Nanoscale

PAPER

Cite this: DOI: 10.1039/c4nr05509h

Received 21st September 2014,Accepted 9th November 2014

DOI: 10.1039/c4nr05509h

www.rsc.org/nanoscale

Cationic polystyrene nanospheres induceautophagic cell death through the induction ofendoplasmic reticulum stress†

Hui-Wen Chiu,a,e,f Tian Xia,b Yu-Hsuan Lee,a Chun-Wan Chen,c Jui-Chen Tsai*d andYing-Jan Wang*a,g,h

Nanoparticles (NPs) have been used to produce a wide range of products that have applications in

imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface

charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the

possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has

been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with

different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly

toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore,

NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen

species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation.

The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition,

the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered auto-

phagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death

and provide mechanistic insights into the inhibition of the toxicity and safe material design.

1. Introduction

Applications of nanoparticles (NPs) are prevalent and continueto grow rapidly. Industrial and commercial applications ofNPs include catalysis, sensors, environmental remediation,personal care products and cosmetics, and NPs show greatpromise in the field of medicine, including imaging and drugdelivery.1 The development of nanotechnology calls for a com-prehensive understanding of the impact of NPs on biological

systems. However, the understanding of the interactions ofNPs with biological systems is still rudimentary.2 Polystyrene(PS) NPs can be easily synthesized in a wide range of sizes,which facilitates their application as biosensors as well asin photonics and self-assembling nanostructures.3,4 Specific-surface modification, high drug-loading capacity and colloidalstability in biological media also contribute to their appli-cation as experimental drug carrier systems.5 Our group hasshown that NH2-labeled PS (NH2-PS) nanospheres can inducecell death in macrophage and lung epithelial cells with apop-totic and necrotic features, respectively.6 Furthermore, wefound that macrophage cells used an endosomal–lysosomalroute of uptake, while lung epithelial cells used a caveolaruptake mechanism.6 Considering the proposed nanomedicalapplications of PS NPs, an evaluation of these processesbecomes even more important as these processes may result inpotentially unwanted consequences.

It is now known that different modalities of cell death(apoptosis, necrosis, autophagy) contribute to the patho-physiology of different human disorders.7 Autophagy is a proteindegradation system in which cellular proteins and organellesare sequestered, delivered to lysosomes, and digested by lyso-somal hydrolases. In normal cells, autophagy functions tomaintain homeostasis by eliminating excessive or unnecessaryproteins.8 In recent years, the role of autophagy as an alterna-tive cell death mechanism has been a topic of debate. A clearer

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05509h

aDepartment of Environmental and Occupational Health, National Cheng Kung

University, Tainan, Taiwan. E-mail: yjwang@mail.ncku.edu.tw;

Fax: +886-6-2752484; Tel: +886-6-235-3535 ext. 5804bDivision of NanoMedicine, Department of Medicine, University of California,

Los Angeles, California, USAcInstitute of Labor, Occupational Safety and Health, Ministry of Labor, Executive

Yuan, TaiwandInstitute of Clinical Pharmacy and Pharmaceutical Sciences, National Cheng Kung

University, Tainan, Taiwan. E-mail: jctsai@mail.ncku.edu.tw; Fax: +886-6-237-3149;

Tel: +886-6-235-3535 ext. 5689eDivision of Nephrology, Department of Internal Medicine, Shuang Ho Hospital,

Taipei Medical University, TaiwanfGraduate Institute of Clinical Medicine, Taipei Medical University, Taipei, TaiwangDepartment of Biomedical Informatics, Asia University, Taichung, TaiwanhDepartment of Medical Research, China Medical University Hospital, China

Medical University, Taichung, Taiwan

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understanding of the signaling pathways involved in auto-phagy as well as its role in inducing cell death is highly desir-able. Recently, treatment with NPs from various sources hasbeen shown to induce autophagy in cell lines.9–12 However, theroles of NP-induced autophagy in cell survival or cell death arestill unclear. Thus, a better understanding of the implicationand biological significance of PS NP-induced cell death willhelp us understand the risks associated with its uses anddevelop safer nanotechnology.

Among the factors contributing to autophagy, there isincreasing evidence that endoplasmic reticulum (ER) stressplays an important role.13 The ER, which functions in proteinfolding and assembly, lipid biosynthesis, vesicular traffic andcellular calcium storage, is sensitive to alterations in homeo-stasis.14 Proper functioning of the ER is essential to autophagyand cell survival.15 Reactive oxygen species (ROS), misfoldedprotein aggregates, DNA damage, hypoxia and hypocalcemiacan induce ER stress.16,17 If the cells are exposed to prolongedor robust ER stress, they die by apoptosis.18 Previous studieshave demonstrated that the ER stress response in combinationwith autophagy represents an adaptive mechanism for sup-porting cell survival in response to a great variety of detrimen-tal conditions.19 Recently, silver NP-induced apoptosis hasbeen reported to be mediated by the ER stress-signalingpathway.20 Christen and Fent indicated that silica NPs andsilver-doped silica NPs induced the ER stress response, asdemonstrated in induced expression of BiP and splicing ofXBP1 mRNA, two selective markers of ER stress in human livercells.21 However, whether ER stress is induced by PS NPs isstill unknown.

