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Time-dependent Toxic Effect and Distribution of Silver Nanoparticles

Compared to Silver Nitrate after Intratracheal Instillation in Rats " Time-
dependent Toxic Effect and Distribut...

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DOI: 10.12691/ajn-4-1-3

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American Journal of Nanomaterials, 2016, Vol. 4, No. 1, 12-19
Available online at http://pubs.sciepub.com/ajn/4/1/3
© Science and Education Publishing
DOI:10.12691/ajn-4-1-3

Time-dependent Toxic Effect and Distribution of Silver

Nanoparticles Compared to Silver Nitrate after
Intratracheal Instillation in Rats
Alaa Fehaid1,3, Mohamed F. Hamed4, Mamdouh M. Abouelmagd3, Akiyoshi Taniguchi1,2,*
1
Cellular Functional Nanobiomaterials Group, Research Center for Functional Materials, National Institute for Materials Science
(NIMS), Tsukuba, Ibaraki, Japan
2
Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan
3
Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt
4
Department of Pathology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt
*Corresponding author: [email protected]

Abstract Silver nanoparticles (AgNPs) are widely used because of their anti-bacterial and anti-inflammatory
properties; however, the adverse health effects of these nanoparticles, especially to the lungs, have been less studied.
We thus investigated the inflammatory response of polyvinylpyrrolidone (PVP)-coated AgNPs and silver nitrate
(AgNO3) after 24 h, 14 days and 28 days of single intratracheal instillation in rats. The bronchoalveolar lavage fluid
(BALF) samples were collected and analyzed; a significant influx of neutrophils into the lung was found in both
treated groups after 24 h with a presence of AgNPs in the alveolar macrophages after 24 h, 14 days and 28 days of
instillation. Pro-inflammatory cytokines and enzymatic activities showed a significant increase after 24 h in both
treated groups with a higher significance in the AgNO3-treated group than the AgNPs-treated group. After 28 days,
these increases were completely recovered in the AgNO3-treated group but were still present in the AgNPs-treated
group. The gross examination of lung tissues revealed a clear focal inflammation in the AgNPs-treated group after
28 days. More than 29% and 9% of the initial dose of AgNPs were recovered in lung tissues after 1 day and 28 days,
respectively. Comparatively, the AgNO3-treated group recovered only 16.5% and 1%, suggesting that the silver ions
are easily absorbed into the circulation and distributed to different tissues more than the nanoparticles. Our results
indicated that the PVP-AgNPs caused a subchronic pulmonary inflammation compared to the acute one induced by
the ionic form, which can be recovered easily.
Keywords: silver nanoparticles, pulmonary inflammation, silver distribution, toxicity
Cite This Article: Alaa Fehaid, Mohamed F. Hamed, Mamdouh M. Abouelmagd, and Akiyoshi Taniguchi,
“Time-dependent Toxic Effect and Distribution of Silver Nanoparticles Compared to Silver Nitrate after
Intratracheal Instillation in Rats.” American Journal of Nanomaterials, vol. 4, no. 1 (2016): 12-19.
doi: 10.12691/ajn-4-1-3.

interact with the epithelial cells and lung macrophages

leading to pulmonary disorders [9]. Previous studies have
1. Introduction demonstrated the toxic effect of AgNPs on lung cells.
Polyvinylpyrrolidone (PVP)-AgNPs revealed a cytotoxic
Silver (Ag) has been reported to have antimicrobial and anti-proliferative effect on human lung carcinoma cell
effects. Therefore, silver nanoparticles (AgNPs) are line by ROS-dependent and independent pathways [10].
incorporated in several medical products such as wound Another study showed the size-dependent cytotoxic effect
dressing [1] and catheters coatings [2]; this is supported concluding that only 10 nm AgNPs affected the viability
by their tiny size (1 to 100 nm in diameter of one of normal human bronchial epithelial cell line (BEAS-2B)
dimension at least) [3] that helps create a very high by intracellular ion release effect [11]. Moreover, AgNPs
surface area [4]. In addition, they are also used in other and Ag ions could induce oxidative stress followed by
consumer products such as deodorant sprays [5], textiles genotoxic and cytotoxic effects [12].
[6], cosmetics, and electronics [7]. However, the Bronchoalveolar lavage (BAL) is a suitable way to
consumption of these products exhibits a potential health detect the respiratory tract toxicity of inhaled materials by
risk (i.e., silver toxicity) in humans and livestock; this providing information on both biochemical and
should be evaluated to obtain more data about silver cytological biomarkers of the inflammatory response
toxicity. induced by these materials [13]. Studies have shown that
Lungs are considered as the target organ for aerosolized the intratracheal instillation of 250 µg PVP-AgNPs leads
AgNPs in spray products due to their large surface area [8]. to a cytotoxic and inflammatory response in the lungs of
It has been found that the deposited particles in the lungs rats after 24 h as indicated by bronchoalveolar lavage fluid
 American Journal of Nanomaterials 13

