Ecotoxicology and Environmental Safety 198 (2020) 110674

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Silver nanoparticles induce size-dependent and particle-specific T

neurotoxicity to primary cultures of rat cerebral cortical neurons
Bingjie Zhanga,b, Na Liua,c, Qian S. Liua, Jianqing Zhangd,∗∗, Qunfang Zhoua,b,e,∗, Guibin Jianga,b
a
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085,
China
b
College of Resources and Environment, University of Chinese Academy of Sciences, Beijing, 100049, China
c
School of Life Science, Shanxi University, Taiyuan, 030006, China
d
Shenzhen Center for Disease Control and Prevention, Shenzhen, 518055, China
e
School of Environment, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310000, China

A R T I C LE I N FO A B S T R A C T

Keywords: Silver nanoparticles (AgNPs) are widely applied in many fields because of their excellent antibacterial activities.
Silver nanoparticles Toxicological studies have showed that AgNPs can cross the blood-brain barrier and exhibit high retention in the
Size-dependent effect brain. Therefore, the potential neurotoxicity of AgNPs is raising serious concerns. This study investigated the
Particle-specific effect neurotoxicological effects of AgNPs with two different sizes (20 nm and 70 nm, AgNPs-20 and AgNPs-70) using
Neurotoxicity
primary cultures of rat cerebral cortical neurons in mature and developing stages. The contribution of silver ion
Primary cultures of cerebral cortical neurons
release was investigated by testing the effects of ionic silver in parallel. The results showed that both AgNPs-20
and AgNPs-70 significantly decreased neuronal cell viability, and AgNPs-20 had stronger toxicity compared with
AgNPs-70. AgNP applications caused the granulated skeleton structure of the mature neurons with some broken
synapses after a 24-h exposure, and inhibited neuronal growth during a 7-day exposure. Intracellular silver
accumulation at non-cytotoxic exposure levels inhibited dopamine efflux, which was particle-specific and free of
released silver ions. The findings herein can aid in guiding the proper applications of AgNPs in different areas,
especially in medical use.

1. Introduction
Silver nanoparticles (AgNPs) are a kind of metallic silver with at
least one dimension at nano scale (< 100 nm) in three-dimensional
space. These are widely used as antimicrobial agents in various com-
modities, such as wound dressings and antibacterial clothes. AgNPs can
be released into the environment during the processes of manufacture,
use, and disposal (McShan et al., 2014). They may migrate and trans-
form in the atmosphere, soil, surface water and ground water (Pachapur
et al., 2016). The increase of AgNP release in the environment poses a
threat to wildlife and human health. A large number of studies have
shown that AgNPs can cause damage to organs, including the kidneys,
heart, and lungs (Kaewamatawong et al., 2014), and induce adverse
effects on immune and reproductive systems (Ema et al., 2017).
Recent studies have indicated that AgNPs can penetrate the blood-
brain barrier (Chen et al., 2016; Dan et al., 2018) and exert high re-
tention in the brain (Wen et al., 2016). Neurobehavioral studies showed
that the exposure of AgNPs inhibited locomotive activities, and caused
cerebellar ataxia-like symptoms in Sprague Dawley (SD) rats (Zhang
et al., 2013; Yin et al., 2015). Histopathological studies showed that
AgNPs caused obvious distortions in both Purkinje and granular layers
of rat brains, and the granular layer was degenerated with loosened and
separated structure (Yin et al., 2015). AgNPs were also reported to in-
duce astrocytic proliferation, neuronal degeneration, and demyelina-
tion (Ahmed and Hussein, 2017).
Although there are a large quantity of existing neurotoxicological
studies of AgNPs based on in vivo experiments and in vitro immortalized
cell tests (Zhang et al., 2018), investigations into how AgNPs affect
primary cortical neurons remain limited. A previous study demon-
strated a direct effect of AgNP upon neurotoxicity by showing that
AgNPs caused the loss of cytoskeleton components and neurite out-
growth inhibition in premature cortical neurons (Xu et al., 2013).
Nevertheless, more investigations are warranted regarding the neuro-
logical alterations of primary cultures of mature and developing

∗
Corresponding author. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy
of Sciences, Beijing, 100085, China.
∗∗
Corresponding author.
E-mail addresses: jianqingzh@szcdc.net (J. Zhang), zhouqf@rcees.ac.cn (Q. Zhou).

https://doi.org/10.1016/j.ecoenv.2020.110674
Received 19 July 2019; Received in revised form 20 April 2020; Accepted 21 April 2020
Available online 05 May 2020
0147-6513/ © 2020 Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
B. Zhang, et al.
neuronal cells.
