Trojan-Horse Mechanism in the Cellular Uptake of Silver

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Trojan-horse Mechanism in the Cellular Uptake of Silver Nanoparticles Verified by Direct Intra- and Extracellular Silver Speciation Analysis I-Lun Hsiao, Yi-Kong Hsieh, Chu-Fang Wang, I-Chieh Chen, and Yuh-Jeen Huang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es504705p • Publication Date (Web): 18 Feb 2015 Downloaded from http://pubs.acs.org on February 22, 2015

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Environmental Science & Technology

Trojan-horse Mechanism in the Cellular Uptake of Silver Nanoparticles Verified by Direct Intra- and Extracellular Silver Speciation Analysis I-Lun Hsiao, Yi-Kong Hsieh, Chu-Fang Wang, I-Chieh Chen, Yuh-Jeen Huang*

Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, Taiwan 30013

Corresponding author: Yuh-Jeen Huang E-mail: [email protected] Tel.: (886) 3-571-5131 ext.35496 Fax: (886) 3-571-8649

E-mail for all authors: I-Lun Hsiao, [email protected] Yi-Kong Hsieh, [email protected] Chu-Fang Wang, [email protected] I-Chieh Chen, [email protected] Yuh-Jeen Huang, [email protected]

Keywords: Ag nanoparticles, Trojan-horse mechanism, X-ray absorption near edge structure, Laser ablation-ICPMS, intracellular ROS 1

ACS Paragon Plus Environment


1

Abstract

2

The so-called “Trojan-horse” mechanism, in which nanoparticles are internalized within cells,

3

then release high levels of toxic ions, has been proposed as a behavior in the cellular uptake of

4

Ag nanoparticles (AgNPs). While several reports claim to have proved this mechanism by

5

measuring AgNPs and Ag ions (I) in cells, it cannot be fully proven without examining those

6

two components in both intra- and extracellular media. In our study, we found that even

7

though cells take up AgNPs similarly to (microglia (BV-2)) or more rapidly than (astrocyte

8

(ALT)) Ag (I), the ratio of AgNPs to total Ag (AgNPs+Ag (I)) in both cells was lower than

9

that in outside media. It could be explained that H2O2, a major intracellular reactive oxygen

10

species (ROS), reacts with AgNPs to form more Ag (I). Moreover, the major speciation of Ag

11

(I) in cells was Ag(cysteine) and Ag(cysteine)2, indicating the possible binding of monomer

12

cysteine or vital thiol proteins/peptides to Ag ions. Evidence we found indicates that the

13

“Trojan-horse” mechanism really exists.

14 15 16 17 18 2


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Introduction

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Due to broad applications in textiles, wound dressings, food packaging and other medical

21

devices,1-4 silver nanoparticles (AgNPs) have appeared rapidly in our lives, but the study of

22

the relationship of silver nanotechnology with health issues is relatively new. Long-term

23

exposure and mechanism-related studies are still in progress.5

24

Many studies have found that AgNPs

gradually dissolve to Ag ions in the

25

presence of oxygen,6-7 which can lead to significant cytotoxicity in mammalian cells.8-9

26

Current studies claim that the toxicity mechanism of AgNPs on mouse macrophage

27

(RAW264.7) and human bronchial epithelial cells (BEAS-2B) might arise from a

28

“Trojan-horse” mechanism,10-12 in which toxic substances, such as nanoparticles (NPs),

29

serve as carriers that penetrate cell membranes and release high levels of toxic ions.13 Some

30

claim to have proved this mechanism by observing AgNPs and Ag ions (I) simultaneously

31

in cells, even while it is difficult for the ionic form of metal to penetrate cell membranes.

32

For instance, Wang et al. used cloud-point extraction to separate AgNPs and Ag ions in

33

mouse erythroid (MEL) cells and found Ag ions (17.9%) and AgNPs (82.1%) together in

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cells.14 However, AgNP dissolution could occur or even be enhanced in biological media.15

35

Therefore, ions detected in cells might also come from extracellular media, not only from

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the carrier (AgNPs). To demonstrate the existence of a mechanism, we should not only

3



37

determine the amount of AgNPs and ions in cells, but also compare their ratio to

38

extracellular media. Another way to prove it is to find enhanced intracellular ion release

39

from AgNPs, i.e., net conversion of AgNPs to silver ions in cells. Recent studies indicate

40

that the generation of peroxide intermediates, such as H2O2 or acidic surroundings of the

41

lysosomal cellular compartment, might enhance AgNP oxidation.6,16 However, the evidence

42

was obtained in a simple system (e.g., water), not in cells or culture media.

43

Many studies have tried to detect Ag ion release in environmental matrices or

44

growth media for microorganisms.15,17-22 Fewer report ion release in biological media for

45

mammalian cells and intracellular surroundings.14-15,18,22 Generally, previous studies used

46

two strategies to detect Ag ions (I) and/or AgNPs:

47

cloud-point extraction to separate AgNPs and Ag ions, then measure them separately with

48

inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy

49

(AAS);14,22-24 2) directly analyze AgNPs and/or Ag ions with no pre-treatment. For example,

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UV-vis spectroscopy and ion-selective electrode (ISE) are used to measure AgNPs and free

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Ag ions, respectively; and/or directly separate AgNPs and Ag (I) by reverse-phase liquid

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chromatography coupling with ICP-MS.15,19,21 The former strategy may be inadequate for

53

measuring Ag ions (I) in culture media, because AgCl and Ag2S particles, formed when Ag

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ions (I) combine with components of the medium, cannot be separated from AgNPs.

1) centrifugation, ultrafiltration or

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Underestimation of Ag (I) concentration may occur.18 Also, when free Ag ions are released

56

to culture media, many kinds of Ag (I) compounds or complexes generate in intra- and

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extracellular surroundings. Most studies calculated equilibrium of silver speciation,15,23,25

58

but nearly none report real detection of Ag species in biological systems. Recently, Ag

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L3-edge X-ray absorption near-edge spectroscopy (XANES) was used to analyze Ag

60

speciation in microcosm surface water, a method that can recognize not only the oxidative

61

state of Ag, but also species formed in Ag (I).24,26 To further describe the Trojan-horse

62

mechanism,

63

in cells.

the present study used this method to obtain information about silver species

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Animal studies have found that AgNPs can accumulate in many parts of the brain,

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especially the hippocampus.27 The potential neurotoxicity of NPs attracts researcher

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attention because reactive oxygen species (ROS) induced by NPs could be associated with

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neurodegenerative disorders. Studies have found that nanoparticles are mainly taken up by

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microglia and astrocytes in the central nervous system and damage to those cells may affect

69

normal neuron cells.28,29 In our study, microglia and astrocytes were used to help determine:

70

1) the ratio of AgNPs to total Ag (AgNPs+Ag (I)) between single cells and outside media

71

after 24 and 48 h of exposure; 2) if H2O2, an intracellular ROS, participates in AgNP

72

dissolution in cells; 3) Ag species analysis in cells; and 4) whether or not the “Trojan-horse” 5



73

mechanism exists. Five measuring techniques (UV-vis spectrometer, ISE and XANES for

74

Ag species analysis, flow cytometry and Laser Ablation (LA)-ICP-MS30 for uptake

75

quantification) were used to decrease interference caused by pre-treatment procedures.