Our previous study has demonstrated that NH2-PS nano-spheres were highly toxic to RAW 264.7 cells (a mouse macro-phage cell line) and BEAS-2B cells (a human bronchialepithelial cell line).6 In this study, RAW 264.7 and BEAS-2B cellswere used to investigate the autophagic effects and ER stressinduced by NH2-PS nanospheres. We examined whether NH2-PS-induced autophagy could serve as a pro-survival or cell deathmechanism. Furthermore, we also examined the signaling path-ways associated with the process of autophagy induced by NH2-PS in RAW 264.7 and BEAS-2B cells. Our data demonstratedclearly the involvement of ER stress and the importance of theAkt/mTOR and AMPK signaling pathways in NH2-PS-inducedautophagy, which served primarily as a pro-death pathway.

2. Experimental2.1 Cell culture and co-incubation with NPs

The mouse macrophage cell line RAW 264.7 (ATCC TIB-71) andthe human bronchial epithelial cell line BEAS-2B (ATCCCRL-9609) were obtained from the American Type Culture Col-lection (ATCC). RAW 264.7 cells were cultured in Dulbecco’smodified essential medium (DMEM) (Gibco BRL, GrandIsland, NY), containing 4.5 g l−1 D-glucose, supplemented withantibiotics containing 100 U ml−1 penicillin, 100 μg ml−1

streptomycin (Gibco BRL, Grand Island, NY) and 10% fetal

bovine serum (FBS) (HyClone, South Logan, UT). BEAS-2B cellswere cultured in LHC-9 serum-free medium (Gibco BRL, GrandIsland, NY). The cells were incubated under a humidifiedatmosphere containing 5% CO2 at 37 °C. Exponentiallygrowing cells were detached with 0.05% trypsin-EDTA (GibcoBRL, Grand Island, NY) in DMEM or LHC-9 medium. PS nano-spheres were obtained from Bangs Laboratory (Fishers, IN).These include 60 nm unmodified (PS), 60 nm carboxylated(COOH-PS) and 60 nm amino-modified (NH2-PS) polystyreneparticles. Fluorescent PS and COOH-PS were obtained fromBangs Laboratory and fluorescent NH2-PS was obtained fromNanocs Inc. (New York, NY). For all experiments and analyses,water was deionized and filtered with 0.45 μm pore size Acro-disc Syringe Filters (Pall Corporation, NY). All of the NP solu-tions were freshly prepared from stock solutions (5 mg mL−1)and sonicated for 30 s before addition to cell cultures.

2.2 Physicochemical characterization

The average hydrodynamic size, zeta potential and polydisper-sity index (PDI) of all NPs were determined by dynamic laserscattering (Delsa™ Nano C, Beckman Coulter, Inc., USA). Thisinstrument is capable of measuring particles in the size rangeof 0.6 nm to 7 μm. The size measurements were performed ondilute NP suspensions in aqueous solution and DMEM(including 10% FBS).

2.3 Cell viability assay

The cellular viability was measured by the MTS assay, whichlooks at the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carb-oxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) toformazan in viable cells. Briefly, cells were plated onto 96-wellplates (Thermo, MA, USA). After incubation with the indicateddose of NPs for various lengths of time at 37 °C, the formazanabsorbance was measured at 490 nm. The mean absorbance ofnon-exposed cells was the reference value for calculating 100%cellular viability.

2.4 PS NP uptake using fluorescence confocal microscopyand flow cytometry

We plated cells onto 6-well plates with a glass coverslip perwell. After PS NP exposure, cells were fixed with 4% para-formaldehyde. After three washes in PBS, the cells were stainedwith 4′-6-diamidino-2-phenylindole (DAPI) (Sigma, MO, USA).Fluorescence confocal images were obtained using a confocalmicroscope (Carl Zeiess LSM780, Instrument DevelopmentCenter, NCKU). The uptake of particles by cells was also ana-lyzed by flow cytometry (Becton Dickinson, San Jose, Califor-nia). The side scatter (SSC) data were analyzed usingCELLQuest™ software (Becton Dickinson). Ten thousand cellswere acquired for each measurement.

2.5 ROS production measurement

To measure ROS generation, a fluorometric assay using theintracellular oxidation of 2,7-dichlorofluorescein diacetate(DCFH-DA, Sigma, MO, USA) was performed.22 The cells weretreated with different concentrations of the NH2-PS nano-

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spheres for 2, 4 or 6 hours and were then incubated with10 μM DCFH-DA for 30 min. After washing with PBS, theDCFH fluorescence of the cells from each well was measuredon a fluorescence microplate reader (Thermo, MA, USA) at anexcitation wavelength of 485 nm and emission at 530 nm. Theintensity of fluorescence reflects the extent of oxidative stress.

