lable at ScienceDirect

Biomaterials 35 (2014) 4706e4715

Contents lists avai

Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Cytotoxicity, oxidative stress, apoptosis and the autophagic effectsof silver nanoparticles in mouse embryonic fibroblasts

Yu-Hsuan Lee a, Fong-Yu Cheng b, Hui-Wen Chiu a, Jui-Chen Tsai c, Chun-Yong Fang a,Chun-Wan Chen d,**, Ying-Jan Wang a,*

aDepartment of Environmental and Occupational Health, National Cheng Kung University, Medical College, 138 Sheng-Li Road, Tainan 704, Taiwanb Institute of Oral Medicine, National Cheng Kung University, Tainan, Taiwanc Institute of Clinical Pharmacy, National Cheng Kung University, Tainan, Taiwand Institute of Labor, Occupational Safety and Health, Ministry of Labor, 99, Lane 407, Hengke Road, Shijr, Taipei 22143, Taiwan

a r t i c l e i n f o

Article history:Received 17 December 2013Accepted 12 February 2014Available online 13 March 2014

Keywords:NanoparticleCytotoxicityCellular uptakeROSAutophagyApoptosis

* Corresponding author. Tel.: þ886 6 235 3535x58** Corresponding author. Tel.: þ886 2 2660 7600x2

E-mail addresses:wann@mail.iosh.gov.tw (C.-W. Chebmm175@gmail.com (Y.-J. Wang).

http://dx.doi.org/10.1016/j.biomaterials.2014.02.0210142-9612/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

With the advancement of nanotechnology, nanomaterials have been comprehensively applied in ourmodern society. However, the hazardous impacts of nanoscale particles on organisms have not yet beenthoroughly clarified. Currently, there exist numerous approaches to perform toxicity tests, but commonand reasonable bio-indicators for toxicity evaluations are lacking. In this study, we investigated the ef-fects of silver nanoparticles (AgNPs) on NIH 3T3 cells to explore the potential application of thesenanoparticles in consumer products. Our results demonstrated that AgNPs were taken up by NIH 3T3cells and localized within the intracellular endosomal compartments. Exposure to AgNPs is a potentialsource of oxidative stress, which leads to the induction of reactive oxygen species (ROS), the up-regulation of Heme oxygenase 1 (HO-1) expression, apoptosis and autophagy. Interestingly, AgNPsinduced morphological and biochemical markers of autophagy in NIH 3T3 cells and induced autopha-gosome formation, as evidenced by transmission electron microscopic analysis, the formation ofmicrotubule-associated protein-1 light chain-3 (LC3) puncta and the expression of LC3-II protein. Thus,autophagy activation may be a key player in the cellular response against nano-toxicity.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Nanoparticles (NPs) are an emerging class of functional mate-rials. Application fields for NP range frommedical imaging and newdrug delivery technologies to various industrial products. Recentadvances in particle-forming chemistries used for developingnanotechnology has not only widened applications for nanoscalematerials but has also induced significant concern regarding theiradverse biological effects [1e3]. With the rapid development ofnanotechnology and the widespread use of nano-products, the riskof human exposure rapidly increases, and reliable toxicity testsystems are urgently needed [3,4]. Evidence is accumulating thatNPs differ significantly from traditional materials and may causedifferent toxicities [5]. Therefore, understanding the detailed

04; fax: þ886 6 275 2484.07.n), yjwang@mail.ncku.edu.tw,

mechanism of cell-specific cytotoxicity of NPs will be helpful forassessing their risk [6,7]. Nevertheless, some of the important andbasic characteristics of NPs, including the process of cellular uptake,the mechanism of cytotoxicity, the intracellular location and thetranslocation of NPs, remain unclear [8]. More studies are neededto focus on the processes of nanoparticleecell interactions, theirintracellular fate and their relationships. Silver nanoparticles(AgNPs) are used in many consumer products. Because of superiortheir anti-microbial activity, AgNPs are involved in the productionof several medical products, including catheters, implants andother materials to prevent infection [9,10]. In addition to theirmedical uses, AgNPs are used in clothing, the food industry, paints,household products and other fields [11e13]. Although the use ofAgNPs is increasingly widespread in medicine and in dailylife, comprehensive biologic and toxicological information is lack-ing [10].

Several different mechanisms contributed to AgNPs toxicity,notably the production of excess reactive oxygen species (ROS). ROSare both physiologically necessary and potentially destructive. ROSmay contribute to tissue damage in many pathophysiological

Y.-H. Lee et al. / Biomaterials 35 (2014) 4706e4715 4707

conditions and participate in several cellular events, includingsignal transduction, proliferative response, gene expression andprotein redox regulation [14,15]. High ROS levels are indicative ofoxidative stress and can influence cellular signal transductionpathways, such as proinflammatory signaling pathways, and canmodulate the expression of numerous genes [16]. In mammaliansystems, the heme oxygenase-1 (Ho-1) gene represents one of themost widely studied examples of a redox-regulated gene [17]. ROScan induce HO-1, the rate-limiting enzyme in heme degradation, aswell as steal electrons from lipids in the cell membrane, resulting incellular injuries and cell death. Excessive production of ROS in thecell is known to induce apoptosis [18,19]. ROS generation has beenshown to play an important role in apoptosis induced by AgNPs[20e22]. It is now known that different types of cell death(apoptosis, necrosis and autophagy) contribute to the pathophysi-ology of different human disorders [23]. One of the cell death typesthat has received much attention in the recent years is autophagy.Autophagy is a process of bulk degradation of toxic protein aggre-gates and damaged organelles in which portions of the cytoplasmare sequestered into double-membrane vesicles known as auto-phagosomes, which then fused with lysosomes to form single-membrane autolysosomes; ultimately, the contents of the autoly-sosomes are degraded by lysosomal hydrolases and recycled forenergy utilization [24]. Unlike apoptosis, autophagy acts as either asurvival or death safeguard mechanism on different environmentalstresses and cell types. Several markers of the autophagic processhave been discovered and various strategies have been reported forstudying this molecular process in different biological systems inboth physiological and stress condition. In this study, we selectedAgNPs, which have been commonly used in the industry, ascandidate toxicants and looked for the nanoparticle toxicity bio-indicators in a mammalian cell line.