76

Ascorbic acid (AA) was used as a reducing agent to protect AgNPs from rapid dissolution

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and was expected to get a high ratio of AgNPs to total Ag inside cells. Lipopolysaccharide

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(LPS), an endotoxin, was used to induce inflammation in normal animal immune systems.31

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LPS could also activate microglia and astrocytes32,33 and increase nanoparticle uptake,

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particularly in N11 microglia,34 thus, potentially aiding observation of a Trojan-horse

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mechanism in those cell types under inflammation.

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Materials and Methods

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Silver nanoparticle sources

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Obtained from Gold Nanotech, Inc., Taiwan, the AgNPs were produced by a proprietary

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molecular beam epitaxy process and did not contain surfactants or stabilizers.35,36 Briefly,

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bulk silver was cut into the target material, then the Ag target was vaporized to the atomic

87

level by an electrically gasified method under vacuum. The vapor was condensed in the

88

presence of an inert gas, then accumulated to form AgNPs. Nanoparticle size was effectively

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controlled by evaporation time and electric current. The nanoparticles were collected in a cold

90

trap and dispersed in sterile water. A concentration of 10 µg/mL was determined by ICP-MS

91

(Agilent 7500a, USA). The stock solution had good stability in storage for six months at 4 °C.

92 93

AgNPs characterization

94

The morphology of the AgNP stock solution was analyzed using a JEM2100 transmission

95

electron microscope (TEM) (JEOL, Japan). The hydrodynamic size and surface charge of the

96

AgNPs in stock solution and culture medium were monitored using a Zetasizer Nano ZS

97

apparatus (Malvern Instruments, UK). Details of sample preparation are described in the

98

Supporting Information (SI).

99 7



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AgNP dissolution measurement

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AgNPs have a localized surface plasmon resonance (LSPR) peak at ca. 400 nm, which can be

102

utilized for quantifying AgNPs in solutions. By using the Zook et al.,15 method, AgNP

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solution absorbance between 300 and 900 nm was determined using UV–Vis

104

spectrophotometry (Varian 50 Bio, CA, USA). The AgNP stock solutions were diluted with

105

water or cell media immediately before each absorbance measurement. The background

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absorbance of Dulbecco's modified Eagle's medium (DMEM)/10% FBS was subtracted from

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AgNP spectra dispersed in DMEM. Additionally, interference due to AgCl NPs was

108

approximated by assuming Rayleigh scattering and setting the absorbance at 321 nm (Abs321)

109

to

110

AbsAgCl=(Abs321−0.086×Abs407)×(λ/321)−4. The calibration curve of AgNPs at each

111

concentration was derived from the integrated area (320 to 610 nm) or the absorbance peak

112

intensity at ~ 407 nm. Fig. S1 shows AgNP calibration curves using these two methods from

113

0.2 to 5 µg/mL. The limit of detection (LOD) of the latter method was 0.18 µg/mL, which was

114

calculated from 3 σ/s; where σ is the standard deviation of 7 replicated blank absorption, and s

115

is the slope of the calibration curve.

8.6%

of

the

peak

absorbance

Abs407,

resulting

116 117

Free silver ion activity measurement

8


in

the

equation

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For analyzing free silver ion activity in: (a) AgNP stock solution; and in (b) DMEM medium

119

(pH 7.2), a commercial silver ISE (perfectION™ comb Ag/S2 Combination Electrode,

120

Mettler-Toledo) was used. Using the method developed by Koch et al.20 the system was

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calibrated: (a) in the medium-activity range with a series of AgNO3 standards (10–300

122

ng/mL); (b) in the high-activity range with a series of AgNO3 standards (1–1,000 µg/mL); (c)

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in the low-activity range by titration of 2 mL AgNO3 10.2 mM against NaCl 28.2 mM, KBr

124

12.5 mM and KI 7.9 mM solutions. As an ionic strength adjuster, NaNO3 was added to all

125

samples and standards.

126

ISE.

Fig. S2 shows titration and calibration curves of silver ions using

127 128

Ag species in DMEM medium simulation

129

Simulations were conducted by a GeoChem-EZ program. The program models Ag speciation

130

in a water-based environment at equilibrium. We input the exact concentration of amino acids

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and salts in DMEM medium and assumed: 1) There were 2.3 µg/mL of silver ions (I) in

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medium derived from subtracting AgNP stock concentration with a mean AgNP concentration

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at 24 h; 2) The pH value was fixed at 7.2; 3) CO2 partial pressure was 0.05 L/L; 4) Ionic

134

strength was 0.13 M; and 5) Solids can precipitate.

135 9



136

Cell culture

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The immortalized murine microglial cell line (BV-2) (established by Cancer Gene Therapy

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Laboratory, National Tsing Hua University, Hsin-Chu, Taiwan) and murine brain astrocyte

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cells (ALT) (BCRC-60581, Hsin-Chu, Taiwan) were cultured in high-glucose DMEM

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supplemented with 10% FBS and 1% Antibiotic–Antimycotic, and cultivated in T25 flasks at

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37 °C in a humidified atmosphere of 5%CO2/95% air.

142 143

NP exposure condition

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Before all exposure processes took place, cells were seeded in culture plates 30 h for

145

attachment. A serum-free medium was pretreated for 6 h with or without (w/wo) LPS from

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Escherichia coli 055:B5 (Sigma, St. Louis, MO, USA) (100 ng/mL for BV-2 and 1000 ng/mL

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for ALT). The cells were then treated with 5 µg/mL of AgNPs in suspension for 24 or 48 h.

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The 5 µg/mL of AgNP suspensions were prepared by mixing the stock solution 1:1 with 2×

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DMEM/20%FBS. To prevent AgNP oxidation, the diluted suspensions were added to the

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culture plate immediately without any dispersion treatment. AgNPs also were co-treated with

151

ascorbic acid (100 µM) (Sigma, St. Louis, MO) to observe the protection of Ag dissolution in

152

the system.

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AgNP cytotoxicity

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The viability of ALT and BV-2 cells after 24 and 48 h of AgNP exposure was measured by

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AlamarBlue® assay (Invitrogen, CA, USA). Measurement and data calculations are detailed

157

in the SI.

158 159

Intracellular ROS release

160

Production of H2O2, a major intracellular ROS, was measured using 2’,7’ -dichlorofluorescin

161

diacetate (DCFH-DA, Sigma-Aldrich, MO) as a reactive fluorescent probe. When DCFH-DA

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is absorbed by cells, esterase transforms it into nonfluorescent HDCF, which is then oxidized

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to fluorescent dichlorofluorescin (DCF) by intracellular ROS. The fluorescence is then

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detected at 528 nm after excitation at 488 nm. cells (1 × 104 cells/cm2) were seeded in 12-well

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plates for cell viability analysis. After exposing cells to NP solutions at 5 µg/mL, suspensions

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were removed, then the cells were rinsed three times with phosphate buffered saline (PBS).