In addition to studies regarding the neurotoxic effects of AgNPs, it
remains unclear whether the mechanism occurs via particulate or ionic
properties. Some studies reported that the toxicological effect of silver
ions (Ag+) on cell apoptosis was stronger than that of AgNPs (Hadrup
et al., 2012). Other studies showed that AgNPs induced cytotoxicity by
a Trojan-horse type mechanism (Eun-Jung et al., 2010), which meant
that AgNPs would further release silver ions to cause toxicity after
entering cells. Nevertheless, several recent studies have indicated that
AgNPs may have special particulate effects (Sun et al., 2016). The
characteristics of AgNPs, such as particle size, are also considered to be
important factors in the regulation of their biological activities (Gliga
et al., 2014). Therefore, it would be important to demonstrate how
AgNPs of different sizes induce neurotoxic effects on primary neuronal
cells.
In the present study, AgNPs with different particle sizes (20 nm and
70 nm, AgNPs-20 and AgNPs-70) and silver ion were applied to cultures
of primary rat cerebral cortical neurons at early development and
mature stages in order to evaluate cell viability, neuron-specific skeletal
protein expression, intracellular silver accumulation, and neuro-
transmitter secretion. The findings of the present study based on pri-
mary cultures of mature and developing neurons may provide im-
portant insight into the neurotoxicity of AgNPs of different sizes and
their particle-specific effects.
2. Materials and methods
2.1. Reagents
Two kinds of polyvinylpyrrolidone (PVP)-coated AgNP suspensions,
including AgNPs-20 (20 nm, 1 mg/mL as silver) and AgNPs-70 (70 nm,
1 mg/mL as silver) were commercially purchased from Shanghai
Huzheng Nano Technology Co., Ltd. (Shanghai, China) and
nanoComposix, Inc. (USA), respectively. Silver nitrate (AgNO3, > 99%)
was obtained from Sigma-Aldrich (USA), and its stock solution (1 mg/
mL as silver) in distilled water was used as the control of ionic silver.
The working solutions of three silver species were prepared by suitable
dilution with neurobasal medium (Gibco, Thermo Fisher Scientific,
USA) under sonication for cell exposure experiments.
2.2. AgNP characterization
The physicochemical properties of AgNPs-20 and AgNPs-70 used in
this study were characterized after ultrasonication in aqueous solution
for 5 min. The morphology of AgNPs in both water and cell culture
medium were observed by transmission electron microscopy (TEM,
JEOL, 2100F, Japan). Briefly, several droplets of AgNP suspensions
were dispersed on carbon membrane-coated copper nets and air dried.
The representative images were then photographed, and the sizes of
AgNPs were analyzed based on counting 200 particles in four views
using Nano Measurer software (Version 1.2, China). The hydrodynamic
particle sizes as well as ζ-potentials were determined by a dynamic light
scattering (DLS) technique using Malvern Zetasizer Nano ZS (Malvern,
UK) at room temperature, and the measurements were repeated for
three times. Total silver levels in two kinds of AgNPs were measured by
inductively coupled plasma mass spectrometry (ICP-MS, Agilent 8800,
USA) after digestion with the mixture of HNO3:H2O2 (3:1) at 95 °C for
2 h. Silver ion release was evaluated by setting different concentrations
of AgNP suspensions in neurobasal medium, which were incubated at
37°C with 5% CO2 for 24 h and 7 days, respectively, to mimic the fol-
lowing exposure systems. The levels of Ag+ released from AgNP sus-
pensions were analyzed by ICP-MS after ultracentrifugation (Millipore,
Amicon 10 kDa, 16,000 g, 30 min).
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Ecotoxicology and Environmental Safety 198 (2020) 110674
2.3. Primary cultures of cortical neurons
All experimental procedures involving animals were performed in
accordance with the protocols approved by the Committee of Scientific
Research in the Research Center for Eco-Environmental Sciences,
Chinese Academy of Sciences. Pure cortical neurons were obtained from
SD rat embryos on embryonic day 18 as previously reported (Hossain,
2016). Briefly, the embryonic cortices dissected from the pregnant fe-
male SD rats (Beijing Vital River Laboratory Animal Technology Co.,
Ltd., China) were cut into small pieces and enzymatically digested.
After dissociation and filtration, the single cell suspension was seeded
in poly-L-lysine-coated plates and cultured in plating medium at 37°C
with 5% CO2 for 24 h. Then, the cells were incubated in neurobasal
medium supplemented with 0.25% GlutaMAX and 2% B27, and 2.5 μM
cytosine arabinoside (Sha et al., 2019), a mitotic inhibitor, was used on
the second day to eliminate glial cells. The primary cultures of cortical
neurons were maintained with half of the culture medium renewed
every 4 days afterward.