2. Materials and methods

2.1. Preparation and characterization of AgNPs

AgNPs were prepared from the NaBH4 reduction of AgNO3. Briefly, aqueoussodium citrate solution (1.2 ml, 80 mM) and AgNO3 solution (1.5 ml, 20 mM) weremixed with H2O (26.7 ml). Ice-cold aqueous NaBH4 solution (0.6 ml, 100 mM) wasadded dropwise under vigorous stirring. The rapid formation of AgNPs was indi-cated by the color change of the mixture from colorless to yellow. The Ag hydrosolwas then aged for 24 h to completely decompose the residual NaBH4. Character-ization of the AgNPs was performed using transmission electron microscopy (TEM)(JEOL Co., MA, USA). AgNPs were examined after suspension in M.Q. water or DMEMmedium and subsequently deposition onto copper-coated carbon grids. TEM soft-ware was calibrated to measure the sizes of the AgNPs. The composition of theAgNPs was determined by electron dispersive X-ray (EDX) analysis. The hydrody-namic sizes, zeta potential and polydispersity index (PDI) of AgNPs examined bydynamic light scattering (DLS) (Delsa�Nano C, Beckman, U.S.A.). The zeta potential ofthe AgNPs was analyzed in aqueous dispersion using a phase analysis light scat-tering (PALS) (Delsa� Nano C, Beckman, U.S.A.).

2.2. Cell culture and co-incubation with nanoparticles

The NIH 3T3 mouse embryonic fibroblast cell line was obtained from theAmerican Type Culture Collection (ATCC). NIH 3T3 cell lines were cultured in DMEM(Gibco BRL, Grand Island, NY) supplemented with antibiotics containing 100 U/mlpenicillin, 100 mg/ml streptomycin (Gibco BRL, Grand Island, NY) and 1% fetal bovineserum (HyClone, South Logan, UT, USA). Cells were incubated in a humidified at-mosphere containing 5% CO2 at 37 �C. Exponentially growing cells were detachedwith 0.05% trypsin-EDTA (Gibco BRL, Grand Island, NY) in DMEM. All of the AgNPssolutions were fresh prepared from stock solutions and sonicated for 5 min beforeaddition to cell cultures.

2.3. Electron microscopy

After NIH 3T3 cells were incubated for 24 h with silver nanoparticles (2 or 5 mg/ml), the cells were washed with PBS and then centrifuged at 2000 r/min for 5 min.The supernatants were removed. The cell pellets were fixed with a solution con-taining 2.5% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH7.3, for 1 h. After fixation, the samples were postfixed in 1% OsO4 in the same bufferfor 30 min. Ultrathin sections were then observed under a transmission electronmicroscope (JEOL JEM-1200EX, Japan) at 100 kV.

2.4. Cell morphology by phase-contrast microscopy

Cells were seeded on a 6-well plate at a density of 1.5�105 cells per well in 2 mlof growthmedium. After overnight growth, the culture supernatants were aspirated,and fresh growth medium containing AgNPs at the indicated concentration (0e30 mg/ml) was added. After incubation for 24 h, the cells werewashedwith 0.1 M PBS,pH 7.4, and morphological changes were observed under an inverted phase contrastmicroscope at 200� magnification.

2.5. Cell viability assay

Cellular viability was determined by the MTS assay, which observes thereduction of (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) to formazan in viable cells. Briefly, cells wereplated onto 96 multiwell plates (Costar, Corning, NY). After incubation with theindicated dose of AgNPs for various lengths of time at 37 �C, formazan absorbancewas measured at 490 nm. The mean absorbance of the non-exposed cells was thereference value for calculating 100% cellular viability.

Cytotoxicity was quantified using a Live/Dead viability/cytotoxicity kit (Invi-trogen, CA, USA), according to themanufacturer’s protocol. Briefly, cells were treatedwith AgNPs for 24 h and the combined Live/Dead assay reagents were added. Thecells were incubated with the assay reagents for 30e45 min at room temperature.The labeled cells were analyzed by a fluorescence microscope (Olympus, Japan) andflow cytometry (BD Biosciences, Germany).

2.6. Intracellular reactive oxygen species (ROS) and glutathione (GSH) measurement

ROS production was monitored by flow cytometry using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). This dye is a stable, nonpolarcompound that readily diffuses into cells and is hydrolyzed by intracellular esteraseto yield the DCHF, which trapped within the cells. Hydrogen peroxide (H2O2) or lowmolecular weight peroxides produced by the cells oxidize DCHF to the highlyfluorescent compound 20 ,70-dichlorofluorescein (DCF). Thus, the fluorescence in-tensity is proportional to the amount of hydrogen peroxide produced by the cells.The thioreactive fluorescent dye 5-chloromethylfluorescein diacetate (CMFDA) wasused for GSH determination. CFMDA forms a GSH adduct in a reaction catalyzed byglutathione-S-transferase. After conjugation with GSH, CMFDA is hydrolyzed to thefluorescent 5-chloromethylfluorescein by cellular esterase [25]. After treatmentwith AgNPs for 3 and 6 h, cells were incubated with DCFHDA (20 mM) or CFMDA(30 mM) for a further 30 min. The cells were harvested, washed once and resus-pended in PBS. Fluorescence was monitored by flow cytometry. Histograms wereanalyzed using Winmdi software and were compared with the histograms of un-treated control cells.

2.7. Detection of apoptosis using Annexin V/PI and DAPI staining

Apoptosis was assessed by observing the translocation of phosphatidyl serineto the cell surface, as detected with an Annexin V apoptosis detection kit (Cal-biochem, San Diego, CA) as described previously [26]. Cells were treated withAgNPs in a concentration-and time-dependent manner. After the exposure time,cells were trypsinised, washed with 1 X PBS and centrifuged at 3000 rpm for 5 min.Cells were resuspended in 100 ml of 1 X Annexin V-binding buffer (10 mM HEPES(pH 7.4), 0.14 M NaCl and 2.5 mM CaCl2) that contained 5 ml of Annexin V-FITC(Becton Dickinson, San Jose, CA, USA) alone or in combination with 10 ml of PI(50 mg/ml) and were incubated at room temperature for 15 min. The 1� bindingbuffer (400 ml) was added to stop the reaction, and the stained cells were collectedfor flow cytometry analyses. For the observation of nuclear morphology, cellstreated under the indicated conditions were fixed in methanol, incubated with40 ,6-diamidino-2-phenylindole (DAPI) (Sigma Chemical Co.), and analyzed using afluorescence microscope.

