beer 2012 agnp ions published
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Toxicology Letters 208 (2012) 286292
Contents lists available at SciVerse ScienceDirect
Toxicology Letters
journal homepage: www.elsevier .com/ locate / toxlet
Toxicity ofsilver nanoparticlesNanoparticle or silver ion?
Christiane Beer a,, Rasmus Foldbjerga, Yuya Hayashi b, Duncan S. Sutherland b, Herman Autrup a
a Department of Public Health, AarhusUniversity, Bartholins All 2, DK-8000 Aarhus C, Denmarkb InterdisciplinaryNanoscienceCenter, Aarhus University,Ny Munkegade 120, DK-8000 Aarhus C, Denmark
a r t i c l e i n f o
Article history:
Received 7 September 2011
Received in revised form 3 November 2011
Accepted 4 November 2011Available online 11 November 2011
Keywords:
Nanoparticle
Silver
Toxicity
Silver ion
Reactive oxygen species
Nanosilver
a b s t r a c t
The toxicity ofsilver nanoparticles (AgNPs) has been shown in many publications. Here we investigated
to which degree the silver ion fraction ofAgNP suspensions, contribute to the toxicity ofAgNPs in A549
lung cells. Cell viability assays revealed that AgNP suspensions were more toxic when the initial silverion fraction was higher. At 1.5g/ml total silver, A549 cells exposed to an AgNP suspension containing
39% silver ion fraction showed a cell viability of 92%, whereas cells exposed to an AgNP suspension
containing 69% silver ion fraction had a cell viability of54% as measured by the MTT assay. In addition,
at initial silver ion fractions of 5.5% and above, AgNP-free supernatant had the same toxicity as AgNP
suspensions. Flow-cytometric analyses ofcell cycle and apoptosis confirmed that there is no significant
difference between the treatment with AgNP suspension and AgNP supernatant. Only AgNP suspensions
with silver ion fraction of2.6% or less were significantly more toxic than their supernatant as measured
by MTT assays. From our data we conclude that at high silver ion fractions (5.5%) the AgNPs did not add
measurable additional toxicity to the AgNP suspension, whereas at low silver ion fractions (2.6%) AgNP
suspensions are more toxic than their supernatant.
2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Thesmallsizeof nanoparticles (NPs),defined as particleswith at
leastonedimensionbetween1and100nm,results in unique chem-
ical and physical characteristics leading to advanced magnetic,
electrical, optical, mechanical and structural properties compared
to the original bulk substance. However, the same characteristics
making NPs so attractive for their exploitation in new products
have led to concerns that NPs may pose a risk for humans and
the environment. For example, the remarkably higher surface to
volume ratio of NPs enhances their surface properties thereby
increasing the interaction with serum, saliva, mucus, or lung lin-
ing fluid components, and makes NPs potentially more reactive
than larger particles. This and other physicochemical properties
of NPs have raised the concern that NPs also may interact in new
unpredicted ways with biological systems (Landsiedel et al., 2010;
Maynard et al., 2011; Oberdrster et al., 2005). Although there
have been quite a number of toxicological studies published on
NPs, it is still difficult to draw definite conclusions about their
Abbreviations: NP, nanoparticles; AgNP, silver nanoparticles; ROS, reactiveoxy-
gen species. Corresponding author. Tel.: +45 8942 6181; fax: +4589426200.
E-mail addresses: [email protected] (C. Beer), [email protected] (R. Foldbjerg),
[email protected] (Y. Hayashi), [email protected] (D.S. Sutherland),
[email protected] (H. Autrup).
toxicity as a number of the studies have been performed without
thorough characterization and description of the investigated NPs
and the solutions used under experimental conditions. This is also
reflected in an opinion published by the European Commission Sci-
entific Committee on Emerging and Newly Identified Health Risks
(SCENIHR) who concluded fromthe existing studies, thatNPs might
have different toxicological properties from the bulk substance, but
their risks should be assessed on a case-by-case basis (SCENIHR,
2006, 2009).