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Next, trypsin-EDTA (0.5 mL) was added to remove adherent cells from the flask surface. The

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DCFH-DA working solution, obtained after diluting 20 mM DCFH-DA of stock solution (in

169

methanol) to 10 µM in serum and phenol red-free medium, was added to each well, followed

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by incubation for 30 min at 37 °C. The solutions were then centrifuged (200g, 5 min) and the

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supernatant medium was removed. Prior to measurement, the cells were resuspended in PBS.

11



172

Fluorescence was determined using a BD FACSCanto flow cytometer (BD Science, San Jose,

173

CA), with 10,000 cells collected. The geometric mean fluorescence intensity was analyzed

174

using Flowing software 2.

175 176

Uptake potential from flow cytometry analysis

177

In this study, uptake potential of AgNPs was measured by flow cytometry light scatter

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analysis developed by Suzuki et al.37 Based on their idea, once particles are taken up into

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cells, side scatter (SSC) intensity increases with the increased cell density.38 Evaluation of

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SSC intensity was carried out using a FACSCanto II (BD Biosciences, San Jose, CA), and

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data were analyzed using Flowing software 2. The relative amount of particles taken up by the

182

cells were analyzed using geometric mean of SSC intensity. Uptake and intracellular ROS

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data could be measured simultaneously38; therefore, the sample preparation procedure was the

184

same as intracellular ROS analysis.

185 186

Quantification of uptake by LA-ICP-MS

187

For LA-ICP-MS experiments, cells were seeded on sterile coverslips in 6-well plates and

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incubated with 5 µg/mL of AgNPs or co-treatment samples. After exposure, cells were washed

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three times with PBS (4 °C) and fixed with 4% para-formaldehyde (Sigma-Aldrich, St. Louis,

12


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MO) overnight, then dehydrated in a graded series (30%, 50%, 70%, 95%) of ethanol for

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LA-ICP-MS analysis. Samples were stored in absolute ethanol at -20 °C until analysis. The

192

LA-ICP-MS experimental condition and data treatment are described in SI.

193 194

Ag Speciation in cells

195

To prepare the samples for Ag speciation, cells (1 × 104 cells/cm2) were seeded in a T175

196

flask for 30 h. After treatment with 5 µg/mL of AgNPs or co-treatment samples, the

197

suspensions were removed and the cells were rinsed three times with 1 × PBS. Next, 1 ×

198

trypsin-EDTA (0.5 mL) was used to harvest cells. The cell suspensions were then centrifuged

199

(200 g, 5 min) and the supernatants were removed. The samples were stored in liquid nitrogen

200

until analysis.

201

The speciation of AgNPs was evaluated using silver LIII-edge X-ray absorption near-edge

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spectroscopy (XANES) which can analyze the oxidation state and speciation of silver because

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silver, a transition metal, gives a strong edge peak in the LIII absorption edge, which is

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assigned to the 2p-4d dipole transition.39 XANES spectra were collected at the BL-16A

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beamlines of the National Synchrotron Radiation Research Center (NSRRC) of Taiwan. All

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reference materials and samples were deposited on a single side of Kapton tape and placed in

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front of the X-ray beam to minimize self-adsorption. Furthermore, the sample chamber was

13



208

purged in 99.999% N2 for 1 h before any data collection to minimize background absorption

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of Ar. XANES data were measured at room temperature in fluorescence mode.

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Reference materials for XANES data analysis included AgNPs, AgCl, Ag2O, Ag2S,

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Ag(I)-cysteine powders formulated with 1:1 and 1:2 silver/cysteine molar ratios (Ag(CYS),

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and Ag(CYS)2), and Ag (I)-histidine powders formulated with 1:1 silver/histidine molar ratios

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(Ag(His)). Reference material preparation procedures are described in the SI.

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Data analysis was performed using the Athena software package. Linear combination

215

fitting (LCF) of XANES data for the AgNPs-in-cell samples was performed by four

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components fitting in the range of -15~+28 eV below and above the Ag LIII-edge (3351 eV).

217

Relative proportions and errors were determined by the software’s least-squares fitting

218

module. Fitting residual parameters are described in the SI.

219 220

Statistics

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Two-tailed Student’s t-test was used to evaluate for the significance between control and each

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of the other samples.

223 224 225 14


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Results and discussion

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Physico-chemical characterization of AgNPs

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Characteristics of AgNPs (size and morphology, secondary size and surface charge, and free

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ion concentration in stock solution), are discussed in the SI. Briefly, the average AgNP size

230

was 10.2 nm, with uniform spheres (Fig. S3). Dynamic laser scattering (DLS) measurement

231

showed that AgNPs had good stability within 48 h in DMEM/10%FBS medium (Table S1,

232

Figs. S4 and S5).

233

Some reports have found Ag ions are released during storage and initial Ag ion

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concentration plays an important role in AgNP toxicity.40,41 Using ISE, we found a low initial

235

concentration of Ag ions in our AgNP stock solution (2%). From UV-vis spectra, the

236

absorption peak slightly red shifted as AgNPs were in cell medium at 48 h (Fig. 1), indicating

237

that particles were stable with very slight agglomeration in cell exposure conditions at

238

concentrations used in our experiments.

239 240

AgNP oxidation in culture medium: H2O2 promotes, ascorbic acid (AA) inhibits oxidation

241

dissolution

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Fig. 1 shows dissolutions of AgNPs w/wo additives (H2O2 (150 µM), LPS (1000 ng/mL), AA

243

(100 µM)) in DMEM/10% FBS measured by UV-vis spectra. Original absorbance spectra

15



244

data of AgNPs over time, used to construct part of Fig. 1 are shown on the upper right. Based

245

on opinions from Zook et al., the peak absorbance intensity can probably be calculated as an

246

upper bound of the Ag dissolution. The area under the absorbance curve shows a better

247

evaluation (closer to ICP-MS data) and can be regarded as a lower bound.15 Using this

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strategy, the order of the ratio of AgNPs to total Ag (AgNPs + Ag (I)) in medium is

249

AA+AgNPs > AgNPs≒AgNPs+LPS > H2O2+AgNPs. The dissolution of AgNPs in

250

DMEM/10%FBS strongly increased in the first two hours, then the rate slowed over longer

251

times. Previous studies have found the same result in an oxygen-containing, pure-water

252

system.7,40 Oxygen depletion may explain the slowdown of dissolution over time. However,

253

we demonstrate that H2O2 will accelerate AgNP dissolution. In ROS species, H2O2 will react

254

with AgNPs because it has a higher redox potential (+1.763 V) than Ag (+0.799 V). Ho et al.