2.4. Cell morphology and purity evaluation
The neuron cells plated in 6-well plates at the density of
4.5 × 105 cells/well were cultured for 7 days, and then fixed with 4%
paraformaldehyde for 30 min. After washing three times with phos-
phate-buffered saline (PBS), the cells were incubated in PBS solution
supplemented with 10% FBS and 1% Triton X-100 at 37°C for 2 h, and
then immunoreacted with anti-neuron-specific β-III tubulin (TuJ1) an-
tibody (R&D, USA, 1:100, monoclonal mouse IgG2A) for 3 h at room
temperature in the dark. After rinsing with PBS, the cells were subse-
quently stained with 4,6-diamino-2-phenylindole (DAPI, Solarbio, USA)
for 1 h, and observed using the inverted fluorescence microscope
(Olympus, IX73, Japan).
After a 7-day culture, the cells were fixed and washed in 6-well
plates, then permeabilized by 0.2% Triton X-100, and washed with PBS.
Then, the cells were incubated with anti-NeuN antibody (Abcam, USA,
1:300) at 4°C overnight, followed by incubation with goat anti-rabbit
IgG (H + L) (Thermo Fisher Scientific, USA, 1:500) at 37°C for 1 h and
final DAPI staining. The images were taken by the inverted fluorescence
microscope based on the random selection of the visual fields and
analyzed by Image J software (Ver 1.4.3, National Institutes of Health).
2.5. Cell viability assay
To study the neurotoxicity of AgNPs, mature neuronal cells after a 7-
day culture were exposed to chemicals for 24 h (method 1). To study
the neurodevelopmental toxicity of AgNPs, neuronal cells were cultured
for 24 h, exposed to chemicals for 7 days (method 2), and the neurite
outgrowth of the cells was carefully observed. Half of the exposure
medium (the culture medium containing different concentrations of
chemicals) was renewed every 4 days afterward.
The cells plated in 24-well plates at the density of 3.0 × 105 cells/
well were exposed to different concentrations of AgNPs and AgNO3. In
method 1, the exposure concentrations were 0.01, 0.1, 0.5, 1, 2, 5, 10,
20, 30, and 40 μg/mL, whereas in method 2, the concentrations were
0.01, 0.1, 0.5, 1, 2, and 5 μg/mL. When the exposure was terminated,
half of the exposure medium (400 μL) was aspirated, and 40 μL of
100 μM alarmarBlue (AB) reagent (Sigma-Aldrich, USA) was added to
each well. The plates were incubated at 37°C with 5% CO2 for 2 h in the
dark. The cell viability was evaluated by measuring fluorescence in-
tensities at 530 nm/590 nm (λexcitation/λemission) using the multi-
function microplate reader (Thermo Scientific, VARIOSKAN FLASH,
USA). The EC50 values of the test chemicals were calculated by
Nonlinear Curve Fit (Logistic) using Origin 2017 (Version 94E).
B. Zhang, et al.
Fig. 1. AgNP characterization in medium. (A, B) TEM images. (C, D) The particle size distribution. (E, F) The hydrodynamic particle sizes.
2.6. Immunocytochemical staining
The cells plated in 6-well plates at the density of 9.0 × 105 cells/
well were stimulated by different concentrations of AgNPs and AgNO3
(1, 2, and 5 μg/mL for method 1, and 0.1, 0.5, and 2 μg/mL for method
2). After exposure, the cells were fixed with 4% paraformaldehyde and
washed three times with PBS. They were incubated in PBS solution
supplemented with 10% FBS and 1% Triton X-100 for 2 h at 37°C, and
immunoreacted with TuJ1 antibody (R&D, USA) at the dilution ratio of
1:100 for 3 h at room temperature in the dark. The cytoskeleton mor-
phology was observed using the inverted fluorescence microscope, and
photographed at 493 nm/514 nm (λexcitation/λemission), based on
tracking the fluorescence of fluorochrome NL493 conjugated directly
on the purified TuJ1 antibody. The total fluorescence densities of dif-
ferent 55,000 μm2 were analyzed by ImageJ software.
2.7. Analysis of intracellular silver
The cells plated in 100 mm dishes at the density of 4.5 × 106 cells/
dish were exposed to 0.5 μg/mL AgNPs and AgNO3 for 24 h in method
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Ecotoxicology and Environmental Safety 198 (2020) 110674
1, and 0.5 μg/mL AgNPs and AgNO3 for 24 h, 72 h, and 168 h, re-
spectively in method 2. After exposure, the cells were washed for three
times with cold PBS, and lysed for 30 min. The cell lysates were col-
lected after centrifugation (9,600 g, 5 min) to remove cell debris,
avoiding non-specific attachment of substantial amounts of chemicals
on cell surface. Total silver concentrations in cell lysates were measured
by ICP-MS (Agilent 8800, USA) after digestion in the mixture of
HNO3:H2O2 (3:1) at 95°C for 2 h. Untreated cell lysates were analyzed
as negative control, and the undetectable silver content confirmed no
cross contamination during exposure experiments. The protein con-
centrations of cell lysates measured by Pierce BCA kit (Thermo, USA)
were used to calibrate final results of intracellular silver contents.