2.6 Staining of misfolded proteins

The misfolded proteins were measured using a ProteoStatAggresome Detection Kit (Enzo Life Sciences, NY, USA) accord-ing to the manufacturer’s protocol. Briefly, the cells were fixedwith 4% paraformaldehyde and then the permeabilizing solu-tion was added over 30 min. The cells were washed twice andthe substrate solution was added to each sample over 30 min.Finally, the cells were washed twice and the Dual DetectionReagent was added over 30 min. After staining, the cells wereanalyzed under a fluorescence microscope (Olympus, Japan).

2.7 Staining of ER

The cells were treated with NH2-PS nanospheres. Sixteen hourslater, the ER-Tracker Blue-White DPX (Molecular Probes,Eugene, OR) probe was added to the cells and incubated for30 min under the same growth conditions. The loading solu-tion was removed, and the cells were then washed with PBS.Microscopic images were collected using the fluorescencemicroscope. Cells were pretreated with the ER stress inhibitor,tauroursodeoxycholic acid (TUDCA) (Merck KGaA, Darmstadt,Germany), for 1 h before NH2-PS treatment.

2.8 Immunofluorescence microscopy

The cells were cultured on coverslips. After NH2-PS nanospheretreatment, the cells were fixed in 4% paraformaldehyde andblocked with 1% BSA for 30 min. This was followed by incu-bation with a specific antibody against LC3 (MBL, Japan) for1 h. After washing, the cells were labeled with a DyLight™ 488-conjugated affinipure goat anti-rabbit IgG (Jackson Immuno-Research Laboratories, PA, USA) for 1 h and with DAPI. Finally,the cells were washed in PBS, covered with a coverslip, andexamined with a fluorescence microscope or confocal micro-scope (Carl Zeiess LSM780, Instrument Development Center,NCKU). To quantify LC3-positive cells, a minimum of 50 cellsper sample was counted, and the number of LC3 aggregateswas enumerated. The data are presented as a percentage ofLC3-positive cells within the total of the cells examined.

2.9 Western blot analysis

Total cellular protein lysates were prepared by harvesting cellsin protein extraction buffer for 1 h at 4 °C as described pre-viously.23 The densities of the bands were quantified with acomputer densitometer (AlphaImager™ 2200 System AlphaInnotech Corporation, San Leandro, CA, USA). The expressionof GAPDH was used as the protein loading control. The anti-bodies for detecting Akt, phospho-Akt, phospho-p70S6 K,phospho-AMPK, AMPK and Beclin 1 were obtained from CellSignaling Technology (Ipswich, MA, USA); anti-phospho-mTORwas obtained from Millipore (Billerica, MA, USA); anti-GAPDH

was obtained from Abcam (Cambridge, MA, USA); anti-LC3 was obtained from Abgent (San Diego, CA, USA); anti-p62/SQSTM1 was obtained from MBL (Nagoya, Japan); anti-mTORand anti-p70S6 K were obtained from Epitomics (Burlingame,CA, USA).

2.10 RNA interference (RNAi)

We used the Arrest-In Transfection Reagent (Thermo, MA,USA) to transfect cells according to the manufacturer’s proto-col. RNAi reagents were obtained from the National RNAi CoreFacility located at the Institute of Molecular Biology/GenomicResearch Center, Academia Sinica, supported by the NationalCore Facility Program for Biotechnology Grants of NSC(NSC 100-2319-B-001-002). The human library should bereferred to as TRC-Hs 1.0. Individual clones are identified asshRNA TRCN0000010171, shRNA TRCN0000000859, shRNATRCN0000002168 and shRNA TRCN0000072178.

3. Results and discussion3.1 PS NP characterization

Non-labeled (PS), NH2-PS and carboxy-labeled (COOH-PS) witha primary particle size of 60 nm were purchased from BangsLaboratory. The detailed physicochemical characteristics of PSNPs after suspension in cell culture medium or water are pre-sented in Table 1. Non-labeled (PS), NH2-PS and carboxy-labeled (COOH-PS) NPs have similar hydrodynamic sizes ofapproximately 60 nm in water, confirming their primary par-ticle size as provided by the manufacturer. The zeta potentialof NH2-PS in water is positive (+34.97), whereas plain PS andCOOH-PS have negative charges. As previously reported, all ofthe NPs have negatively charged surfaces in DMEM due to theformation of a corona of negatively charged proteins.6 NPswith a zeta potential above (±)30 mV have been shown to bestable in suspension because the surface charge preventsaggregation of the particles.24 Thus, the zeta potential ofNPs in water indicates that they have surface propertiesthat provide good stability in suspension. All NPs showed arelatively low polydispersity index (PDI): the values for PS,NH2-PS and COOH-PS in water were 0.049, 0.113 and 0.112,respectively. Previous studies have indicated that a PDI lowerthan 0.2 is associated with a high homogeneity in the particlepopulation.25 In summary, these particles formed a stablesuspension in aqueous media.

