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Intracellular Dissolution of Silver Nanoparticles: Evidence from Double Stable Isotope Tracing Sujuan Yu, Yujian Lai, Lijie Dong, and Jing-fu Liu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b03251 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 6, 2019

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Environmental Science & Technology

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Intracellular Dissolution of Silver Nanoparticles: Evidence

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from Double Stable Isotope Tracing

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Su-juan Yu, Yu-jian Lai, Li-jie Dong, Jing-fu Liu*

4

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-

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Environmental Sciences, Chinese Academy of Sciences, P. O. Box 2871, Beijing, 100085

6 7 8 9 10 11 12 13 14 15

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* Corresponding author.

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Tel.: +86-10-62849192

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E-mail: [email protected]

19

ACS Paragon Plus Environment


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ABSTRACT

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To track transformations of silver nanoparticles (AgNPs) in vivo, HepG2 and A549 cells were co-

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cultured with two enriched stable Ag isotopes (107AgNPs and

23

enzymatic digestion,

24

quantified by liquid chromatography combined with ICP-MS. We found that ratios of 107Ag+ to total

25

107Ag

26

Trojan-horse mechanism occurred, i.e. AgNPs released high contents of Ag+ after internalization.

27

While the presence of 109Ag+ (5 and 100 µg/L) have little influences on the uptake of 107AgNPs (0.1

28

and 2 mg/L), the presence of 107AgNPs at a high dose (2 mg/L) dramatically increases the ingestion

29

of

30

internalization of

31

107AgNPs,

32

experiments in toxicology studies, culturing organisms with AgNO3 at the same concentration of Ag+

33

in the AgNP exposure medium, may underestimate uptake of Ag+ and thus cannot exclude suspected

34

toxic effects of Ag+ at high AgNP exposure doses.

107AgNPs,

and proportions of

109Ag+,

even though

ionic

107Ag+

107Ag+/ 109Ag+

107AgNPs

109Ag+.

and

109Ag+

109AgNO

3)

at nontoxic doses. After

in exposed cells could be separated and

in cells increased gradually after exposure, proving that the

at a low dose (100 µg/L) showed negligible effects on the

Cellular homeostasis may be perturbed under sublethal exposure of

and thus enhanced uptake of 109Ag+. Our findings suggest that the widely adopted control


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35

Introduction

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The superior antibacterial properties of silver nanoparticles (AgNPs) made them among one of the

37

most commercialized nanoparticles (NPs). AgNPs exist in about 24% of the consumer products that

38

listed to contain NPs1, including clothing, food packaging materials, laundry detergents, and personal

39

care products2. Given the increasing production and their widespread applications, the inevitable

40

release of AgNPs into the environment would be expected, and their influences on the ecosystem

41

health are concerned3-5.

42

The toxicity of AgNPs has been extensively studied,6-13 and some papers reported that ionic Ag

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species were detected in cells after AgNP exposure14-17. The Trojan-horse mechanism that AgNPs

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release high contents of toxic silver ions after ingested by cells was regarded as the main mechanism

45

for AgNP toxicity. However, there is still a knowledge gap on understanding this mechanism. Since

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AgNPs are highly dynamic, the pristine exposure medium of AgNPs often contains a low amount of

47

Ag+. It is hard to confirm whether ionic Ag species found in cells are derived from the uptake of Ag+

48

in the exposure medium or the dissolution of AgNPs in cells18.

49

In order to fill the knowledge gap, a good approach is to label the AgNPs and Ag+ in the pristine

50

medium to make them distinctive before exposure, and then using a proper analytical method to

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selectively determine AgNPs and ionic Ag+ species in biological matrices. The common used labeling

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methods include enriched stable isotope labeling, radioisotope labeling, and fluorescent dyes

53

labeling19. However, handling and disposal issues associated to radioisotope tracers and the

54

requirement of post surface modification of NPs with fluorescent dyes limit the use of the latter two

55

methods20. Stable isotope traces are free from these drawbacks and have been used to study the toxicity

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and bioavailability of several NPs with high sensitivity19-26. Our previous study also showed that the

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transformation of AgNPs and Ag+ in aquatic environments and plants could be well monitored by

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using isotopically enriched AgNPs and Ag+ 27-29. ACS Paragon Plus Environment


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To detect AgNPs and ionic Ag species in biological samples, proper decomposition methods are

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required to liberate AgNPs and ionic Ag species from the complex matrices and overcome the

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interference effects. Enzymatic treatment and alkaline digestion methods have been widely used in the

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literature. For example, tetramethylammonium hydroxide (TMAH) based alkaline digestion was

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developed to extract NPs from animal tissues, Daphnia magna, Lumbriculus variegatus, and HepG2

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cells30-35. However, long time digestion by TMAH may lead to fast dissolution of AgNPs.34 Unlike

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alkaline digestion, the enzymatic treatment is much milder. For instance, proteinase K based extraction

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of AgNPs from chicken meat and rat tissues are reported30, 36-38, albeit low analyte recovery was stated

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as its main shortcoming30. In this study, to eliminate the possible dissolution of AgNPs during the

68

extraction procedure, enzymatic digestion was preferred.

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Several techniques have been utilized for the selective determination of AgNPs and Ag+ in

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biological samples, including single particle ICP-MS32, 37-38, cloud point extraction combined with

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ICP-MS determination18, and other hyphenated techniques with ICP-MS detection such as field flow

72

fractionation (FFF)33-34,

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hyphenated ICP-MS method (LC-ICP-MS) was reported for the accurate characterization of core size

74

and corona thickness of different silver species in biological tissues, which provides a good solution

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for the in vivo speciation analysis of Ag35.