2.8. Measurement of dopamine efflux
The cells plated in 6-well plates at the density of 9.0 × 105 cells/
well were exposed to different concentrations of AgNPs (0.01, 0.1, 0.25,
and 0.5 μg/mL) and 0.05 μg/mL AgNO3 for 24 h (method 1) and 7 days
(method 2), respectively. When the exposure was terminated, the
medium in each well was collected for the evaluation of dopamine
B. Zhang, et al.
efflux. The cells were washed three times with cold PBS, and submitted
to subsequent analysis of protein contents. The concentration of dopa-
mine was analyzed by a rat dopamine enzyme-linked immunosorbent
assay (ELISA) kit (Shanghai Yanjin Biological, China). The final values
were calibrated by cell protein concentrations in different conditions,
and the results relative to the control were expressed to adjust the
variations from different batches of the experiments.
2.9. Statistical analysis
All results were expressed as the mean values ± standard devia-
tions (SDs) of at least three independent experiments for each assay.
The statistical analysis was conducted for different groups by one-way
analysis of variance (ANOVA) with the Bonferroni multi-group com-
parison test. The significant differences were considered when the p-
value was less than 0.05 (*) or 0.01 (**).
3. Results
3.1. AgNP characterization
It has been shown that the particle size (Wang et al., 2018), shape
(Jasinski et al., 2018), and coating (Zheng et al., 2018) can affect the
biological effects of nanomaterials. To correlate the toxicity data with
the specific properties of nanoparticles, two kinds of AgNPs were
comprehensively characterized in this study. TEM results showed that
the test AgNPs were uniform in size and well dispersed in both water
(Fig. S1A, Fig. S1B) and cell culture medium (Fig. 1A, Fig. 1B). No
obvious agglomeration was observed in any of the image fields. By
fitting AgNP sizes with normal distribution function (Fig. S1C, Fig. 1C),
the particle sizes of AgNPs-20 were 27.67 ± 3.39 nm and
15.56 ± 1.53 nm in water and culture medium, respectively. Those of
AgNPs-70 were 69.86 ± 6.97 nm and 84.39 ± 10.92 nm in water and
culture medium, respectively (Fig. S1D, Fig. 1D). These results were
consistent with the product information provided by the manufacturers.
DLS measurement indicated that the hydrodynamic particle sizes of
AgNPs-20 were 38.47 ± 5.68 nm with a polydispersity index (PDI) of
0.239 in water, and 49.35 ± 3.74 nm with a PDI of 0.264 in the
culture medium (Fig. S1E, Fig. 1E). As for AgNPs-70, the hydrodynamic
sizes were 87.12 ± 1.61 nm with a PDI of 0.294 in water and
91.71 ± 1.58 nm with a PDI of 0.342 in the culture medium (Fig. S1F,
Fig. 1F). The zeta potentials of AgNPs-20 and AgNPs-70 in water were
−15.30 ± 0.08 mV and −14.33 ± 1.48 mV, respectively. No ob-
vious aggregation was observed during any of the following exposure
experiments.
Silver ion release would definitely influence the biological effects of
AgNPs, and this process could be affected by many factors, such as
AgNP concentration, storage condition and pH of the dispersion (Choi
et al., 2018). The determination of silver ion release ratios was thus
Fig. 2. Effects of AgNPs and AgNO3 on neuronal cell viability (n = 3). (A) Mature cells on day 7 treated for 24 h. (B) Developing cells treated for 7 days.
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Ecotoxicology and Environmental Safety 198 (2020) 110674
performed for AgNPs dispersed in cell culture medium at different
concentrations. The results in Table S1 show that the silver ion release
ratios of AgNPs-20 and AgNPs-70 (1.0–5.0 μg/mL) were 0.9%–5.3%
and 2.4%–7.9% during 24 h, respectively. Regarding 7 days of ex-
posure, silver ion release ratios of AgNPs-20 and AgNPs-70 (0.1–1.0 μg/
mL) were 2.8%–9.9% and 2.3%–11.4%, respectively. The two sizes of
AgNPs had similar silver ion release ratios. Comparatively, Ag+ release
ratios were relatively higher in lower concentrations of AgNP disper-
sions.