255

demonstrated that the reaction is of the first order with respect to both Ag (0) and H2O2 and

256

was independent of the NP size.42 Ascorbic acid, a well-known antioxidant, can be a ROS

257

scavenger and reducing agent due to its lower redox potential (+0.28V). In this study, AA was

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observed to slow down the dissolution. However, our finding differs from Liu et al., who

259

found, surprisingly, that 100 µM of AA greatly increases Ag ion release in modified acetate

260

buffer media.25

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Free silver ion activity in medium: most silver ions exist in complex with other components

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Free silver ion activity of AgNPs in DMEM/FBS is shown in Fig. S6. After adding AgNPs in

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medium, free silver ion activity decreased over time. The pAg value at 24 h was 10.1, which

265

accounted for only 1.57 × 10-4 % of total Ag in medium. The low silver ion activity can be

266

explained by the existence of constituents, such as amino acids (Cysteine or Histidine) or

267

vitamins, which have a high affinity to Ag ions. Simulation with the chemical speciation

268

program Geochem-EZ shows that free activity of Ag ions was 9.5, 1.82 % as AgClx1-x (x > 1)

269

and 98.17 % as Ag-histidine when 2.3 µg/mL of Ag ions in DMEM medium (24 h).

270

Precipitate AgCl formed only as a Ag ion concentration was higher than 5 µg/mL. The results

271

did not give Ag-cysteine complex, because the modeling program considers cysteine SH

272

groups (pKa=8.3) to be in a protonated and nonreactive state at pH 7.2. However, Adams and

273

Kramer found high affinity of Ag+ and cysteine (Kf=11.9) in the range of pH 4-8.43 Moreover,

274

nearby positive-charged amino acids, N-terminal end of α-helix proteins or peptides can lower

275

the pKa of cysteine SH groups.44,45 Thus, a Ag-cysteine complex cannot be ignored in the

276

biological system.

277 278

AgNPs caused mild cytotoxicity to cells, AA co-treated with AgNPs led to high toxicity

279

Fig. S7 shows the cytotoxicities of AgNPs and co-treatment additives to ALT and BV-2 cells.

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LPS caused slight toxicity to BV-2 cells for 24 and 48 h exposures (~80% of viability). Cell

281

viability also decreased in AgNP treatment groups for 24 h (82%), LPS co-treated with

282

AgNPs for 24 h (94%) and AA co-treated with AgNPs for 48 h (50%) (Fig. S7A). AA only

283

retarded AgNP toxicity in BV-2 at 24 h. In contrast, significant toxicity was shown in ALT

284

cells exposed to AA for 48 h (73%), AgNPs for 24 and 48 h (88% and 74% respectively), LPS

285

co-treated with AgNPs at 48 h (68%) and AA co-treated with AgNPs for 24 and 48 h (65%

286

and 40% respectively) (Fig. S7B). Toxicity of AA to ALT might come from high ROS release

287

(Fig. 2B). Reports have found that AA induced oxidation of glutathione (GSH) to its oxidized

288

form (GSSG), leading to H2O2 accumulation and apoptosis induction.46,47

289 290

AgNPs can be internalized into cells at different levels

291

In this study we used the LA-ICP-MS and flow cytometry SSC intensity to quantify cellular

292

uptake.30,37 It is worth noting whether or not the detected Ag comes from within cells. We

293

compared removal efficiency of PBS washing with PBS-Fe3+- S2O32- procedure (mixture of

294

10 mM tripotassium hexacyanoferrate (III) (K3Fe(CN)6) and 10 mM of sodium thiosulfate

295

pentahydrate

296

membrane-bound AgNPs.48 Our results show that uptake decreased about 44% in BV-2 after

297

PBS-Fe3+- S2O32- washing. This procedure did not significantly affect ALT (Fig. S8). Thus,

(Na2S2O3•5H2O)

in

PBS).

The

latter

procedure

18


rapidly

removed

Page 19 of 34


298

the amount of Ag detected in ALT should be from inside cells and BV-2 may be

299

overestimated. Figs. 3A and 3B show examples of overlapping images from bright field

300

microscopy and silver quantification imagery from LA-ICP-MS. Other sample images are

301

shown in the SI (Figs. S9 and S10). From the chemical images, we clearly see that the amount

302

of Ag in cells is not homogeneous. Silver was not detected in some cells (Figs. 3A, S9),

303

especially BV-2. In order to quantify the Ag uptake in both cell types, we randomly selected

304

15 cells from each CCD image and integrated the indicated area with chemical images to get

305

the Ag amount value. Figs. 3C and 3D present the amount of Ag in BV-2 and ALT cells in

306

various conditions in a box chart. For BV-2 cells, a large amount of AgNPs can be detected in

307

the LPS co-treatment group (Fig. 3C). For ALT cells, more AgNPs were taken up than in

308

BV-2, in general, but LPS did not enhance the uptake of AgNPs (Fig. 3D). Compared with the

309

flow cytometry approach, most of the potential uptake results correspond to the LA-ICP-MS

310

data, except AA co-treated group in ALT cells (Fig. S11).

311 312

Ratio of AgNPs to total Ag (AgNPs+Ag (I)) in both cells was lower than that in outside media

313

To distinguish AgNPs and Ag (I) species in cells, Ag LIII-edge X-ray absorption near-edge

314

spectroscopy was used. The strategy to determine Ag species in cells for linear combination

315

fitting (LCF) is described in the SI. Detailed LCF results for Ag (0) and Ag (I) species are

19



316

shown in Table S2. The best LCF fitting results are shown in Figs. 4A and 4C. As AgNPs

317

were treated to BV-2 cells, Ag (0) accounted for 30% and 38% of total Ag after 24 h and 48 h

318

of exposure. As the LPS was co-treated with AgNPs, a higher percentage of Ag (0) (64% in

319

24 h and 40% in 48 h) existed in the cells. With the co-treatment of the AA, AgNPs in total Ag

320

accounts for 73% in 24 h, but decreased in 48 h (44 %) (Fig. 4A). For ALT cells, the total

321

amounts of Ag(0) in cells after 24 and 48 h of exposure are similar, which accounts for ca.

322

31% of total Ag. As the LPS was co-treated with AgNPs, the cells exhibited a higher

323

percentage of Ag(0) (48% in 24 h and 42% in 48 h). With AA co-treatment, the AgNPs in total

324

Ag accounted for 51% in 24 h and 47% for 48 h (Fig. 4C). Figs. 4B and 4D show the amount

325

of AgNPs and Ag (I) (sum of all Ag (I) species) in single cells for each condition, which was

326

calculated from combining median uptake quantification data (LA-ICP-MS) and the ratio of

327

Ag species (XANES analysis) results.