2.8. Detection and quantification of acidic vesicular organelles with acridine orangestaining

Cell staining with acridine orange (Sigma Chemical Co.) was performed ac-cording to the published procedures [27,28], adding a final concentration of 1 mg/mlfor a period of 20 min. Photographs were obtained with a fluorescence microscope(Axioscop) equipped with a mercury 100-W lamp, 490-nm band-pass blue excita-tion filters, a 500-nm dichroic mirror, and a 515-nm long-pass barrier filter. Flowcytometric analysis was used to detect acidic vesicular organelles (AVOs), which area characteristic of autophagy [27].

2.9. Immunofluorescence microscopy for LC3

After NIH 3T3 cells were incubated for 18 h with silver nanoparticles (10 mg/ml), the cells were fixed in 4% paraformaldehyde and blocked with 1% BSA for30 min. Fixed cells were incubated with primary antibodies specific for LC3 (MBL,Japan) for 1 h. After washing, cells were labeled with a DyLight� 488-conjugatedaffinipure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, PA, USA) for1 h. After washing again, the cells were counterstained with DAPI and visualizedwith a confocal microscope (Carl Zeiss LSM780, Instrument Development Center,NCKU).

Table 1Characterization of AgNPs.

Characterization Method Condition Results

Size TEM Dry 26.2 � 7.6 nmMorphology TEM Dry SphericalComposition and purity EDX Dry Ag (99.6%)Zeta potential PALS Water �28.4 mVHydrodynamic size DLS Water 40.1 � 1.7 nmPolydispersity index (PDI) DLS Water 0.204lmax UVeVis Water 389 nmHydrodynamic size DLS DMEM 92.9 � 2.1 nmPolydispersity index (PDI) DLS DMEM 0.289lmax UVeVis DMEM 398 nm

Y.-H. Lee et al. / Biomaterials 35 (2014) 4706e47154708

2.10. Western blot analysis

Total cellular protein lysates were prepared by harvesting cells in proteinextraction buffer for 1 h at 4 �C as described previously [29]. The expression ofGAPDH was used as the protein loading control. The antibodies for detecting anti-poly-(ADP-ribose) polymerase (PARP) antibody were obtained from Millipore(Billerica, MA); anti-caspase-3 and anti-cleaved-caspase-3 antibodies were obtainedfrom Epitomics (Burlingame, CA); anti-Beclin 1 antibody was obtained from Cell

Fig. 1. Characterization of AgNPs. (a) UVevis spectra of AgNPs inwater or DMEM plus 1% FBSwere mainly spherical in shape with a mean diameter of 26 � 7.6 nm (mean � SD) (scale

Signaling Technology (Ipswich, MA, USA); anti-GAPDH antibody was obtainedfrom Abcam (Cambridge, MA, USA); anti-LC3 antibody was obtained from Abgent(San Diego, CA, USA); and anti-p62/SQSTM1 antibody was obtained from MBL(Nagoya, Japan).

2.11. Statistical analysis

All data represented the mean � SD of at least 3 independent culture experi-ments. Statistical significance was determined using Student’s t-test for comparisonbetween themeans or using a one-way analysis of variancewith a post-hoc Dunnett’stest. Differences were considered significant when p < 0.05. Images are represen-tative of three or more experiments.

3. Results

3.1. AgNPs physical-chemical characterization and dispersion inwater or cell culture media

The physicalechemical properties of the AgNPs used in thisstudy are summarized in Table 1. Size, morphology, and composi-tion were characterized by TEM and EDX analysis. AgNPs weredemonstrated to exhibit negative surface charges by PALS.

. (b) Transmission electron microscope (TEM) documentation of NP morphology: AgNPsbar representing 20 nm). (c) An X-ray diffraction (XRD) pattern of AgNPs.

Y.-H. Lee et al. / Biomaterials 35 (2014) 4706e4715 4709

The particle of hydrodynamic size ranged from 40.1 � 1.7 nm to92.9 � 2.1 nm in the water or DMEM supplemented with 1% FBS atpH 7.4. These values are higher than those obtained by TEM,possibly due to some precipitation or agglomeration of silvernanoparticles in the solution. The value of PDI indicated thedispersion stability and solubility of silver nanoparticles inwater orDMEM. Previous studies have indicated that PDI lower than 0.2might be associated with high homogeneity in the particle popu-lation [30]. The synthesized AgNPs were analyzed using UVevisabsorbance spectrophotometry. The plasmon absorption is clearlyvisible at lmax 389 nm or 398 nm in the water or DMEM (Fig. 1a).Because hydrodynamic size in DMEM was larger than in the water,the maximum UVevis absorption also shifted with increasing hy-drodynamic size. The TEM image suggests that the particles arepolydispersed and are mostly spherical in shape (Fig. 1b). The darkspots represent regularly shaped AgNPs. X-ray diffraction (XRD)pattern analysis was performed to assess the face center cubiccrystalline nature of the nanoparticles. The face center cubicstructure of elemental silver can be clearly observed in Fig. 1c.

3.2. Cellular uptake of silver nanoparticles

The uptake of NPs by cells is an important factor to assessnanotoxicity. To confirm that AgNPs entered cells, we used TEM toobserve the cellular responses. In Fig. 2, NIH 3T3 cells treated withAgNPs clearly manifested the accumulation of autophagic vacuoles(Fig. 2 b, c & e) in comparison with untreated cells (Fig. 2a). AgNP-treated cells evidently internalized AgNPs aggregates via largeintracellular cytoplasmic vacuoles. Additionally, some of thecellular materials were found in characteristic double membranevesicles, which are typical features of autophagosomes (Fig. 2 d & f).