SilverNPs (AgNPs) are, dueto their antimicrobial properties, the
most widely used NPs in commercial products. AgNPs are incorpo-
rated into medical products like bandages as well as textiles and
house hold items (Marambio-Jones and Hoek, 2010; The project
of emerging nanotechnologies at Woodrow Wilson International
Center of Scholars, http://www.nanotechproject.org/inventories/
consumer/analysis draft/). Besides its antimicrobial effect, AgNPs
are known to induce toxicity in many different species (Bilberg
et al., 2011; Navarro et al., 2008) and chronic exposure to silver
is known to cause argyria and/or argyrosis in humans (reviewed in
Drake and Hazelwood, 2005).
The toxicity of AgNPs has also been shown by a number of
in vitro studies (Foldbjerg et al., 2011, 2009; Kawata et al., 2009;
Kim et al., 2009). Toxicological investigations of NPs imply that,
e.g., size, shape, chemical composition, surface charge, solubility,
their ability to bind and affect biological sites as well as their
metabolismand excretion influence the toxicityof NPs(Castranova,
2011; Schrand et al., 2010). The high surface area of metal-based
0378-4274/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved.
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http://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.toxlet.2011.11.002http://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.toxlet.2011.11.002http://www.sciencedirect.com/science/journal/03784274http://www.elsevier.com/locate/toxletmailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://www.nanotechproject.org/inventories/consumer/analysis_draft/http://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.toxlet.2011.11.002http://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.toxlet.2011.11.002http://www.nanotechproject.org/inventories/consumer/analysis_draft/mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://www.elsevier.com/locate/toxlethttp://www.sciencedirect.com/science/journal/03784274http://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.toxlet.2011.11.002 -
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an equal aliquot of NIM-DAPI solution was added to the cells and kept on ice until
flowcytometryanalysis(CellLab QuantaSCMPL,Beckman Coulter).The UVarc lamp
witha 355/37BP filterwas usedfor excitationand fluorescence wasdetectedin FL-1
using 465/30BPfilter. For each sample 1104 cells were investigated.
2.12. Reactive oxygen species (ROS) assay
The intracellular generation of ROS was measured by using the fluorescent
markerH2DCF-DAas previouslydescribed byKrejsaand Schieven(2000)withminor
modifications. The cells were incubated with 5M 2,7-dichlorodihydrofluorescin
diacetate (DCF) in PBS for 20min and subsequently washed with PBS and resus-pended at thedesiredconcentration in phenol-red freeDMEMmedium with1% FBS.
Cells were exposed to AgNPs andAg+ in 6-well plates, andafter24 h thecells were
detached from the culture plates and 300l cell suspensions were transferred to
sample cupsand immediately analyzed by flow cytometry (CellLab Quanta SCMPL,
Beckman Coulter). A 488nm wavelength laser was used for excitation and fluores-
cencewas detectedin FL-1usinga 525/30BP filter. Themean fluorescenceof 2104
cellswas determined foreach sampleby FlowJosoftware ver. 7.6.4(Tree Star, Inc.).
2.13. Annexin V/PI assay
The ratio of apoptotic and necrotic cells were measured with the annexin
V/propidium iodide assay (van Engeland et al., 1996). As a marker of early-stage
apoptosis the externalization of phosphatidylserine was measured by binding of
Alexa488-conjugatedannexin V proteinto phosphatidylserine. Late-stage apoptosis
and necrosis was detected bythe binding of PI to nuclear DNA dueto cellularmem-
branedamage. After exposure,cells were washed in binding buffer (10 mM HEPES,
pH 7.4; 140mM NaCl; 2.5mM CaCl2) and incubated in the dark for 10min at roomtemperaturein 100l bindingbuffercontainingannexinV-Alexa488(40l/ml)and
PI (1g/ml). Subsequently, 400l bindingbufferwas added toeach sampleand cells
were kept on ice until analysis. A 488nm wavelengthlaserwas used forexcitation.