36

and liquid chromatography (LC)39-40. Very recently, a versatile LC

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The presence of ionic Ag species in organisms might be due to the uptake of Ag+ from the

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exposure medium and in vivo dissolution of AgNPs. To delineate the source of Ag+ and

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transformations of AgNPs in vivo, in this study, cells were co-cultured with two enriched stable Ag

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isotopes (107AgNPs and

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nanoparticulate, while 109AgNO3 was used to trace the fate of Ag+. As the amount of Ag+ in the initial

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exposure medium of AgNPs is usually no more than 10% of the total content of Ag38, 41-42, 109AgNO3

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was mixed with

107AgNPs

109AgNO

3),

in which

107AgNPs

were used to trace the fate of Ag

at a concentration of 5% of the total concentration of ACS Paragon Plus Environment

107AgNPs,

and co-

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exposed to cells. Previous studies have found that AgNPs can be detected in many organs after AgNP

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exposure, of which liver and lung are the main targets for the accumulation of Ag after oral and

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inhalation exposure38, 43-45. Thus, HepG2 cells, a human hepatocarcinoma cell line, and A549 cells, an

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adenocarcinomic human alveolar basal epithelial cell line, were selected in this study. An optimized

87

enzymatic digestion method was used to extract AgNPs and ionic Ag species from cells, and various

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Ag forms were separated and determined by LC-ICP-MS. The ratios of ionic 107Ag+ species to total

89

107Ag

90

detailed insights into the transformation of AgNPs in cells.

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Materials and methods

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Materials. AgNO3 (purity, >99.5%) was purchased from Sinopharm Chemical Reagent Co. Ltd.

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(Shanghai, China). 107Ag (99.5% enriched) and 109Ag isotopes (99.81% enriched) in solid form were

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obtained from Trace Sciences International Inc. (Texas, USA). Trisodium citrate dihydrate

95

(purity, >99%), sodium borohydride (>99.99%, trace metals basis), sodium dodecyl sulfate (SDS),

96

potassium hexacyanoferrate(III) (K3Fe(CN)6), sodium thiosulfate pentahydrate (Na2S2O3 · 5H2O),

97

and Proteinase K (from Engyodontium album) were obtained from Sigma Aldrich (St. Louis, MO,

98

USA). High purity nitric acid was purchased from Merck (Darmstadt, Germany). The Ag+ standard

99

(1000 mg/L) used for ICP-MS determination was purchased from National Institute of Metrology

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(Beijing, China). All the other chemicals were obtained from Sinopharm Chemical Reagent Co.

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(Beijing, China) with analytical grades or better. Ultrapure water (18.3 MΩ.cm) produced from a

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Milli-Q Gradient system (Millipore, Billerica, MA, USA) was used throughout the experiments.

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Synthesis of the Isotopically Labeled AgNPs and Ag+. Citrate-coated AgNPs with natural isotope

104

abundances, which were used to optimize the enzymatic digestion parameters, were prepared by a

105

previously reported method22, 27. Typically, 0.43 mL of 58.8 mM AgNO3 and 3.7 mL of 34 mM

106

trisodium citrate dihydrate were added into 100 mL of boiling distilled water. To this solution, 1 mL

and the proportions of

107Ag+/109Ag+

were calculated at different exposure times to achieve



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107

of 50 mM sodium borohydride was added drop-wise under vigorous stirring. Then the mixture was

108

stirred for another 30 min, and allowed to cool to room temperature. AgNPs were purified by

109

centrifugal ultrafiltration (Amicon Ultra-15 100 kD, Millipore, MA), and further washed with

110

ultrapure water three times, after which the stock suspension was stored at 4 °C in the dark for later

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use. For the preparation of citrate-coated 107AgNPs, 107Ag isotope metal was first dissolved in HNO3

112

to get its nitrate salt and then used for the synthesis of the NPs according to the procedure mentioned

113

above. 109AgNO3 was also obtained by dissolving 109Ag isotope metal in HNO3.

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Characterization of AgNPs. Transmission electron microscopy (TEM) was performed with an H-

115

7500 (Hitachi, Japan) at 80 kV. TEM samples were prepared by placing 5 μL aliquots of AgNP

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suspensions onto an ultrathin carbon-coated copper grid and vacuum dryed at room temperature. The

117

hydrodynamic diameter and ζ potential of AgNPs were measured by dynamic light scattering (DLS)

118

with a Zetasizer Nano (Malvern Instruments Ltd. Malvern, UK) at 25 °C. Briefly, stock AgNP

119

suspensions were diluted with ultrapure water to a final concentration of 10 mg/L, and three

120

measurements were conducted to get the average value.

121

Cell Culture and Treatments. As liver and lung are the main targets for the accumulation of Ag after

122

oral and inhalation exposures, HepG2 and A549 cells were used to study the transformations of AgNPs

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in the cells. Cells were grown in minimum essential medium (MEM) (Hyclone) supplemented with

124

10% fetal bovine serum (FBS) (Hyclone), 0.1 mM nonessential amino acids (Invitrogen), 100 U/mL

125

penicillin and 100 mg/mL streptomycin (Invitrogen) in a humidified incubator with 5% CO2.

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When grown to 70% confluency in 100 mm culture plates, cells were incubated with 10 mL of

127

cell culture medium spiked with 107AgNPs and 109Ag+ alone or mixtures of 107AgNPs and 109Ag+. At

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different exposure times (3 h, 6 h, 9 h, 11.5 h, 24 h), the medium was removed, and then cells were

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washed with 3 mL of phosphate buffered saline (PBS) solution three times or the etching solution (see

130

the following section). After that, the washed cells were detached from the culture plates by adding 1 ACS Paragon Plus Environment

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mL of 0.25% enzyme trypsin and kept at 37 °C for 3 min. Subsequently, cells were harvested and

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centrifuged at 1500 rpm for 5 min at 4 °C, and cell number was counted.