(BALF) analysis. The BALF analysis revealed elevated instilled with 400 µl aliquot BSA 1% solution (control
neutrophilic number, lactate dehydrogenase (LDH), and group), AgNPs suspension (AgNPs-treated group) and
protein and cytokines levels [14]. Another study on AgNO3 solution (AgNO3-treated group) at a dose of 25,
AgNPs intratracheal instillation demonstrated the 50 and 100 µg Ag/rat. For this purpose, the anesthetized
concentration-dependent manner of pulmonary responses rats were fixed in supine position by their incisors to a
of mice in the acute stage that involves IL-1β and TNF-α string on a board slanted at 60° angle to the bench. Under
in the pathogenesis of the induced toxicity [15]. To a good light source, a flexible cannula was inserted into
examine the mechanism of AgNPs toxicity and investigate the tracheal orifice, and the solution was gently instilled in
whether it is particle-specific or mediated by released the upper third of the trachea followed by 400 µl air by
silver ions, some researchers studied the difference using an insulin syringe. AgNO3 was purchased from
between the acute toxicity of Ag ions and particles. Some Wako, Japan (CAT no: 196-00831).
reports recommended that AgNPs toxicity is mediated by
the released silver ions ([14,16,17]). One of these studies 2.4. Bronchoalveolar Lavage
concluded that the ionic form caused more potent cellular
inflammatory response than the nanoparticulate form after After instillation for 24 h, 14 days, and 28 days, 4 rats
1 day of intratracheal instillation in mice and also in from each group were anesthetized with Na pentobarbital
J774.1 murine macrophages cells [16]. (40 mg/kg body weight) and euthanized by exsanguination
Many studies have investigated the toxic effect of via the abdominal aorta. The lungs were collapsed by
AgNPs, but most of them have focused on acute toxic puncturing the thoracic cavity. A cannula was inserted
effects. Only a few studies have focused on the subchronic into the upper third of the trachea, and the lungs were
and chronic effects of nanoparticles, which are important lavaged 3 times by infusion and aspiration of saline (0.035
for assessing the health risks of AgNPs when used in ml/g body weight) using a syringe.
advanced technologies. This assessment is particularly
important when daily exposed to very few concentrations 2.5. BALF Analysis
of these nanoparticles for a long period via antimicrobial
and deodorant sprays. Moreover, no studies have 2.5.1. Cell Count
compared the pulmonary toxicity induced by Ag ions and BALF was centrifuged (4 °C, 1000 rpm, 5 min), and the
nanoparticles after a subchronic exposure with the acute first BALF supernatants were used for measuring
stage of toxicity. Therefore, the aim of this study was to cytokines concentration and enzymatic activity. The
differentiate between the time-dependent (acute and cellular pellets were resuspended in BALF supernatant,
subchronic) toxic effects of PVP-AgNPs and compare it to stained with crystal violet and used for the total cell count
that of ions after single intratracheal instillation in rats. using a hemocytometer. BAL smears were prepared by
cytocentrifugation and stained with Diff-Quik®
(International Reagents, Kobe, Japan) for differential cell
2. Materials and Methods count.

