Silver Nanoparticle-Induced Autophagic-Lysosomal Disruption and NLRP3-Inflammasome Activation in HepG2 Cells Is Size-Dependent.

ToxSci Advance Access published January 21, 2016

Silver Nanoparticle-Induced Autophagic-Lysosomal Disruption and NLRP3-Inflammasome Activation in HepG2 Cells is Size-Dependent

Division of Biology, Chemistry, and Materials Science, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, MD

(*Corresponding author)

Peter L Goering Ph.D.

[email protected]

Address: USFDA-CDRH 10903 New Hampshire Avenue Bldg 64, Rm 4064 Silver Spring MD 20904

Running title Silver nanoparticles and autophagy disruption

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Downloaded from http://toxsci.oxfordjournals.org/ at New Mexico State University Library on January 25, 2016

Anurag Mishra, Jiwen Zheng, Xing Tang, Peter L. Goering*

Abstract Silver nanoparticles (AgNPs) are incorporated into medical and consumer products to exploit their excellent antimicrobial properties; however, potential mechanisms of toxicity of AgNPs in mammalian cells are not fully understood. The objective of this study was to determine the mechanism of size- and concentration-dependent cytotoxicity of AgNPs in human liver derived hepatoma (HepG2) cells.

autophagy induction, lysosomal activity, inflammasome-dependent caspase-1 activation and apoptosis were examined. Using enhanced dark-field light microscopy hyperspectral imaging, electron microscopy, and EDS, AgNPs were shown to rapidly accumulate in cytoplasmic vesicles for up to 24 hr exposure and 10nm AgNPs exhibited the highest uptake and accumulation. Autophagy and enhanced lysosomal activity were induced at non-cytotoxic concentrations (1 µg/ml; primary particle size:10nm>50nm>100nm), whereas increased caspase-3 activity (associated with apoptosis) was observed at cytotoxic concentrations (10, 25 and 50 µg/ml). Sub-cytotoxic concentrations of AgNPs enhanced expression of LC3B, a proautophagic protein, CHOP, an apoptosis inducing ER-stress protein, and activation of NLRP3inflammasome (caspase-1, IL-1β). Disrupting the autophagy-lysosomal pathway through chloroquine or ATG5-siRNA exacerbated AgNPs-induced caspase-1 activation and LDH release, suggesting that NLRP3-inflammasome plays an important role in AgNPs-induced cytotoxicity. Overall, 10nm-AgNPs showed the highest cellular responses compared to 50nm AgNPs and 100nm-AgNPs based on equal mass dosimetry. The results indicate the potential of vesicle-engulfed 10nm-AgNPs to induce cytotoxicity by a mechanism involving perturbations in the autophagy-lysosomal system and inflammasome activation. Key words silver nanoparticle, nanomaterials, autophagy, inflammasome, apoptosis, caspase-1

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Mechanisms of toxicity were explored at sub-cytotoxic concentrations (≤ 10 µg/ml AgNPs) and

Introduction Silver nanoparticles (AgNPs) are engineered nanomaterials (ENMs) with excellent antimicrobial activity (Kim et al., 2007). The broad-spectrum antimicrobial action of AgNPs is lethal against several pathogens such as E. coli (Radzig et al., 2013), hepatitis B and HIV virus (Lu et al., 2008), with no known development of resistance against its mode of action. The protracted antimicrobial action of

oxidative damage to the microorganism (Chernousova and Epple, 2013). AgNPs-containing medical products, such as wound dressings (Maneerung et al., 2008), catheters (Lai and Fontecchio, 2002), implants (Secinti et al., 2011), and medical textiles (Raza et al., 2015), promise reductions in both microbial contamination and health-care related costs; however, questions remain regarding human health risks (Ahamed et al., 2010). The unique properties of nanoscale silver (Ag) such as nanometer size, large surface area per mass volume, and ionic dissolution properties raise concerns about human exposure since long- and short-term health effects are not completely known (Nel et al., 2006b). In addition to physicochemical factors, the toxicity of ENMs are influenced by variety of biological factors, such as route of entry, tissue distribution, and target organs (Oberdörster et al., 2005). Among off-target organs, liver is one of the primary sites of systemic nanoparticle accumulation and biotransformation. Recent studies examining tissue distribution of injected nanoparticles, e.g., gold nanparticles and carbon nanotubes, have confirmed their rapid accumulation in liver followed by significant changes in hepatic gene expression, metabolism, enzyme activity, and inflammation-induced tissue damage (Balasubramanian et al., 2010; Wei et al., 2012). Previous reports from our group showed that liver is a primary organ for AgNPs accumulation after intravenous administration of low-level citratecoated 10nm- and 50nm-AgNPs to pregnant mice (Austin et al., 2012). Remarkably, some studies have shown that AgNPs administered by inhalation (Sung et al., 2009), ingestion (Yun et al., 2015) or dermal

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AgNPs are due to the slow dissolution of Ag+ ions from the particle surface, causing non-specific