Table 1 Physical characteristics of PS NPs

Hydrodynamicdiameter (nm) PDIa

Zeta potential(mV)

Water DMEM Water DMEM Water DMEM

PS 61.3 86.7 0.049 0.080 −33.99 −14.01NH2-PS 62.1 206.1 0.113 0.240 +34.97 −12.33COOH-PS 62.0 97.1 0.112 0.143 −40.06 −14.01

a PDI is the polydispersity index.

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3.2 Cytotoxic effects and cellular uptake of PS NPs in RAW264.7 and BEAS-2B cells

To confirm that the observed toxic effects are specificallyrelated to the NH2 substitution on the particle surface, wecompared NH2-PS with COOH-PS and PS nanospheres(Fig. 1A). The results demonstrated that only NH2-PS butnot PS or COOH-PS particles decreased the cell viability.In addition, we further examined the viability of the cellstreated with different doses and time points for NH2-PS andfound that cell death was dependent on both the concen-tration and time in both RAW 264.7 and BEAS-2B cells (Fig. 1Band C). The uptake of NPs by cells is an important factor inthe assessment of their toxicity.26 To evaluate the entry of the

PS NPs into the cells, we used fluorescent PS NPs (Fig. 1D).Confocal microscopic analysis showed that exposure to NH2-PSincreased the fluorescence intensity of both RAW 264.7 andBEAS-2B cells. However, only weak intracellular fluorescencewas observed when the cells were treated with PS or COOH-PS.Furthermore, the side scatter (SSC) intensity analyzed by flowcytometry also confirmed that the NH2-PS was apparentlyengulfed by the BEAS-2B cells, whereas low levels of cellularuptake of PS and COOH-PS were observed (Fig. 1E and F). Con-sistent with our findings, numerous reports in the literatureshow that the rate of uptake is significantly higher for the posi-tively charged NPs than for their negatively charged counter-parts.27,28 Previous studies have demonstrated that surfacefunctionalization of NPs is crucial for particle durability, sus-

Fig. 1 Cell viability detection by the MTS assay and cellular uptake of PS NPs. (A) After stimulation of RAW 264.7 and BEAS-2B cells with 20 μg ml−1

of the different types of PS nanospheres for 16 hours, cell viability was determined using the MTS assay. *, p < 0.05, PS versus control. (B and C) Con-centration- and time-dependent effects of NH2-PS on the viability of RAW and BEAS-2B cells. The cells were treated with 1, 5, 10, 20 or 40 μg ml−1

NH2-PS for 4, 8, 16 or 24 hours. After stimulation with NH2-PS particles, the cells were incubated with the MTS reagent for 30 min, and the absor-bance was measured at 490 nm. All of the MTS values of different doses were normalized according to the control values (no particle exposure),which were regarded as 100% cell viability. (D) Uptake of PS NPs detected by fluorescence confocal microscopy. The different types of PS NPs areshown in red and DAPI (blue) is a nuclei-specific marker. The cells were treated with 20 μg ml−1 PS NPs for 8 hours. (E) The results of SSC light offlow cytometry demonstrated that PS NPs were apparently engulfed by BEAS-2B cells. The cells were treated with PS NPs at 10, 20 or 40 μg ml−1 for8 hours. (F) Quantification of the scatter intensity in BEAS-2B cells with PS NP treatment. The data are presented as the mean ± standard deviationof three independent experiments.

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pensibility in biological media, biocompatibility, and biodistri-bution.29 Recent findings, including ours, have indicated thatcationic particles have more adverse effects than anionicparticles.6,30–32 Moreover, it is known that the cellular toxicityand apoptosis induced by many of these non-viral transfectionsystems can be attributed to their cationic charge and inter-ference in proton pump activities.33,34 The toxicity of cationicNPs has been reported to be related to their enhanced inter-actions with the cell membrane, a feature initially mediated bytheir strong electrostatic attraction to the negatively chargedbilayer.31,35 The currently available evidence indicated thatthe protein corona plays an important role in the interactionsbetween NPs and biological systems.36,37 Living systems usuallyinteract with protein-coated NPs rather than bare NPs, and thestructure, dynamics and stability of the corona can be decisivefactors governing the biological response of cells and organismsto NP exposure.38,39 Furthermore, the cellular uptake of NPs isstrongly affected by the presence and the physicochemical pro-perties of the protein corona around these NPs.37 NPs that carrya positive surface charge attract different protein components ofthe medium from those proteins carrying a negative surfacecharge. Nevertheless, the underlying mechanisms of how cat-ionic NPs disrupt cellular signaling and the role of the proteincorona are still unclear and require further investigation.