328

Studies to detect AgNP dissolution inside cells are quite rare. Wang et al., using

329

cloud-point extraction plus ICP-MS, demonstrated that over 82.1% of Ag existed in the form

330

of nanoparticles, with 17.9% as Ag ions inside MEL cells, which is higher than the proportion

331

of Ag ions (11.6%) in diluted AgNP stock solution.14 Using the same method, Yu and

332

colleagues documented that about 10.3% of Ag was present as ions in exposed HepG2 cells,

333

with only 5.2% Ag ions in pristine AgNPs.22 Singh and Ramarao used autometallography

20


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(AMG) with quantitative image analysis and found only 5% of AgNPs had dissolved.11 The

335

discrepancy in the ratio of Ag dissolution in these studies could be due to different

336

physico-chemical properties of AgNPs used and different cell types. Unfortunately, these

337

studies only compare the AgNPs and Ag ions inside cells with the original stock solution, not

338

with cell medium. In our study, the ratio of AgNPs to total Ag inside BV-2 and ALT cells

339

(30~38%) was lower than that in extracellular medium (45~52%) after 24 h and 48 h of

340

exposure. However, AgNPs were detected in the medium in the absence of cells, which might

341

be an overestimation because they would be lost via cellular uptake. Thus, we compared the

342

dissolution of AgNPs in medium in the presence and absence of astrocytes in 24 h, and found

343

only 3% difference between each other at 2 µg/mL of AgNPs exposure (Fig. S12). Moreover,

344

only 0.19 µg of Ag was detected from all astrocytes in each well (at 5 µg/mL of AgNPs, 24 h

345

of exposure), which accounted for 3.8% of applied AgNPs (Fig. S8). These results indicate

346

that AgNP uptake does not significantly account for the total amount of AgNPs in medium.

347 348

Released H2O2 may react with AgNPs in both cells

349

To explain why intracellular AgNP ratios could be lower than extracellular, factors that can

350

promote Ag dissolution in cells, e.g., reactions of H2O2 with AgNPs, or AgNPs located in

351

lysosome (organelle which has a lower pH value), should be proved. Our UV-vis data

21



352

confirmed H2O2-enhanced AgNP dissolution in cell media (Fig. 1). Our intracellular ROS

353

results represent the balance between the generation of H2O2 from cells and its consumption

354

by reacting to AgNPs (Fig. 2). In our study, each cell type showed clues to suggest that H2O2

355

reacts with AgNPs in cells. For BV-2 cells, ROS was inhibited after exposure to AgNPs (Fig.

356

2A), which differs from previous reports (e.g., AgNPs induced more ROS in mixed rat neural

357

cells and mouse macrophages (RAW 264.7)).11,28 LPS induced ROS release in 48 h of

358

exposure, while ROS was inhibited when co-treating with AgNPs. For ALT cells, AA alone

359

can induce more ROS in 24 and 48 h, while ROS can be significantly inhibited from

360

co-treating AA with AgNPs (Fig. 2B). In summary, the decrement of ROS in BV-2 and ALT

361

suggests oxidation of AgNPs in cells.

362 363

Intracellular AgNP ratio can be explained by various factors

364

A lower ratio of AgNPs to total Ag in cell cannot be explained only by enhanced AgNP

365

dissolution, but also by faster uptake of ions than AgNPs. However, we treated 1 µg/mL of

366

AgNPs and Ag ions (using AgNO3) to the two cell types for 4 h, and found a similar uptake

367

level between two species in BV-2 while more efficient AgNPs internalization than Ag ions in

368

ALT cells (Fig. S13). The free metal ions could not enter the cells freely due to the membrane

369

barrier.49 Cronholm et al. also demonstrated a lower uptake rate of Ag ions than AgNPs in two

22


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370

lung cell lines (A549 and BEAS-2B).50 Therefore, the latter explanation can be excluded. On

371

the other hand, as LPS was co-treated with AgNPs in BV-2 cells, the AgNP ratio (74%) was

372

higher than that in cell medium at 24 h (Fig. 4A). LPS-activated BV-2 cells were able to

373

perform macrophage-like activities including scavenging and phagocytosis.51 Thus, the above

374

result might be attributable to enhanced AgNP internalization. AA induced the cellular uptake

375

of AgNPs in BV-2 cells, and showed similar AgNPs to total Ag ratio to LPS-activated cells

376

(Fig. 3C), thus it is hard to confirm its ability to protect the Ag from oxidation inside cells.

377

However, even though AA induced a high ROS release, which would enhance the dissolution

378

of AgNPs in ALT, the AA co-treatment group still showed a higher AgNP ratio than the

379

non-cotreatment group (Fig. 4C), indicating that Ag oxidation was blocked. After 48 h of

380

exposure, the uptake of Ag (AgNPs + Ag (I)) in LPS-activated BV-2 cells was lower than that

381

in 24 h (Fig. 3C). In that group, the decreased level of AgNPs was greater than the increased

382

level of Ag (I) after 48 h (Fig. 4B). This indicated that AgNP exocytosis plays a more crucial

383

role than intracellular AgNP dissolution at 48 h. On the other hand, in AgNPs treated or

384

LPS+AgNPs co-treated ALT cells, the uptake of Ag also decreased at 48 h (Fig. 3D).

385

Interestingly, the decrease of both AgNPs and Ag (I) was observed (Fig. 4D). It seems that for

386

ALT, not only AgNP exocytosis, but ion penetration to extracellular media determined the

387

AgNP ratio in longer exposures.

23



388 389

Ag speciation (I) in cells: Ag-thiol group complex accounts for the major species

390

It is important to measure Ag species (I) distribution in cells because the binding of Ag ions

391

may be related to the final toxicity. From Figs. 4A, 4C and Table S2, Ag-thiol binding species,

392

Ag(CYS) and Ag(CYS)2, account for most of the Ag (I) due to the fact that Ag has a high

393

affinity to binding with a thiol group. One report states that thiol targets can be sufficiently

394

abundant (typical total intracellular thiol concentration is 12 mM) to receive most of the silver

395

ions.25 AgCl was the second highest ratio of Ag species in Ag (I), owing to the high chloride

396

concentration (~30 mM) in the cytoplasm.25 Interestingly, the lower content of Ag2S can be

397

found in some treatment groups to ALT while not nearly so much to BV-2. This might be

398

related to H2S in astrocytes, which reportedly protects cells from injury during H2O2

399

production.52 Ag2O, which forms only on the surface of NPs and subsequently dissolves to an

400

ionic state, also accounts for a small ratio in Ag (I). AgCl and Ag2S are reported to have low

401

toxicity in mammalian cells.53-54 However, the binding of thiol proteins (e.g.,

402

cysteine/peptides) with Ag ions, such as GSH, may reduce the ability to neutralize ROS.55

403

Thus, future work should focus on the cytotoxicity of those species in order to find more

404

evidence of whether or not the Trojan-horse mechanism causes cytotoxicity of AgNPs.

405 24


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406

In this study, we compared the various ratios of AgNPs to total Ag in single cells (microglia

407

and astrocytes) and outside media, and found that the ratio of AgNPs to total Ag in cells is

408

lower than in outside media in AgNP treatment groups. This can be explained by enhanced

409

dissolution of AgNPs in cells, and not due to faster ion uptake. The intracellular ROS species,

410

H2O2, plays a significant role in enhanced AgNP dissolution. Furthermore, by using XANES,

411

we even found that the Ag-thiol species, Ag(CYS) and Ag(CYS)2, are major speciations of

412

silver ions (I) in cells. This indicates that the binding of vital thiol proteins/peptides to Ag ions

413

in cells is possible. All of this evidence demonstrates that the “Trojan-horse” mechanism

414

really exists.