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Removal of Cell Surface-attached Ag by Chemical Etching. A significant amount Ag may attach

134

on the external surface of cell membranes after Ag exposure14, and this may lead to a misleading result

135

while evaluating in vivo transformation of AgNPs. A chemical etching method was used to remove

136

the surface-bound Ag according to previous studies14, 46. In short, after cells were exposed to Ag, the

137

medium was discarded, and cells were washed with 3 mL of PBS twice. Then 5 mL of the etching

138

solution (mixture of 10 mM K3Fe(CN)6 and 10 mM Na2S2O3 ·5H2O in PBS) was added in the cell

139

culture plates，and incubated with cells for 30 s. Subsequently, the etching solution was discarded,

140

and then cells were washed with 3 mL of PBS three times. The morphology of exposure cells before

141

and after chemical etching washing was observed using an optical microscope to make sure that the

142

integrity of cells did not change after washing. Meanwhile, a control experiment was also carried out

143

to investigate if there was any elimination of internalized Ag during the washing procedure. Briefly,

144

after washed with the etching solution and PBS, exposed cells were incubated with another 5 mL of

145

the etching solution for 30 s, and then the washing solution was collected. The concentration of Ag in

146

the washing solution was determined by ICP-MS. After the washing procedure, cells were collected

147

by trypsinization.

148

The leftover of etching solutions may induce the dissolution of intracellular AgNPs during the

149

enzymatic digestion process. To this end, a control experiment was conducted according the following

150

procedure. Two groups of non-exposed HepG2 cells, one group washed with PBS and the other

151

washed with the chemical etching solution were collected, spiked with 200 μg/L AgNPs, and then

152

digested with Proteinase K for analysis.

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Cytotoxicity Assessment. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)

154

assay was carried out to examine the cell viability. Briefly, HepG2 and A549 cells were seeded in 96ACS Paragon Plus Environment


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well plates (8  103 cells/well), and then exposed to different concentrations of 107AgNPs, 109Ag+ or

156

mixtures of 107AgNPs and 109Ag+ for 24 h. After the exposure, 10 µL of 5 mg/mL MTT in PBS was

157

added into each well, and cells were incubated for another 5 h. Then, the culture medium was discarded,

158

and cells were treated with 100 µL dimethyl sulfoxide (DMSO). The plates were slightly shaken for

159

10 min. Absorbance was measured at 490 nm using a multimode microplate spectrophotometer

160

(Thermo Electron, Varioskan Flash, USA).

161

Enzymatic Digestion. The enzyme solution was prepared by dissolving Proteinase K to a final

162

concentration of 10 mg/mL in water. To optimize the enzymatic digestion parameters, cells without

163

Ag exposure were cultured and collected. In short, 2 mL of ultrapure water was added into the

164

collected cells, and sonicated for 5 min to homogenize the cell lysates. Then, the suspension was

165

spiked with aliquots of AgNPs and/or Ag+ and mixed by vortexing for 15 s, and 2 mL digestion buffer

166

(20 mM Tris, 1% SDS, pH 7.5) was added. Finally, 20 μL of enzyme solution was added and vortexed

167

for 30 s. Although Proteinase K exhibited maximum activity at 37 oC, high temperature may accelerate

168

the dissolution of AgNPs47-48, thus the digestion process was performed at room temperature

169

throughout the experiment to reduce the release of Ag+. The suspension was kept at room temperature

170

for 30 min and diluted with the mobile phase for LC-ICP-MS analysis.

171

Instrumentation. An Agilent 1200 series LC system (Agilent Technologies, Palo Alto, CA, USA)

172

hyphened with Agilent 7700 ICP-MS (Santa Clara, CA, USA) was used for the separation and

173

quantification of AgNPs and ionic Ag species.35, 39-40. LC separation (10 μL sample) was performed

174

with an amino column (Venusil XBP NH2, 5 μm particle size, 1000 Å pore size, 4.6 × 250 mm, Bonna-

175

Agela Technologies Inc., Tianjin, China) and a mobile phase consisting of 2% (v/v) FL-70 and 2 mM

176

Na2S2O3 at a flow rate of 0.5 mL/min. ICP-MS analysis was conducted with 1500 W RF power, 8 mm

177

sampling depth, 1.0 L/min carrier gas, and 0.5 sec integration time. Both isotopes of 107Ag and 109Ag

178

were monitored. ACS Paragon Plus Environment

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Environmental Science & Technology 107Ag

Quantification and Calculation of Tracer Concentration. The concentrations of

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of the stock solution of AgNPs and Ag+ were analyzed by Agilent 7700 ICP-MS (limit of detection of

181

0.009 μg/L) with Ag standards. Before determination, the stock solution of 107AgNPs was digested by

182

concentrated HNO3. Briefly, to a glass vessel were added 100 µL of 107AgNPs and 2 mL of high purity

183

HNO3, and the mixture were heated on a hot plate at 120 °C for 15 min. Then the solution was diluted

184

with ultrapure water for ICP-MS analysis. The concentration of the stock solution was calculated as

185

the product of the natural relative abundances of 107Ag (0.5184) and the total Ag concentration given

186

by the ICP-MS software from the Ag intensity. In our previous study, we have demonstrated that with

187

the proper mobile phase (mixture of 2% FL-70 and 2 mM Na2S2O3), AgNPs showed similar

188

sensitivities with their ionic counterparts by LC-ICP-MS40. Therefore, a series of Ag+ standards (0.5,

189

1, 2, 5, 10, 20, 50 μg/L) were analyzed to get a calibration curve by plotting the total peak areas of Ag

190

against the concentration of Ag+. The concentration of 107AgNPs, 107Ag+ and 109Ag+ can be calculated

191

according to their corresponding peak areas. The total contents of

192

digested cell samples were also analyzed by ICP-MS after microwave-assisted digestion (CEM Mars

193

5, Xpress, Matthews, NC) to compare with the results calculated by the LC-ICP-MS calibration curve.