Alexa488was detected inFL-1usinga 525/30 BPfilterwhilePI wasdetected in FL-2
using a 575/30 BP filter. Using single-stained andunstained cells, standardcompen-
sationwas donein theQuantaSC MPLAnalysissoftware(BeckmanCoulter).For each
sample,2 104 cellswereanalyzed andearlyapoptotic (annexinV+, PI), lateapop-
totic/necrotic (annexin V+, PI+) and live (annexin V, PI) cells were expressed as
percentages of the measured2 104 cellsusing the FlowJo softwarever. 7.6.4 (Tree
Star, Inc.).
2.14. Statistical analysis
Data are expressed as mean standard deviation (SD). The statistical signifi-
cance was determined by theStudents t-test (p< 0.05).
3. Results
3.1. Cytotoxicity of AgNP suspensions strongly depends on the
silver ion concentration
Several studies have previously shown that AgNP suspensions
are toxic (Foldbjerg et al., 2009, 2011; Kim et al., 2009; Navarro
etal.,2008). However, therehave beenno systematic studies, which
analyze to which degree varying amounts of silver ions present
in AgNP suspensions contribute to their toxicity. Thus, A549 cells
were treated for24 h with twodifferentbatchesof AgNP suspension
which contained 39% (Batch 2) and 69%(Batch 3) silver ion fraction
(Table 1 and supplementary data). The cell viability was measured
by the MTT assay. As expected, the AgNP suspension with lower
silver ion fraction (39%, Batch 2) was less toxic compared to theAgNP suspension with highersilverion fraction (69%, Batch 3) (e.g.,
94% cell viability compared to 54% cell viability at 1.5g/ml total
silver, respectively) (Fig.1). In comparison, viabilityof cellsexposed
0
20
40
60
80
100
120
0 0,5 1 1,5 2MTT
-Cellviability[%]
Total Ag [g/ml]
39% Ag+ 69% Ag+
* **
Fig.1. Thetoxicityof AgNP suspensionsis dependent on thesilverion content.A549
cells were treated with different concentrations total silver of AgNP suspensions
(NanoAmor,batch 2and 3)containing39% or69% silverions for24 h andcell viability
was measured using MTT assay.
to the laboratory-synthesized AgNP suspension with much lower
silver ion fraction (2.6%, Batch 4) decreased only at much higher
total silver concentrations e.g., 45% cell viability at 20g/ml total
silver (Fig. 2B). This suggests that the toxicity of AgNP suspensions
strongly depends on the initial silver ion content.
3.2. Proportion of silver ions influences the cytotoxicity of AgNP
suspensions
To further investigate to what extent silver ions account for the
overall toxicityof AgNPsuspensions, the majorityof AgNPswas pel-
leted by ultracentrifugation and the supernatant used for cell via-
bilitystudies.A549cells were incubatedfor 24h with thesame vol-
ume of AgNP supernatant or AgNP suspension andthereby with the
same amount of silver ions corresponding to 0.21.6g/ml silver
ions. It has to be kept in mind that the total amount of silver added
to the cells ranged from0.5 to 4g/ml in the case of AgNP suspen-
sion dueto thepresence of AgNPs.Any additional toxicity when thecells were treated with AgNP suspension would thereforeoriginate
from the AgNPs. In Fig. 2A, an example is shown were the silver ion
fraction was 69% in the AgNP suspension/supernatant (Batch 3). At
this high amount of silver ions there was no difference in the cell
viability of the A549 cells treated with the AgNP suspension or its
supernatant.The same wastruefor alltested AgNP suspensions and
the corresponding supernatant when the preparations using com-
mercially available AgNPs were used (data not shown). The silver
ion fraction of the tested AgNP suspensions was between 39 and
71%. To exclude the possibility that PVP in the AgNP suspension
leads to theobservedtoxicityof thesupernatant, thetoxicityof PVP
was investigatedin the MTT assay. However, no significant toxicity
of PVP concentrations of up to 0.5% PVP, which would correspond
to 50% (wt) PVP in the AgNP powder, were found (data not shown).When laboratory-synthesized AgNP suspensions with initial silver
ion fraction between 1 and 2.6% were used the AgNP suspension
was significantly more toxic than its corresponding supernatant
Table 1
Characterization of AgNPs obtained from NanoAmor and laboratory-synthesized AgNPs.