415 416

Acknowledgment

417

We thank Dr. Ling-Yun Jang, spokesperson of Beamline 16A at the National Synchrotron

418

Radiation Research Center (NSRRC), Taiwan, for conducting the XANES experiments. We

419

also thank Gold Nanotech, Inc. Taiwan, for providing AgNPs materials.

420 421

Associated content

422

Supporting Information

423

Additional experimental data and tables are discussed and available free of charge via the

25



424

Internet at http://pubs.acs.org

425 426

References

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1. Dworniczek, E.; Nawrot, U.; Seniuk, A.; Wlodarczyk, K.;Bialynicki-Birula, R. The in vitro effect of a silver−containing dressing on biofilm development. Adv. Clin. Exp. Med. 2009, 18, 277–281. 2. Azeredo, H. Nanocomposites for food packaging applications. Food. Res. Int. 2009, 42 (9), 1240–1253; DOI 10.1016/j.foodres.2009.03.019 3. Dubas, S. T.; Kumlangdudsana, P.;Potiyaraj, P. Layer-by-layer deposition of antimicrobial silver nanoparticles on textile fibers. Colloid. Surface. A 2006, 289, (1), 105–109; DOI 10.1016/j.colsurfa.2006.04.012 4. Chen, X.;Schluesener, H. Nanosilver: a nanoproduct in medical application. Toxicol. lett. 2008, 176, (1), 1–12; DOI 10.1016/j.toxlet.2007.10.004 5. Stensberg, M. C.; Wei, Q.; McLamore, E. S.; Porterfield, D. M.; Wei, A.;Sepúlveda, M. S. Toxicological studies on silver nanoparticles: challenges and opportunities in assessment, monitoring and imaging. Nanomedicine-UK 2011, 6 (5), 879–898; DOI 10.2217/nnm.11.78 6. Liu, J.;Hurt, R. H. Ion release kinetics and particle persistence in aqueous nano-silver colloids. Environ. Sci. Technol. 2010, 44 (6), 2169–2175; DOI 10.1021/es9035557 7. Loza, K; Diendorf, J; Sengstock, C ; Ruiz-Gonzalez, L ; Gonzalez-Calbet, JM ; Vallet-Regi, M; Koller, M; Epple, M. The dissolution and biological effects of silver nanoparticles in biological media. J. Mater. Chem. B 2014, 2 (12), 1634–1643; DOI 10.1039/c3tb21569e. 8. Herzog, F.; Clift, M. J.; Piccapietra, F.; Behra, R.; Schmid, O.; Petri-Fink, A.;Rothen-Rutishauser, B. Exposure of silver-nanoparticles and silver-ions to lung cells in vitro at the air-liquid interface. Part. Fibre. Toxicol. 2013, 10 (1), 11; DOI 10.1186/1743-8977-10-11 9. Powers, C. M.; Badireddy, A. R.; Ryde, I. T.; Seidler, F. J.;Slotkin, T. A. Silver nanoparticles compromise neurodevelopment in PC12 cells: critical contributions of silver ion, particle size, coating, and composition. Environ. Health Persp. 2011, 119 (1), 37; DOI 10.1289/ehp.1002337 10. Park, E.-J.; Yi, J.; Kim, Y.; Choi, K.;Park, K. Silver nanoparticles induce cytotoxicity by a Trojan-horse type mechanism. Toxicol. in Vitro 2010, 24 (3), 872–878; DOI 10.1016/j.tiv.2009.12.001 11. Singh, R. P.;Ramarao, P. Cellular uptake, intracellular trafficking and cytotoxicity of silver nanoparticles. Toxicol. lett. 2012, 213, (2), 249–259; DOI 10.1016/j.toxlet.2012.07.009 12. Lubick, N. Nanosilver toxicity: ions, nanoparticles or both? Environ. Sci. Technol. 2008, 42 (23), 8617–8617; DOI 10.1021/es8026314 13. Limbach, L. K.; Wick, P.; Manser, P.; Grass, R. N.; Bruinink, A.; Stark, W. J. Exposure of engineered nanoparticles to human lung epithelial cells: influence of chemical composition and catalytic activity on oxidative stress. Environ.Sci. Technol. 2007, 41(11), 4158–4163; DOI 10.1021/es062629t 14. Wang, Z.; Liu, S.; Ma, J.; Qu, G.; Wang, X.; Yu, S.; He, J.; Liu, J.; Xia, T.;Jiang, G.-B. Silver Nanoparticles Induced RNA Polymerase-Silver Binding and RNA Transcription Inhibition in Erythroid Progenitor Cells. ACS nano 2013, 7 (5), 4171–4186; DOI 26