3.3 ROS generation, misfolded protein aggregation and ERstress in RAW 264.7 and BEAS-2B cells treated with NH2-PS

Induction of ROS-mediated cell death has been reported in avariety of cells treated with different NPs.8,40,41 Our currentfindings showed higher ROS levels in the RAW 264.7 andBEAS-2B cells exposed to NH2-PS than in the controls (Fig. 2A).The generation of ROS in RAW 264.7 cells was significantlyenhanced by 116%, 127%, 144% and 185% after 6 hours oftreatment with 5, 10, 20 and 40 μg ml−1 of NH2-PS, respecti-vely. Accumulating evidence suggests that ROS directly orindirectly affects ER homeostasis and protein folding.17 There-fore, we further analyzed the misfolded protein aggregation.Hoechst 33342 staining and ProteoStat Aggresome DetectionKit were applied to measure the aggregated protein using afluorescence microscope, through detection of denaturedand/or misfolded protein cargo in fixed and permeabilizedcells.40,42 We found an increased red signal representinghigher levels of misfolded protein aggregates, in the NH2-PS-treated cells (Fig. 2B). However, the red signal did not increaseafter treatment with PS or COOH-PS (Fig. S2A†). ER stress canactivate signaling pathways involved in apoptosis and auto-phagy.13 In mammalian cells, ER stress has been shown tofacilitate the formation of autophagosomes, and induction ofautophagy enables the removal of toxic misfolded proteins.43

Because autophagy has been invoked as a means of cell deathunder higher ER stress,44 we investigated whether the NH2-PScould induce ER stress and subsequent autophagic cell death.Detection of ER stress can be achieved using ER-Tracker Blue-White DPX, an ER-specific dye.45 The results shown in Fig. 2Cdemonstrated that the treatment of the cells with NH2-PS sig-nificantly increased the fluorescence staining intensity of this

dye, suggesting a remarkable induction of ER stress. However,the fluorescence staining intensity did not increase after treat-ment with PS or COOH-PS (Fig. S2B†). Furthermore, ER stressis initiated by three ER transmembrane proteins: RNA-depen-dent protein kinase (PKR)-like ER kinase (PERK), inositol-requiring protein 1 (IRE1) and activating transcription factor 6(ATF6). These three ER stress sensors trigger divergent andconvergent signaling cascades that lead to adaptation or celldeath.46 We therefore measured the expression of ER stress-related proteins in RAW 264.7 and BEAS-2B cells and founddose-dependent increases in the expression of IRE1α in thecells treated with NH2-PS compared with the control cells(Fig. 2D). However, the expression of IRE1α did not changeafter treatment with PS or COOH-PS (Fig. S2C & S2D†). Inaddition, to determine whether ROS generated by NH2-PScontributes directly to ER stress, we applied the anti-oxidantN-acetylcysteine (NAC). Cells pretreated with NAC and then sub-jected to NH2-PS treatment were analyzed to determine ROSlevels. We observed that pretreatment with NAC decreasedNH2-PS-induced ROS and misfolded protein aggregates (Fig. 2Eand F). Additionally, NAC pretreatment significantly attenuatedthe NH2-PS-induced upregulation of IRE1α expression (Fig. 2G).The observation confirms the induction of misfolded proteinaggregation and ER stress via an oxidative stress mechanism.

3.4 Measurement of autophagy in RAW 264.7 and BEAS-2Bcells treated with NH2-PS

Various NPs have been well demonstrated to induce autophagicevents in cultured cells.47,48 Microtubule-associated proteinlight chain 3 (LC3) is widely used to monitor autophagy.49

Thus, we applied fluorescence microscopy to determine thepercentage of cells with punctate LC3 staining (Fig. 3) andfound that NH2-PS increased the LC3 signals in RAW 264.7and BEAS-2B cells in a time- and concentration-dependentmanner. To detect the expression of the autophagic-relatedproteins (LC3, Beclin 1 and p62), we performed western blot-ting with lysates from RAW 264.7 and BEAS-2B cells treatedwith different concentrations of NH2-PS (Fig. 4A). Theexpression levels of the LC3-II, p62 and Beclin 1 proteinsincreased with NH2-PS treatment. However, the levels of LC3-IIdid not change when the cells were exposed to PS or COOH-PS(Fig. S2C & S2D†). In addition, we applied bafilomycin A1(BAF) to inhibit autophagic flux and found elevated levels ofLC3-II in cells treated with NH2-PS but not in those treatedwith PS or COOH-PS (Fig. 4B & S3†), implying that NH2-PS-induced autophagosome synthesis is regulated at a pointupstream of the autophagosome–lysosome fusion. Previousstudies have demonstrated that poly(amidoamine) (PAMAM)dendrimers induced both cytotoxicity and autophagic flux in apanel of human glioma cell lines.50 Interestingly, our previousstudy also found that NH2-PS increased lysosomal permeabili-zation, permitting escape by lysosomal rupture in RAW 264.7cells.6 The dual roles of NH2-PS in both autophagosomesynthesis and damage to the lysosomes, leading to blockage ofautophagosome–lysosome fusion, may explain in part thecomplex role of autophagy in cell death.51 In addition, it is