194

Briefly, to 1 mL of enzymatic digested cell samples were added 4 mL concentrated HNO3 and 1 mL

195

of concentrated H2O2. The mixture was irradiated at 120 °C (800 W) for 10 min, followed by 180 °C

196

(1600 W) for 30 min. After digestion, samples were diluted with ultrapure water and analyzed by ICP-

197

MS. The dogfish liver certified reference material (DOLT-5) was used to control the digestion

198

procedure. The certified values of Ag is 2.05±0.08 mg/kg. The detected value of Ag in the experiment

199

was 1.98±0.04 mg/kg, with a recovery was 96.6%. Blank analysis, which used to investigate the

200

possible contamination from reagents, containers and cells, showed that the concentrations of Ag in

201

all the blank samples were below the limit of detection of ICP-MS. As a result, we assume that all the

202

Ag detected in exposed cells was from Ag we added in the cell culture medium. Meanwhile, although ACS Paragon Plus Environment

107Ag

and

109Ag

and

109Ag

179

in enzymatic


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the enrichment of the two isotopes we used was not 100% (99.5% for 107Ag and 99.81% for 109Ag),

204

the ultratrace impurities would have very limited influences on the final results. Therefore, to simplify

205

the calculation, we also assume that 100% 107Ag was from 107AgNPs and 100% 109Ag was from 109Ag+

206

in exposed cells.

207

Results and Discussion

208

Characterization of AgNPs. Citrate-coated

209

used in this study, as shown in Figure S1a. DLS measurements revealed that 107AgNPs carried negative

210

surface charges (zeta potential of -11.9±2.6 mV) with a hydrodynamic diameter of 25.6±0.4 nm.

211

After three months’ storage, TEM images (Figure S1c) and DLS analysis (hydrodynamic diameter of

212

24.9±0.6 nm) of the stock suspension demonstrated that the size and morphology did not change

213

significantly, indicating that the synthesized citrate-coated

214

stock suspension was prepared every three months.

215

Optimization of the Enzymatic Digestion Parameters. Proteinase K is a highly active and broad

216

spectrum proteinase, which can degrade proteins into amino acids. Proteinase K remains active over

217

a wide range of pH values (7.5-12)，and is extensively used to digest proteins during the purification

218

of DNA or RNA in molecular biology applications30,

219

enzymatic digestion with Proteinase K would be a promising method for sample preparation. A

220

digestion buffer consisting 10 mM Tris, 0.5% SDS at pH 7.5 was prepared for enzymatic digestion.

221

The influence of digestion time on the recovery of AgNPs and Ag+ was studied by respective spiking

222

unexposed HepG2 cells with AgNPs, Ag+, or mixtures of AgNPs and Ag+. As shown in Figure 1a,

223

highest recovery values of Ag+ and AgNPs were obtained in the first 30 min of digestion time, and

224

longer digestion time did not improve the Ag+ recovery. As for AgNPs, further prolonged digestion

225

time may promote the dissolution of AgNPs, resulting in a little decrease of the recovery. Moreover,

226

there is no substantial difference in AgNP recovery between the cells spiked with AgNPs alone and

107AgNPs

with an average size of 12.5±3.6 nm were

107AgNPs

36-37.

were stable. In this study, the

For samples consisting of proteins,


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mixtures of AgNPs and Ag+, indicating that AgNPs and Ag+ were separated successfully with the LC-

228

ICP-MS method. The representative LC-ICP-MS chromatograms of AgNPs and Ag+ in cells after

229

digestion were shown in Figure 1b.

230

Analytical Performance.

231

Different concentrations of AgNPs (20-2000 μg/L) and Ag+ (2-2000 μg/L) were spiked into blank

232

HepG2 cells to evaluate the analytical performance of this method. The linear correlation coefficients

233

were 0.9897 and 0.9599 for AgNPs and Ag+, respectively. The concentrations of AgNPs and Ag+

234

detected in Ag exposed cells were in the spiking range. The detection limit (LOD) defined as 3 times

235

of signal to noise ratio (S/N =3) was 0.53 μg/L. The precision of this optimized method was

236

investigated by analyzing cells spiked with 100 μg/L AgNPs. The relative standard deviation (RSD)

237

of retention time was 0.3% and RSD of peak area was 1.4% (n=7) for the peak of AgNPs.

