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Cationic polystyrene nanospheres induce autophagic cell death through the

induction of endoplasmic reticulum stress

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DOI: 10.1039/c4nr05509h · Source: PubMed

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Cationic polystyrene nanospheres induce

Cite this: Nanoscale, 2015, 7, 736
autophagic cell death through the induction of
endoplasmic reticulum stress†
Hui-Wen Chiu,a,e,f Tian Xia,b Yu-Hsuan Lee,a Chun-Wan Chen,c Jui-Chen Tsai*d and
Ying-Jan Wang*a,g,h

Received 21st September 2014,
Accepted 9th November 2014
DOI: 10.1039/c4nr05509h
www.rsc.org/nanoscale
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 systems. However, the understanding of the interactions of

Applications of nanoparticles (NPs) are prevalent and continue
to grow rapidly. Industrial and commercial applications of
NPs include catalysis, sensors, environmental remediation,
personal care products and cosmetics, and NPs show great
promise in the field of medicine, including imaging and drug
delivery.1 The development of nanotechnology calls for a com-
prehensive understanding of the impact of NPs on biological
a
Department 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. 5804
b
Division of NanoMedicine, Department of Medicine, University of California,
Los Angeles, California, USA
c
Institute of Labor, Occupational Safety and Health, Ministry of Labor, Executive
Yuan, Taiwan
d
Institute 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. 5689
e
Division of Nephrology, Department of Internal Medicine, Shuang Ho Hospital,
Taipei Medical University, Taiwan
f
Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan
g
Department of Biomedical Informatics, Asia University, Taichung, Taiwan
h
Department of Medical Research, China Medical University Hospital, China
Medical University, Taichung, Taiwan
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4nr05509h
NPs 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 as
in photonics and self-assembling nanostructures.3,4 Specific-
surface modification, high drug-loading capacity and colloidal
stability in biological media also contribute to their appli-
cation as experimental drug carrier systems.5 Our group has
shown that NH2-labeled PS (NH2-PS) nanospheres can induce
cell death in macrophage and lung epithelial cells with apop-
totic and necrotic features, respectively.6 Furthermore, we
found that macrophage cells used an endosomal–lysosomal
route of uptake, while lung epithelial cells used a caveolar
uptake mechanism.6 Considering the proposed nanomedical
applications of PS NPs, an evaluation of these processes
becomes even more important as these processes may result in
potentially 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 protein
degradation system in which cellular proteins and organelles
are sequestered, delivered to lysosomes, and digested by lyso-
somal hydrolases. In normal cells, autophagy functions to
maintain homeostasis by eliminating excessive or unnecessary
proteins.8 In recent years, the role of autophagy as an alterna-
tive cell death mechanism has been a topic of debate. A clearer