(Yeo et al., 2015) routes can also translocate from the primary site of exposure to the liver; thus, increasing the likelihood of hepatotoxicity. Nanoparticle-induced autophagy has attracted much attention recently and is considered as a potential molecular target for nanoparticle-based chemotherapy (Adiseshaiah et al., 2013; Stern et al., 2008). Autophagy in healthy eukaryotic cells contributes to both survival and disease through a highly

vesicles called autophagosomes and its delivery to lysosomes for degradation and recycling (Jiang and Mizushima, 2014). Under most circumstances, autophagy activation ensures cell survival during starvation or stressful conditions; however, nanoparticle activated autophagy has been associated with inflammation, oxidative stress and activation of apoptosis (Johnson-Lyles et al., 2010; Stern et al., 2012). Activation of both autophagy and apoptosis-induced cell death by nanoparticles suggests that nanoparticle-induced autophagy causes irreversible cellular damage, apoptotic and necrotic cell death (Roy et al., 2014). Lysosomal membrane permeabilization (LMP) has been implicated in the pathogenesis of variety of diseases (Guicciardi et al., 2004) and can be a therapeutic tool to induce apoptotic-mediated cell death (Domenech et al., 2013). Recent studies have shown that LMP and the lysosomal protease cathepsin-B are involved in inflammasome activation (Weber and Schilling, 2014). The NLRP3-inflammasome is a multi-protein complex which recruits and activates caspase-1. Inflammasome-dependent caspase-1 is responsible for activation of the highly inflammatory cytokines IL1-β and IL-18 and induction of pyroptosis, a non-apoptotic programmed cell death (Latz et al., 2013). Caspase-1 mediated cell death is distinct from other programmed cell death such as apoptosis and necrosis (Denes et al., 2012). Several particulate materials, such as silica, asbestos, and carbon nanotubes, are known activators of the NLRP3 inflammasome (Baron et al., 2015; Duewell et al., 2010); however, participation of the autophagylysosomal pathway in inflammasome activation in nanoparticle-induced cell death is poorly understood. 4


conserved process which involves sequestration of unwanted cytoplasmic content into double-membrane

Therefore, elucidation of the mechanisms involved in nanoparticle-induced autophagy-LMP and inflammasome activation is needed to better understand AgNP toxicity. Investigations of molecular mechanisms of toxicity of AgNPs in liver cells will provide key information for understanding the toxicological impacts of nanoparticle tissue accumulation in vivo. In this study, the molecular mechanisms of the in vitro toxicity of AgNPs (primary particle sizes:10nm,

evaluated. We hypothesized that AgNPs induce apoptosis via autophagy disruption, lysosomal damage and pro-inflammatory caspase-1 activation. The results showed size- and concentration-dependent toxicity of AgNPs in HepG2 cells. Further, a novel molecular mechanism of AgNPs-induced cellular stress was elucidated, whereby sub-lethal concentrations of AgNPs activate pro-inflammatory caspase-1dependent signaling by disrupting the autophagy-lysosomal pathway. Taken together, these results suggest that AgNPs can cause cell injury by destabilizing the autophagy-lysosomal system leading to NLRP3 inflammasome dependent casaspe-1 activation, endoplasmic reticulum stress, LDH release and apoptosis. The results will help in bioengineering safer ENMs for therapeutic, medical, and consumer applications and also aid in developing novel molecular biomarkers for predicting potential adverse responses associated with ENMs.

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50nm and 100nm diameter, polyvinylpyrrolidone(PVP)-coated) in human liver derived HepG2 cells were

Materials and Methods Characterization of silver nanoparticles: Biopure polyvinylpyrrolidone (PVP)-coated AgNPs were purchased from nanoComposix, Inc (San Diego, CA). To confirm the characterization data from the manufacturer, the AgNPs were initially characterized for primary particle size, morphology and agglomeration by a transmission electron microscopy (TEM, JEOL JEM 2010F, Jeol USA Inc., Peabody,

shape, agglomeration and endotoxin contamination. Briefly, diluted suspensions of AgNPs in water and EMEM media were lightly sonicated at a power of 130 W, frequency of 20 kHz, and amplitude settings of 50% for 5-10 sec and were applied on carbon-coated copper grids and air dried at room temperature for 3 hr. Particles were directly examined by TEM operated at an accelerating voltage of 200 keV and diameters of 300 particles were measured for particle size distributions. Hydrodynamic size and zetapotential of AgNPs was measured in milli-Q water using a Malvern Zetasizer Nano ZS with a 633 nm laser source (Malvern Instruments Ltd., Southborough, MA, USA). Briefly, AgNPs were diluted in either mill-Q water. For hydrodynamic diameter, 5 separate measurements were performed with each measurement of 12 sub-runs of 10 sec duration. Intensity of scattered light was detected at 1730. All measurements were conducted at 250 C. For AgNPs surface zeta-potential measurement, particles were diluted in EMEM media (0.1 µg/µl, pH 7.4) and analyzed soon after dilution. Endotoxin levels were measured using kinetic turbidity based limulus amebocyte lysate (LAL) assay (Life technologies, Grand Island, NY). Ultraviolet-visible (UV-vis) absorption spectra of the AgNPs dispersed in EMEM (10 µg/ml) was obtained using a Spectramax-UV/Vis-spectrophotometer (Molecular Devices, Sunnyvale, CA) Cell culture. HepG2 cells were cultured in EMEM (ATCC, Manassas, VA) supplemented with fetal bovine serum (10%), streptomycin (100 µg/ml) and penicillin (100 units/ml) (Sigma-Aldrich Corp, St. Louis, MO). For all experiments cells were seeded in cell culture treated 150 cm2 flasks, 96, 12 and 6well plates (Corning, NY) and maintained at 37 0 C in a CO2 (5%) incubator with 90% humidity. Cells 6


MA, USA). Each batch of particles received from the manufacturer was immediately checked for size,

were sub-cultured to 80% confluence and medium was changed every 2-3 days. For experiments, cells from only passage number 5-15 were used. For each experiment, cells were seeded in culture plates and allowed to adhere for 24 hr and medium was replaced with fresh medium containing AgNPs. For treatment, AgNPs were lightly sonicated using a probe (60% amplitude) and diluted from the stock solution just before the treatment using endotoxin-free cell culture supplies. Treatments with AgNPs during all the experiments were performed when the cells were in the growth phase.