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Removal of Surface-attached Ag. To remove the membrane-bound Ag species, a chemical etching

239

method (washing with a mixture containing 10 mM K3Fe(CN)6 and 10 mM Na2S2O3·5H2O in PBS)

240

was used. Effect of the washing procedure was also evaluated. No cell rupture or distinct change in

241

cellular morphology was observed after the chemical washing (Figure S2), indicating that this washing

242

process did not destroy the cell integrity. Moreover, a control experiment that incubated the chemically

243

washed cells with another 5 mL of the etching solution showed that the concentration of Ag in the

244

etching solution was below the detection limit of ICP-MS. Thus, the leakout of internalized Ag was

245

excluded during the etching procedure. Meanwhile, the PBS washing was also conducted to compare

246

the removal efficiency. The detected intracellular contents of Ag reduced markedly after the chemical

247

etching. The uptake of Ag by HepG2 cells decreased about 20% and 35% when exposed to mixtures

248

of 5 mg/L of AgNPs and 250 μg/L Ag+ and 2 mg/L of AgNPs and 100 μg/L Ag+ (Figure S3),

249

respectively, indicating that earlier studies may overestimate the intracellular amount of AgNPs in

250

cells after PBS washing18. ACS Paragon Plus Environment


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Non-exposed HepG2 cells washed with PBS solutions or etching solutions were collected and

252

spiked with 200 μg/L AgNPs to investigate the effects of leftover etching solutions on the enzymatic

253

digestion of AgNPs. Results showed that there was no significant difference between the two groups

254

(AgNP recovery was 85.0% for PBS washing and 84.8% for chemical etching solution washing),

255

implying that the solution residue had little influence on the AgNP dissolution.

256

Uptake and Transformation of 107AgNPs and 109 Ag+ in Cells. In this report, the uptake and possible

257

transformation of citrate coated AgNPs and Ag+ in HepG2 and A549 cells were studied at nontoxic

258

doses. Citrate coated AgNPs do not exist in nature and that AgNPs in nature and in biological matrices

259

undergo fast (surface) transformation, which for reasons of simplification have not been taken into

260

account in the present work. According to an earlier study on the biological effects of AgNPs under

261

sublethal dosage, HepG2 cells showed minimal cytotoxicity at exposure concentrations of 2-8 mg/L41.

262

We further assessed the cytotoxicity of HepG2 and A549 cells exposed to 2 mg/L of 107AgNPs, 100

263

μg/L 109Ag+ and a mixture of the two solutions. Since no significant cytotoxicity was observed (Figure

264

S4), the cells were incubated with a mixture of 2 mg/L 107AgNPs and 100 μg/L 109Ag+ in the following

265

experiment. The cells were collected at predetermined exposure times, subjected to enzymatic

266

digestion, and nanoparticulate and ionic Ag species were separated and determined by LC-ICP-MS

267

(Figure S5). Compared with the pristine

268

intracellular

269

As the separation followed a size exclusion mechanism39-40, the longer retention time of

270

implied that the size of intracellular

271

107AgNPs

272

with metalloproteins49, and the decrease in retention time suggested that Ag(I) might liberated as the

273

form of Ag(I)-biomolecule complex after enzymatic digestion35. As shown in Figure 2a and 3a, the

274

ingestion of 107Ag and 109Ag by HepG2 cells increased with incubating time, from 371.0 pg 107Ag/104

107AgNPs

107AgNP

stock solution (Figure S5a), the retention time of

delayed, while the retention time of

107AgNPs

107Ag(I)

and

109Ag(I)

species decreased. 107AgNPs

decreased, which may ascribe to the dissolution of

in cells. Once ingested by cells or intracellular released from AgNPs, Ag+ would complex


Page 13 of 28

Environmental Science & Technology 109Ag/104

276

after 24 h. The total amount of Ag uptake by HepG2 cells was comparable with that of A549 cells

277

(Figure 4), and was lower than a previous study16 (15240 pg total Ag/104 cells after 24 h exposure) in

278

which CHO-K1 cells were used to evaluate the cytotoxicity of citrate-coated AgNPs. The different

279

sizes and types of AgNPs, diverse cell lines, and varying exposure concentrations may explain the

280

discrepancy in the amount of Ag detected in cells. On the other hand, the total amount of Ag in HepG2

281

cells was also determined by ICP-MS after microwave digestion. The recoveries, calculated by

282

dividing the sum of

283

directly analyzed by ICP-MS, were in the range of 86.7% - 115%, showing that the LC-ICP-MS

284

method was accurate to quantify the uptake of Ag in cells.

285

The fractions of

and

109Ag

107AgNPs

cells and 288.3 pg

109Ag/104

cells and 79.1 pg

107Ag

cells after 3 h to 2594.8 pg

107Ag/104

275

cells

contents derived from LC-ICP-MS by the total amount of Ag

and ionic

107Ag

species in total

107Ag

in HepG2 cells at different

286

incubation time were also calculated (Figure 2b). In the pristine exposure solution, only 3.7% of 107Ag

287

existed as 107Ag+; however, the proportions of 107Ag+ in HepG2 cells increased to 82.7% just after 3

288

h, and raised to 93.3% after 24 h, which was much higher than the fraction of 107Ag+ in the cell culture

289

medium (6.9%) kept under the same condition for 24 h.

290

Due to the lack of proper analytical methods, the speciation analysis of Ag in cells was rarely

291

reported in the literature. Recently, X-ray absorption near-edge spectroscopy (XANES) has emerged

292

as a powerful and nondestructive tool to investigate the chemical species of Ag in cells. Based on

293

XANES fingerprints, several studies have found an increase of the proportion of ionic Ag species in

294

cells relative to the original exposure medium after AgNP exposure14, 16-17, 49, and claimed that they

295

proved that the Trojan-horse mechanism existed. However, the progressively growing proportion of

296

Ag+ in cells might be from the possible dissolution of AgNPs in cells and/or faster ingestion of Ag+

297

than AgNPs18. Control experiments were conducted in some studies14 by incubating cells with the

298

same concentration of AgNPs and Ag+, and they observed higher amounts of Ag in the AgNP exposed ACS Paragon Plus Environment