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Nanoscale
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 has
been shown to induce autophagy in cell lines.9–12 However, the
roles of NP-induced autophagy in cell survival or cell death are
still unclear. Thus, a better understanding of the implication
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and biological significance of PS NP-induced cell death will
help us understand the risks associated with its uses and
develop safer nanotechnology.
Among the factors contributing to autophagy, there is
increasing evidence that endoplasmic reticulum (ER) stress
plays an important role.13 The ER, which functions in protein
folding and assembly, lipid biosynthesis, vesicular traffic and
cellular calcium storage, is sensitive to alterations in homeo-
stasis.14 Proper functioning of the ER is essential to autophagy
and cell survival.15 Reactive oxygen species (ROS), misfolded
protein aggregates, DNA damage, hypoxia and hypocalcemia
can induce ER stress.16,17 If the cells are exposed to prolonged
or robust ER stress, they die by apoptosis.18 Previous studies
have demonstrated that the ER stress response in combination
with 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 has
been reported to be mediated by the ER stress-signaling
pathway.20 Christen and Fent indicated that silica NPs and
silver-doped silica NPs induced the ER stress response, as
demonstrated in induced expression of BiP and splicing of
XBP1 mRNA, two selective markers of ER stress in human liver
cells.21 However, whether ER stress is induced by PS NPs is
still 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 bronchial
epithelial cell line).6 In this study, RAW 264.7 and BEAS-2B cells
were used to investigate the autophagic effects and ER stress
induced by NH2-PS nanospheres. We examined whether NH2-
PS-induced autophagy could serve as a pro-survival or cell death
mechanism. 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 demonstrated
clearly the involvement of ER stress and the importance of the
Akt/mTOR and AMPK signaling pathways in NH2-PS-induced
autophagy, which served primarily as a pro-death pathway.
2. Experimental
2.1 Cell culture and co-incubation with NPs
The mouse macrophage cell line RAW 264.7 (ATCC TIB-71) and
the human bronchial epithelial cell line BEAS-2B (ATCC
CRL-9609) were obtained from the American Type Culture Col-
lection (ATCC). RAW 264.7 cells were cultured in Dulbecco’s
modified essential medium (DMEM) (Gibco BRL, Grand
Island, NY), containing 4.5 g l−1 D-glucose, supplemented with
antibiotics containing 100 U ml−1 penicillin, 100 μg ml−1
streptomycin (Gibco BRL, Grand Island, NY) and 10% fetal
This journal is © The Royal Society of Chemistry 2015
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Paper
bovine serum (FBS) (HyClone, South Logan, UT). BEAS-2B cells
were cultured in LHC-9 serum-free medium (Gibco BRL, Grand
Island, NY). The cells were incubated under a humidified
atmosphere containing 5% CO2 at 37 °C. Exponentially
growing cells were detached with 0.05% trypsin-EDTA (Gibco
BRL, 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) polystyrene
particles. Fluorescent PS and COOH-PS were obtained from
Bangs Laboratory and fluorescent NH2-PS was obtained from
Nanocs 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 laser
scattering (Delsa™ Nano C, Beckman Coulter, Inc., USA). This
instrument is capable of measuring particles in the size range
of 0.6 nm to 7 μm. The size measurements were performed on
dilute NP suspensions in aqueous solution and DMEM
(including 10% FBS).
2.3 Cell viability assay
The cellular viability was measured by the MTS assay, which
looks at the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carb-
oxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) to
formazan in viable cells. Briefly, cells were plated onto 96-well
plates (Thermo, MA, USA). After incubation with the indicated
dose of NPs for various lengths of time at 37 °C, the formazan
absorbance was measured at 490 nm. The mean absorbance of
non-exposed cells was the reference value for calculating 100%
cellular viability.
2.4 PS NP uptake using fluorescence confocal microscopy
and flow cytometry
We plated cells onto 6-well plates with a glass coverslip per
well. After PS NP exposure, cells were fixed with 4% para-
formaldehyde. After three washes in PBS, the cells were stained
with 4′-6-diamidino-2-phenylindole (DAPI) (Sigma, MO, USA).
Fluorescence confocal images were obtained using a confocal
microscope (Carl Zeiess LSM780, Instrument Development
Center, 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 using
CELLQuest™ software (Becton Dickinson). Ten thousand cells
were acquired for each measurement.
2.5 ROS production measurement
To measure ROS generation, a fluorometric assay using the
intracellular oxidation of 2,7-dichlorofluorescein diacetate
(DCFH-DA, Sigma, MO, USA) was performed.22 The cells were
treated with different concentrations of the NH2-PS nano-
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spheres for 2, 4 or 6 hours and were then incubated with
10 μM DCFH-DA for 30 min. After washing with PBS, the
DCFH fluorescence of the cells from each well was measured
on a fluorescence microplate reader (Thermo, MA, USA) at an
excitation wavelength of 485 nm and emission at 530 nm. The
intensity of fluorescence reflects the extent of oxidative stress.
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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-mTOR
and anti-p70S6 K were obtained from Epitomics (Burlingame,
CA, USA).