3.2. Cell morphology and purity
The freshly isolated cortical neuronal cells were counted and eval-
uated by the cell counting instrument (Life Technologies, AMQAX1000,
USA), and the batches with living cell proportions higher than 90%
were submitted to further culture. The cell growth observation by the
inverted microscope indicated that neurons were well spread and ad-
hered in dishes 4 h after plating. After the 7-day culture, cells differ-
entiated into mature neurons, and the tight neuronal network structure
was well developed (Figs. S2A–S2C), which could also be evidently
characterized by the structural protein, TuJ1, expressed in neuronal cell
bodies and axons (Fig. S3A). Mature neuronal cells could well survive
for 1 more week (Figs. S2D–S2F), thus providing an attractive experi-
mental model of primary cultures with an approximately 2-week testing
window for neurotoxicity studies.
To characterize the purity of primary cultures, the NeuN-stained
neuronal cells (red fluorescence) and total cells indicated by a DAPI-
counterstained nucleus (blue fluorescence) were evaluated after the 7-
day culture, and the results are shown in Figs. S3D–S3F. The consistent
overlay of NeuN and DAPI-stained neuronal nuclei in the merged
images shows the high purity of neuronal cultures. Quantitative ana-
lysis based on counting the numbers of both NeuN- and DAPI-stained
cells in three independent cell culture batches (four fields per batch)
showed that the average purity of neuronal cells was greater than
90.7% (Table S2), confirming that the lab-prepared cell models were
free of glial cell contamination (Lavezzi et al., 2013).
3.3. Cell viability
To investigate the potential neurotoxicity of AgNPs and silver ions,
the viability of neuronal cells exposed to different concentrations of the
test agents were evaluated using AB assay. The results in Fig. 2A
showed that all test silver species caused exposure concentration-de-
pendent inhibition in cell proliferation after 24-h exposure. Based on
the calculation of logistic fitting curves for their dose responses, the 24-
h EC50 values were obtained for AgNPs-20 (6.61 ± 0.28 μg/mL),
AgNPs-70 (38.4 ± 1.61 μg/mL), and AgNO3 (2.86 ± 0.12 μg/mL),
respectively. The cytotoxicity order of the test silver species of
AgNO3 > AgNPs-20 > AgNPs-70 was thus concluded. It was
B. Zhang, et al.
apparent that ionic silver was more toxic than particulate silver, and
AgNPs with smaller size exerted higher neurotoxicity than did those
with larger size, potentially due to the fact that small particulate silver
was easier for cells to take up (Chen et al., 2015) and possessed higher
bioactivity (Riaz Ahmed et al., 2017). The no-observed-adverse-effect
level (NOAEL) for the test silver species was approximately 1 μg/mL.
Regarding the strategy of 7-day exposure, the results in Fig. 2B
showed typical exposure concentration-related decreases in cell viabi-
lity for all three test silver species, wherein, the logistic fitting curves
for silver ion and AgNPs-20 were very similar. The calculated 7-day
EC50 values were 1.08 ± 0.4 μg/mL, 2.02 ± 0.07 μg/mL and
1.22 ± 0.04 μg/mL for AgNPs-20, AgNPs-70, and AgNO3, respectively.
The toxicity order based on 7-day exposure experiments was AgNPs-20,
AgNO3 > AgNPs-70, which was different from that observed in the 24-
h experiments. The NOAEL of the test silver species was approximately
0.5 μg/mL, and it was apparent that their cytotoxicity was obviously
elevated when the exposure period was extended to 7 days. Obvious
decreases in cell viability were observed even when the exposure con-
centrations were 1 μg/mL or less. In addition, the cytotoxic effect of
AgNPs-70 increased sharply in the 7-day exposure group when com-
pared with that in the 24-h treatment, and its EC50 value decreased
from 38.4 ± 1.61 μg/mL to 2.02 ± 0.07 μg/mL, which suggested the
importance of studying the long-term and low-dose effects of chemicals,
although the low toxicity of AgNPs with large sizes has been commonly
reported (Gottschalk et al., 2009).
3.4. Cytoskeleton changes
To characterize cellular morphological changes induced by the ex-
posures of three silver species at cytotoxic and non-cytotoxic levels,
neuronal cells with different silver treatments (1–5 μg/mL for 24 h, or
0.1–2 μg/mL for 7 days) were submitted to inverted microscopy ob-
servation under both bright and fluorescent modes based on TuJ1 im-
munostaining.
According to the images under bright field (Fig. S4A), the well-de-
veloped neuronal networks were formed in negative controls. The 24-h
treatments of AgNPs-20 and AgNO3 induced breakages in neuronal fi-
laments (indicated by black arrows) to different extents, whereas
AgNPs-70 did not (Figs. S4B–S4D). According to TuJ1 im-
munocytochemical staining images, the neuronal cells were evenly
distributed with big and intact cell bodies located in filament networks,
and the synapses of different neurons interlaced and joined into the
reticular structure in negative controls (Fig. 3A). Comparatively,
AgNPs-20 at 1 μg/mL or 2 μg/mL caused few changes in the neuronal
Fig. 3. The influences of three silver species on cytoskeleton structures of neuronal cells. (A–D) Fluorescent images for TuJ1 expression in mature neuronal cells
treated with 2 μg/mL silver species for 24 h. (E–H) Fluorescent images for TuJ1 expression in developing neuronal cells treated with 2 μg/mL silver species for 7 days.