for 12 and 24 hr. Cells were treated with final concentration of 0.01-50 µg/ml AgNPs. Cell viability was determined after 12 hr and 24 hr using the water-soluble WST-1 [(2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium)] dye assay (Roche Diagnostics, Mannheim, Germany), which is based on the conversion of tetrazolium salt into water soluble yellow colored formazon product by mitochondrial dehydrogenases in metabolically active cells. Briefly, at the conclusion of the 12 hr and 24 hr exposures, 50µl of WST-1 dye was added to each well containing 100 µl media and the plate was incubated at 370 C for 4 hr for maximum sensitivity. The absorbance was measured at 450 nm (A450nm) by a multi-well spectrophotometer (Molecular Devices, Sunnyvale, CA). Absorbance from particle-only controls was subtracted from each treatment to account for natural AgNPs absorbance interference. Cell viability was determined relative to that of control cells and data was plotted as 100% of control. IC50 values (half maximal inhibitory concentration) were then calculated from the dose-response curve using Graph Pad Prism 5.1 (GraphPad Software Inc., La Jolla, CA). Each sample was assayed in triplicate and results are presented as the mean ± standard deviation (SD) of three independent experiments. LDH assay: Cellular membrane damage following AgNPs exposure was determined by measuring the amount of lactate dehydrogenase (LDH) released in the cell culture supernatant. LDH release was measured following the formation of formazon according to manufacture kit (Promega, 7


Cell viability assay. HepG2 cells (20 x 103 cells/well) were seeded in 96-well plate and cultured

Madison, WI). Briefly, HepG2 cells (2 X 104) were seeded in 96 well plates for 24 hr and exposed to AgNPs. After 12 hr supernatant was collected and LDH assays were performed according to the manufacturer's instructions. Positive control (Cells treated with 0.1% triton X), particle control (AgNP in EMEM media) and EMEM media only control were used to avoid any particle interference with the LDH dye. The absorbance was measured at 450nm by a multi-well spectrophotometer (Molecular Devices, Sunnyvale, CA). Each sample was assayed in triplicate and results are presented as the mean ± standard

Cellular staining for AgNPs uptake. HepG2 cells were seeded at low densities on collagen coated round glass coverslips (Corning BioCoat, Corning, MA) and allowed to attach for 24 hr. Cells were washed and incubated with AgNPs (1µg/ml) for 6 hr and 12 hr. After incubation time, coverslips were rinsed extensively in 370C warmed phosphate buffer saline (PBS) to remove unbound nanoparticles. Coverslips were incubated with 3.7% formaldehyde solution in PBS for 10 minutes at room temperature and subsequently, washed with PBS (5x, 5 min), and again incubated for 30 min with PBS containing 1% bovine serum albumin (BSA) to reduce nonspecific binding. Cell were incubated with actin staining solution containing AF-488-Phalloidin (Life technologies, Grand Island, NY,) 1% BSA and PBS for 30 min at room temperature. Subsequently, coverslips were washed with PBS (3x, 5min) and mounted with ProLong® Gold Antifade Mount with DAPI (Molecular Probes Inc., Eugene, OR)) on ultrasonically cleaned glass slides (Nexterion Glass B, Applied Microarray, Tempe, AZ). Slides were dried at room temperature in dark overnight before hyperspectral imaging. CytoViva hyperspectral imaging. CytoViva technology was specifically designed for optical observation and spectral confirmation of NPs as they interact with cells and tissues. Using integrated CytoViva Hyperspectral Imaging (CytoViva, Auburn AL), reflectance spectra from specific materials can be acquired and analyzed. SAM (Spectral Angle Mapping) is an automated procedure used to determine whether AgNPs are present in the image and identifies which pixels contain the chemical identity of the 8


deviation (SD) of three independent experiments.

material of interest. SAM accomplishes this task by comparing unknown spectra in hyperspectral imaging with known spectra of the material of interest (AgNPs in this case). Briefly, AgNPs spectral library was generated using enhanced-dark field imaging. Cells with AgNPs were scanned for spectra generation. Each pixel of a scanned image was compared with the AgNPs spectra library and color-coded for pixel location. Finally, images were compared with dual mode fluorescence images stained for F-actin (phalloidin) and nucleus (DAPI). The images were compared to determine the location of intracellular

TEM analysis for AgNP uptake in HepG2 cells. Briefly, HepG2 cells were treated with 1 µg/mL of 10nm-50nm- and 100-AgNP for 12 h and 24 h. After incubation, cells were washed with PBS (3X, 5min) and then were harvested with trypsin. The cells were pelleted by centrifugation at 1000x rpm and immediately fixed with 2.5% glutaraldehyde and formaldehyde in cacodylate buffer for 2 h. Cells were then post-fixed in 1% OsO4 for 1 h, After being progressively dehydrated by increasing concentrations of ethanol, the cell pellets were infiltrated with mixture of resin and acetonitrile (1:1 ratio) for 2hr. The cells were finally incubated with resin overnight under 4º C and then kept in oven (55º C) for 48 h to allow the resin to be hardened. Ultra-thin sections (70–90nm) were prepared with an ultramicrotome (Leica Microanalysis) and examined with the TEM and Energy Dispersive X-Ray Spectroscopy (EDS). Lysosomal activity assay. Lysotracker assay was based on the protocol obtained from the National Characterization Laboratory-National Cancer Institute website and previously published studies [34, 35]. Lysotracker is a weakly basic amine dye that selectively accumulates and labels acidic compartments in cells such as lysosomes. Briefly, HepG2 cells (5X104) cells were seeded in 96-well plates and treated with 10nm-, 50nm- and 100 nm-AgNPs (1 and 10 µg/ml). After 12 hr and 24 hr exposures, cells were washed twice with phenol-free -EMEM media containing 1% FBS and incubated with 100 µl of 50 nM Lysotracker Red DND-99 (Life technologies Inc., Carlsbad, CA) and 10 µM Celltracker Green CMFDA (Life technologies Inc., Carlsbad, CA) co-stain in phenol-free EMEM media. 9


AgNPs.