2.10 RNA interference (RNAi)

2.6 Staining of misfolded proteins
The misfolded proteins were measured using a ProteoStat
Aggresome Detection Kit (Enzo Life Sciences, NY, USA) accord-
ing to the manufacturer’s protocol. Briefly, the cells were fixed
with 4% paraformaldehyde and then the permeabilizing solu-
tion was added over 30 min. The cells were washed twice and
the substrate solution was added to each sample over 30 min.
Finally, the cells were washed twice and the Dual Detection
Reagent was added over 30 min. After staining, the cells were
analyzed under a fluorescence microscope (Olympus, Japan).
2.7 Staining of ER
The cells were treated with NH2-PS nanospheres. Sixteen hours
later, the ER-Tracker Blue-White DPX (Molecular Probes,
Eugene, OR) probe was added to the cells and incubated for
30 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 fluorescence
microscope. 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 nanosphere
treatment, the cells were fixed in 4% paraformaldehyde and
blocked with 1% BSA for 30 min. This was followed by incu-
bation with a specific antibody against LC3 (MBL, Japan) for
1 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, and
examined with a fluorescence microscope or confocal micro-
scope (Carl Zeiess LSM780, Instrument Development Center,
NCKU). To quantify LC3-positive cells, a minimum of 50 cells
per sample was counted, and the number of LC3 aggregates
was enumerated. The data are presented as a percentage of
LC3-positive cells within the total of the cells examined.
2.9 Western blot analysis
Total cellular protein lysates were prepared by harvesting cells
in protein extraction buffer for 1 h at 4 °C as described pre-
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 Core
Facility located at the Institute of Molecular Biology/Genomic
Research Center, Academia Sinica, supported by the National
Core Facility Program for Biotechnology Grants of NSC
(NSC 100-2319-B-001-002). The human library should be
referred to as TRC-Hs 1.0. Individual clones are identified as
shRNA TRCN0000010171, shRNA TRCN0000000859, shRNA
TRCN0000002168 and shRNA TRCN0000072178.
3. Results and discussion
3.1 PS NP characterization
Non-labeled (PS), NH2-PS and carboxy-labeled (COOH-PS) with
a primary particle size of 60 nm were purchased from Bangs
Laboratory. The detailed physicochemical characteristics of PS
NPs 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 of
approximately 60 nm in water, confirming their primary par-
ticle size as provided by the manufacturer. The zeta potential
of NH2-PS in water is positive (+34.97), whereas plain PS and
COOH-PS have negative charges. As previously reported, all of
the NPs have negatively charged surfaces in DMEM due to the
formation of a corona of negatively charged proteins.6 NPs
with a zeta potential above (±)30 mV have been shown to be
stable in suspension because the surface charge prevents
aggregation of the particles.24 Thus, the zeta potential of
NPs in water indicates that they have surface properties
that provide good stability in suspension. All NPs showed a
relatively 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 lower
than 0.2 is associated with a high homogeneity in the particle
population.25 In summary, these particles formed a stable
suspension in aqueous media.
Table 1 Physical characteristics of PS NPs

viously.23 The densities of the bands were quantified with a Hydrodynamic Zeta potential
computer densitometer (AlphaImager™ 2200 System Alpha diameter (nm) PDIa (mV)
Innotech Corporation, San Leandro, CA, USA). The expression
Water DMEM Water DMEM Water DMEM
of GAPDH was used as the protein loading control. The anti-
bodies for detecting Akt, phospho-Akt, phospho-p70S6 K, PS 61.3 86.7 0.049 0.080 −33.99 −14.01
phospho-AMPK, AMPK and Beclin 1 were obtained from Cell NH2-PS 62.1 206.1 0.113 0.240 +34.97 −12.33
COOH-PS 62.0 97.1 0.112 0.143 −40.06 −14.01
Signaling Technology (Ipswich, MA, USA); anti-phospho-mTOR
a
was obtained from Millipore (Billerica, MA, USA); anti-GAPDH PDI is the polydispersity index.

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3.2 Cytotoxic effects and cellular uptake of PS NPs in RAW
264.7 and BEAS-2B cells
To confirm that the observed toxic effects are specifically
related to the NH2 substitution on the particle surface, we
compared NH2-PS with COOH-PS and PS nanospheres
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PS NPs into the cells, we used fluorescent PS NPs (Fig. 1D).
Confocal microscopic analysis showed that exposure to NH2-PS
increased the fluorescence intensity of both RAW 264.7 and
BEAS-2B cells. However, only weak intracellular fluorescence
was observed when the cells were treated with PS or COOH-PS.
Furthermore, the side scatter (SSC) intensity analyzed by flow