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299

groups. However, different from the control experiment, the concentration of AgNPs was much higher

300

than Ag+ in the exposure medium, which may largely alter the uptake behavior of cells. Thus, the

301

possibility of faster uptake of Ag+ than AgNPs should not also be excluded. To achieve detailed

302

insights into the mechanism, we further calculated the ratios of 107Ag+/109Ag+ at different incubation

303

time, and then calculated Δ(107Ag+/109Ag+) values by subtracting the ratio of 107Ag+/109Ag+ in the initial

304

cell culture medium. As can be seen in Figure 2c, Δ(107Ag+/ 109Ag+) values in HepG2 cells increased

305

gradually over time. As the cell uptake rate constant would be equal for

306

obviously raised value of Δ(107Ag+/ 109Ag+) in HepG2 cells gave the direct evidence of the intracellular

307

release of 107Ag+ from 107AgNPs.

308

107Ag+

and

109Ag+,

the

Though HepG2 cells showed no obvious cytotoxicity at the exposure level, the concentration 107AgNPs

and 100 μg/L

109Ag+)

309

(mixtures of 2 mg/L of

used was relatively high. In the real

310

environment, the predicted concentrations of AgNPs were in the range of ng/L to pg/L50-51; however,

311

the LOD of existing methods hinder the accurate detection of AgNPs under such low exposure

312

concentration. Therefore, a higher exposure concentration (100 μg/L 107AgNPs and 5 μg/L 109Ag+) was

313

selected to make the exposure as environmentally relevant as possible, while ensuring sufficient

314

signal-to-noise ratio to accurately quantify AgNPs and Ag+. In previous studies, comparative

315

concentrations of AgNPs were also used. For example, when evaluating the bioaccumulation of

316

nanosilver by Chlamydomonas reinhardtii, the green alga was exposed to AgNPs in the range of 0-

317

100 μg/L52. Murine microglial cells and murine brain astrocyte cells were incubated with 5 mg/L

318

AgNPs in a study to reveal the toxicity mechanism of AgNPs14. To monitor the morphology change

319

of alga after AgNP exposure and evaluate the contribution of released silver ions to the toxicity, algal

320

cells were treated with 40 μg/L Ag+ or 200 μg/L AgNPs53. Another study used AgNPs in the range of

321

0.8-200 μg/L to investigate the toxicity of AgNPs to algal and reveal the behaviors of AgNPs after

322

exposure to organisms54. Meanwhile, another cell line A549 was also treated with mixtures of ACS Paragon Plus Environment

Page 15 of 28


323

107AgNPs

324

observed or not. As shown in Figure 4c, Δ(107Ag+/ 109Ag+) values increased after 24 h in both cells at

325

two exposure levels, which confirmed the intracellular release of 107Ag+ from 107AgNPs.

326

and 109Ag+ following the same procedure to evaluate whether similar fate of AgNPs can be

The proportions of ionic 107Ag species in the total 107Ag vary significantly at different exposure 107Ag+

327

levels (Figure 4b). Though distinct ratios of

were detected in HepG2 and A549 cells, which

328

may ascribe to natural differences in the cell lines, AgNPs were more likely to release Ag+ at higher

329

exposure concentrations in both cells. Cells have their own regulatory mechanism, and would adapt

330

when suffering from external stimuli to maintain the internal homeostasis41. Although no direct

331

cytotoxicity was observed after different Ag treatment (Figure S4), we speculated that different

332

dosages of Ag may incur distinct responds in cells, resulting in the reprogramming of some protein

333

expression to satisfy the demand for cell survival, such as intracellular metallothionein 1 (MT1), a key

334

protein in maintaining the metal homeostasis in cells. As reported in a previous study, MT1 could

335

surround the internalized AgNPs to form a protein corona and induce the near-total dissolution55, and

336

the differential secretion of MT1 might trigger the diverse intercellular dissolution of AgNPs.

337

Uptake of 109Ag+ in Cells Incubated with 109Ag+ Alone. To study the role of AgNPs on the uptake

338

of Ag+ in cells, HepG2 and A549 cells were treated with 100 μg/L or 5 μg/L 109Ag+ alone, the same

339

concentration of 109Ag+ as the co-exposure group. The contents of 109Ag+ in cells were also determined

340

and compared with that of the co-exposure group. The ingestion of 109Ag+ in the sole 109Ag+ treatment

341

group was much lower than that of the co-exposure group in both cells at the high exposure

342

concentration (Figure 3a), while was comparable or even lower than that of the co-exposure group at

343

the low exposure level (Figure 3b), exhibiting different influences of AgNPs on the uptake of Ag+.

344

Previous studies have reported that even at non-toxic exposure levels, AgNPs may perturb the cellular

345

homeostasis, such as the abnormal expression of some key proteins and the attenuation of respiratory

346

chain41, 56. The obviously enhanced uptake of 109Ag+ in the presence of 107AgNPs at a high exposure ACS Paragon Plus Environment


Page 16 of 28

347

level may ascribe to increased membrane permeability of cells56 or the overexpression of ion

348

transporters stimulated by the co-existence of 107AgNPs. In previous toxicology studies41-42, 57-58, due

349

to the co-occurrence of AgNPs and Ag+ in the exposure medium, in order to answer the question that

350

whether the observed toxicity was induced by the nanoparticulate form or the co-presence of Ag+,

351

control experiments were often conducted by exposure organisms with Ag+ alone at the same

352

concentration as present in the exposure medium. As no damage was found in the Ag+ treatment group,

353

they concluded that the toxicity was ascribed to AgNPs themselves41-42. The observed higher uptake

354

of 109Ag+ in the presence of 107AgNPs at high AgNP dosages in this study may show that prior results

355

of toxicity studies should be more stringently scrutinized.