After 1 hr incubation, cells were washed twice with phenol-free EMEM media and fluorescence for Lysotracker Red and Celltracker Green were read at 544 nm/590 nm and 492 nm/517 nm, respectively. The Lyso-tracker response was reported as percent control response normalized to percent control cell tracker response. Fluorescence Microscopy. Cells were grown in 4-well chamber slides at low densities (5 X 103

AgNPs (1 µg/ml). After 12 hr, medium was removed and cells were washed (2xPBS, 5 min) with phenolfree EMEM medium and incubated with phenol-free RPMI 1640 medium containing 50nM Lysotracker Red DND-99 dye and DAPI (3µM). After 1 hr, loading medium was replaced with fresh medium and cell were examined under fluorescence microscope (Nikon TI 2000, Nikon, Okinawa, Japan) using appropriate filter for Lysotracker red DND-99. Nuclei were counterstained with DAPI. Confocal Microscopy. LC3B punctate per cells were analyzed by confocal microscopy (Olympus FluoView FV1000, Waltham, MA). Briefly, cells were grown on cover slips and treated with 10nm, 50 nm and 100 nm AgNPs (1 µg/ml). After 12 hr, cells were washed with PBS (3X) for 5 min and fixed with 4% formaldehyde solution. Subsequently, cells were permeabilized with 0.5% Triton X-100 for 15 min, washed with PBS and blocked with 1% goat serum for 1 hr at room temperature. Cells were incubated with LC3B antibody (Life Technologies, Grand Island, NY) in antibody dilution buffer overnight at 40 C. Cells were washed in PBS (3X) and incubated with Alexa Fluor® 488 conjugated antirabbit IgG secondary antibody, (Santa Cruz Biotechnology, Dallas, TX) for 4 hr followed by washing and cells were mounted on coverslip using ProLong® Gold Antifade Mountant with DAPI (Life Technologies Corporation, Grand Island, NY)and analyzed with confocal microscope. CytoID green staining and flow cytometry. Autolysosomes were detected by flow cytometry using Cyto-ID autophagy detection kit (Enzo life Sciences, NY). HepG2 cells were exposed to AgNPs for 18 hr. Cells were trypsinized, washed in PBS and re-suspended in 100ul assay buffer containing 5 µl dye. 10


cells/ml) and after cells were attached (24 hr), medium was replaced with fresh medium containing

After 10 min, cells were washed with assay buffer and analyzed using flow cytometry (BD FACSCanto™ II, BD Biosciences, San Jose, CA). Representative images from N = 5 replicate experiments with at least 100 cells per condition in each experiment. Western blotting. Cells were harvested and lysed with M-PER® Mammalian protein extraction reagent containing Halt Protease Inhibitor Cocktail (Thermo Scientific, Waltham, MA). The lysate was

Hercules, CA). 40 µg of protein was resolved under denaturing conditions on a 12 % polyacrylamide gel at 125V and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) at 45V. The membrane was blocked by incubating with 5% milk in TBST (25 mM Tris–HCl, pH 7.4, 125 mM NaCl, 0.05% Tween 20) for 1 hr at room temperature and then incubated with primary antibody (1/1000) overnight at 40 C. Membranes were washed 3X with TBST and incubated with HRP-conjugated isotypespecific secondary antibody (Santa Cruz Biotechnology, Dallas, TX) for 1 hr at room temperature. The immune complexes were detected by a ChemiDoc MP System and quantified using analyst/PC densitometry software (Bio-Rad Laboratories, Hercules, CA). Caspase-1 activity assay. Active caspase-1 was measured with Caspase-1 fluorometric assay kit (BioVision, Milpitas, CA). Briefly, 1x106 HepG2 cells were stimulated 1 hr before the exposure with lipopolysaccharide (LPS, 2 ng/ml) and incubated with AgNPs for indicated period of time and lysed at the end of incubation period. Cell lysates were mixed with substrate for caspase-1 in reaction buffer containing dithiothreitol (10 µm). The plate was incubated for 2 hr at 370 C. After incubation, the absorbance was read with Spectramax-fluorescence-multiwell plate reader (Molecular Devices, Sunnyvale, CA) at 400 nm excitation filter and 505 nm emission filter. ELISA for IL-1β. IL-1β was measured by ELISA per manufacturer’s instruction (R&D Systems, Minneapolis, MN). Briefly, cells were stimulated with LPS (10 ng/ml) for 1hr and treated with different concentration of AgNPs for 24 hr. ATP (300 mM) was used as a positive control. After the 11


collected and total protein content was determined by the Bradford method (Bio-Rad Laboratories,

exposure, supernatants were collected, centrifuged to remove any dead cells, and then assayed. IL-1β assay concentrations in treated cells were compared with results from control HepG2 cells. Apoptosis Assay. Caspase-3 activation was determined by the cleavage of enzyme substrate, Ac DEVE-pNA, by active Caspase-3. Cell lysate aliquots (50 µl) were mixed with 5µl substrate in 50 µl assay buffer. After 2 hr incubation, the fluorescence was read using Spectramax-fluorescence-multiwell

Apoptosis activation by AgNPs was also confirmed by Annexin-V dye staining. Briefly, HepG2 cells were exposed to AgNPs and after 24 hr, cells were harvested and washed with PBS (2X). Cells were incubated with assay buffer containing 5 µl of FITC-Annexin-V dye followed by washing with PBS (2X). Cells were analyzed by flow cytometry (BD FACSCanto™ II, BD Biosciences, San Jose, CA) and data plotted as mean fluorescence intensity (MFI).