(Fig. 1A). The results demonstrated that only NH2-PS but
not PS or COOH-PS particles decreased the cell viability.
In addition, we further examined the viability of the cells
treated with different doses and time points for NH2-PS and
found that cell death was dependent on both the concen-
tration and time in both RAW 264.7 and BEAS-2B cells (Fig. 1B
and C). The uptake of NPs by cells is an important factor in
the assessment of their toxicity.26 To evaluate the entry of the
cytometry also confirmed that the NH2-PS was apparently
engulfed by the BEAS-2B cells, whereas low levels of cellular
uptake of PS and COOH-PS were observed (Fig. 1E and F). Con-
sistent with our findings, numerous reports in the literature
show 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 surface
functionalization 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 are
shown 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 of
flow 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 for
8 hours. (F) Quantification of the scatter intensity in BEAS-2B cells with PS NP treatment. The data are presented as the mean ± standard deviation
of three independent experiments.

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pensibility in biological media, biocompatibility, and biodistri-
bution.29 Recent findings, including ours, have indicated that
cationic particles have more adverse effects than anionic
particles.6,30–32 Moreover, it is known that the cellular toxicity
and apoptosis induced by many of these non-viral transfection
systems can be attributed to their cationic charge and inter-
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ference in proton pump activities.33,34 The toxicity of cationic
NPs has been reported to be related to their enhanced inter-
actions with the cell membrane, a feature initially mediated by
their strong electrostatic attraction to the negatively charged
bilayer.31,35 The currently available evidence indicated that
the protein corona plays an important role in the interactions
between NPs and biological systems.36,37 Living systems usually
interact with protein-coated NPs rather than bare NPs, and the
structure, dynamics and stability of the corona can be decisive
factors governing the biological response of cells and organisms
to NP exposure.38,39 Furthermore, the cellular uptake of NPs is
strongly affected by the presence and the physicochemical pro-
perties of the protein corona around these NPs.37 NPs that carry
a positive surface charge attract different protein components of
the medium from those proteins carrying a negative surface
charge. Nevertheless, the underlying mechanisms of how cat-
ionic NPs disrupt cellular signaling and the role of the protein
corona are still unclear and require further investigation.
3.3 ROS generation, misfolded protein aggregation and ER
stress in RAW 264.7 and BEAS-2B cells treated with NH2-PS
Induction of ROS-mediated cell death has been reported in a
variety of cells treated with different NPs.8,40,41 Our current
findings showed higher ROS levels in the RAW 264.7 and
BEAS-2B cells exposed to NH2-PS than in the controls (Fig. 2A).
The generation of ROS in RAW 264.7 cells was significantly
enhanced by 116%, 127%, 144% and 185% after 6 hours of
treatment with 5, 10, 20 and 40 μg ml−1 of NH2-PS, respecti-
vely. Accumulating evidence suggests that ROS directly or
indirectly affects ER homeostasis and protein folding.17 There-
fore, we further analyzed the misfolded protein aggregation.
Hoechst 33342 staining and ProteoStat Aggresome Detection
Kit were applied to measure the aggregated protein using a
fluorescence microscope, through detection of denatured
and/or misfolded protein cargo in fixed and permeabilized
cells.40,42 We found an increased red signal representing
higher levels of misfolded protein aggregates, in the NH2-PS-
treated cells (Fig. 2B). However, the red signal did not increase
after treatment with PS or COOH-PS (Fig. S2A†). ER stress can
activate signaling pathways involved in apoptosis and auto-
phagy.13 In mammalian cells, ER stress has been shown to
facilitate the formation of autophagosomes, and induction of
autophagy enables the removal of toxic misfolded proteins.43
Because autophagy has been invoked as a means of cell death
under higher ER stress,44 we investigated whether the NH2-PS
could 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. 2C
demonstrated that the treatment of the cells with NH2-PS sig-
nificantly increased the fluorescence staining intensity of this
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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 stress
is 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 and
convergent signaling cascades that lead to adaptation or cell
death.46 We therefore measured the expression of ER stress-
related proteins in RAW 264.7 and BEAS-2B cells and found
dose-dependent increases in the expression of IRE1α in the
cells treated with NH2-PS compared with the control cells
(Fig. 2D). However, the expression of IRE1α did not change
after treatment with PS or COOH-PS (Fig. S2C & S2D†). In
addition, to determine whether ROS generated by NH2-PS
contributes directly to ER stress, we applied the anti-oxidant
N-acetylcysteine (NAC). Cells pretreated with NAC and then sub-
jected to NH2-PS treatment were analyzed to determine ROS
levels. We observed that pretreatment with NAC decreased
NH2-PS-induced ROS and misfolded protein aggregates (Fig. 2E
and F). Additionally, NAC pretreatment significantly attenuated
the NH2-PS-induced upregulation of IRE1α expression (Fig. 2G).
The observation confirms the induction of misfolded protein
aggregation and ER stress via an oxidative stress mechanism.
3.4 Measurement of autophagy in RAW 264.7 and BEAS-2B
cells treated with NH2-PS
Various NPs have been well demonstrated to induce autophagic
events in cultured cells.47,48 Microtubule-associated protein
light chain 3 (LC3) is widely used to monitor autophagy.49
Thus, we applied fluorescence microscopy to determine the
percentage of cells with punctate LC3 staining (Fig. 3) and
found that NH2-PS increased the LC3 signals in RAW 264.7
and BEAS-2B cells in a time- and concentration-dependent
manner. To detect the expression of the autophagic-related
proteins (LC3, Beclin 1 and p62), we performed western blot-
ting with lysates from RAW 264.7 and BEAS-2B cells treated
with different concentrations of NH2-PS (Fig. 4A). The
expression levels of the LC3-II, p62 and Beclin 1 proteins
increased with NH2-PS treatment. However, the levels of LC3-II
did 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 of
LC3-II in cells treated with NH2-PS but not in those treated
with PS or COOH-PS (Fig. 4B & S3†), implying that NH2-PS-
induced autophagosome synthesis is regulated at a point
upstream of the autophagosome–lysosome fusion. Previous
studies have demonstrated that poly(amidoamine) (PAMAM)
dendrimers induced both cytotoxicity and autophagic flux in a
panel of human glioma cell lines.50 Interestingly, our previous
study also found that NH2-PS increased lysosomal permeabili-
zation, permitting escape by lysosomal rupture in RAW 264.7
cells.6 The dual roles of NH2-PS in both autophagosome
synthesis and damage to the lysosomes, leading to blockage of
autophagosome–lysosome fusion, may explain in part the
complex 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- and
time-dependent quantification of ROS generation in cells that were treated with NH2-PS and were then incubated with DCFH-DA for 30 min. The
fluorescence of the cells was immediately assayed using a fluorescence microplate reader. (B) The cells were treated with NH2-PS at 20 μg ml−1 for
16 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 at
20 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 and
BEAS-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. The
cells 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 cells
were 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 ProteoStat
Aggresome 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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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 images
showing 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 examined
using a confocal microscope. (C and D) Concentration dependence of NH2-PS-induced punctate LC3 staining in RAW and BEAS-2B cells. The cells
were 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-2B
cells. 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.