356

Uptake of

357

conducted to evaluate the influence of 109Ag+ on the uptake of 107AgNPs. Cells were incubated with 2

358

mg/L or 100 μg/L of 107AgNPs alone, the same concentration of 107AgNPs in the co-exposure group.

359

As shown in Figure 4, the ingestion of

360

group and the co-exposure group for both cells at the two exposure levels, indicating that the presence

361

of small amounts of 109Ag+ did not significantly affect the internalization of 107AgNPs.

362

107AgNPs

in Cells Incubated with

107AgNPs

107AgNPs

Alone. Further experiments were also

did not vary largely between the single exposure

In summary, our results showed that the fractions of

107Ag+

of total

107Ag

in exposed cells

363

increased over time, accompanied by the gradual rise of Δ(107Ag+/109Ag+) ratios. Taking that the cell

364

uptake rate constant would be equal for

365

can be considered as a direct evidence for the intracellular dissolution of

366

treated cell lines with 107AgNPs or 109Ag+ alone at the same dosage of 107AgNPs or 109Ag+ in the co-

367

exposure group showed that the presence of

368

assisted the uptake of 109Ag+, while did not affect the ingestion of 109Ag+ at a low concentration (100

369

µg/L) significantly. The diverse behaviors of 107AgNPs on the internalization of 109Ag+ indicated that

370

cellular homeostasis may be perturbed even at sublethal exposure levels, which highlight the necessity

107Ag+

and

109Ag+,

107AgNPs

the increased ratios of Δ(107Ag+/109Ag+) 107AgNPs.

Moreover, the

at a high concentration (2 mg/L) remarkably


Page 17 of 28


371

to focus on biological effects of AgNPs at nontoxic concentrations. Meanwhile, previous toxicology

372

studies often conducted control experiments by incubating organisms with AgNO3 at the same

373

concentration as present in the AgNP exposure medium to exclude suspected toxic effects of Ag+. Our

374

observation proved that this is not a proper comparison strategy at high AgNP exposure doses.

375 376

ASSOCIATED CONTENT

377

Supporting Information

378

Additional results are provided in Supporting Information, including TEM images and size

379

distribution of as synthesized 107AgNPs, morphology of HepG2 and A549 cells before and after PBS-

380

Fe3+-S2O32- etching solution washing, cellular uptake of Ag in HepG2 cells with PBS washing or with

381

PBS-Fe3+-S2O32- etching solution washing, cell viability of HepG2 and A549 cells after exposure to

382

different concentrations of 107AgNPs and 109Ag(I), and LC-ICP-MS chromatograms of Ag species in

383

HepG2 cells at different exposure times. This material is available free of charge via the Internet at

384

http://pubs.acs.org.

385

The authors declare no competing financial interest.

386 387

Acknowledgements

388

This work was supported by the the National Key R&D Program of China (2016YFA0203102), and

389

the National Natural Science Foundation of China (21507147, 201620102008, and 21527901). Special

390

thanks to Dr. Meseret Amde for revising the article.

391 392

References

393 394 395 396

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541 542


(58) Comfort, K. K.; Braydich-Stolle, L. K.; Maurer, E. I.; Hussain, S. M. Less Is More: Long-term in vitro exposure to low levels of silver nanoparticles provides new insights for nanomaterial evaluation. ACS Nano 2014, 8 (4), 3260-3271.

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544

Figure Captions

545

Figure 1. Optimization of the enzymatic digestion method. (a) Effects of digestion time on the recovery

546

of Ag+ and AgNPs; (b) representative LC-ICP-MS chromatograms of Ag+, AgNPs, and mixtures of

547

Ag+ and AgNPs. Blank HepG2 cells were respectively spiked with 200 μg/L Ag+, AgNPs or mixtures

548

of 200 μg/L Ag+ and 200 μg/L AgNPs.

549 550

Figure 2. Cellular uptake profiles of 107AgNPs, 107Ag+ and 109Ag+. (a) Contents of 107AgNPs and 107Ag+,

551

(b) ratios of 107AgNPs and 107Ag+ to total 107Ag, and (c) relative ratios of Δ(107Ag+/109Ag+) at different

552

exposure times. HepG2 cells were cultured with 2 mg/L of 107AgNPs and 100 μg/L 109Ag+.

553 554

Figure 3. Cellular uptake contents of 109Ag+ when cells were incubated with mixtures of 107AgNPs and

555

109Ag+

556

μg/L 109Ag+ for the co-exposure group, and were cultured with 100 μg/L 109Ag+ alone for the single

557

Ag exposure group; (b) HepG2 and A549 cells were cultured with 100 μg/L of 107AgNPs and 5 μg/L

558

109Ag+

559

exposure group.

or

109Ag+

alone. (a) HepG2 and A549 cells were cultured with 2 mg/L of

109Ag+

for the co-exposure group, and were cultured with 5 μg/L

107AgNPs

and 100

alone for the single Ag

560 107AgNPs, 107Ag+

Figure 4. Cellular uptake profiles of

562

107AgNPs

563

Δ(107Ag+/109Ag+). HepG2 and A549 cells were cultured with different concentrations of 107AgNPs and

564

109Ag+

and

107Ag+,

(b) ratios of

107AgNPs

and

and

109Ag+.

561

107Ag+

to total

for 24 h.