ATG5-siRNA Transfection. ATG5 siRNA specifically inhibits Atg5 (autophagy-related 5) expression, an autophagosome forming protein, which prevents autophagosome formation and autophagy activation. HepG2 cells (50X103) were grown in 6-well plate and after 24 h, transfected with 100nM of ATG-5 SiRNA (Cell Signaling Technology, Danvers, MA) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Cells were harvested after 24-48 h post transfection and used for assays. Autophagy was also chemically disrupted by 3 methyladenine (3-MA) and chloroquine (CQ), a known inhibitor of autophagosome-lysosome fusion and lysosomal protein degradation. HepG2 cells were pretreated with either chloroquine (30 µM) for 1 hr or 3-MA (5µM) for 4 hr to disrupted autophagy and treated with NPs.

Statistical analysis. One- and two-way ANOVAs followed by post-hoc test were performed to determine statistical significance between treatments using Graph Pad Prism 5.1 (GraphPad Software Inc., La Jolla, CA) at a confidence level of p < 0.05. Either a Dunnett’s or Tukey-Kramer HSD post-hoc 12


plate reader (Molecular Devices, Sunnyvale, CA) at 400-nm excitation filter and 505-nm emission filter.

test was conducted when the ANOVA result indicated a significant difference among means. For IC50 calculations, dose-response curve was transformed and the best fit value for IC50 was calculated using Graph Pad Prism 5.1 (GraphPad Software Inc., La Jolla, CA).


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Results Silver nanoparticle characterization and cytotoxicity. The physicochemical characteristics of 10nm-, 50nm, and 100nm-AgNPs were fully characterized for size, morphology and endotoxin contamination and are summarized in Supplemental Fig. S1 and Fig. S2. AgNPs appeared to have spherical morphology with no significant differences in diameter and shape within each group. AgNPs

102±7nm for 10nm-, 50nm- and 100nm-AgNPs, respectively. No large agglomerated particles were observed and all particles appeared as mono-dispersed in buffer. Cell viability was measured by analyzing the mitochondrial activity (WST-1 assay) in AgNPs-treated HepG2 cells and compared to non-treated control cells. As shown in Fig. 1, cytotoxicity was observed to be dependent on AgNPs size, concentration, and exposure time. 10nm-AgNPs reduced cell viability to less than 70% as early as 6 hr post exposure (Fig. 1a) and 50% at 12 hr post-exposure at the highest exposure concentrations of 10 and 50 µg/ml (Fig. 1b). At 24 hr exposure (Fig. 1c), viability was reduced below 50% for all AgNPs sizes at 10 and 50 µg/ml concentrations. The concentration-response curve was used to calculate the inhibitory concentration (IC) at which 50% reduction in cell viability was observed. The IC50 values of 10nmAgNPs, 50nm-AgNPs and 100nm-AgNPs at 24 hr post exposure were 5.1, 7.6 and 6.4 µg/ml, respectively. No toxicity was observed at lower exposure concentrations of 0.01 to 5 µg/ml by AgNPs at any exposure time. No significant reduction in cell viability was observed in 50nm-AgNPs and 100nmAgNPs treatment group at 6 hr and 12 hr post-exposure. Based on the cell viability data and IC50 values, non-toxic as well as sub-toxic exposure doses of 1-5 µg/ml and 12 hr to 18 hr exposure-time was selected for further studies to evaluate molecular responses. Cellular uptake of AgNPs using dark-field microscope and hyperspectral imaging. Cellular uptake of 10nm-, 50nm- and 100nm-AgNPs in HepG2 cells after 6 hr and 12 hr exposure is shown in Fig. 2. Spectral libraries for 10nm-, 50nm-, and 100nm-AgNPs were created using hyperspectral imaging 14


exhibited narrow size distribution. The average diameter from TEM images were 14±6nm, 52±2nm and

where each spectrum in the library represents a single pixel (Fig. 2a). Cells in the same field of view are also stained for f-actin using the dual fluorescence-dark field mode and exhibited healthy morphology and adherence. Cellular uptake data confirmed direct cell-nanoparticle interaction and internalization (Fig 2b). Uptake of AgNPs was observed for 10nm-AgNPs as early as 6 hr (left columns) after exposure and peaked at 12 hr (right columns). These results correlate with the cell viability data, suggesting that 10nmAgNPs are rapidly internalized followed by 50nm and 100nm-AgNPs. TEM images confirm the

exposure. AgNPs internalization in cytosolic vesicle and disintegration. To further examine the fate of AgNPs after uptake, we imaged intracellular AgNPs using TEM (Fig. 3). As shown in Fig. 3a, majority of AgNPs were found in cytosol surrounded by either single or double membrane vesicles. The particles can be seen in cytosolic vesicles at 24 hr post exposure (right columns). Large numbers of cytosolic vacuoles containing 10nm-AgNPs can be seen at 24 hr exposure suggesting that a vacuole based mechanism is involved in the intracellular trafficking of AgNPs. Interestingly, as shown in Fig 4b, 100nm-AgNPs were found disintegrating into smaller size particles inside some vesicles. EDS analysis confirmed presence of AgNP in cytoplasmic vesicles (figure S3). AgNPs induce autophagic flux. We next investigated the concentration-dependent increase in “autophagic flux” by AgNPs in HepG2 cells. The microtubule-associated protein 1A/1B-light chain 3 (LC3) is a 17kDa ubiquitin-like protein that is recruited during the formation of autophagosomes. The turnover of LC3-II (phosphatidylethanolamine conjugated form, 14kD) from LC3-I (cytosolic form, 16kD) is considered one of the common methods to detect autophagy flux (Barth et al., 2010). The activation of autophagic flux was observed in HepG2 cells at the sub-cytotoxic exposure concentrations (1-10 µg/ml) of AgNPs. Confocal imaging showed that AgNPs (1 µg/ml) induce significantly higher levels of LC3B punctate formation in HepG2 cells (Fig 4a). To further confirm the autophagic response, 15


hyperspectral imaging results showing particle internalized in different cytoplasmic vesicles after 12 post-