Fig. 2. Cellular uptake of AgNPs by NIH 3T3 cells. TEM images demonstrated the interactionAutophagic vacuoles were observed in cells treated with AgNPs at 2 mg/ml for 24 h (c and elysosomes. The image also exhibits a large vacuole containing AgNPs and cellular debris, posin AgNPs-treated cells as indicated by the label “AP”. Lysosomes are indicated by the label “Lother subcellular organelles (e.g. nucleus, mitochondria and Golgi complex). Scale bars: 2 mm15 mg/ml for 24 h. The results of side-scatter(ed) light of flow cytometry demonstrated tha

AgNP-treated NIH 3T3 cells revealed increases in the number oflysosomes and the disappearance of some cytoplasmic organelles,which indicated the onset of autophagy. Furthermore, the results ofside-scatter(ed) light of flow cytometry also demonstrated thatAgNPs were apparently engulfed by NIH 3T3 cells (Fig. 2g). Theseresults definitively demonstrated an interaction between nano-particles and cells that resulted in cellular damage and autophagyin NIH 3T3 cells.

3.3. Effects of AgNPs on cellular morphology and cytotoxicity

After 24 h of AgNPs treatment, the changes in cell morphologywere examined using phase-contrast microscopy. Compared withuntreated cells (Fig. 3a), significant morphological changes char-acteristic of cell death, including cell shrinkage, few cellular ex-tensions, increased floating cells, and AgNPs cluster accumulationin the cytoplasm, were observed in NIH 3T3 cells exposed to AgNPs.In addition, our results demonstrated that AgNPs reduced theviability of NIH 3T3 cells in a concentration-and time-dependentmanner (Fig. 3b). To assure the validity of the assay used to deter-mine cell death, we applied additional experiments using a recentlydeveloped cytotoxicity assay (“Live/Dead,” Molecular Probes, Inc.)that utilizes two-color fluorescence dyes to discriminate betweenlive and dead cells. This assay identifies dead cells by a red fluo-rescent fluorophore ethidium homodimer (EthD-1), which enterscells with damaged membranes and binds to nucleic acids. Viablecells are identified by a green (fluorescein) stain generated by theenzymatic hydrolysis of calcein-AM that only occurs in live cells as aresult of esterase activity. As shown in Fig. 3c, results from thosestudies were straightforward as cells exhibit difference colors un-der the fluorescence microscope, green (live) or red (dead) cells.

0

0

1023

1023

FSC-H

0 1023FSC-H

0 1023FSC-H

SSC-H

01023

SSC-H

01023

SSC-H

of AgNPs with cells. (a) Cellular structures were unchanged in the untreated control. (b)) The treated cells (10 mg/ml, 24 h) contained large numbers of dense endosomes andsibly autophagosomes. (d and f) High magnification view of autophagosome formationY”. Black arrows point to AgNPs. AgNPs were observed inside vesicles and did not enter(a, b, c and e); 0.5 mm (d and f). (g) NIH 3T3 cells were treated with AgNPs at 0, 7.5, or

t AgNPs were apparently engulfed by NIH 3T3 cells.

Y.-H. Lee et al. / Biomaterials 35 (2014) 4706e47154710

Quantifications of cell death with the Live/Dead viability/cytotox-icity kit were performed using flow cytometry (Fig. 3d). NIH 3T3cell treated with 10 mg/ml AgNPs for 24 h exhibited substantialnumbers of dead cells (56.8%), as similarly examined byMTS. Therewas a dose-related increase in cytotoxicity.

3.4. Effects of AgNPs on cellular reactive oxygen species (ROS),glutathione (GSH) and the expression of heme oxygenase 1 (HO-1)

Oxidative stress has been reported as one of the most importantmechanisms of toxicity related to nanoparticle exposure [31]. Toinvestigate the potential role of oxidative stress induced by AgNPs,ROS generation was measured. As shown in Fig. 4a and b, the ROSlevels generated in response to AgNPs were significantly increasedat 15 mg/ml for 3 and 6 h. In the meantime, alterations in GSH levelscan be monitored as an indication of oxidative stress in cells. AgNPstreatment decreased GSH levels as measured by flow cytometry.GSH is considered an important antioxidant to protect cells againstapoptosis by removing toxic hydrogen peroxide from cells [32,33]

Fig. 3. Cytotoxicity of AgNP-treated NIH 3T3 cells. Cells were grown for 1 day in 6-well platecellular morphology. Cells were observed by phase-contrast microscopy. (b) Cell viability wconcentration-and time-dependent manner. *p < 0.05 versus 24 h Control; #p < 0.05 versuscanned at low power (100�). The green color and the red color indicate the fluorescence of dcytometry. AgNPs reduced the viability of NIH 3T3 cells in a concentration-dependent mareferred to the web version of this article.)

and the depletion of GSH within cells appears to promote intra-cellular ROS accumulation and the expression of heme oxygenase-1(HO-1) mRNA [34]. HO-1, whose induction responds to multipleforms of chemical and physical cellular stress, represents a generalmarker of oxidative cellular stress and confers cytoprotection innumerous models of oxidative injury [35]. At concentrations of2 mg/ml and above, AgNPs stimulated HO-1 mRNA expression(Fig. 4c). The reverse transcription quantitative PCR (RT-qPCR) re-sults indicated that the relative expression levels after 12 h treat-ment with AgNPs increased from 33- to 70-fold in comparisonwiththose without AgNPs treatment.