worth mentioning that gold nanorods (AuNRs) can escape
from the lysosomes occasionally and the escaped AuNRs are
recycled back into the lysosomal system through autophagy.52
Recent evidence shows that NPs not only enter cells but also
trigger a disturbance of intracellular component. It has been
reported 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 cause
lysosomal dysfunction, and then induce toxicity.54,55 There-
fore, PS NPs may induce autophagy through the disruption of
the intracellular organelles. In addition, apoptosis and necro-
sis were examined using Annexin V-FITC/propidium iodide
staining. Using this technique, we found that NH2-PS not only
induced autophagy but also apoptosis and necrosis (Fig. S1†).
Pretreatment with a pan-caspase inhibitor, Z-VAD, significantly
prevented cell death (Fig. S1E†).
Next, we used 3-methyladenine (3-MA), an inhibitor of
autophagy, 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 cells
when the combined treatment was compared with NH2-PS
treatment alone (Fig. 4C and D). Our observations paralleled
that of other investigators.50 On the one hand, autophagy plays
a role as a cell survival mechanism that allows cells to remove
damaged cytoplasmic proteins and organelles through lyso-
somal degradation and thus to survive metabolic stress.56 On the
other hand, autophagy has also been found to contribute to
programmed cell death in response to various stimuli.57 It is
now known that autophagy can promote cell death by the
selective removal of survival factors and/or prolonged removal
of cellular constituents, resulting in the demise of cells.58 Pre-
vious studies have indicated that the autophagy triggered by a
variety of NPs might be due in part to the early adaptive
responses 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