Page 26 of 34

Page 27 of 34

468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517


10.1021/nn400594s 15. Zook, J. M.; Long, S. E.; Cleveland, D.; Geronimo, C. L. A.;MacCuspie, R. I. Measuring silver nanoparticle dissolution in complex biological and environmental matrices using UV–visible absorbance. Anal. Bioanal. Chem. 2011, 401 (6), 1993–2002; DOI 10.1007/s00216-011-5266-y 16. Sabella, S; Carney, RP ; Brunetti, V ; Malvindi, MA; Al-Juffali, N ; Vecchio, G; Janes, SM; Bakr, OM ; Cingolani, R ; Stellacci, F ; Pompa, PP. A general mechanism for intracellular toxicity of metal-containing nanoparticles. Nanoscale 2014, 6(12), 7052–7061; DOI 10.1039/c4nr01234h 17. Sotiriou, G. A.; Meyer, A.; Knijnenburg, J. T.; Panke, S.;Pratsinis, S. E. Quantifying the origin of released Ag+ ions from nanosilver. Langmuir 2012, 28 (45), 15929–15936; DOI 10.1021/la303370d 18. Chao, J.B.; Liu, J.F.; Yu, S.J.; Feng, Y.D.; Tan, Z.Q.; Liu, R.;Yin, Y.G. Speciation analysis of silver nanoparticles and silver ions in antibacterial products and environmental waters via cloud point extraction-based separation. Anal. Chem. 2011, 83 (17), 6875–6882; DOI 10.1021/ac201086a 19. Soto-Alvaredo, J.; Montes-Bayón, M.;Bettmer, J. Speciation of silver nanoparticles and silver (I) by reversed-phase liquid chromatography coupled to ICPMS. Anal. Chem. 2013, 85 (3), 1316–1321; DOI 10.1021/ac302851d 20. Koch, M.; Kiefer, S.; Cavelius, C.;Kraegeloh, A. Use of a silver ion selective electrode to assess mechanisms responsible for biological effects of silver nanoparticles. J. Nanopart. Res. 2012, 14 (2), 1–11; DOI 10.1007/s11051-011-0646-y 21. Maurer-Jones, M. A.; Mousavi, M. P.; Chen, L. D.; Bühlmann, P.;Haynes, C. L. Characterization of silver ion dissolution from silver nanoparticles using fluorous-phase ion-selective electrodes and assessment of resultant toxicity to Shewanella oneidensis. Chem. Sci. 2013, 4 (6), 2564–2572; DOI 10.1039/C3SC50320H 22. Yu, S.J.; Chao, J.B.; Sun, J.; Yin, Y.G.; Liu, J.F.;Jiang, G.B. Quantification of the uptake of silver nanoparticles and ions to HepG2 cells. Environ. Sci. Technol.2013, 47 (7), 3268–3274; DOI 10.1021/es304346p 23. Liu, J.; Wang, Z.; Liu, F. D.; Kane, A. B.;Hurt, R. H. Chemical transformations of nanosilver in biological environments. ACS nano 2012, 6 (11), 9887–9899; DOI 10.1021/nn303449n 24. Gondikas, A. P.; Morris, A.; Reinsch, B. C.; Marinakos, S. M.; Lowry, G. V.;Hsu-Kim, H. Cysteine-induced modifications of zero-valent silver nanomaterials: implications for particle surface chemistry, aggregation, dissolution, and silver speciation. Environ. Sci. Technol. 2012, 46 (13), 7037–7045; DOI 10.1021/es3001757 25. Liu, J.; Sonshine, D. A.; Shervani, S.;Hurt, R. H. Controlled release of biologically active silver from nanosilver surfaces. ACS nano 2010, 4 (11), 6903–6913; DOI 10.1021/nn102272n 26. Bone, A. J.; Colman, B. P.; Gondikas, A. P.; Newton, K. M.; Harrold, K. H.; Cory, R. M.; Unrine, J. M.; Klaine, S. J.; Matson, C. W.;Di Giulio, R. T. Biotic and abiotic interactions in aquatic microcosms determine fate and toxicity of Ag nanoparticles: Part 2–Toxicity and Ag speciation. Environ. Sci. Technol.2012, 46 (13), 6925–6933; DOI 10.1021/es204682q 27. Liu, P.; Huang, Z.;Gu, N. Exposure to silver nanoparticles does not affect cognitive outcome or hippocampal neurogenesis in adult mice. Ecotox.Environ. Safe.2012, 87, 124–130; DOI 10.1016/j.ecoenv.2012.10.014 28. Haase, A.; Rott, S.; Mantion, A.; Graf, P.; Plendl, J.; Thünemann, A. F.; Meier, W. P.; Taubert, A.; Luch, A.;Reiser, G. Effects of silver nanoparticles on primary mixed neural cell cultures: uptake, oxidative stress and acute calcium responses. Toxicol. Sci. 2012, 126 (2), 457–468; DOI 10.1093/toxsci/kfs003 27



518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567

29. Wang, Y.; Wang, B.; Zhu, M.T.; Li, M.; Wang, H. J.; Wang, M.; Ouyang, H.; Chai, Z.F.; Feng, W.Y.;Zhao, Y.L. Microglial activation, recruitment and phagocytosis as linked phenomena in ferric oxide nanoparticle exposure. Toxicol. lett. 2011, 205 (1), 26–37; DOI 10.1016/j.toxlet.2011.05.001. 30. Drescher, D.; Giesen, C.; Traub, H.; Panne, U.; Kneipp, J.;Jakubowski, N., Quantitative imaging of gold and silver nanoparticles in single eukaryotic cells by laser ablation ICP-MS. Anal. Chem. 2012, 84 (22), 9684–9688; DOI 10.1021/ac302639c 31. Liu, B; Hong, JS. Role of microglia in inflammation-mediated neurodegenerative diseases: Mechanisms and strategies for therapeutic intervention. J. Pharmacol. Exp. Ther. 2003, 304(1),1–7; DOI 10.1124/jpet.102.035048 32. Neher, JJ; Neniskyte, U ; Zhao, JW; Bal-Price, A; Tolkovsky, AM; Brown, GC. Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death. J. Immunol. 2011, 186(8),4973–4983; DOI 10.4049/jimmunol.1003600 33. Tarassishin, L; Suh, H.S.; Lee, S.C. LPS and IL-1 differentially activate mouse and human astrocytes: role of CD14. Glia 2014, 62(6), 999–1013; DOI 10.1002/glia.22657 34. Cengelli, F; Maysinger, D; Tschudi-Monnet, F; Montet, X; Corot, C; Petri-Fink, A; Hofmann, H; Juillerat-Jeanneret, L. Interaction of functionalized superparamagnetic iron oxide nanoparticles with brain structures. J. Pharmacol. Exp. Ther. 2006, 318(1),108–116; DOI 10.1124/jpet.106.101915 35. Yen, H. J.; Hsu, S. h.;Tsai, C. L. Cytotoxicity and immunological response of gold and silver nanoparticles of different sizes. Small 2009, 5 (13), 1553–1561; DOI 10.1002/smll.200900126 36. Huang, Y.H .; Chen, C.Y. ; Chen, P.J. ; Tan, S.W. ; Chen, C.N. ; Chen, H.M. ; Tu, C.S.; Liang, Y.J. Gas-injection of gold nanoparticles and anti-oxidants promotes diabetic wound healing. RSC Adv. 2014, 4, 4656–4662; DOI 10.1039/C3RA44359K 37. Suzuki, H.; Toyooka, T.;Ibuki, Y. Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis. Environ. Sci. Technol. 2007, 41(8), 3018–3024; DOI 10.1021/es0625632 38. Toduka, Y.; Toyooka, T.;Ibuki, Y. Flow cytometric evaluation of nanoparticles using side-scattered light and reactive oxygen species-mediated fluorescence–correlation with genotoxicity. Environ. Sci. Technol. 2012, 46(14), 7629–7636; DOI 10.1021/es300433x 39. Miyamoto, T.; Niimi, H.; Kitajima, Y.; Naito, T.;Asakura, K. Ag L3-edge X-ray absorption near-edge structure of 4d10 (Ag+) compounds: origin of the edge peak and its chemical relevance. J. Phys. Chem. A 2010, 114 (12), 4093–4098; DOI 10.1021/jp907669s 40. Kittler, S.; Greulich, C.; Diendorf, J.; Koller, M.;Epple, M. Toxicity of silver nanoparticles increases during storage because of slow dissolution under release of silver ions. Chem. Mater. 2010, 22 (16), 4548–4554; DOI 10.1021/cm100023p 41. Beer, C.; Foldbjerg, R.; Hayashi, Y.; Sutherland, D. S.;Autrup, H. Toxicity of silver nanoparticles—nanoparticle or silver ion? Toxicol. lett. 2012, 208 (3), 286–292; DOI 10.1016/j.toxlet.2011.11.002 42. Ho, C.M.; Yau, S. K.W.; Lok, C.N.; So, M.H.;Che, C.M. Oxidative dissolution of silver nanoparticles by biologically relevant oxidants: A kinetic and mechanistic study. Chem. Asian J. 2010, 5, (2), 285–293; DOI 10.1002/asia.200900387. 43. Adams, N. W.;Kramer, J. R. Potentiometric determination of silver thiolate formation constants using a Ag2S electrode. Aquat. Geochem. 1999, 5 (1), 1–11; DOI 10.1023/A:1009699617808 44. Lutolf, M.; Tirelli, N.; Cerritelli, S.; Cavalli, L.;Hubbell, J. Systematic modulation of Michael-type reactivity of thiols through the use of charged amino acids. Bioconjugate Chem. 2001, 12 (6), 1051–1056; DOI 10.1021/bc015519e 45. Iqbalsyah, T. M.; Moutevelis, E.; Warwicker, J.; Errington, N.;Doig, A. J. The CXXC 28