Batch # Origin Silver ion fraction [% of total Ag] TEM size [nm] n Calculated SSA [m2/g] DLS z -average [ nm]
1 Commercially available powder 59 69 49a 229a 121.0a
2 Commercially available powder 39 69 49a 229a 121.0a
3 Commercially available powder 69 69 49a 229a 121.0a
4 Laboratory-synthesized 2.6 15.9 7.6 490 24.5 39.7
5 Laboratory-synthesized 5.5 19.8 10.7 507 18.3 41.6
6 Laboratory-synthesized 5.9 17.4 8.8 502 22.1 40.2
a
Data from Foldbjerget al.(2009).
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C. Beer et al. / Toxicology Letters208 (2012) 286292 289
0 10 20 30 40
0
20
40
60
80
100
120
0 0,2 0,4 0,6 0,8 1
Total Ag concentraon in AgNP suspension [g/ml]
MTT-C
ellviability[%]
Ag+ concentraon [g/ml]
Supernatant AgNP
C
B
D
0 1 2 3 4 5
0,0
20,0
40,0
60,0
80,0
100,0
120,0
0 0,5 1 1,5 2
Total Ag concentraon in AgNP suspension [g/ml]
MTT-c
ellviability[%]
Ag+ concentraon [g/ml]
Supernatant AgNP H2O*
**
*
**
0 10 20 30
0
20
40
60
80
100
120
0 0,5 1 1,5 2
Total Ag concentraon in AgNP suspension [g/ml]
MTT-Cellviability[%]
Ag+ concentraon [g/ml]
Supernatant AgNP H2O
0 10 20 30
0
20
40
60
80
100
120
140
0 0,5 1 1,5 2
Total Ag concentraon in AgNP suspension [g/ml]
WST8-Cellviability[%]
Ag+ concentraon [g/ml]
Supernatant AgNP H2O
*
A
Fig. 2. Effect of AgNPsuspensionand AgNP supernatant on the cell viability.(A) AgNP suspension andAgNP supernatant areequally toxic. A549 cellswere treated withAgNP
suspension or AgNP supernatant corresponding to the same concentrations of silver ions or water as control for 24h and cell viability was measured using MTT assay. The
experiment wasdone in 6 independent experiments done in 8 replicates. Onerepresentative experiment is shown forNanoAmor batch 3, silver ionconcentration was 69%.
The data are expressed as meanSD. (B) AgNPs add additional toxicity at low initial silver ion fraction in the AgNP suspension of laboratory synthesized AgNPs.A549 cells
were treated with AgNP suspension or AgNP supernatant corresponding to the same concentrations of silverions for24 h and cell viability was measured using MTT assay.
The experiment was done in 2 independent experiments done in 8 replicates. Onerepresentative experiment is shown forlaboratory synthesized AgNPs batch 4. Silver ionconcentration was 2.6%. The data are expressed as meanSD. (C and D) AgNPs does not add significant additional toxicity at silver ion concentrations of 5.9%. A549 cells
were treated with laboratory synthesized AgNP suspension (batch 6) or AgNP supernatant corresponding to the same concentrations of silver ions or water as control for
24 h and cell viability was measured using MTT (C)and WST8 (D)assay. The data areexpressed as meanSD of three independent experiments.
(one representative example is shown in Fig. 2B). However, except
from one data point we found no significant difference in the toxic-
ity of laboratory-synthesized AgNP suspension and its supernatant
for a batch with a silver ion fraction of 5.9% (Batch 6, Fig. 2C).
Another batch with 5.5% silver ion fraction showed no significant
differenceof the toxicity of AgNPsuspension and AgNPsupernatant
(Batch 5, data not shown). To complement the obtained data for
batch 6, the toxicity of the AgNP suspension and its correspond-
ing supernatant was measured by the WST-8 assay. As for the MTT
assay, not significant difference in the toxicity of AgNP suspensionand supernatant for batch 6 could be detected (Fig. 2D).