2.1. Characterization of AgNPs 2.5.2. Measurement of Enzymatic Activity in BALF

LDH activity was determined spectrophotometrically at
PVP-coated AgNPs (Sigma-Aldrich, Cat. No. 576832)
a wavelength of 580 nm using LDH cytotoxic test (Wako,
have a particle size of less than 100 nm. To characterize
Japan). Alkaline phosphatase (ALP) activity was measured
the morphology and size of AgNPs, transmission electron
spectrophotometrically at a wavelength of 405 nm
microscope (TEM; JEM-2000FX, JEOL) and electronic
following Lab AssayTM ALP (Wako, Japan). β-
light scattering detector (ELS; zeta potential and particle
Glucuronidase (βG) activity was measured
size analyzer ELSZ-2000, Otsuka Electronics, Japan) were
spectrophotometrically at a wavelength of 540 nm
used. AgNPs were sterilized by autoclaving (121 °C for
according to the Enzymatic Assay of ß-Glucuronidase (EC
20 min) and suspended in sterile deionized water
3.2.1.31, Sigma-Aldrich).
containing 1% BSA by ultrasonication.
2.5.3. Measurement of Cytokines Concentrations in
2.2. Animals BALF
Specific pathogen-free male Sprague-Dawley rats (4 IL-1β concentration was measured using an enzyme-
weeks old) were purchased from Clea Japan (Tokyo, linked immunosorbent assay kit (ELISA; DuoSet, R&D
Japan). The rats were housed in a clean room with Systems, Japan), and IL-18 concentration was measured
controlled conditions (23±1 °C, 12 h light and 12 h dark using ELISA kit (Invitrogen). The absorbance was
cycle) and were fed on standard laboratory pelleted diet measured at a wavelength 450 nm for both concentrations
and water ad libitum. The animal study was approved by the as mentioned in the manufacturer’s protocols.
Ethics Committee for Animal Care and Experimentation
of the National Institute for Environmental Studies (Japan). 2.6. Ag Concentrations Measurement
The whole lung, liver, kidneys, and testis tissues were
2.3. Intratracheal Instillation
wet-digested using nitric acid and hydrogen peroxide
At 5 weeks of age, the rats were divided into three main (0.75 and 0.25 ml, respectively for 0.2 g tissue) at 135 °C
groups (control, AgNPs-treated group and AgNO3-treated for 48 h. The digested samples were diluted with
group) and anesthetized with 2.5% halothane in a carrier deionized water to measure Ag concentrations using
gas (nitrous oxide:oxygen=1:2). They were intratracheally
14 American Journal of Nanomaterials