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Fig. 2 ROS generation, misfolded protein aggregation and ER stress induced by NH2-PS in RAW 264.7 and BEAS-2B cells. (A) Concentration- andtime-dependent quantification of ROS generation in cells that were treated with NH2-PS and were then incubated with DCFH-DA for 30 min. Thefluorescence of the cells was immediately assayed using a fluorescence microplate reader. (B) The cells were treated with NH2-PS at 20 μg ml−1 for16 hours and stained with Hoechst 33342 and then with a ProteoStat Aggresome Detection Kit. The red color and the blue color indicate the fluore-scence of the detected aggregates and stained nuclei, respectively. (C) ER staining enhanced by NH2-PS. The cells were treated with NH2-PS at20 μg ml−1 for 16 hours and were treated with the ER Tracker Blue-White DPX probe for ER staining. (D) Western blotting for IRE1α in RAW 264.7 andBEAS-2B cells. The cells were treated with NH2-PS for 16 hours. (E) Effects of NAC on ROS generation induced by NH2-PS in RAW 264.7 cells. Thecells were pretreated with NAC for 1 h before NH2-PS treatment (20 μg ml−1) for 6 hours. *, p < 0.05, NH2-PS versus NH2-PS + NAC. (F) The cellswere pretreated with NAC (5 mM) for 1 h before NH2-PS at 20 μg ml−1 for 16 hours and stained with Hoechst 33342 and then with a ProteoStatAggresome Detection Kit in RAW 264.7 cells. (G) Western blotting for IRE1α. The cells were pretreated with NAC (5 mM) for 1 h before NH2-PS treat-ment (20 μg ml−1) for 16 hours. The data are presented as the mean ± standard deviation of three independent experiments.

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worth mentioning that gold nanorods (AuNRs) can escapefrom the lysosomes occasionally and the escaped AuNRs arerecycled back into the lysosomal system through autophagy.52

Recent evidence shows that NPs not only enter cells but alsotrigger a disturbance of intracellular component. It has beenreported that NPs caused massive disruption of the intracellu-lar microtubule assembly and thereby led to limited cell moti-lity.53 Furthermore, NPs can damage mitochondria and causelysosomal dysfunction, and then induce toxicity.54,55 There-fore, PS NPs may induce autophagy through the disruption ofthe intracellular organelles. In addition, apoptosis and necro-sis were examined using Annexin V-FITC/propidium iodidestaining. Using this technique, we found that NH2-PS not onlyinduced autophagy but also apoptosis and necrosis (Fig. S1†).Pretreatment with a pan-caspase inhibitor, Z-VAD, significantlyprevented cell death (Fig. S1E†).

Next, we used 3-methyladenine (3-MA), an inhibitor ofautophagy, to determine whether inhibition of autophagy

alters NH2-PS-induced cytotoxicity. The results revealed a sig-nificant decrease in LC3-II expression and cytotoxicity in cellswhen the combined treatment was compared with NH2-PStreatment alone (Fig. 4C and D). Our observations paralleledthat of other investigators.50 On the one hand, autophagy playsa role as a cell survival mechanism that allows cells to removedamaged cytoplasmic proteins and organelles through lyso-somal degradation and thus to survive metabolic stress.56 On theother hand, autophagy has also been found to contribute toprogrammed cell death in response to various stimuli.57 It isnow known that autophagy can promote cell death by theselective removal of survival factors and/or prolonged removalof cellular constituents, resulting in the demise of cells.58 Pre-vious studies have indicated that the autophagy triggered by avariety of NPs might be due in part to the early adaptiveresponses to stress. However, these responses might sub-sequently lead to cytotoxicity.8,9,48,59,60 In our current study,the role of autophagy as a contributor to cell death was further

Fig. 3 Immunofluorescence staining of the LC3 protein in RAW 264.7 and BEAS-2B cells treated with NH2-PS. (A and B) Representative cell imagesshowing punctate LC3 distribution after NH2-PS treatment. The cells were treated with 20 μg ml−1 of NH2-PS for 16 hours and were then examinedusing a confocal microscope. (C and D) Concentration dependence of NH2-PS-induced punctate LC3 staining in RAW and BEAS-2B cells. The cellswere incubated with 0–40 μg ml−1 NH2-PS for 16 hours. (E and F) The time course of NH2-PS-induced punctate LC3 staining in RAW and BEAS-2Bcells. The cells were incubated with 20 μg ml−1 NH2-PS for 0–24 hours. *, p < 0.05, NH2-PS versus control. The data are presented as the mean ±standard deviation of three independent experiments.

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confirmed by the autophagy inhibitor 3-MA. The resultsdemonstrated that 3-MA has a protective effect on NH2-PS-induced cell death (Fig. 4D), suggesting that autophagy plays apro-death role in this case.