Page 28 of 34

Page 29 of 34

568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605


motif at the N terminus of an α‐helical peptide. Protein Sci. 2006, 15 (8), 1945–1950; DOI 10.1110/ps.062271506 46. Park, S.; Han, S.S.; Park, C.H.; Hahm, E.R.; Lee, S.J.; Park, H.K.; Lee, S.H.; Kim, W.S.; Jung, C.W.; Park, K. Riordan, H.D.; Kimler, B.F.; Kim, K; Lee, J.H. L-Ascorbic acid induces apoptosis in acute myeloid leukemia cells via hydrogen peroxide-mediated mechanisms. Int. J. Biochem. Cell Biol. 2004, 36, 2180–2195; DOI 10.1016/j.biocel.2004.04.005 47. Park, S. The effects of high concentrations of vitamin C on cancer cells. Nutrients 2013, 5(9), 3496–3505; DOI 10.3390/nu5093496 48. Braun, G.B.; Friman, T; Pang, H.B.; Pallaoro, A.; de Mendoza, T.H.; Willmore, A.M.A.; Kotamraju, V.R.; Mann, A.P.; She, Z.G.; Sugahara, K.N.; Reich, N.O.; Teesalu, T.; Ruoslahti, E. Etchable plasmonic nanoparticle probes to image and quantify cellular internalization. Nat. Mater. 2014, 13(9), 904–911; DOI 10.1038/NMAT3982 49. Studer, A. M.; Limbach, L. K.; Van Duc, L.; Krumeich, F.; Athanassiou, E. K.; Gerber, L. C.; Moch, H.;Stark, W. J. Nanoparticle cytotoxicity depends on intracellular solubility: comparison of stabilized copper metal and degradable copper oxide nanoparticles. Toxicol. lett. 2010, 197 (3), 169–174; DOI 10.1016/j.toxlet.2010.05.012 50. Cronholm, P.; Karlsson, H. L.; Hedberg, J.; Lowe, T. A.; Winnberg, L.; Elihn, K.; Wallinder, I. O.;Möller, L. Intracellular uptake and toxicity of Ag and CuO nanoparticles: a comparison between nanoparticles and their corresponding metal ions. Small 2012, 9 (7), 970–982; DOI 10.1002/smll.201201069 51. Hanisch, U.K. and Kettenmann, H. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci. 2007, 10(11), 1387–1394; DOI 10.1038/nn1997 52. Lu, M.; Hu, L.F.; Hu, G.;Bian, J.S. Hydrogen sulfide protects astrocytes against H2O2-induced neural injury via enhancing glutamate uptake. Free Radical Bio. Med. 2008, 45 (12), 1705–1713; DOI 10.1016/j.freeradbiomed.2008.09.014 53. Suresh, A. K.; Doktycz, M. J.; Wang, W.; Moon, J. W.; Gu, B.; Meyer III, H. M.; Hensley, D. K.; Retterer, S. T.; Allison, D. P.;Phelps, T. J. Monodispersed biocompatible Ag2S nanoparticles: Facile extracellular bio-fabrication using the gamma-proteobacterium, S. oneidensis; Oak Ridge National Laboratory (ORNL); Center for Nanophase Materials Sciences; High Temperature Materials Laboratory: 2011. 54. Zhang, S.; Du, C.; Wang, Z.; Han, X.; Zhang, K.;Liu, L. Reduced cytotoxicity of silver ions to mammalian cells at high concentration due to the formation of silver chloride. Toxicol.in Vitro 2012, 27 (2), 739–744. DOI 10.1016/j.tiv.2012.12.003 55. Nguyen, K. C.; Seligy, V. L.; Massarsky, A.; Moon, T. W.; Rippstein, P.; Tan, J.;Tayabali, A. F. In Comparison of toxicity of uncoated and coated silver nanoparticles, J. Phys.: Conf. Ser. 2013, 429, 012025; DOI 10.1088/1742-6596/429/1/012025

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For Table of Content only 253x166mm (96 x 96 DPI)


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Figure 1 Dissolution of AgNPs, LPS + AgNPs (1000 ng/mL), ascorbic acid (AA)+AgNPs (100 µM) and H2O2 + AgNPs (150 µM) in DMEM/10%FBS were measured at 1 min, and 2, 4, 6, 8, 24 and 48 h, by calculated decrement in absorbance. The upper bound (filled symbols) and the lower bound (empty symbols) of the dissolution rate were calculated by decrements of the peak intensity at 407 nm and of the integrated area under the absorbance curve, respectively. The dashed line shows the average dissolution rate between upper and lower bounds. On the upper right side, an example of original absorbance spectra at several time points illustrates how absorbance decreases as the AgNPs dissolve. 61x44mm (300 x 300 DPI)



Figure 2 Intracellular ROS releases from the following treatments (LPS, AA, AgNPs, LPS+AgNPs and AA+AgNPs) after 24- and 48-h exposure to (A) Microglia (BV-2) (calculated by geometric mean) (B) Astrocyte (ALT) cells (calculated by geometric mean). Significant effects: * p < 0.05 vs control cells. # p< 0.05 between two treatment groups. 122x178mm (600 x 600 DPI)


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Figure 3 Quantification of Ag from cellular uptake by LA-ICP-MS. CCD images of fixed (A) Microglia (BV-2) and (B) Astrocyte (ALT) cells overlap with their corresponding LA-ICP-MS images of the 107Ag intensity distribution (in fg). Cells were incubated with AgNPs in a concentration of 5 µg/mL for 24 h. Scale bars represent 100 µm. Quantification results of Ag element in single BV-2 (C) and ALT cell (D) by LA-ICP-MS. Data for each group was derived from 15 randomly selected single cells, and were expressed in boxchart. The mark * stands for maximum and minimum value, error bar stands for standard deviation. The box is determined by the 25th and 75th percentiles. The median line and closed circle represent median and mean, respectively. 127x97mm (300 x 300 DPI)



Figure 4 Ag speciation analysis results in a four-component LCF for exposure of different treatment groups (AgNPs, LPS + AgNPs, AA + AgNPs) and duration (24 and 48 h) in (A) Microglia (BV-2) and (C) Astrocyte (ALT) cells. The amount of AgNPs and Ag ions (I) species in each single (B) BV-2 and (D) ALT cell, derived from total Ag (LA-ICP-MS quantification) and ratio of Ag species (XANES analysis). 142x101mm (300 x 300 DPI)


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Trojan-Horse Mechanism in the Cellular Uptake of Silver

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