The white arrows indicate the fragmentation of neuronal networks.
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Ecotoxicology and Environmental Safety 198 (2020) 110674
networks, whereas the obvious granulated skeleton structure with
broken synapses was observed when the exposure concentration was
increased to 5 μg/mL (indicated by white arrows, Fig. 3B, Figs. S5A and
S5B). As for AgNPs-70, they caused few deleterious effects on neuronal
morphology in any of the exposure groups (Fig. 3C, Figs. S5C and S5D).
In view of AgNO3 treatments, the observable damages to neuronal
networks occurred in the 2 μg/mL exposure group, and more severe
granulation in neuronal cells with ruptured structures appeared when
the exposure concentration was increased to 5 μg/mL (Fig. 3D, Figs.
S5E and S5F). Quantitative analysis of fluorescence intensities in dif-
ferent exposure groups showed that 2 μg/mL AgNPs-20 and 2–5 μg/mL
AgNO3 significantly compromised the expressions of TuJ1 (p < 0.01),
whereas the fluorescence intensities of the other groups showed no
difference from that of the negative control (Fig. 4A).
Compared with the results from the 24-h exposure experiments, 7-
day treatments of AgNPs and AgNO3 caused more severe degeneration
in the cytoskeleton structures. More specifically, the granular and
fractured cytoskeleton structures were observed in different treatments.
The delayed neuronal development was observed due to the prolonged
chemical treatments, resulting in sparse filaments in both bright fields
(Figs. S4F–S4H) and fluorescent views (Fig. 3F–H, S6). The im-
munostaining was apparently decreased, showing that the neurotoxi-
city could be induced by all test silver species at both cytotoxic and non-
cytotoxic exposure levels. The quantitative analysis for the im-
munostaining of TuJ1 expression showed significant decreases in all
test groups (p < 0.01), which were exhibited in exposure concentra-
tion-related manners (Fig. 4B).
3.5. Intracellular silver contents
Recent studies have indicated that NPs enter into cells mainly
through endocytosis (Greulich et al., 2011), and this process can be
affected by the characteristics of NPs, such as surface charge (Bannunah
et al., 2014) and particle size (Iversen et al., 2011). The intracellular
AgNPs can be expelled out of cells through slow exocytosis (AshaRani
et al., 2009), thus forming a dynamic equilibrium process. The eva-
luation of intracellular silver levels could aid in understanding cyto-
toxicity induced by AgNPs. The concentration of 0.5 μg/mL was se-
lected in follow-up experiments to avoid excessive damage to cells.
Investigations on silver contents in mature neuronal cells after a 24-
h exposure of AgNPs showed that treatments of three silver species at
the concentration of 0.5 μg/mL all increased intracellular silver levels
ranging from 152 μg/g to 197 μg/g protein (Fig. 5A), and the accu-
mulation followed the order of AgNPs-20 > AgNPs-70 > AgNO3,
B. Zhang, et al.
Fig. 4. Quantification of neuron beta-tubulin III by fluorescent TuJ1 expression in response to increasing doses of AgNPs-20, AgNPs-70, and AgNO3 exposures for (A)
24 h, and (B) 7 days. n = 3, **p < 0.01 versus the control.
showing the relatively higher bioavailability of particulate silver com-
pared with ionic silver. The time course shown in Fig. 5B demonstrates
that the particulate-derived intracellular silver concentrations increased
over time compared with ionic-derived silver. In regard to the accu-
mulation capacity of different silver species, the exposure of particulate
silver caused relatively higher intracellular silver levels than did ionic
silver, which was in accord with the result in Fig. 5A. In addition, the
intracellular silver levels in the AgNPs-20 exposure groups were gen-
erally higher than those in AgNPs-70 treatments, suggesting that the
penetration of AgNPs through the cell membrane could be particle size-
related. Moreover, intracellular silver levels in developing neurons ex-
posed to AgNPs for 7 days did not increase in contrast to increased
levels observed in mature neurons exposed for 24 h. This indicates that
prolonged exposure time does not contribute to intracellular silver ac-
cumulation, suggesting that the dynamic equilibrium of silver between
endocytosis and exocytosis may exist to maintain metal homeostasis in
neurons.