3.5. AgNPs induced morphological and biochemical markers ofautophagy in NIH 3T3 cells

Autophagy is characterized by the formation of numerous acidicvesicles that are called acidic vesicular organelles (AVOs) [36,37].Microphotographs of AVOs were observed with green and redfluorescence in acridine orange (AO)-stained cells using a

s and exposed to AgNPs at the different concentrations for 24 h. (a) Effects of AgNPs onas measured using the MTS assays. AgNPs decreased the viability of NIH 3T3 cells in as 48 h Control. (c) Cell death was observed by fluorescence microscopy. Each slide wasetected live cells and dead cells, respectively. (d) Cell viability was measured using flownner. (For interpretation of the references to color in this figure legend, the reader is

Y.-H. Lee et al. / Biomaterials 35 (2014) 4706e4715 4711

fluorescence microscope (Fig. 5a). Untreated NIH 3T3 cells exhibi-ted limited AVOs in the cytoplasm. On the contrary, cells treatedwith AgNPs exhibited more AVOs in the perinuclear region of thecytoplasm. Quantitative analysis of AO staining (Fig. 5b) verifiedthat AgNPs triggered a significantly higher percentage of cells withAO than the untreated group after 18 h exposure. Microtubule-associated protein light chain 3 (LC3) is now widely used tomonitor autophagy (Mizushima and Yoshimori, 2007). During theactivation of autophagy, LC3 is converted from LC3-I (18 kD) to LC3-II (16 kD), accumulates on the autophagosome membrane andappears as punctae [38]. After 18 h of AgNPs treatment, immuno-fluorescence microscopy for LC3 (Fig. 5c) demonstrated that AgNPsprovoked the appearance of many green punctae, which were notin the untreated cells. Furthermore, we performed western blotting

Fig. 4. Effects of AgNPs on cellular reactive oxygen species (ROS), glutathione (GSH) and thNIH 3T3 cells treated with AgNPs for 3, 6 h and with DCFH-DA or CFMDA for an additional 3AgNPs increased HO-1 expression at the transcription level in NIH 3T3 cells treated with AgNControl; **p < 0.01 versus Control; ***p < 0.001 versus Control.

with lysates from NIH 3T3 cells receiving different concentrationsof AgNPs (Fig. 5d). The expression levels of the LC3-II and p62proteins increased with AgNPs treatment. However, Beclin-1 pro-tein did not change in these treatments. These data togetherdemonstrate the accumulation of autophagosomes in AgNP-treatedcells which was consistent with the observation of TEM images.

3.6. Measurements of apoptosis in NIH 3T3 cells treated with AgNPs

Previous reports have demonstrated that AgNPs are cytotoxicthrough their interactions with mitochondria [20,39] and theirinduction of the apoptosis pathway [39] via the production of ROS,which leads to cell death. To evaluate the cytotoxic effects of AgNPsin terms of apoptosis, cellular nuclei were stained with DAPI and

e expression of Heme oxygenase 1 (HO-1). (a, b) ROS generation and GSH reduction in0 min. The fluorescence in the cells was immediately assayed using flow cytometry. (c)Ps at 0, 2, 5, 10, or 15 mg/ml for 12 h, as measured by quantitative PCR. *p < 0.05 versus

Y.-H. Lee et al. / Biomaterials 35 (2014) 4706e47154712

assessed by microscopy. The AgNPs-treated cells exhibited signifi-cant nuclear fragmentation, which is indicative of apoptosis(Fig. 6a). In addition, early apoptosis in NIH 3T3 cells was measuredby flow cytometry with the Annexin V apoptosis detection kit.AgNPs treatment increased the percentage of apoptotic cell deathwhen compared with a control at any dose for 24 h. However, therewere no changes in the percentage of necrotic cell death (Fig. 6b).As shown in Fig. 6c, AgNPs led to the accumulation of active formsof PARP and caspase 3 in a dose-dependent manner (from 2 to15 mg/ml). PARP cleavage was stimulated at 2 mg/ml AgNPs expo-sure but diminished at 15 mg/ml. These results suggest that AgNP-induced apoptosis signals through a caspase-dependent pathwaywith mitochondrial involvement.

4. Discussion

The increasing use of nanomaterials in consumer and industrialproducts has aroused global concern regarding their toxicity andtheir fate in biological systems. While the toxicity of many bulk

Fig. 5. AgNPs induced morphological and biochemical markers of autophagy in NIH 3T3 cel5 mg/ml for 18 h. Detection of green and red fluorescence in acridine orange (AO)-stained celcells. Detection of green and red fluorescence in AO-stained cells using flow cytometry. Quamean � standard deviation from three independent experiments. *p < 0.05 versus Control; *(10 mg/ml) for 18 h and then processed for the immunofluorescence detection of LC3 (revealein the cytoplasm when cells were exposed to AgNPs. Scale bars: 20 mm (up); 10 mm (down)cells. Cells were treated with AgNPs at 0, 2, 5, 10, or 15 mg/ml for 18 h. Data are presented frofigure legend, the reader is referred to the web version of this article.)

materials is well understood, it is not known at what concentrationor size they begin to exhibit new toxicological properties due tonanoscopic dimensions [40]. Because the toxicity of nanoparticlesis affected by their composition and physico-chemical properties,an initial characterization of the test substance is imperative beforeany toxicity screening is commenced [5,41]. In the present study,AgNPs were coated by sodium citrate to maintaining their stabilityand dispersity in the culture medium. As shown in Table 1, we usedfive different techniques to characterize the physico-chemicalproperties of AgNPs. It should be noted that it is absolutelynormal to observe a difference in particle size measurements madein a vacuum by TEM and those made in solution using methodssuch as DLS because the physico-chemical parameter measured isfundamentally different in each case. In the culture medium, thereare various components (such as serum proteins, essential aminoacids, etc.) that interact with the AgNPs surface, thus influencingtheir hydrodynamic size and UVevis spectra.

In terms of mechanisms of toxicity, several studies havedemonstrated that the main mechanism of silver nanoparticles

Aut

opha

gy (%

)

12 18 24 (hrs)

Control2 µg/ml5 µg/ml15 µg/ml

0

10

20

30

40

50

60

70

*

**

****

*

ls. (a) Microphotograph of AVOs in NIH 3T3 cells. Cells were treated with AgNPs at 0 orls was performed using a fluorescence microscope. (b) Development of AVOs in NIH 3T3ntification of AVOs with AO-stained cells treated with AgNPs. Data are presented as the*p < 0.01 versus Control. (c) Cells were left untreated as a control or subjected to AgNPsd in green) and nuclear chromatin (stained in blue by DAPI). LC3 proteins were detected. (d) Western blotting of Beclin-1, p62/SQSTM1, LC3-I and LC3-II expression in NIH 3T3m three independent experiments. (For interpretation of the references to color in this