inductively coupled plasma-mass spectrometry (ICP-MS; same doses of AgNPs, we established a dose-dependent
7500c, Agilent Technologies, Tokyo) at m/z 107. experiment. Figure 2 (A-F) shows the dose-dependent
effect of AgNPs compared to AgNO3 on the inflammatory
2.7. Histopathological Studies and cytotoxic markers in BALF samples. As shown in
Figure 2 (A), neutrophils ratio was significantly increased
Specimens from the lungs were preserved in 10% in both treated groups compared to the control. However,
neutral buffer formalin. Sections of 5-micron thickness there was a significant increase in the case of AgNO3-
were prepared from all specimens, stained with treated group with different doses (25, 50 and 100 µg
hematoxyline and eosin (H&E) and examined Ag/rat) compared to the same doses in the AgNPs-treated
microscopically. group. The results indicated that ions were more toxic than
particles. The highest percent of neutrophils was induced
2.8. Statistical Analysis by the highest doses in both treated groups up to 76.4% in
Data were subjected to statistical analysis using a AgNO3 and 64.8% in AgNPs-treated groups.
statistical software program (GraphPad InStat version 3.00 The enzymatic activities of LDH, ALP and βG are
for Windows 95, GraphPad Software, USA). The shown in Figure 2 (B, C and D) and cytokines concentrations
differences between the mean values of different groups (IL-1β and IL-18) are shown in Figure 2 (E and F). These
were determined using one-way ANOVA with Duncan parameters showed a significant increase in 25, 50 and
multiple comparison tests. A P value less than 0.05 was 100 µg Ag/rat AgNO3-treated groups compared to the
considered to be statistically significant. Data were control. The AgNPs-treated group showed significant
presented as mean ± standard deviation (SD). increase only in the case of 50 and 100 µg Ag/rat, but not
in the lowest dose group. In addition, there was a
significant increase in AgNO3-treated groups compared to
3. Results AgNPs–treated groups in case of LDH, βG and IL-1β.
These findings indicate that the ions were more toxic than
3.1. Characterization of AgNPs particles, and the lowest dose of AgNPs did not induce
any toxic effect compared to the same dose of ions.
In order to characterize the morphology, size and zeta Moreover, the toxicity was dose-dependent and increased
potential of NPs, TEM and ELS detector were used. on increasing the dose.
Figure 1 shows the TEM image of AgNPs suspended in
BSA 1%; NPs were slightly aggregated and spherical in 3.2.2. Time-dependent Effect
shape with the mean size being 50–90 nm. The average In order to know the effect of time on these toxic signs
hydrodynamic diameter of AgNPs sonicated for 10 min in and whether it will be recovered or not, we analyzed
deionized water was 167.1 nm ± 53.1 and the
BALF samples after 24 h, 14 days and 28 days of
polydispersity index was 0.202. The zeta potential of instillation of 100 µg Ag/rat. Figure 3 (A-F) shows the
AgNPs in water was −53.2 mv as measured by ELSZ- time-dependent effect of AgNPs compared to AgNO3 on
2000.
the inflammatory and cytotoxic markers in BALF samples
after single intratracheal instillation of 100 µg Ag/rat. As
shown in Figure 3 (A), the neutrophils ratio was
significantly decreased in BALF samples after 14 days
and 28 days of the instillation compared to the percent
after 24 h. The results showed that the neutrophils ratio
was significantly increased in both treated groups
compared to the control. Furthermore, the AgNO3-treated
group showed significantly higher neutrophils ratio
compared to the AgNPs-treated group after 24 h. The
results indicate the toxic effects of Ag ions than the
AgNPs during the first 24 h.
The enzymatic activities of LDH, ALP and βG are
shown in Figure 3 (B, C and D), and cytokines concentrations
(IL-1β and IL-18) are shown in Figure 3 (E and F). In the
case of AgNO3-treated group, all these parameters showed
a significant increase only after 24 h of the instillation
compared to the control and showed no any significant
Figure 1. Representative TEM image (250.000X magnification) of PVP- differences after 14 and 28 days. Compared with the
AgNPs. AgNPs were suspended in BSA 1% and sonicated for 30 min. A control and AgNO3-treated group, the AgNPs-treated
drop was placed onto a collodion membrane-attached sheet mesh (150-A) group showed a continuous significant increase in
and dried at room temperature. AgNPs are spherical and approximately activities of ALP and βG and in cytokines concentrations
50–90 nm in diameter
after 14 and 28 days of the instillation. These findings
indicate the complete and quick recovery of rats after the
3.2. BALF Analysis acute toxicity caused by silver ions. Moreover, NPs lead
to a subchronic toxic effect that persisted up to 28 days
3.2.1. Dose-dependent Effect after the instillation.
To detect the differences between the toxic effects of To examine the possibility of finding the nanoparticles
different doses (low, medium and high) of AgNO3 and the in the lungs after 28 days of instillation, we examined the
 American Journal of Nanomaterials 15

BALF cells microscopically. Figure 4 shows a with internalized AgNPs. The results indicate the presence
representative BALF cells image after intratracheal of AgNPs in the activated alveolar macrophages even after
instillation of AgNPs (100 µg/rat) after 28 days of 28 days. These results confirmed the previous data
instillation. The black arrows show alveolar macrophages showing the persistence of NPs in lungs for a longer time.

Figure 2. Dose-dependent effect of AgNPs compared to AgNO3 on inflammation and cytotoxic markers in BALF samples. Lungs were lavaged after 24
h of single intratracheal instillations of 25, 50 and 100 µg Ag/rat. (A) Neutrophils ratio (%), (B) LDH activity, (C) ALP activity, (D) βG activity, (E) IL-
1β and (F) IL-18 concentrations. The results are shown as mean ± SD., n=4 for each group. (*) represented the significant difference from control group.
(#) showed the significant difference from the corresponding AgNO3-treated group

