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Article
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
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Abstract
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The so-called “Trojan-horse” mechanism, in which nanoparticles are internalized within cells,
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then release high levels of toxic ions, has been proposed as a behavior in the cellular uptake of
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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
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cysteine or vital thiol proteins/peptides to Ag ions. Evidence we found indicates that the
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“Trojan-horse” mechanism really exists.
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Introduction
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Due to broad applications in textiles, wound dressings, food packaging and other medical
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devices,1-4 silver nanoparticles (AgNPs) have appeared rapidly in our lives, but the study of
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the relationship of silver nanotechnology with health issues is relatively new. Long-term
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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
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“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
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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
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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
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determine the amount of AgNPs and ions in cells, but also compare their ratio to
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extracellular media. Another way to prove it is to find enhanced intracellular ion release
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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.
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Many studies have tried to detect Ag ion release in environmental matrices or
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growth media for microorganisms.15,17-22 Fewer report ion release in biological media for
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mammalian cells and intracellular surroundings.14-15,18,22 Generally, previous studies used
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two strategies to detect Ag ions (I) and/or AgNPs:
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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
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(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
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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
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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
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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
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speciation in microcosm surface water, a method that can recognize not only the oxidative
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state of Ag, but also species formed in Ag (I).24,26 To further describe the Trojan-horse
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mechanism,
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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
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normal neuron cells.28,29 In our study, microglia and astrocytes were used to help determine:
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1) the ratio of AgNPs to total Ag (AgNPs+Ag (I)) between single cells and outside media
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after 24 and 48 h of exposure; 2) if H2O2, an intracellular ROS, participates in AgNP
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dissolution in cells; 3) Ag species analysis in cells; and 4) whether or not the “Trojan-horse” 5
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mechanism exists. Five measuring techniques (UV-vis spectrometer, ISE and XANES for
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Ag species analysis, flow cytometry and Laser Ablation (LA)-ICP-MS30 for uptake
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quantification) were used to decrease interference caused by pre-treatment procedures.
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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
78
(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
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level by an electrically gasified method under vacuum. The vapor was condensed in the
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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
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trap and dispersed in sterile water. A concentration of 10 µg/mL was determined by ICP-MS
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(Agilent 7500a, USA). The stock solution had good stability in storage for six months at 4 °C.
92 93
AgNPs characterization
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The morphology of the AgNP stock solution was analyzed using a JEM2100 transmission
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electron microscope (TEM) (JEOL, Japan). The hydrodynamic size and surface charge of the
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AgNPs in stock solution and culture medium were monitored using a Zetasizer Nano ZS
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apparatus (Malvern Instruments, UK). Details of sample preparation are described in the
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Supporting Information (SI).
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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
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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
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spectrophotometry (Varian 50 Bio, CA, USA). The AgNP stock solutions were diluted with
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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
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approximated by assuming Rayleigh scattering and setting the absorbance at 321 nm (Abs321)
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to
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AbsAgCl=(Abs321−0.086×Abs407)×(λ/321)−4. The calibration curve of AgNPs at each
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concentration was derived from the integrated area (320 to 610 nm) or the absorbance peak
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intensity at ~ 407 nm. Fig. S1 shows AgNP calibration curves using these two methods from
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0.2 to 5 µg/mL. The limit of detection (LOD) of the latter method was 0.18 µg/mL, which was
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calculated from 3 σ/s; where σ is the standard deviation of 7 replicated blank absorption, and s
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is the slope of the calibration curve.
8.6%
of
the
peak
absorbance
Abs407,
resulting
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Free silver ion activity measurement
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For analyzing free silver ion activity in: (a) AgNP stock solution; and in (b) DMEM medium
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(pH 7.2), a commercial silver ISE (perfectION™ comb Ag/S2 Combination Electrode,
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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
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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
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12.5 mM and KI 7.9 mM solutions. As an ionic strength adjuster, NaNO3 was added to all
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samples and standards.
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ISE.
Fig. S2 shows titration and calibration curves of silver ions using
127 128
Ag species in DMEM medium simulation
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Simulations were conducted by a GeoChem-EZ program. The program models Ag speciation
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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
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strength was 0.13 M; and 5) Solids can precipitate.
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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
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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
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ascorbic acid (100 µM) (Sigma, St. Louis, MO) to observe the protection of Ag dissolution in
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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
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in the SI.
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Intracellular ROS release
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Production of H2O2, a major intracellular ROS, was measured using 2’,7’ -dichlorofluorescin
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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
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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.
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Fluorescence was determined using a BD FACSCanto flow cytometer (BD Science, San Jose,
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CA), with 10,000 cells collected. The geometric mean fluorescence intensity was analyzed
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using Flowing software 2.