3.6. Dopamine efflux
Dopamine is one of the most important neurotransmitters, and it is
critical to exercise regulation, learning, memory, and addictive beha-
vior (Langdon et al., 2018). In vitro studies have shown that excessive
dopamine secretion can cause cytotoxic effects, whereas insufficient
dopamine release may result in disease risks, such as Parkinson's dis-
ease (Hisahara and Shimohama, 2011). AgNP exposure has been re-
ported to alter dopamine levels in Wistar rat brains (Hadrup et al.,
2012). The changes in dopamine release from the primary cultures of
neuronal cells upon AgNP exposure were studied herein to find the
potential influences of the test silver species on neuronal functions. The
Fig. 5. Intracellular silver accumulation in mature neuronal cells after 24-h exposure (A), and developing neuronal cells during 7-day exposure (B). The exposure
concentration of each chemical was 0.5 μg/mL. n = 3, *p < 0.05, and **p < 0.01.
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Ecotoxicology and Environmental Safety 198 (2020) 110674
result (Fig. 6A) shows that 24-h exposure of AgNPs-20 and AgNPs-70 to
mature neurons both caused significant concentration-dependent de-
creases in extracellular dopamine levels (p < 0.01). Comparatively,
AgNPs-20 exerted stronger inhibition effect on dopamine efflux than
did AgNPs-70. Considering the potential contribution of silver ions re-
leased from AgNPs to the disturbed dopamine efflux, an equivalent
concentration of AgNO3 (0.05 μg/mL) comparable to 0.5 μg/mL AgNPs
with an Ag+ release ratio less than 10%, was evaluated for its effect on
dopamine level. The result (Fig. 6A) shows no significant effect on
neurons with AgNO3 when compared to control (p > 0.05), indicating
that AgNP-induced inhibition in dopamine efflux is particle-specific
rather than ionic.
For developing neuronal cells after a 7-day exposure to AgNPs, the
data in Fig. 6B shows that inhibition of dopamine levels exhibits a U-
shaped curve with increasing AgNP levels (0.01–0.5 μg/mL). The dif-
ferent dopamine release responses in developing neuronal cells
(Fig. 6B) compared with those in mature ones (Fig. 6A) represented the
complicated regulation of neuronal function in different primary cul-
tures. When compared with AgNPs-70, AgNPs-20 exerted relatively
stronger effects on altering dopamine efflux, showing a similar particle
size-related effect to that observed in Fig. 6A. The test for 7-day ex-
posure of 0.05 μg/mL AgNO3 also showed no changes in dopamine
efflux (Fig. 6B), further confirming the particle-specific effect of AgNPs
on developing neurons. Altogether, AgNP-caused inhibition in dopa-
mine efflux in both mature and developing neurons suggested their
potential disturbances in neuronal function.
4. Discussion
The biosafety of AgNPs is of great importance, considering their
B. Zhang, et al.
Fig. 6. AgNPs disturbed dopamine release from primary cultures of cerebral cortical neurons (n = 3). (A) Mature cells at day 7 treated for 24 h. (B) Developing cells
treated for 7 days. *p < 0.05, and **p < 0.01.
wide application in diverse fields. And tuning the characteristics of
AgNPs would influence their bioactivities and toxicities, including
neurotoxicity. In the present study, two kinds of AgNPs with different
sizes, together with AgNO3, were evaluated for their potential size-re-
lated and particle-specific effects on primary cultures of cortical neu-
ronal cells. The findings provided new insights for toxicity-directed
AgNP synthesis strategy and risk assessment on commerically available
AgNPs.
With regard to AgNP-induced neurotoxicity in primary cultures of
neuronal cells, the cell viability assay showed the toxicity order of the
three silver species (Fig. 2), which was visually demonstrated by cell
morphological alterations (Fig. S4). The cytoskeleton characterized by
TuJ1 protein indicated the deleterious effect caused by the test silver
species (Figs. 3 and 4), which was in accordance with previous findings
of degeneration of neuronal structures and dissolution of β-tubulin
proteins in AgNP-exposed corticle neurons (Xu et al., 2013). In-
tracellular silver existence (Fig. 5) caused the internal exposure of
neuronal cells, which would be responsible for the incidence of neu-
rotoxicological effects. The decreased dopamine efflux in AgNP ex-
posure groups (Fig. 6) showed the potential purturbation of neuronal
function, which would be clinically related to neurodegenerative dis-
eases, such as Parkinson's disease (Hisahara and Shimohama, 2011).
Concerning the neurotoxicity of AgNPs, the role of silver ion release,
and particulate- and size-related effect required clarification.