group, only trace amounts of Ag were detected in the liver,

3.3. Ag Concentration Measurements in kidney and testis after 24 h. In contrast, 9.75%, 0.59% and
Different Tissues 0.15% of the initial dose of silver were recovered in liver,
Ag concentrations were measured in different tissues to kidney and testis, respectively, in the AgNO3-treated
know the distribution and fate of silver inside the body. group. After 14 and 28 days, there was no any significant
Table 1 shows the percentage (%) of Ag remained in presence of Ag in the tissues of the AgNO3-treated group.
different tissues after single intratracheal instillation of In contrast, 15.97% and 9.29% of Ag were recovered in
AgNO3 and AgNPs (100 µg Ag/Rat) as an initial dose. liver tissues of the AgNPs-treated group after 14 and 28
The concentration of Ag in lung tissue of AgNPs-treated days, respectively. Moreover, there was a significant
group was significantly higher than that in the AgNO3- increase of Ag percentage in kidney and testis of the
trated group after 24 h and 14 days. In the AgNPs-treated AgNPs-treated group after 14 and 28 days. These data
indicate that ions were easily absorbed into the circulation
16 American Journal of Nanomaterials

and distributed to the different tissues during the first 24 h. and we could not find any NPs in the organs other than the
In contrast, NPs were slowly absorbed and distributed, lung except after 14 days of instillation.

Figure 3. Time-dependent effect of AgNPs compared to AgNO3 on inflammation and cytotoxic markers in BALF samples. BALF samples were
collected 24 h, 14 days and 28 days after 100 µg Ag/rat exposure. (A) Neutrophils ratio (%), (B) LDH activity, (C) ALP activity, (D) βG activity, (E)
IL-1β and (F) IL-18 concentrations. The results are shown as mean ± SD., n=4 for each group, (*) represented the significant difference from control
group, (#) showed the significant difference from the corresponding AgNO3-treated group

Table 1. Percentage (%) of Ag remained in different tissues. Numbers indicate % of Ag in the whole organ after single intratracheal instillation
of AgNO3 and AgNPs (100 µg Ag/Rat) as an initial dose. Tissues were collected after 24 h, 14 days and 28 days and digested by acids. Then Ag
concentrations were measured by ICP-MS at m/z 107. The results are shown as mean ± SD., n=4 for each group. (a) Significantly different
from control group. (b) Significantly different from the corresponding AgNO3-treated group
Group 24 h. 14 days 28 days
Organ AgNO3 AgNPs AgNO3 AgNPs AgNO3 AgNPs
16.5 29.39a 4.64 29.09a 1.08 9.01
Lung
±4.75 ±7.55 ±1.69 ±16.5 ±0.47 ±10.18
9.75a 0.03 3.41 15.97ab 2.09 9.29a
Liver
±2.61 ±0.01 ±1.85 ±4.63 ±1.69 ±11.1
0.59a 0.004b 0.07 0.69ab 0.06 0.22
Kidney
±0.09 ±0.0004 ±0.02 ±0.77 ±0.04 ±0.13
0.15a 0.0009b 0.14a 0.28ab 0.06 0.1a
Testis
±0.05 ±0.0005 ±0.02 ±0.1 ±0.01 ±0.03
 American Journal of Nanomaterials 17

3.4. Gross and Histopathological Examination

of Lung Tissues
To see the effect on the histology of lung tissues, we
examined the tissues macroscopically and microscopically.
The gross examinations of lung tissues showed very clear
congestion and diffuse inflammation of lung tissues of
both treated groups after 24 h of instillation of 100 µg
Ag/rat. After 28 days, the AgNPs-treated group showed
the persistence of clear focal inflammation of lung tissue
as shown in Figure 5 (C). In contrast, AgNO3-treated
group lung tissues were completely recovered as shown in
Figure 4. Representative BALF cells image after intratracheal Figure 5 (B) compared with the control lung tissues in
instillation of AgNPs (100 µg/rat) after 28 days of instillation. Black
arrows show alveolar macrophages with internalized PVP-AgNPs
Figure 5 (A).