3.3. Induction of apoptosis and necrosis by silver ions and AgNPs
In addition to the MTT and WST8 assays an annexin V/PI
assay was used to determine the proportion of cells undergoing
apoptosis/necrosis. A549 cells were treated for 24h with AgNP
suspension or AgNP supernatant and subsequently the amount of
early apoptotic, late apoptotic and necrotic as well as living cells
were measured using flow cytometry. Although there was a slight
reductionin living cells anda slightincrease in early apoptotic cells
compared to control this was not statistically significant except for
2g/ml total silverof AgNP supernatant (Fig.3A). In addition,there
was no significant difference between the treatments with AgNP
suspension and supernatant.
3.4. Influence of silver ions and AgNPs on ROS production
AgNPs and silver ions have been shown earlier to induce reac-
tive oxygen species (ROS) in A549 cells (Foldbjerg et al., 2011). To
investigate to which degree AgNPs and silver ions induce produc-
tion of ROS, A549 cells were treated for 24h with AgNP suspension
or AgNP supernatant corresponding to 1, 2, or 3g/ml total silver.Interestingly, AgNP supernatant induces between 2.6- and 6-fold
more ROS than AgNP suspension (Fig. 3B), which suggests that ROS
production is mainlydue to thepresence of silver ions as thesuper-
natantcontains only silver ions andtherefore more silverions were
added compared to the AgNP suspension (Batch 3).
3.5. Influence of silver ions and AgNPs on the cell cycle
A549 cells were incubated with the same volumes of AgNP sus-
pension orAgNPsupernatantwhich correspondedto 2,3 or4g/ml
silver ions. After 24h incubation the cells were trypsinized, their
DNA stained with NIM-DAPI and subsequently analyzed by flow
cytometry (Fig. 4A and B). There was no significant difference in
the number of cells in the G2/M phase of the cell cycle when AgNP
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B
0
200
400
600
800
1000
1200
1400
1600
0 0,5 1 1,5 2 2,5 3 3,5
DCF[%ofcontrol]
Total Ag concentraon [g/ml]
AgNP SupernatantA
*
*
0
20
40
60
80
100
120
1 2 3 1 2 3 0AgNP Supernatant Control
Cells[%]
Total Ag concentraon [g/ml]
Early apoptosis Late apoptosis / necrosis Live
*
Fig. 3. A549cellswereexposed for24 h to cooledAgNPsuspensioncontaining NPs from NanoAmorBatch or thesupernatant.(A) Thepercentage of viable(annexin V/PI),
early apoptotic (annexin V+/PI) and late apoptotic/necrotic (annexin+/PI+) cells at each concentration was determined by the annexin V/PI assay. (B) The increase in DCF
fluorescence was measured as a marker of ROS and is presented as percent of control. Control cells were cultured in NP-free growth media. The data are expressed as
meanSD of three independent experiments.
Fig. 4. Cell cycle analysis of cells treated with AgNP suspension or AgNP supernatant from NanoAmor (Batch 3). A549 cells were treated with AgNP suspension or AgNP
supernatant corresponding to the same amount of silver ions for 24h. The cells were harvested; stained using NIM-DAPI and their DNA content measured using flow
cytometry. Silver ion concentration was 69%. (A) Flow cytometry histograms comparing the DNA content at different concentrations silver ions from AgNP suspension or
AgNP supernatant. (B)Flow cytometryhistogramscomparing the DNA content of AgNP suspension or AgNP supernatanttreated cells at thesame silverion concentrations.
(C) Amount of cells in the G1, S or G2 phase of the cell cycleafter 24h treatment with AgNP suspension or AgNP supernatant. The data are expressed as meanSDof two
independent experiments.
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suspensionsbe used as standard additional control to makereliable
statements of the toxicity of AgNPs, and to discriminate between
silver ions toxicity and AgNP-induced toxicity e.g.,those performed
in Bouwmeester et al. (2011). In addition, the same may be true for
other metal-based NPs having relatively high solubility rate such
as CuONPs.
Conflict of interest statement
All authors declare not to have any conflicts of interest.
Acknowledgements
The authors would like to thank Duy Anh Dang for his excel-
lent technical assistance and Per Guldhammer Henriksen for his
help with the AAs measurements. The research was funded by the
Danish Council for Strategic Research.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.toxlet.2011.11.002.
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