It is well known that Ag+ release is an important mechanism of
AgNPs' bactericidal effect (van Aerle et al., 2013). As a slow and con-
tinuous release source of Ag+, AgNPs have more lasting bactericidal
effects (Zhou et al., 2015; Wang et al., 2013). Most studies reported that
Ag+ exerted higher toxicity than did AgNPs. For example, at 1.5 μg/mL
total silver, A549 cells exposed to an AgNP suspension containing 39%
Ag+ caused a cell viability of 92%, whereas cells exposed to an AgNP
suspension containing 69% Ag+ caused a cell viability of 54% (Beer
et al., 2012). The viability of planktonic N. europaea cells was inhibited
by 95% upon 0.5 μg/mL Ag+ exposure and by 87% upon 20 μg/mL
AgNP exposure (Barker et al., 2018). The 24-h EC50 values were ob-
tained for AgNPs-20 (6.61 ± 0.28 μg/mL), AgNPs-70
(38.4 ± 1.61 μg/mL), and AgNO3 (2.86 ± 0.12 μg/mL), respectively,
in this study, which were consistent with previous findings that Ag+
was more toxic than AgNPs. Moreover, 7-day EC50 values were
1.08 ± 0.4 μg/mL, 2.02 ± 0.07 μg/mL and 1.22 ± 0.04 μg/mL for
AgNPs-20, AgNPs-70, and AgNO3 respectively, which indicated that the
ability of AgNPs to degrade cell viability was increased after chronic
exposure. Similarly, long-term continuous exposure to AgNPs lowered
the effective inhibitory concentration by 1–2 orders of magnitude when
compared with those observed in short-term exposures (Barker et al.,
2018).
Further, the unique particle-specific effects of AgNPs have also been
extensively reported, which could be different from Ag+-induced ef-
fects. For instance, it was reported that the cell cycle pathway in larval
7
Ecotoxicology and Environmental Safety 198 (2020) 110674
zebrafish could be affected by both AgNPs and dissolved Ag+, but the
regulation patterns by these two silver species were opposite (Kang
et al., 2016). AgNPs could accumulate in the nucleus of NIH3T3 cells
and induce DNA damage, compared with Ag+ (Jiravova et al., 2016).
Different neurotoxic mechanisms were found for AgNPs and AgNO3 in a
recent study on human embryonic stem cell-derived neuron and as-
trocyte network (Repar et al., 2018). Regarding intracellular silver ac-
cumulation and dopamine release, the findings in the present study also
showed a series of distinct effects induced by AgNPs and AgNO3 on
primary neuron cells, suggesting that particle-specific effects induced
by AgNPs could be more than what the released Ag+ could explain.
When it comes to biological effects of AgNPs, particle size is usually
recognized as a crucial factor. For instance, smaller AgNPs more easily
entered cells (Chen et al., 2015), and had relatively higher biological
activity due to higher specific surface areas. According to the results
obtained herein, AgNPs-20 exposure caused relatively stronger effects
on primary neuronal cultures than did AgNPs-70, in view of cytotoxi-
city, cell morphology, and dopamine efflux. Nevertheless, neurotoxic
effects induced by AgNPs-70 were obviously increased when exposure
time was prolonged. In addition, the vulnerability of mature and de-
veloping neurons to AgNP exposure could also be different, indicating
the potential neurodevelopmental toxicity of AgNPs, which was in ac-
cordance with previous findings (Ema et al., 2017; Yin et al., 2018).
Collectively, the neurotoxicity of two kinds of AgNPs (AgNPs-20 and
AgNPs-70) was evaluated using primary cultures of mature and devel-
oping neuronal cell models. AgNP exposures caused particle size-re-
lated cytotoxicity, cellular silver accumulation, and dopamine release.
The developing neurons upon 7-day exposure responded more mark-
edly to cytotoxic and non-cytotoxic exposure levels of three silver
species than did mature neurons based on cell viability assay and TuJ1-
probed cytoskeleton structure observation. The disturbances in dopa-
mine efflux induced by AgNP exposure was particle-specific, although
silver ions exerted higher toxicity to neuronal cells. These findings
provide substantial data on neurotoxicological effects of two different
sizes of AgNPs, and this information will be very helpful in their bio-
safety assessment.
CRediT authorship contribution statement
Bingjie Zhang: Methodology, Investigation, Writing - original draft.
Na Liu: Software, Validation. Qian S. Liu: Formal analysis, Data
curation. Jianqing Zhang: Resources. Qunfang Zhou:
Conceptualization, Writing - review & editing. Guibin Jiang:
Supervision.
Declaration of competing interest
The authors declare that they have no conflicts of interest to this
B. Zhang, et al.
work.
Acknowledgments
We thank Dr. Qunhui Xie for the technique support on primary
cultures of rat cerebral cortical neurons. This study was financially
supported by the National Key Research and Development Program of
China (2018YFA0901101), Chinese Academy of Sciences (QYZDJ-SSW-
DQC017), National Natural Science Foundation of China (21876195,
21806178, 21527901), and Sanming Project of Medicine in Shenzhen
(SZSM201811070).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ecoenv.2020.110674.
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