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Uptake potential from flow cytometry analysis
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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
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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
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same as intracellular ROS analysis.
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Quantification of uptake by LA-ICP-MS
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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,
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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
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LA-ICP-MS experimental condition and data treatment are described in SI.
193 194
Ag Speciation in cells
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To prepare the samples for Ag speciation, cells (1 × 104 cells/cm2) were seeded in a T175
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flask for 30 h. After treatment with 5 µg/mL of AgNPs or co-treatment samples, the
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suspensions were removed and the cells were rinsed three times with 1 × PBS. Next, 1 ×
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trypsin-EDTA (0.5 mL) was used to harvest cells. The cell suspensions were then centrifuged
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(200 g, 5 min) and the supernatants were removed. The samples were stored in liquid nitrogen
200
until analysis.
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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
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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
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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).
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Relative proportions and errors were determined by the software’s least-squares fitting
218
module. Fitting residual parameters are described in the SI.
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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.
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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
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was 10.2 nm, with uniform spheres (Fig. S3). Dynamic laser scattering (DLS) measurement
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showed that AgNPs had good stability within 48 h in DMEM/10%FBS medium (Table S1,
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Figs. S4 and S5).
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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
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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
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data of AgNPs over time, used to construct part of Fig. 1 are shown on the upper right. Based
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on opinions from Zook et al., the peak absorbance intensity can probably be calculated as an
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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
248
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
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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
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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
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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).
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Precipitate AgCl formed only as a Ag ion concentration was higher than 5 µg/mL. The results
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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
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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
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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
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viability also decreased in AgNP treatment groups for 24 h (82%), LPS co-treated with
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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
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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
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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
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the amount of Ag detected in ALT should be from inside cells and BV-2 may be
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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
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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
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shown in Table S2. The best LCF fitting results are shown in Figs. 4A and 4C. As AgNPs
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were treated to BV-2 cells, Ag (0) accounted for 30% and 38% of total Ag after 24 h and 48 h
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of exposure. As the LPS was co-treated with AgNPs, a higher percentage of Ag (0) (64% in
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24 h and 40% in 48 h) existed in the cells. With the co-treatment of the AA, AgNPs in total Ag
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accounts for 73% in 24 h, but decreased in 48 h (44 %) (Fig. 4A). For ALT cells, the total
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amounts of Ag(0) in cells after 24 and 48 h of exposure are similar, which accounts for ca.
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31% of total Ag. As the LPS was co-treated with AgNPs, the cells exhibited a higher
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percentage of Ag(0) (48% in 24 h and 42% in 48 h). With AA co-treatment, the AgNPs in total
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Ag accounted for 51% in 24 h and 47% for 48 h (Fig. 4C). Figs. 4B and 4D show the amount
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of AgNPs and Ag (I) (sum of all Ag (I) species) in single cells for each condition, which was
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calculated from combining median uptake quantification data (LA-ICP-MS) and the ratio of
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Ag species (XANES analysis) results.
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Studies to detect AgNP dissolution inside cells are quite rare. Wang et al., using
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cloud-point extraction plus ICP-MS, demonstrated that over 82.1% of Ag existed in the form
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of nanoparticles, with 17.9% as Ag ions inside MEL cells, which is higher than the proportion
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of Ag ions (11.6%) in diluted AgNP stock solution.14 Using the same method, Yu and
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colleagues documented that about 10.3% of Ag was present as ions in exposed HepG2 cells,
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with only 5.2% Ag ions in pristine AgNPs.22 Singh and Ramarao used autometallography
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(AMG) with quantitative image analysis and found only 5% of AgNPs had dissolved.11 The
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discrepancy in the ratio of Ag dissolution in these studies could be due to different
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physico-chemical properties of AgNPs used and different cell types. Unfortunately, these
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studies only compare the AgNPs and Ag ions inside cells with the original stock solution, not
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with cell medium. In our study, the ratio of AgNPs to total Ag inside BV-2 and ALT cells
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(30~38%) was lower than that in extracellular medium (45~52%) after 24 h and 48 h of
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exposure. However, AgNPs were detected in the medium in the absence of cells, which might
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be an overestimation because they would be lost via cellular uptake. Thus, we compared the
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dissolution of AgNPs in medium in the presence and absence of astrocytes in 24 h, and found
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only 3% difference between each other at 2 µg/mL of AgNPs exposure (Fig. S12). Moreover,
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only 0.19 µg of Ag was detected from all astrocytes in each well (at 5 µg/mL of AgNPs, 24 h
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of exposure), which accounted for 3.8% of applied AgNPs (Fig. S8). These results indicate
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that AgNP uptake does not significantly account for the total amount of AgNPs in medium.