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Fig. 4 Autophagic cell death and autophagic flux induced by NH2-PS in
RAW 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 western
blotting 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) for
16 hours. (C) Western blotting of LC3-I and LC3-II expression in the
absence or presence of 3-methyladenine (3-MA). (D) Cytotoxic effects
in the absence or presence of 3-MA. The cells were pretreated with
3-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 as
the mean ± standard deviation of three independent experiments.
confirmed by the autophagy inhibitor 3-MA. The results
demonstrated that 3-MA has a protective effect on NH2-PS-
induced cell death (Fig. 4D), suggesting that autophagy plays a
pro-death role in this case.
3.5 The Akt/mTOR and AMPK signaling pathways are
involved in the NH2-PS-induced autophagy in RAW 264.7 and
BEAS-2B cells
Previous studies have demonstrated that the Akt/mammalian
target of rapamycin (mTOR) and the adenosine monophos-
This journal is © The Royal Society of Chemistry 2015
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Paper
phate-activated protein kinase (AMPK) pathway are involved in
regulating autophagy.61 Therefore, to investigate whether the
Akt/mTOR and AMPK signaling pathways were involved in the
NH2-PS-induced autophagy, we performed western blotting to
evaluate the protein phosphorylation status (Fig. 5). The
results indicated that phosphorylation of Akt, mTOR and
p70S6 K decreased and phosphorylation of AMPK increased in
cells treated with NH2-PS compared with the control in both
RAW 264.7 and BEAS-2B cells. However, the expression of the
proteins of the Akt/mTOR and AMPK signaling pathways did
not change in the RAW 264.7 or BEAS-2B cells treated with PS
or COOH-PS (Fig. S4A & S4B†). In our previous study, we
observed a steep decline in the cellular ATP levels in cells
treated with NH2-PS.6 Activation of AMPK has been observed
to increase with ROS generation and ATP depletion.8,62 Under
various stresses, such as ATP depletion, AMPK is activated by
increased catabolism. Our current findings further revealed
that 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 and
AMPK could change the levels of autophagic cell death
induced by NH2-PS. We applied the shRNA technology to
inhibit either Akt or AMPK expression. As shown in Fig. 6A
and 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 then
examined whether the reduced expression of Akt or AMPK
altered the NH2-PS-induced cytotoxicity (Fig. 6C). Transfection
with Akt shRNA significantly enhanced the cytotoxicity
induced by the NH2-PS treatment. Transfection with AMPK
shRNA significantly reduced the toxic effect of the NH2-PS
treatment in BEAS-2B cells. Furthermore, BEAS-2B cells trans-
Fig. 5 Akt/mTOR (A) and AMPK (B) signaling pathway protein
expression in RAW 264.7 and BEAS-2B cells treated with NH2-PS. The
cells were treated with 0–40 μg ml−1 NH2-PS for 16 hours.
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Fig. 6 The Akt/mTOR and AMPK signaling pathways are involved in
NH2-PS-induced autophagy in BEAS-2B cells. (A and B) Western blotting
for Akt or AMPK. The cells were transfected with control, Akt or AMPK
shRNA for 48 hours. (C) Measurement of LC3-punctate cells in the
absence or presence of Akt or AMPK shRNA. (D) Cytotoxic effects in the
absence or presence of Akt shRNA or AMPK shRNA. The cells were
transfected with control, Akt or AMPK shRNA for 48 hours and were
then incubated with 20 μg ml−1 of NH2-PS for 16 hours. *, p < 0.05
versus control shRNA + NH2-PS.
fected with AMPK shRNA showed a significant decrease in the
percentage of LC3 punctate cells compared with the NH2-PS
treatment, whereas cells transfected with Akt shRNA showed a
significant increase in the percentage of LC3 punctate cells
(Fig. 6D). These results suggest that one of the mechanisms of
NH2-PS-induced autophagy could be mediated by inhibition of
Akt/mTOR and activation of AMPK signaling pathways in RAW
264.7 and BEAS-2B cells. Recent studies have found that the
Akt/mTOR and AMPK pathways play crucial roles in the regu-
lation of both apoptosis and autophagy.63,64 Akt can activate