Figure 5. Lung tissues after 28 days of instillation of 100 µg Ag/rat. Panel (C) shows persistence of clear focal inflammation in the AgNPs-treated
group (B) recovery of the AgNO3-treated group compared to panel (A), which shows the normal tissue of control group

Figure 6. Histopathology of lung tissues (H&E staining). (A and B) show lung tissue of control group after 24 h and 28 days respectively with normal
histological architecture, and normal intrapulmonary bronchioles, 4x. (C) Lung tissue of AgNO3-treated group after 24 h shows acute pneumonia where
active hyperemia of interstitial capillaries and marked neutrophilic recruitment alveoli (arrow), 10x. (D) Lung tissue of AgNO3-treated group after 28
days shows normal pulmonary alveoli and normal intrapulmonary bronchioles, 4x. (E) Lung tissue of AgNPs-treated group after 24 h shows marked
diffuse thickening in interstitial tissue with eosinophilic (arrow) and histiocytic infiltration and fibroblastic proliferation, 10x. (F) Lung tissue of AgNPs-
treated group after 28 days shows focal recruitment of eosinophils (arrows) and histiocytic replacing pulmonary tissue, 10x
18 American Journal of Nanomaterials

Figure 7. Hypothesis illustrated the events that were induced by AgNPs compared to Ag ions after 24 h, 14 and 28 days

of the initial dose of silver were recovered in lung tissues

4. Discussion and Conclusion of AgNPs-treated group after 24 h, 14 days, and 28 days
respectively while it was only 1.08%, 4.6%, and 16.5%,
In this work, we studied the time-dependent pulmonary respectively, in the case of AgNO3-treated group.
toxic effect and distribution of AgNPs compared to Ag Furthermore, there were traces of Ag in liver, kidney and
ions after single intratracheal instillation in rats. First, we testis of the AgNO3-treated group after 14 and 28 days
exposed rats to different doses of AgNPs and ions (25, 50 while it was significantly increased in the AgNPs-treated
and 100 µg Ag/rat) for 24 h to determine the dose group.
appropriate for studying the time-dependent toxic effect. The findings of our study concluded that ions were
From the enzymatic activities of LDH, ALP and βG, and easily absorbed into the blood circulation and distributed
cytokines concentrations (IL-1β and IL-18) in BALF to the different tissues during the first 24 h causing acute
samples, we found that the ions were more toxic than the toxicity, which was recovered easily within less time. In
particles. The lowest dose of AgNPs did not induce any contrast, PVP-AgNPs were slowly absorbed and
toxic effect compared to the same dose of ions. In addition, distributed leading to a subchronic toxicity after single
the toxicity was dose-dependent and increased when the intratracheal instillation in rats.
dose of both ions and particles were increased. Therefore, To explain the reason that delayed the toxicity of
we decided to use the highest dose (100 µg Ag/rat) to AgNPs after that of Ag ions, as shown in Figure 7, we
establish the time-dependent experiment. hypothesized that during the first 24 h, Ag ions could
We found that the ions were more toxic than easily go inside the pulmonary epithelial cells leading to
nanoparticles after 24 h of instillation. However, after 14 cell death. This was then engulfed by activated
and 28 days, AgNPs were still found to be toxic, causing a macrophages, which subsequently released the cytokines
significant increase in inflammatory biomarkers compared and induced the inflammatory process. This event could
to control and Ag ions-treated group, which was almost be stopped by the cellular defense. While in the case of
totally recovered after 14 days. The presence of PVP-coated AgNPs, depending on their size, it might go
internalized AgNPs in the alveolar macrophages (Figure 4) inside the epithelial cells either without any active process
with the gross and histopathological examinations of lung and stayed free in the cytoplasm with very poor release of
tissues confirmed the same results as shown in Figures 5 silver ions because of the cytoplasmic environment, or by
(C) and 6. It is well known that the target organs of endocytosis forming phagolysosomes / endosomes and
AgNPs are lung and liver as confirmed by a subchronic stayed there to release the silver ions slowly by the
inhalation study in rats in which AgNPs also accumulated lysosomal environment, or others can be trapped by the
in kidney tissues showing some changes in the macrophages and enter the lysosomes and also stayed
histological structure [18]. Moreover, a previous study has there for a time for slowly release of silver ions which
concluded that AgNPs led to a deleterious effect on the then induce the cytokines production and inflammatory
testes that increased progressively with time after oral response. This hypothesis will guide our future work to
administration of 20 µg/kg B/W for 90 days [19]. In investigate the mechanism of cell toxicity induced by
another recent study, the tissue distribution of Ag AgNPs by using in-vitro studies in different pulmonary
measured by ICP_MS was in the same line with the other epithelial cell lines.
mentioned data showing that 29.39%, 29.09%, and 9.01%
 American Journal of Nanomaterials 19

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