3.5 The Akt/mTOR and AMPK signaling pathways areinvolved in the NH2-PS-induced autophagy in RAW 264.7 andBEAS-2B cells

Previous studies have demonstrated that the Akt/mammaliantarget of rapamycin (mTOR) and the adenosine monophos-

phate-activated protein kinase (AMPK) pathway are involved inregulating autophagy.61 Therefore, to investigate whether theAkt/mTOR and AMPK signaling pathways were involved in theNH2-PS-induced autophagy, we performed western blotting toevaluate the protein phosphorylation status (Fig. 5). Theresults indicated that phosphorylation of Akt, mTOR andp70S6 K decreased and phosphorylation of AMPK increased incells treated with NH2-PS compared with the control in bothRAW 264.7 and BEAS-2B cells. However, the expression of theproteins of the Akt/mTOR and AMPK signaling pathways didnot change in the RAW 264.7 or BEAS-2B cells treated with PSor COOH-PS (Fig. S4A & S4B†). In our previous study, weobserved a steep decline in the cellular ATP levels in cellstreated with NH2-PS.

6 Activation of AMPK has been observedto increase with ROS generation and ATP depletion.8,62 Undervarious stresses, such as ATP depletion, AMPK is activated byincreased catabolism. Our current findings further revealedthat ATP depletion by NH2-PS could enhance the phosphoryl-ation of AMPK in RAW 264.7 and BEAS-2B cells, which trig-gered autophagy via the suppression of mTOR.62

Next, we investigated whether the inhibition of Akt andAMPK could change the levels of autophagic cell deathinduced by NH2-PS. We applied the shRNA technology toinhibit either Akt or AMPK expression. As shown in Fig. 6Aand B, the expression of the Akt and AMPK proteins was mark-edly decreased by Akt and AMPK shRNA, respectively, com-pared with those treated with control shRNA. We thenexamined whether the reduced expression of Akt or AMPKaltered the NH2-PS-induced cytotoxicity (Fig. 6C). Transfectionwith Akt shRNA significantly enhanced the cytotoxicityinduced by the NH2-PS treatment. Transfection with AMPKshRNA significantly reduced the toxic effect of the NH2-PStreatment in BEAS-2B cells. Furthermore, BEAS-2B cells trans-

Fig. 4 Autophagic cell death and autophagic flux induced by NH2-PS inRAW 264.7 and BEAS-2B cells. (A) Western blotting for LC3-I, LC3-II,p62/SQSTM1 and Beclin 1. The cells were treated with 0–40 μg ml−1

NH2-PS for 16 hours. (B) Autophagic flux was determined by westernblotting with anti-LC3 antibody. The cells were pretreated with bafilo-mycin A1 (BAF) (10 nM) for 1 h before NH2-PS treatment (20 μg ml−1) for16 hours. (C) Western blotting of LC3-I and LC3-II expression in theabsence or presence of 3-methyladenine (3-MA). (D) Cytotoxic effectsin the absence or presence of 3-MA. The cells were pretreated with3-MA (3 mM) for 1 h before NH2-PS treatment (20 μg ml−1) for 16 hours.*, p < 0.05, NH2-PS versus NH2-PS + 3-MA. The data are presented asthe mean ± standard deviation of three independent experiments.

Fig. 5 Akt/mTOR (A) and AMPK (B) signaling pathway proteinexpression in RAW 264.7 and BEAS-2B cells treated with NH2-PS. Thecells were treated with 0–40 μg ml−1 NH2-PS for 16 hours.

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fected with AMPK shRNA showed a significant decrease in thepercentage of LC3 punctate cells compared with the NH2-PStreatment, whereas cells transfected with Akt shRNA showed asignificant increase in the percentage of LC3 punctate cells(Fig. 6D). These results suggest that one of the mechanisms ofNH2-PS-induced autophagy could be mediated by inhibition ofAkt/mTOR and activation of AMPK signaling pathways in RAW264.7 and BEAS-2B cells. Recent studies have found that theAkt/mTOR and AMPK pathways play crucial roles in the regu-lation of both apoptosis and autophagy.63,64 Akt can activatemTOR and lead to inhibition of autophagy.65 By contrast,AMPK is a universal autophagy activator.56 It has recently beenreported that functionalized single-walled carbon nanotubesinduced autophagic cell death in human lung cells throughAkt-TSC2-mTOR signaling.11 However, the role of the AMPKpathway in the activation of autophagy in cells treated withNPs has not previously been reported. To our knowledge, thisis the first study demonstrating that NH2-PS exposure leads toautophagy induction through inhibition of the Akt/mTOR andactivation of the AMPK signaling pathways.