Y.-H. Lee et al. / Biomaterials 35 (2014) 4706e4715 4713

toxicity seems to be mediated by an increase in ROS production[42]. The results in the present study ascertain the capacity ofAgNPs to induce ROS and HO-1mRNA expression in NIH 3T3 cells atan early stage. As shown in Fig. 4, AgNPs were a potent and rapidinducer of ROS. As a result of excessive ROS, which cause injury tovarious cellular constituents such as lipids, proteins and DNA,finally resulting in growth arrest or apoptosis [18], cellular GSHlevels were rapidly depleted to neutralize ROS and HO-1 wasexpressed the increasing AgNPs concentrations. Current in vitrostudies have demonstrated that AgNPs have the potential to induce

Control AgN

60

50

40

30

20

10

012 18 24 (hrs)

Apo

ptos

is (%

)

Control5 µg/ml10 µg/ml15 µg/ml

**

*

**

**

Fig. 6. Measurement of apoptosis in NIH 3T3 cells that received various treatments. (a) Ceobserved under a fluorescent microscope after DAPI staining. The apoptotic bodies are indicacytometry with an Annexin V apoptosis detection kit. Cells were treated with AgNPs for 12,measured by flow cytometry with PI staining. There were no changes after exposure to AgNPlevel of total GAPDH protein was used as the loading control. Cells were treated with AgNP

different types of biological effects in cells. These cellular responsesinclude the altered regulation of signaling pathways that culminatein the regulation of cell survival or cell death pathways. Recentstudies suggest that autophagy, a highly-regulated intracellularprocess for the degradation of long-lived proteins and damagedorganelles, may represent a general cellular and tissue response tooxidative stress [43]. Autophagy could be a cell-survival or cell-death mechanism. Under oxidative stress, autophagy suppressescellular ROS levels to protect cells from ROS-induced damage [44].However, when oxidative stress reaches a level beyond the control

Ps AgNPs/2

12 18 24 (hrs)

50

40

30

20

10

0

Nec

rosi

s (%

)

Control5 µg/ml10 µg/ml15 µg/ml

lls were treated with AgNPs at 0 or 15 mg/ml for 18 h. Apoptotic body formation wasted with arrows. Scale bars: 50 mm. (b) Early apoptosis detection was measured by flow18 or 24 h *p < 0.05 versus Control; **p < 0.01 versus Control. Necrosis detection wass. (c) Western blotting of PARP, cleaved-PARP, procaspase 3 and cleaved-caspase 3. Thes for 18 h. Data are presented from three independent experiments.

Y.-H. Lee et al. / Biomaterials 35 (2014) 4706e47154714

of cellular protective mechanisms, cell death will occur throughnecrosis, apoptosis, or autophagic cell death [45,46].

A number of publications have reported that nanomaterialssuch as quantum dots [47], fullerene [48], gold nanoparticles [49]and carbon nanotubes [50] can induce autophagy. Here we pre-pared citrate-coated AgNPs to examine the induction of autophagy.The formation of autophagosomes and LC3 protein was signifi-cantly increased in AgNP-treated cells (Fig. 2 or Fig. 5). TEM analysisindicated that a double-layered membrane, the autophagosome, isformed that surrounding proteins and damaged organellesdestined for degradation. Lysosomes were also observed aroundthe autophagosomes but autophagosomes did not merge with thelysosomes in the present study. Previous studies have demon-strated that nanoparticles from various sources can induce auto-phagosome accumulation in treated cells. In Fig. 5c, wedemonstrated autophagosome accumulation by LC3 protein ag-gregation in the cytoplasm. The formation of such large aggregatedots similarly occurred in normal rat kidney epithelial cells treatedwith AuNPs [51] and in human keratinocytes treated with titaniumdioxide nanoparticles [52]. Ma et al. [51] reported that the exposureof cells to AuNPs caused the impairment of lysosome degradationcapacity and induced the accumulation of autophagosomes byblocking autophagic flux. Autophagy dysfunction is defined asexcessive autophagy induction or the inhibition of autophagy flux.Autophagy dysfunction is recognized as a potential mechanism ofcell death, resulting in either apoptosis or autophagic cell death[53]. In the beginning, we demonstrated that AgNPs entered intocells (Fig. 2g) and caused fifty percent cell death at 10e15 mg/ml for24 h (Fig. 3b). With the gradual increase in ROS, AgNP-treated cellsexhibited many autophagosomes and LC3 protein in the cytoplasm.The time-dependent increase in AVOs shown in Fig. 5b is likely dueto oxidative stress. Interestingly, although AgNPs exposure led to anincrease in cell autophagy and apoptosis for 18 h, the percentage ofautophagic cells that were exposed to 15 mg/ml of AgNPs for 24 hdid not increase as much as the 18-h treatment. In contrast, therewas a significant increase in apoptosis after treatment with 15 mg/ml of AgNPs for 24 h. It is possible that when cells are exposed toexcessive ROS, autophagic function is impaired, resulting in theaccumulation of damaged organelles, such as mitochondria, thatcan induce oxidative stress, inflammation and DNA damage. Finally,this autophagy dysfunction might induce either apoptosis orautophagic cell death to decrease cell viability as observed in ourstudy.

Recently, dysfunction of the autophagy pathway has been linkedto a variety of diseases [54]. For neurodegenerative diseases such asParkinson’s and Alzheimer’s disease, inhibition of autophagy-mediated elimination of disease-associated proteins or damagedmitochondria may be involved [54e58]. Because autophagy is nowbelieved to have numerous cellular functions and play a role in bothcell-survival and cell death, it is possible that the induction ofautophagy could represent a bioindicator of nanoparticle toxicity inmammalian cell lines.

5. Conclusion

In the present study, we demonstrated that AgNPs induced celldamage via the generation of ROS, which decreased GSH levels andup-regulated HO-1 mRNA expression. Furthermore, we observedthe induction of autophagy and apoptosis. We profiled autophagyactivation in cells treated with AgNPs, using AO staining, LC3expression profiling and TEM ultrastructural analysis. Although theprecise mechanisms by which exposure to AgNPs induces auto-phagy dysfunction is still far from complete, we show the activationof autophagy in NIH 3T3 cells by AgNPs and suggests that

nanoparticle-induced autophagy could have an important influ-ence on determining NIH 3T3 cell survival or death.