(a) Cellular uptake contents of

107Ag,

and (c) relative ratios of

Page 23 of 28


Ag (cps)

(b) 3.0x104

80

107

60

Intensity

Recovery (%)

(a) 100

40 +

20

Ag AgNPs AgNPs and Ag

0

565

0

1

2

3

4

5

AgNPs and Ag AgNPs

4

2.5x10

Ag

dissolved Ag species

+

+

AgNPs

4

2.0x10

4

1.5x10

offset: 10,000 cps 4

1.0x10

offset: 5000 cps

3

5.0x10

+

6

0.0

0

60

Digestion Time (h)

120

180

240

300

360

420

480

Time (sec)

566

Figure 1. Optimization of the enzymatic digestion method. (a) Effects of digestion time on the recovery

567

of Ag+ and AgNPs; (b) representative LC-ICP-MS chromatograms of Ag+, AgNPs, and mixtures of

568

Ag+ and AgNPs. Blank HepG2 cells were respectively spiked with 200 μg/L Ag+, AgNPs or mixtures

569

of 200 μg/L Ag+ and 200 μg/L AgNPs.

570



Page 24 of 28

Uptake of

107

4

Ag / 10 cell (pg)

(a) 3000 107

AgNPs

2500

107

+

Ag

2000 1500 1000 500 0

(b) 120 Percentage of total 107Ag

6h

3h

11.5 h

9h

24 h

Exposure duration 107

107

AgNPs

+

Ag

100 80 60 40 20 0

Ag )

+

8

+ 109

(

9h

6h

11.5 h

24 h

Exposure duration 12

107

3h

14

Ag /

(c)

0h

10

6 4 2 0 0h

571

3h

6h

9 h 11.5 h

24 h

Exposure duration

572

Figure 2. Cellular uptake profiles of 107AgNPs, 107Ag+ and 109Ag+. (a) Contents of 107AgNPs and 107Ag+,

573

(b) ratios of 107AgNPs and 107Ag+ to total 107Ag, and (c) relative ratios of Δ(107Ag+/109Ag+) at different

574

exposure times. HepG2 cells were cultured with 2 mg/L of 107AgNPs and 100 μg/L 109Ag+.

575 ACS Paragon Plus Environment


exposed to 107AgNPs and 109 + exposed to Ag alone

400 350

109

(b)

+

Ag

4

300 250

109

200 150

Uptake of

Uptake of

109

4

Ag / 10 cell (pg)

(a)

100 50 0

Ag / 10 cell (pg )

Page 25 of 28

3h

576

6h

9 h 11.5 h 24 h HepG2

24 h

24 h 24 h A549

8 7

exposed to 107AgNPs and 109 + exposed to Ag alone

109

Ag

+

6 5 4 3 2 1 0

24 h

24 h

24 h

HepG2

Exposure duration

24 h A549

Exposure duration

577

Figure 3. Cellular uptake contents of 109Ag+ when cells were incubated with mixtures of 107AgNPs and

578

109Ag+

579

μg/L 109Ag+ for the co-exposure group, and were cultured with 100 μg/L 109Ag+ alone for the single

580

Ag exposure group; (b) HepG2 and A549 cells were cultured with 100 μg/L of 107AgNPs and 5 μg/L

581

109Ag+

582

exposure group.

or

109Ag+

alone. (a) HepG2 and A549 cells were cultured with 2 mg/L of

for the co-exposure group, and were cultured with 5 μg/L


109Ag+

107AgNPs

and 100

alone for the single Ag


(a)

Page 26 of 28

107

AgNPs

3500

107

+

Ag

A549

HepG2

3000 2500 2000

Uptake of

107

4

Ag / 10 cell (pg)

4000

150 100 50 0

Ps

+ + + + Ps Ps Ps Ps Ps Ps+ Ps N gN gN N N N g A gN g+ A g g g A A A L A + A Ag A + /L /L L L g/ /L Ag mg /L /L /L Ag g/ g m g/ g/L g g  g g  0  L 2 2 m 0 m  /L / 0 0 2 0 10 2 00 10 10 g 10 g 1 10 5 5 A

(b)

gN

Percentage of total

107

Ag

140 120

107

107

AgNPs HepG2

+

Ag

A549

100 80 60 40 20 E m xp ed os iu ur 2 m e m g/ L 2 A gN 10 mg Ps 0 /L g A /L gN 10 Ag + Ps 0 + g 10 /L 0 A gN 5 g / g L Ps /L Ag A N g + Ps 2 + m g/ L 2 A gN 10 mg Ps 0 /L g A /L gN 10 Ag + Ps + 0 g / L 10 A 0 gN 5 g Ps g / L /L A g A g + NP s +

0

(c)

14

A549

HepG2

Ag )

+ + 109

2

10

L g/ m

Ps gN A

8

Ag /

+

107

6

(

(

107

Ag /

+ 109

Ag )

12

4 2 0

+ + Ps + Ps + N N g g Ag g A A A L L /L g/L g/  g/ g   m 0 5 0 2 10 10

+ Ps + N g g A A L L g/  g/ m 0 2 10

1

+ Ps + N g g A A L /L / g g  5 00

583 584

Figure 4. Cellular uptake profiles of

585

107AgNPs

and

107Ag+,

(b) ratios of

107AgNPs, 107Ag+

107AgNPs

and

and

107Ag+

109Ag+.

to total


(a) Cellular uptake contents of

107Ag,

and (c) relative ratios of

Page 27 of 28


586

Δ(107Ag+/109Ag+). HepG2 and A549 cells were cultured with different concentrations of 107AgNPs and

587

109Ag+

for 24 h.



588

For TOC only

589 590 591


Page 28 of 28

Intracellular Dissolution of Silver Nanoparticles ... - ACS Publications

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