mTOR and lead to inhibition of autophagy.65 By contrast,
AMPK is a universal autophagy activator.56 It has recently been
reported that functionalized single-walled carbon nanotubes
induced autophagic cell death in human lung cells through
Akt-TSC2-mTOR signaling.11 However, the role of the AMPK
pathway in the activation of autophagy in cells treated with
NPs has not previously been reported. To our knowledge, this
is the first study demonstrating that NH2-PS exposure leads to
autophagy induction through inhibition of the Akt/mTOR and
activation of the AMPK signaling pathways.
3.6 Inhibition of ER stress decreases cytotoxicity and
autophagy
To test whether NH2-PS-induced ER stress contributes directly
to cell death, we treated BEAS-2B cells with the chemical
chaperone tauroursodeoxycholic acid (TUDCA), which is
known to selectively inhibit ER stress.66 The results indicated
that TUDCA treatment decreased NH2-PS-induced cytotoxicity
and autophagy (Fig. 7A and B). Next, we examined whether
inhibition of ER stress altered the NH2-PS-induced inhibition
744 | Nanoscale, 2015, 7, 736–746
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Nanoscale
Fig. 7 Measurement of autophagy and cytotoxic effects in BEAS-2B
cells pretreated with TUDCA. (A) Effects of TUDCA on cytotoxicity
induced by NH2-PS. The cells were pretreated with TUDCA for 2 hours
before NH2-PS treatment (20 μg ml−1) for 16 hours. *, p < 0.05, NH2-PS
versus NH2-PS + TUDCA. (B) Measurement of LC3-punctate cells in the
absence 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 as
the 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 hours
before NH2-PS treatment (20 μg ml−1) for 16 hours.
of the Akt/mTOR and the activation of the AMPK signaling
pathway. We found that pretreatment with TUDCA retarded
the NH2-PS-decreased expression of Akt phosphorylation
and NH2-PS-increased expression of AMPK phosphorylation
(Fig. 7C). Inhibition of ER stress has recently been suggested
as a novel therapeutic strategy in various diseases.66,67 For
example, TUDCA may inhibit apoptosis by ameliorating ER
stress through the modulation of intracellular calcium and
thus attenuate liver cell death.68 In addition, TUDCA treatment
was reported to attenuate tunicamycin-induced ER stress,
autophagy and cell death in rat hepatocytes,69 implying that
autophagy was involved, at least in part, in ER stress-induced
cell death. Our current results showed that TUDCA increased
Akt activation in NH2-PS-treated macrophage and lung epi-
thelial cells. Consistent with these results, TUDCA was found
to activate Akt in skeletal muscles and myocardium.70,71 The
mechanisms through which TUDCA increases Akt activation
are still unclear. One of the possible mechanisms may be a
G-protein coupled signal cascade.72 Moreover, ER stress leads
to a release of calcium and subsequent activation of AMPK
that inhibits mTOR, thereby promoting autophagy.73 Thus,
NH2-PS-induced ER stress and AMPK activation found in our
current study might play important roles in autophagy induc-
tion and cell death.
This journal is © The Royal Society of Chemistry 2015

Nanoscale
4. Conclusions
Taken together, our results indicate that NH2-PS increased
ROS generation and produced misfolded protein aggregates,
which lead to ER stress that results in autophagic cell death in
RAW 264.7 and BEAS-2B cells. TUDCA, an ER stress inhibitor,
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decreased cytotoxicity and autophagy in the NH2-PS-treated
cells. Furthermore, NH2-PS-induced autophagic cell death may
be 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 was
inhibited by the autophagy inhibitor 3-MA compared with
cells treated with the vehicle, suggesting that autophagy
may serve a pro-death role in NH2-PS-induced cell death.
In addition, NH2-PS increased the autophagic flux. Thus, in
addition 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 US
Public Health Service Grant U19 ES019528 and NSF/EPA DBI
0830117 and 1266377. The Instrument Development Center of
the National Cheng Kung University (NCKU) provided techni-
cal support with the Carl Zeiess LSM780 laser-scanning
microscope.
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