Beer 2012 AgNP Ions Published

8/12/2019 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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288 C. Beer et al. / Toxicology Letters208 (2012) 286292

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



4 Laboratory-synthesized 2.6 15.9 7.6 490 24.5 39.7



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


MTT-c

ellviability[%]


Supernatant AgNP H2O*

**

*

**

0 10 20 30

0

20

40

60

80

100

120

0 0,5 1 1,5 2


MTT-Cellviability[%]


Supernatant AgNP H2O

0 10 20 30

0

20

40

60

80

100

120

140

0 0,5 1 1,5 2


WST8-Cellviability[%]


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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Beer 2012 AgNP Ions Published