammatory properties through both its topical and oral
use, as shown in previous experimental studies.57–60 In these
studies, nanocrystalline silver was shown to significantly
suppress the expression of IL-β, IL-12, metalloproteinase-9,
and TNF-α.57,59 When nanocrystalline silver was introduced
intravesically in a rat cystitis model, it decreased the number
of mast cells and the extent of mast cell degranulation.60
However, there are also experiments showing that silver
NPs induced inflammation.61,62 Proinflammatory cytokines,
such as TNF-α, are rapidly secreted upon stimulation with
various nanoparticles in macrophage RAW 264.7 cells.63,64
It has been proposed that nanosilver modulates cytokine
production in a concentration-dependent manner.62 These
conflict between various studies regarding the properties
of nanoparticles, including silver NPs, illustrate that the
role of the immune system is still being clarified. Among
various inorganic nanoparticles, gold nanoparticles, platinum
nanoparticles, and nano red elemental selenium have been
reported to show antioxidative effects.9–11,21–23 However,
the precise mechanism of quenching induced by platinum
nanoparticles remains unclear, and although selenium is
one of the essential metals in the body, contamination with
sodium serenite makes the use of nanoselenium difficult in
Figure 5 Effect of silver nanoparticles (NPs) on bronchial inflammation in ovalbumin
(OVA)-sensitized and OVA-challenged mice. sampling was performed 72 hours after
the final challenge in saline-inhaled mice administered saline (SAL), OVA-inhaled mice
administered saline (OVA), saline-inhaled mice administered 40 mg/kg of silver NPs
(Ag 40), and OVA-inhaled mice administered 40 mg/kg of silver NPs (OVA + Ag 40).
A−D) representative h&e-stained sections of the lungs. Bars indicate scale of
50 µm. E) Total lung inflammation scores. F) The numbers of total and differential
cellular components of bronchoalveolar lavage (BAL) fluids. Bars indicate the mean
± seM for eight mice per group in four to six independent experiments. *P , 0.05
versus sAL; #P , 0.05 versus OVA.
A
E
F
BC
D
SAL OVAAg 40 OVA + Ag 40
Inflammation score
104 cells/ml
SAL
OVA
Ag 40
OVA + Ag 40
#
#
#
#
*
*
*
*
*
3
2
1
0
Total cells
Macrophage
Eosinophil
Lymphocyte
Neutrophil
30
25
20
15
10
5
0
Figure 6 effect of silver nanoparticles (NPs) on airway responsiveness to inhaled
methacholine in ovalbumin (OVA)-sensitized and OVA-challenged mice. Airway
hyperresponsiveness was measured at 48 h after the final challenge in saline-inhaled
mice administered saline (sAL), OVA-inhaled mice administered saline (OVA), saline-
inhaled mice administered 40 mg/kg of silver NPs (Ag 40), and OVA-inhaled mice
administered 40 mg/kg of silver NPs (OVA + Ag 40). Penh values were obtained
in response to increasing doses (2.5–50 mg/mL) of methacholine. Bars indicate
the mean ± seM for eight mice per group in four to six independent experiments.
*P , 0.05 versus sAL; #P , 0.05 versus OVA.
Penh
Methacholine (mg/mL)
6
5
4
3
2
1
0
SAL
OVA
Ag 40
OVA + Ag 40
Saline 2.55 1025 50
#
#
*
*
*
*

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513
silver nanoparticles in a murine asthma model
biological applications. In the present study, we were unable
to identify the exact mechanism underlying the effects of
silver NP treatment, but the results of our 2′,7′-DCF-DA
and FACS analysis support an antioxidative effect in this
murine model of asthma. To our knowledge, this is the first
report demonstrating the antioxidative effects of silver NPs.
However, studies indicating that silver NPs may generate
free radicals have also been published.65,66
Despite the potential usefulness of silver NPs as anti-
oxidative or anti-inflammatory agents, as indicated by this
study, unresolved issues remain. First, the pharmacokinet-
ics of silver NPs are not fully understood. Second, it is
unclear where silver NPs scavenge ROS in the body of
the mouse. That is, we did not determine the exact loca-
tion of silver NP activity at the cellular level. Third, it may
be important to control the concentration of silver NPs
in order to scavenge only excess ROS and maintain the
optimal ROS level. Fourth, controversy remains regarding
the inflammatory/anti- inflammatory effects of silver NPs.
Recent studies have reported that silver NPs exerted anti-
inflammatory effects through the modulation of cytokine
production, particularly Th1 cytokines, and mast cell
activation.57,60,62 Other studies have suggested that silver
NPs have inflammatory effects.61,63,67 These conflicting
results may be attributable to several confounding factors
such as particle size, surface charge, delivery method, route
of administration, duration of administration, dose, and/or
disease conditions (intra/ extracellular milieu), which may
alter the bioactive characteristics of nanoparticles in
particular cells or tissue types.
In summary, although further studies are needed, our
study suggests that oxidative stress is an important determi-
nant of allergic airway disease and that silver NPs attenu-
ated oxidative stress in the murine asthma model. Also, by
administration of silver NPs, Th2 inflammation, which
is one of the main asthma-inducing immune factors, was
significantly decreased. We suggest that silver NPs may be
useful as a therapeutic strategy through their properties as
antioxidant and anti-inflammatory agents.
Acknowledgments
The English in this document has been checked by at least
two professional editors, both native speakers of English.
We give special thanks to Prof Minho Shong, Chungnam
National University, who supported much of the experi-
mental work. We also thank Prof Yong Chul Lee, Chonbuk
National University, for insightful recommendations and
advice.
Grant support
This research was supported by Chungnam National
University Hospital Research Fund, 2006.
Disclosure
No conflict of interest has been declared.
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