cells were stained with Cyto-ID green dye that specifically binds to pre-autophagic vacuoles like autolysosomes and early autophagosomes. Mean fluorescence intensity (MFI) was measured by flow cytometry and AgNPs (1 µg/ml) treated cells showed significantly higher MFI compared to control cells, confirming the accumulation of autophagosomes (Fig 4b). Accumulation of LC3-II in AgNP treated HepG2 cells (1, 10 and 25 µg/ml) were also confirmed by western blot (Fig. 4c). 10nm-AgNPs induced higher levels of LC3-II expression, starting at 1µg/ml, followed by 50 and 100nm AgNPs, suggesting the

hr) effect of AgNP in LC3-II expression. To confirm autophagic flux, we pretreated HepG2 cells with 3methyladenine (3-MA), a phosphatidylinositol 3-kinases (PI3K) inhibitor, which blocks autophagosome formation (Seglen and Gordon, 1982). As shown in Fig 4e, AgNP-induced LC3-II expression was reduced by 3-MA. Furthermore, TEM (Fig. 4f) images demonstrated the accumulation of double membrane structures (white arrow) loaded with AgNPs (red arrow) that further confirmed the activation of autophagy and presence of autophagic vesicles. AgNPs increase lysosomal activity in HepG2 cells. Lysosomal activity was examined with fluorescence microscopy. As shown in Fig. 5a and 5b, increased lysosomal activity was observed in cells treated with AgNPs compared to non-treated HepG2 cells. To determine the lysosomal injury, AgNPs treated HepG2 cells were evaluated for LysoTracker Red DND-99 dye uptake, the staining of which is proportional to the level of lysosomal dysfunction (Rodriguez-Enriquez et al., 2005). As shown in Fig. 5b, 10nm-AgNPs and 50nm-AgNPs significantly increased dye uptake at concentrations of 5, 10 and 50 µg/ml after 24 hr exposure. Lysosomal activity was highest in cells treated with10nm- and 50nm-AgNPs, whereas 100nm-AgNPs only showed a statistically significant increase at 10µg/ml. AgNPs induce caspase-3 dependent apoptosis. Caspase-3 activates the execution-phase of cell apoptosis and used as a marker for apoptotic cell death. We examined caspase-3 activation and presence of end-stage apoptotic cells by Annexin-V dye staining using flow cytometry. Levels of active caspase-3 16


size-dependent effect of AgNPs in LC3-II expression. Figure 4d shows time-dependent (0, 6, 12 and 24

in HepG2 cells treated with 5, 10, 25 and 50 µg/ml AgNPs were significantly higher than control cells after 24 hr (Fig. 6a). No apoptotic cells death was observed at non-cytotoxic concentrations 0.1-1 µg/ml and at 12 hr post-exposure. Among all three sizes of AgNPs, 10nm-AgNPs induced caspase-3 activation at lowest concentrations (5 and 10µg/ml), whereas 50nm- and 100nm-AgNPs induce caspase-3 at higher concentrations (10, 25 and 50 µg/ml). Fig. 6b shows Annexin-V staining for end-stage apoptosis in cells exposed for 24 hr to concentrations of 5-50 µg/ml AgNPs. To gain further understanding about

stress protein, in AgNPs treated cells. CHOP is one of the components of the autophagy-ER stress mediated apoptosis pathway and strongly induced during apoptosis. As shown in Fig. 6c, CHOP was overexpressed in AgNPs treated cells, which suggests induction of autophagy-mediated cellular stress in AgNPs-treated cells activates programmed cell death. Overall, these results showed that AgNPs induce ER stress and apoptotic cell death at cytotoxic concentrations. AgNPs activate NLRP3 inflammasome dependent Caspase-1 and IL-1β release. We examined the potential of sub-toxic (1 and 10µg/ml) and toxic concentrations (25 µg/ml) of AgNPs to elicit NLRP3 inflammasome- dependent caspase-1activation in treated HepG2 cells. As shown in Fig. 7a, 10nm-AgNPs treated cells have significantly high levels of activated caspase-1 compared to non-treated control cells (CL), suggesting that 10nm-AgNPs induce caspase-1 activation. Inflammasome-dependent caspase-1 is responsible for maturation of highly inflammatory IL-1β and IL-18. AgNPs induce significantly high levels of IL-1β production (Fig. 7b) in AgNPs (10µg/ml) compared to non-treated control cells. These results suggest 10nm-AgNPs is most active in inducing caspase-1and IL-1β, followed by 50nm and 100nm AgNPs. Disruption in autophagy-lysosomal pathway exacerbates AgNPs-induced caspase-1 activation and increased cell death. Autophagy is a multistep process that involves sequestration, transfer and fusion of intracellular components to lysosomes for degradation and digestion. To further 17


autophagy-apoptosis interactions, we analyzed the expression of CHOP protein, a pro-autophagy ER

understand the molecular signaling of AgNPs-induced cytotoxicity, autophagy-lysosomal pathway was blocked in HepG2 cells using ATG5-siRNA or the pharmacological blocker chloroquine diphosphate (CQ). ATG-5 protein is critical for autophagosome formation and CQ prevents autophagosome fusion with lysosomes and autophagosomes accumulation in cytoplasm. As shown in Fig. 8a, induction of significantly higher caspase-1 activation by 10nm-AgNPs (10µg/ml) was observed in cells pretreated with CQ. Figure 8b confirms reduced ATG-5 expression and CQ-induced LC3-II expression in ATG5-siRNA

activation (Fig. 8c) and 2-3 fold increase in LDH release (Fig. 8d). This data suggest protective role of autophagy at sub-cytotoxic concentrations. Also, inhibiting late-stage autophagosome-lysosome fusion by CQ and blocking autophagosome formation by siATG5-RNA exacerbate AgNPs induced caspase-1 activation.