3.6 Inhibition of ER stress decreases cytotoxicity andautophagy

To test whether NH2-PS-induced ER stress contributes directlyto cell death, we treated BEAS-2B cells with the chemicalchaperone tauroursodeoxycholic acid (TUDCA), which isknown to selectively inhibit ER stress.66 The results indicatedthat TUDCA treatment decreased NH2-PS-induced cytotoxicityand autophagy (Fig. 7A and B). Next, we examined whetherinhibition of ER stress altered the NH2-PS-induced inhibition

of the Akt/mTOR and the activation of the AMPK signalingpathway. We found that pretreatment with TUDCA retardedthe NH2-PS-decreased expression of Akt phosphorylationand NH2-PS-increased expression of AMPK phosphorylation(Fig. 7C). Inhibition of ER stress has recently been suggestedas a novel therapeutic strategy in various diseases.66,67 Forexample, TUDCA may inhibit apoptosis by ameliorating ERstress through the modulation of intracellular calcium andthus attenuate liver cell death.68 In addition, TUDCA treatmentwas reported to attenuate tunicamycin-induced ER stress,autophagy and cell death in rat hepatocytes,69 implying thatautophagy was involved, at least in part, in ER stress-inducedcell death. Our current results showed that TUDCA increasedAkt activation in NH2-PS-treated macrophage and lung epi-thelial cells. Consistent with these results, TUDCA was foundto activate Akt in skeletal muscles and myocardium.70,71 Themechanisms through which TUDCA increases Akt activationare still unclear. One of the possible mechanisms may be aG-protein coupled signal cascade.72 Moreover, ER stress leadsto a release of calcium and subsequent activation of AMPKthat inhibits mTOR, thereby promoting autophagy.73 Thus,NH2-PS-induced ER stress and AMPK activation found in ourcurrent study might play important roles in autophagy induc-tion and cell death.

Fig. 6 The Akt/mTOR and AMPK signaling pathways are involved inNH2-PS-induced autophagy in BEAS-2B cells. (A and B) Western blottingfor Akt or AMPK. The cells were transfected with control, Akt or AMPKshRNA for 48 hours. (C) Measurement of LC3-punctate cells in theabsence or presence of Akt or AMPK shRNA. (D) Cytotoxic effects in theabsence or presence of Akt shRNA or AMPK shRNA. The cells weretransfected with control, Akt or AMPK shRNA for 48 hours and werethen incubated with 20 μg ml−1 of NH2-PS for 16 hours. *, p < 0.05versus control shRNA + NH2-PS.

Fig. 7 Measurement of autophagy and cytotoxic effects in BEAS-2Bcells pretreated with TUDCA. (A) Effects of TUDCA on cytotoxicityinduced by NH2-PS. The cells were pretreated with TUDCA for 2 hoursbefore NH2-PS treatment (20 μg ml−1) for 16 hours. *, p < 0.05, NH2-PSversus NH2-PS + TUDCA. (B) Measurement of LC3-punctate cells in theabsence or presence of TUDCA. The cells were pretreated with TUDCA(1 mM) for 2 hours before NH2-PS treatment (20 μg ml−1) for 16 hours.*, p < 0.05, NH2-PS versus NH2-PS + TUDCA. The data are presented asthe mean ± standard deviation of three independent experiments. (C)Western blotting for phosphorylation of Akt, phosphorylation of AMPK,Akt and AMPK. The cells were pretreated with TUDCA (1 mM) for 2 hoursbefore NH2-PS treatment (20 μg ml−1) for 16 hours.

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4. Conclusions

Taken together, our results indicate that NH2-PS increasedROS generation and produced misfolded protein aggregates,which lead to ER stress that results in autophagic cell death inRAW 264.7 and BEAS-2B cells. TUDCA, an ER stress inhibitor,decreased cytotoxicity and autophagy in the NH2-PS-treatedcells. Furthermore, NH2-PS-induced autophagic cell death maybe mediated primarily through the inhibition of the Akt/mTOR signaling pathway and the activation of the AMPK sig-naling pathway. Specifically, cell viability in the NH2-PS treat-ment group was significantly increased when autophagy wasinhibited by the autophagy inhibitor 3-MA compared withcells treated with the vehicle, suggesting that autophagymay serve a pro-death role in NH2-PS-induced cell death.In addition, NH2-PS increased the autophagic flux. Thus, inaddition to apoptosis and necrosis, autophagy should be con-sidered as a potential cell death mechanism, providing intra-cellular selectivity for exposure to NH2-PS nanospheres.

Acknowledgements

This study was supported by the Food and Drug Adminis-tration, Ministry of Health and Welfare, Executive Yuan(DOH101-FDA-41301, DOH102-FDA-41702 and MOHW103-FDA-41407) and Ministry of Labor, Executive Yuan, Taiwan(1013080, 1023038 and 1033059). T.X. was supported by USPublic Health Service Grant U19 ES019528 and NSF/EPA DBI0830117 and 1266377. The Instrument Development Center ofthe National Cheng Kung University (NCKU) provided techni-cal support with the Carl Zeiess LSM780 laser-scanningmicroscope.

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