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Released H2O2 may react with AgNPs in both cells
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To explain why intracellular AgNP ratios could be lower than extracellular, factors that can
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promote Ag dissolution in cells, e.g., reactions of H2O2 with AgNPs, or AgNPs located in
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lysosome (organelle which has a lower pH value), should be proved. Our UV-vis data
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confirmed H2O2-enhanced AgNP dissolution in cell media (Fig. 1). Our intracellular ROS
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results represent the balance between the generation of H2O2 from cells and its consumption
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by reacting to AgNPs (Fig. 2). In our study, each cell type showed clues to suggest that H2O2
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reacts with AgNPs in cells. For BV-2 cells, ROS was inhibited after exposure to AgNPs (Fig.
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2A), which differs from previous reports (e.g., AgNPs induced more ROS in mixed rat neural
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cells and mouse macrophages (RAW 264.7)).11,28 LPS induced ROS release in 48 h of
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exposure, while ROS was inhibited when co-treating with AgNPs. For ALT cells, AA alone
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can induce more ROS in 24 and 48 h, while ROS can be significantly inhibited from
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co-treating AA with AgNPs (Fig. 2B). In summary, the decrement of ROS in BV-2 and ALT
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suggests oxidation of AgNPs in cells.
362 363
Intracellular AgNP ratio can be explained by various factors
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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
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AgNPs and Ag ions (using AgNO3) to the two cell types for 4 h, and found a similar uptake
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level between two species in BV-2 while more efficient AgNPs internalization than Ag ions in
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ALT cells (Fig. S13). The free metal ions could not enter the cells freely due to the membrane
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barrier.49 Cronholm et al. also demonstrated a lower uptake rate of Ag ions than AgNPs in two
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lung cell lines (A549 and BEAS-2B).50 Therefore, the latter explanation can be excluded. On
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the other hand, as LPS was co-treated with AgNPs in BV-2 cells, the AgNP ratio (74%) was
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higher than that in cell medium at 24 h (Fig. 4A). LPS-activated BV-2 cells were able to
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perform macrophage-like activities including scavenging and phagocytosis.51 Thus, the above
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result might be attributable to enhanced AgNP internalization. AA induced the cellular uptake
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of AgNPs in BV-2 cells, and showed similar AgNPs to total Ag ratio to LPS-activated cells
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(Fig. 3C), thus it is hard to confirm its ability to protect the Ag from oxidation inside cells.
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However, even though AA induced a high ROS release, which would enhance the dissolution
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of AgNPs in ALT, the AA co-treatment group still showed a higher AgNP ratio than the
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non-cotreatment group (Fig. 4C), indicating that Ag oxidation was blocked. After 48 h of
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exposure, the uptake of Ag (AgNPs + Ag (I)) in LPS-activated BV-2 cells was lower than that
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in 24 h (Fig. 3C). In that group, the decreased level of AgNPs was greater than the increased
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level of Ag (I) after 48 h (Fig. 4B). This indicated that AgNP exocytosis plays a more crucial
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role than intracellular AgNP dissolution at 48 h. On the other hand, in AgNPs treated or
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LPS+AgNPs co-treated ALT cells, the uptake of Ag also decreased at 48 h (Fig. 3D).
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Interestingly, the decrease of both AgNPs and Ag (I) was observed (Fig. 4D). It seems that for
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ALT, not only AgNP exocytosis, but ion penetration to extracellular media determined the
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AgNP ratio in longer exposures.
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388 389
Ag speciation (I) in cells: Ag-thiol group complex accounts for the major species
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It is important to measure Ag species (I) distribution in cells because the binding of Ag ions
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may be related to the final toxicity. From Figs. 4A, 4C and Table S2, Ag-thiol binding species,
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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
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abundant (typical total intracellular thiol concentration is 12 mM) to receive most of the silver
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ions.25 AgCl was the second highest ratio of Ag species in Ag (I), owing to the high chloride
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concentration (~30 mM) in the cytoplasm.25 Interestingly, the lower content of Ag2S can be
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found in some treatment groups to ALT while not nearly so much to BV-2. This might be
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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
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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.
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In this study, we compared the various ratios of AgNPs to total Ag in single cells (microglia
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and astrocytes) and outside media, and found that the ratio of AgNPs to total Ag in cells is
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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,
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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
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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
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We thank Dr. Ling-Yun Jang, spokesperson of Beamline 16A at the National Synchrotron
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Radiation Research Center (NSRRC), Taiwan, for conducting the XANES experiments. We
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also thank Gold Nanotech, Inc. Taiwan, for providing AgNPs materials.
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Associated content
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Supporting Information
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Additional experimental data and tables are discussed and available free of charge via the
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Internet at http://pubs.acs.org
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References
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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)
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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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Environmental Science & Technology
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)
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Environmental Science & Technology
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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