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treated HepG2 cells. In ATG-5 knockdown HepG2 cells, AgNP induce 3-4 fold increase in casaspe-1

Discussion The present study demonstrated that the cytotoxic response to silver nanoparticles (AgNPs) in metabolically active, human-derived liver (HepG2) cells is size- and concentration-dependent (Knasmüller et al., 1998), and identified autophagy-lysosomal membrane permeabilization and NLRP3inflammasome dependent caspase-1 activation as the mechanism of the observed toxicity. AgNPs size

influence the manner in which it interacts with mammalian cells. As primary particle size decreases, the surface area to volume ratio and surface reactivity markedly increase and could exhibit novel bioactivity (Nel et al., 2006a). Our results support these general findings, as the smallest primary sized particle 10nm AgNPs - were found to be associated with highest toxicity and strong activation of signaling pathways associated with stress, inflammation and cell death. The coating of AgNPs with polymeric materials enhances the stability and slows the dissolution of Ag+ ions from AgNPs surface. However, detachment of coating over time can affect toxicity. Zhao et. al. showed higher toxic effects of sodium-dodecylbenzene sulfonate (S)-coated AgNPs (IC50=1.1µg/ml) compared to same size lactate-coated AgNP (IC50=28.7µg/ml) (Zhao and Wang, 2012). Anderson et.al., showed that PVP-coated AgNPs administered to the lung get cleared more rapidly than citrate-coated AgNPs, suggesting an important role of surface coating on internal clearance and translocation of AgNPs (Anderson et al., 2015). Therefore, we tested PVP-coated AgNPs to avoid any coating dependent variation in cellular response. Additional studies should be done to identify novel coating materials which mask the toxicity without affecting antimicrobial properties, thus making AgNPs safe for use in consumer products. AgNPs are increasingly incorporated into blood contacting devices or implants which can introduce AgNPs to the systemic circulation (Marsich et al., 2013). Liver and spleen are two primary organs for accumulation of circulating nanoparticles (Lankveld et al., 2010) and minimal excretion can lead to 19


influences the rate of Ag+ ion release and the duration of its antimicrobial efficacy; however, size can also

protracted biopersistence in these tissues. Takenaka et al. in their study of AgNPs administered to rats via the pulmonary route, detected AgNPs in blood at 0 days, and after 7 days in liver, lung, kidney and heart, suggesting that AgNPs can migrate to blood and distant organs from site of absorption (Takenaka et al., 2001). Previous published studies from our group showed that liver accumulated the majority of AgNPs dose following intravenous administration (Austin et al., 2012). The present study demonstrated a sizedependent cytotoxicity of AgNPs in cultured human liver cells at 6 hr, 12 hr and 24 hr of exposure (Fig.

nanomaterials to detect toxic endpoints and a corresponding in vivo dose would be very high, raising questions about their clinical relevance. A recent study showed that, in benchtop release analyses, the highest amount of Ag released from several marketed AgNP-enabled medical devices (e.g., wound dressings) was on the order of 1x10-6 ng/cm2 of surface area of device at 370 C (Sussman et al., 2015). This is approximately equivalent to 0.1-100 µg/ml (100-1000 µg/cm2 of cell culture-well plate surface area) in vitro cell culture dose. Our result shows significant bioactivity of AgNPs at non-cytotoxic doses (0.1-10 µg/ml), which suggests that ENMs, specifically AgNPs, at physiologically relevant low exposure concentrations, can activate disease-specific signaling pathways in cells that could lead to cellular stress and toxicity (Unfried et al., 2008). Cellular uptake of nanoparticles can occur by multiple mechanisms but mainly phagocytosis, endocytosis and clathrin-mediated uptake are reported (Murugan et al., 2015). However, the majority of nanoparticles engulfed by the cells were internalized in cytosolic vesicles, and transferred to lysosomes. We showed that the highest accumulation is influenced by the size of the nanoparticle and 10nm-AgNPs were rapidly accumulated in HepG2 cells compared to 50nm- and 100nm-AgNPs (Fig. 2). This result is significant since it is consistent with the cell viability results showing the earliest and highest toxicity in the 10nm-AgNPs exposed treatment group. In addition, 10nm-AgNPs have the largest surface area compared to 50nm- and 100nm-AgNPs on equal mass-dose (µg/ml), and subsequently adsorb relatively large quantities of protein and lipid, forming a biocorona around the particle. TEM images 20


1). A significant number of published in vitro studies incorporated very high concentrations of

illustrated intracellular localization of engulfed particles and disintegration of AgNPs into smaller particles (Fig. 3). We were able to detect disintegration of large 50nm- and 100nm-AgNPs at 24 hr postexposure (Fig 3b and Fig. S3); however, AgNPs sizes less than 10nm diameter could not be detected probably due to rapid dissolution in to soluble Ag+ ions in cytoplasmic vesicles. The fate, stability and toxicity of AgNPs highly depend on the biological environment. Numerous

environments are highly acidic, which facilitates dissolution of Ag+ ions from AgNPs. This effect has been demonstrated by Laura et. al. in which polysaccharide-coated AgNPs (25nm) lost the surface coating more in lysosomal environments resulting in higher toxicity than in water (Braydich-Stolle et al., 2014). Ag+ ion oxidizes thiols groups in cellular protein inducing antimicrobial action, but also toxicity (Jung et al., 2008). To explore consequences of internalization of AgNPs, we examined autophagy and lysosmal uptake of AgNPs in HepG2 cells. Overall, our data demonstrated a size-dependent autophagic response to AgNPs in HepG2 cells, with 10nm-AgNPs most active in inducing autophagic flux. Lysosomal membrane permeabilization (LMP) resulted in acidification and leakage of lytic enzymes into the cytosol, which have been shown to induce cell death via caspase activation and pro-inflammatory signaling (Boya and Kroemer, 2008). These results indicate that AgNPs increase lysosomal activity, becoming more acidic after AgNPs exposure, suggesting that AgNPs are actively transported to the lysosomes by autophagosomes (Fig. 4, 5). This result is significant since the TEM results (Fig. 3b and Fig S3) showed that the majority of engulfed AgNPs are vesicle-bound in cytoplasm. These results suggest that AgNPs induce cytotoxicity by inducing lysosomal membrane permeabilization (LMP). Acidic content of lysosomes facilitates dissolution of Ag+ ions from AgNPs surfaces that can induce lysosomal membrane leakage or lysosomal dysfunction, an effect known to induce cathepsin-mediated apoptosis (Guicciardi, Leist and Gores, 2004; Leist and Jaattela, 2001). Similar effects have been observed with zinc oxide nanoparticles (ZnO) where intra-lysosomal Zn2+ dissolution was shown to induce lung injury