Acknowledgment

This study was supported by the Institute of Labor, OccupationalSafety and Health, Ministry of Labor (1013080 and 1023038), Foodand Drug Administration, Ministry of Health andWelfare, ExecutiveYuan (DOH101-FDA-41301 and DOH102-FDA-41702) and the Na-tional Science Council, Taiwan (NSC-102-2314-B-006-034).

References

[1] Lewinski N, Colvin V, Drezek R. Cytotoxicity of nanoparticles. Small 2008;4:26e49.

[2] Donaldson K, Aitken R, Tran L, Stone V, Duffin R, Forrest G, et al. Carbonnanotubes: a review of their properties in relation to pulmonary toxicologyand workplace safety. Toxicol Sci 2006;92:5e22.

[3] Oberdorster G, Maynard A, Donaldson K, Castranova V, Fitzpatrick J,Ausman K, et al. Principles for characterizing the potential human health ef-fects from exposure to nanomaterials: elements of a screening strategy. PartFibre Toxicol 2005;2:8.

[4] Dreher KL. Health and environmental impact of nanotechnology: toxicologicalassessment of manufactured nanoparticles. Toxicol Sci 2004;77:3e5.

[5] Dhawan A, Sharma V. Toxicity assessment of nanomaterials: methods andchallenges. Anal Bioanal Chem 2010;398:589e605.

[6] Faraji AH, Wipf P. Nanoparticles in cellular drug delivery. Bioorg Med Chem2009;17:2950e62.

[7] Limbach LK, Wick P, Manser P, Grass RN, Bruinink A, Stark WJ. Exposure ofengineered nanoparticles to human lung epithelial cells: influence of chemicalcomposition and catalytic activity on oxidative stress. Environ Sci Technol2007;41:4158e63.

[8] Zhao F, Zhao Y, Liu Y, Chang X, Chen C, Zhao Y. Cellular uptake, intracellulartrafficking, and cytotoxicity of nanomaterials. Small 2011;7:1322e37.

[9] Chen X, Schluesener HJ. Nanosilver: a nanoproduct in medical application.Toxicol Lett 2008;176:1e12.

[10] Ahamed M, Alsalhi MS, Siddiqui MK. Silver nanoparticle applications andhuman health. Clin Chim Acta 2010;411:1841e8.

[11] Cohen MS, Stern JM, Vanni AJ, Kelley RS, Baumgart E, Field D, et al. In vitroanalysis of a nanocrystalline silver-coated surgical mesh. Surg Infect(Larchmt) 2007;8:397e403.

[12] Lee HY, Park HK, Lee YM, Kim K, Park SB. A practical procedure for producingsilver nanocoated fabric and its antibacterial evaluation for biomedical ap-plications. Chem Commun (Camb); 2007:2959e61.

[13] Vigneshwaran N, Kathe AA, Varadarajan PV, Nachane RP, Balasubramanya RH.Functional finishing of cotton fabrics using silver nanoparticles. J NanosciNanotechnol 2007;7:1893e7.

[14] Devasagayam TP, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS, Lele RD. Freeradicals and antioxidants in human health: current status and future pros-pects. J Assoc Physicians India 2004;52:794e804.

[15] Dalle-Donne I, Rossi R, Giustarini D, Colombo R, Milzani A. S-glutathionylationin protein redox regulation. Free Radic Biol Med 2007;43:883e98.

[16] Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing.Nature 2000;408:239e47.

[17] Tyrrell RM. Approaches to define pathways of redox regulation of a eukaryoticgene: the heme oxygenase 1 example. Methods 1997;11:313e8.

[18] Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling forsuicide and survival. J Cell Physiol 2002;192:1e15.

[19] Sastre J, Pallardo FV, Vina J. Mitochondrial oxidative stress plays a key role inaging and apoptosis. IUBMB Life 2000;49:427e35.

[20] Carlson C, Hussain SM, Schrand AM, Braydich-Stolle LK, Hess KL, Jones RL,et al. Unique cellular interaction of silver nanoparticles: size-dependentgeneration of reactive oxygen species. J Phys Chem B 2008;112:13608e19.

[21] AshaRani PV, Low Kah Mun G, Hande MP, Valiyaveettil S. Cytotoxicity andgenotoxicity of silver nanoparticles in human cells. ACS Nano 2009;3:279e90.

[22] Foldbjerg R, Olesen P, Hougaard M, Dang DA, Hoffmann HJ, Autrup H. PVP-coated silver nanoparticles and silver ions induce reactive oxygen species,apoptosis and necrosis in THP-1 monocytes. Toxicol Lett 2009;190:156e62.

[23] Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000;21:485e95.[24] Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adapta-

tions. Nat Cell Biol 2007;9:1102e9.[25] Poot M, Kavanagh TJ, Kang HC, Haugland RP, Rabinovitch PS. Flow cytometric

analysis of cell cycle-dependent changes in cell thiol level by combining a newlaser dye with Hoechst 33342. Cytometry 1991;12:184e7.

[26] Chiu HW, Lin JH, Chen YA, Ho SY, Wang YJ. Combination treatment witharsenic trioxide and irradiation enhances cell-killing effects in human fibro-sarcoma cells in vitro and in vivo through induction of both autophagy andapoptosis. Autophagy 2010;6:353e65.

[27] Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I. Induction of autophagic celldeath in malignant glioma cells by arsenic trioxide. Cancer Res 2003;63:2103e8.

Y.-H. Lee et al. / Biomaterials 35 (2014) 4706e4715 4715

[28] Traganos F, Darzynkiewicz Z. Lysosomal proton pump activity: supravital cellstaining with acridine orange differentiates leukocyte subpopulations.Methods Cell Biol 1994;41:185e94.

[29] Chiu HW, Ho SY, Guo HR, Wang YJ. Combination treatment with arsenictrioxide and irradiation enhances autophagic effects in U118-MG cellsthrough increased mitotic arrest and regulation of PI3K/Akt and ERK1/2signaling pathways. Autophagy 2009;5:472e83.

[30] Saremi S, Atyabi F, Akhlaghi SP, Ostad SN, Dinarvand R. Thiolated chitosannanoparticles for enhancing oral absorption of docetaxel: preparation, in vitroand ex vivo evaluation. Int J Nanomed 2011;6:119e28.

[31] Nel A, Xia T, Madler L, Li N. Toxic potential of materials at the nanolevel.Science 2006;311:622e7.

[32] Fernandez-Checa JC, Garcia-Ruiz C, Colell A, Morales A, Mari M, Miranda M,et al. Oxidative stress: role of mitochondria and protection by glutathione.Biofactors 1998;8:7e11.

[33] Paolicchi A, Dominici S, Pieri L, Maellaro E, Pompella A. Glutathione catabo-lism as a signaling mechanism. Biochem Pharmacol 2002;64:1027e35.

[34] Oguro T, Hayashi M, Nakajo S, Numazawa S, Yoshida T. The expression ofheme oxygenase-1 gene responded to oxidative stress produced by phorone,a glutathione depletor, in the rat liver; the relevance to activation of c-jun n-terminal kinase. J Pharmacol Exp Ther 1998;287:773e8.

[35] Kim HP, Wang X, Chen ZH, Lee SJ, Huang MH, Wang Y, et al. Autophagicproteins regulate cigarette smoke-induced apoptosis: protective role of hemeoxygenase-1. Autophagy 2008;4:887e95.

[36] Bursch W, Ellinger A, Gerner C, Frohwein U, Schulte-Hermann R. Programmedcell death (PCD). Apoptosis, autophagic PCD, or others? Ann N Y Acad Sci2000;926:1e12.

[37] Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E, et al.A novel response of cancer cells to radiation involves autophagy and forma-tion of acidic vesicles. Cancer Res 2001;61:439e44.

[38] Li H, Li Y, Jiao J, Hu HM. Alpha-alumina nanoparticles induce efficientautophagy-dependent cross-presentation and potent antitumour response.Nat Nanotechnol 2011;6:645e50.

[39] Hsin YH, Chen CF, Huang S, Shih TS, Lai PS, Chueh PJ. The apoptotic effect ofnanosilver is mediated by a ROS- and JNK-dependent mechanism involvingthe mitochondrial pathway in NIH3T3 cells. Toxicol Lett 2008;179:130e9.

[40] Sharifi S, Behzadi S, Laurent S, Forrest ML, Stroeve P, Mahmoudi M. Toxicity ofnanomaterials. Chem Soc Rev 2012;41:2323e43.

[41] Bouwmeester H, Poortman J, Peters RJ, Wijma E, Kramer E, Makama S, et al.Characterization of translocation of silver nanoparticles and effects on whole-genome gene expression using an in vitro intestinal epithelium coculturemodel. ACS Nano 2011;5:4091e103.

[42] Christensen FM, Johnston HJ, Stone V, Aitken RJ, Hankin S, Peters S, et al.Nano-silver e feasibility and challenges for human health risk assessmentbased on open literature. Nanotoxicology 2010;4:284e95.

[43] Ryter SW, Choi AM. Regulation of autophagy in oxygen-dependent cellularstress. Curr Pharm Des 2013;19:2747e56.

[44] Kiffin R, Christian C, Knecht E, Cuervo AM. Activation of chaperone-mediatedautophagy during oxidative stress. Mol Biol Cell 2004;15:4829e40.

[45] Chen Y, Azad MB, Gibson SB. Superoxide is the major reactive oxygen speciesregulating autophagy. Cell Death Differ 2009;16:1040e52.

[46] Chen Y, McMillan-Ward E, Kong J, Israels SJ, Gibson SB. Mitochondrialelectron-transport-chain inhibitors of complexes I and II induce auto-phagic cell death mediated by reactive oxygen species. J Cell Sci2007;120:4155e66.

[47] Stern ST, Zolnik BS, McLeland CB, Clogston J, Zheng J, McNeil SE. Induction ofautophagy in porcine kidney cells by quantum dots: a common cellularresponse to nanomaterials? Toxicol Sci 2008;106:140e52.

[48] Zhang Q, Yang W, Man N, Zheng F, Shen Y, Sun K, et al. Autophagy-mediatedchemosensitization in cancer cells by fullerene C60 nanocrystal. Autophagy2009;5:1107e17.

[49] Li JJ, Hartono D, Ong CN, Bay BH, Yung LY. Autophagy and oxidative stressassociated with gold nanoparticles. Biomaterials 2010;31:5996e6003.

[50] Liu HL, Zhang YL, Yang N, Zhang YX, Liu XQ, Li CG, et al. A functionalizedsingle-walled carbon nanotube-induced autophagic cell death in human lungcells through Akt-TSC2-mTOR signaling. Cell Death Dis 2011;2:e159.

[51] Ma X, Wu Y, Jin S, Tian Y, Zhang X, Zhao Y, et al. Gold nanoparticles induceautophagosome accumulation through size-dependent nanoparticle uptakeand lysosome impairment. ACS Nano 2011;5:8629e39.

[52] Zhao Y, Howe JL, Yu Z, Leong DT, Chu JJ, Loo JS, et al. Exposure to titaniumdioxide nanoparticles induces autophagy in primary human keratinocytes.Small 2013;9:387e92.

[53] Kroemer G, Jaattela M. Lysosomes and autophagy in cell death control. NatRev Cancer 2005;5:886e97.

[54] Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW, et al. Regulation of mammalian autophagy in physiology andpathophysiology. Physiol Rev 2010;90:1383e435.

[55] Mak SK, McCormack AL, Manning-Bog AB, Cuervo AM, Di Monte DA.Lysosomal degradation of alpha-synuclein in vivo. J Biol Chem 2010;285:13621e9.

[56] Geisler S, Holmstrom KM, Treis A, Skujat D, Weber SS, Fiesel FC, et al. ThePINK1/Parkin-mediated mitophagy is compromised by PD-associated muta-tions. Autophagy 2010;6:871e8.

[57] Pickford F, Masliah E, Britschgi M, Lucin K, Narasimhan R, Jaeger PA, et al. Theautophagy-related protein beclin 1 shows reduced expression in early Alz-heimer disease and regulates amyloid beta accumulation in mice. J Clin Invest2008;118:2190e9.

[58] Stern ST, Adiseshaiah PP, Crist RM. Autophagy and lysosomal dysfunctionas emerging mechanisms of nanomaterial toxicity. Part Fibre Toxicol2012;9:20.