21


studies have shown that nanoparticles are localized in lysosomal vesicles; however, lysosmal

(Cho et al., 2011). In addition, our hyperspectral imaging and TEM results suggest that AgNPs cytotoxicity is due to the direct interaction of AgNP with cells and dissolution of Ag+ in lysosomes. Particle-induced NLRP3 inflammasome activation is frequently associated with caspase-1 dependent programmed cell death (pyroptosis) in immune cells; however, the role the pathway plays in HepG2 cells is not well known. Recently, caspase-1 activation has been suggested to play a major role in

2015; Yazdi et al., 2010). A recent study by Li et. al. showed that rare-earth nanoparticles and carbon nanotubes destabilize the autophagy pathway causing disruption in the regulation of activated NLRP3 complexes in immune cells (Li et al., 2014). Our study showed that AgNPs induced caspase-1 activation in HepG2 cells and blocking autophagy-lysosomal system exacerbates NLRP3 inflammasome dependent caspase-1 activation (Fig. 8). In many studies, the toxicity of AgNPs has been attributed to Ag+ ion mediated oxidative stress (Aueviriyavit et al., 2014). Prasad et.al. showed that AgNPs size-dependent oxidative stress, glutathione depletion and inflammatory responses in a high-throughput reporter HepG2 cell line (Prasad et al., 2013). However, the molecular mechanisms involved upstream of AgNP-induced inflammation and cell death are poorly understood. Our data points towards an upstream mechanism of AgNP-induced cytotoxicity involving caspase-1 activation in non-immune HepG2 cells. We showed that even at relatively low and non-cytotoxic doses, AgNPs can induce both autophagy and caspase-1 activation (Fig. 4, 7). Our results demonstrate that inhibition of siRNA mediated autophagosome formation or disrupting autophagosome-lysosome fusion by preventing lysosomal acidification exacerbates AgNPs-induced caspase-1 activation and cell death (Fig. 8). The results suggest a protective role of autophagy in AgNPs-induced inflammasome activation at sub-cytotoxic concentrations. We report here a novel mechanism involving the autophagy-lysosomal pathway in AgNPsinduced caspase-1 response in HepG2cells. The results support that AgNPs damage the autophagylysosomal pathway resulting in inflammation, oxidative stress, apoptosis and cytotoxicity. An 22


cellular response to stress in non-immune cells (Denes, Lopez-Castejon and Brough, 2012; Simard et al.,

investigation into AgNPs-induced oxidative stress and NLRP3-inflammasome activation is currently ongoing in our laboratory. Taken together, these results indicate the potential of vesicle engulfed 10nmAgNPs to induce cytotoxicity by a novel mechanism involving perturbations in autophagy-lysosomal system and inflammasome activation. Additionally, the results support that pro-autophagy LC3B and proinflammasome caspase-1 might serve as a sensitive biomarker for rapid screening of the toxic potential of nanoparticles and early detection of toxic nanoparticle exposure.

The precise mechanism involved in AgNPs toxicity is not completely understood. Accumulating evidence suggests that AgNPs induce cytotoxicity by inducing apoptosis and non-apoptotic cell death. We showed that AgNPs (primary particle sizes of 10, 50, and 100nm) induce cytotoxicity in cultured liver cells that is mediated by AgNP-induced lysosomal membrane permeabilization and inflammasome dependent caspase-1 activation, a pro-inflammatory protease which regulates cell death. Blocking AgNP induced autophagy exacerbates caspase-1 activation and cell death. As shown in the schematic diagram depicting the autophagy mechanism (Fig 8), AgNPs induce autophagy and lysosmal membrane permeabilization resulting NLRP3 inflammsome dependent caspase-1activation. Our data also supports the in vitro based mechanistic investigations to predict nanoparticle toxicity, which will help in assessing health risks associated with medical and consumer applications of AgNPs. Acknowledgments This project was supported in part by an appointment to the Research Participation Program at the Center for Devices and Radiological Health administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. The author(s) would like to acknowledge the FDA White Oak Nanotechnology Core Facility and the Flow Cytometry Core Facility for instrument use, scientific and technical assistance. 23


Conclusion

Disclaimer The findings and conclusions in this paper have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any agency determination or policy. The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by Department of Health Downloaded from http://toxsci.oxfordjournals.org/ at New Mexico State University Library on January 25, 2016

and Human Services.

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Figure Legends

Figure 1. Effect of AgNPs on viability of HepG2 cells measured by WST-1 assay. HepG2 cells (20 x 103 cells/well) were grown in 96-well plates and treated with 10, 50 and 100nm AgNPs (0.01-50 µg/mL).

be concentration-, size- and time- dependent. After 24 hr, AgNPs reduced the cell viability to less than 50% at 10 and 50 µg/ml compared to non-treated control. Values represent mean ± SD from N=3 replicate experiments, * represent P

Toxicogenomic responses of human liver HepG2 cells to silver nanoparticles.

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