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Effects of Cerium Oxide Nanoparticles on PC12 Neuronal-Like Cells: Proliferation, Differentiation, and Dopamine Secretion

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Effects of Cerium Oxide Nanoparticles on PC12 Neuronal-Like Cells: Proliferation, Differentiation, and Dopamine Secretion

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Purpose: Oxidative stress has been found to play a key role in several diseases, that range from cancer to neurodegenerative disorders. Besides traditional anti-oxidant agents, in recent years much attention has been focused on nanotechnological solutions, including cerium oxide nanoparticles (nanoceria). Methods: Thanks to its extraordinary catalytic properties, nanoceria mimics the activity of superoxide dismutase and of catalase, therefore acting as a reactive oxygen species (ROS) scavenger in many biological contexts. In this paper, we report on nanoceria interactions with PC12 cell line, that represents a valuable model for many features of central dopaminergic neurons. Results: Nanoceria confirmed a strong anti-ROS action but, most interestingly, also showed beneficial effects on both cell differentiation and dopamine production. Conclusions: Even if deeper examinations will be necessary in order to better clarify the mechanisms at the base of the documented effects, nanoceria demonstrated a significant potential as pharmacological agent in the treatment of neurological disorders.
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RESEARCH PAPER
Effects of Cerium Oxide Nanoparticles on PC12 Neuronal-Like
Cells: Proliferation, Differentiation, and Dopamine Secretion
Gianni Ciofani &Giada G. Genchi &Ioannis Liakos &Valentina Cappello &Mauro Gemmi &Athanassia Athanassiou &Barbara Mazzolai &
Virgilio Mattoli
Received: 7 February 2013 / Accepted: 3 May 2013 /Published online: 10 May 2013
#Springer Science+Business Media New York 2013
ABSTRACT
Purpose Oxidative stress has been found to play a key role in
several diseases, that range from cancer to neurodegenerative
disorders. Besides traditional anti-oxidant agents, in recent years
much attention has been focused on nanotechnological solu-
tions, including cerium oxide nanoparticles (nanoceria).
Methods Thanks to its extraordinary catalytic properties,
nanoceria mimics the activity of superoxide dismutase and of
catalase, therefore acting as a reactive oxygen species (ROS) scav-
enger in many biological contexts. In this paper, we report on
nanoceria interactions with PC12 cell line, that represents a valu-
able model for many features of central dopaminergic neurons.
Results Nanoceria confirmed a strong anti-ROS action but,
most interestingly, also showed beneficial effects on both cell
differentiation and dopamine production.
Conclusions Even if deeper examinations will be necessary in
order to better clarify the mechanisms at the base of the
documented effects, nanoceria demonstrated a significant po-
tential as pharmacological agent in the treatment of neurological
disorders.
KEY WORDS cerium oxide nanoparticles .dopamine .PC12
cells .reactive oxygen species production scavenger
ABBREVIATIONS
EDS energy-dispersive X-ray spectroscopy
ELISA enzyme-linked immunosorbent Assay
NC nanoceria
NGF nerve growth factor
ROS reactive oxygen species
XPS X-ray photoelectron spectroscopy
INTRODUCTION
Cerium oxide nanoparticles (nanoceria, NC) represent an
extremely interesting class of nanomaterials that, thanks to
its intriguing catalytic activity (1), found plenty of applica-
tions in several fields, that range from solid oxide fuel cells to
gas sensors (2).
CeO
2
presents a fraction of Ce in the Ce
3+
form com-
pensated by an analogous number of oxygen vacancies. This
phenomenon is enhanced at the nanoscale level because of
the higher surface/volume ratio, and implies the coexistence
of Ce
3+
and Ce
4+
ions, that are involved in redox reactions
(3). In a biological context, it has been shown that NC is able
to scavenge reactive oxygen species (ROS), mimicking both
the activity of superoxide dismutase (the enzyme that cata-
lyzes the dismutation of the superoxide radical anion), and
of catalase (the enzyme that decomposes H
2
O
2
to O
2
and
H
2
O) (4,5).
The use of NC as a free-radical scavenger has therefore
become popular for biological applications: NC was in fact
proven to be an extremely efficient protective agent for
cardiac (6), neuronal (7), and stem (8) cells. Moreover,
in vivo, NC confirmed to show strong anti-oxidative
properties in mice (9), to prevent loss of retinal function
G. Ciofani (*):G. G. Genchi :B. Mazzolai :V. Mattoli
Istituto Italiano di Tecnologia, Center for Micro-BioRobotics @SSSA
Viale Rinaldo Piaggio 34
56025 Pontedera, Pisa, Italy
e-mail: gianni.ciofani@iit.it
G. G. Genchi
Scuola Superiore SantAnna, The BioRobotics Institute
Viale Rinaldo Piaggio 34
56025 Pontedera, Pisa, Italy
I. Liakos :A. Athanassiou
Istituto Italiano di Tecnologia
Center for Biomolecular Nanotechnologies @UniLe, Via Barsanti
73010 Arnesano, Lecce, Italy
V. Cappello :M. Gemmi
Istituto Italiano di Tecnologia, Center for Nanotechnology Innovation
@NEST, Piazza San Silvestro 12
56127 Pisa, Italy
Pharm Res (2013) 30:21332145
DOI 10.1007/s11095-013-1071-y
in rats (10,11), to promote the regression of pathologic
retinal neovascularization in mice (12), and to promote
wound healing activity (13). A comprehensive review on
potential pharmacological applications of NC is repre-
sented by the work of Celardo and collaborators (14), to
which readers are referred.
The huge interest in antioxidant effects of NC stems for
the essential role of ROS in the development of several
diseases, when they are not adequately buffered by the
natural antioxidant defenses of the cells. It has indeed been
proven, that ROS (like superoxide anion radical O
2
, hy-
droxyl radical OH
,singletoxygen
1
O
2
, and hydrogen
peroxide H
2
O
2
) are extremely reactive chemical species that
play a key role in cancer (15), neurodegenerative disorders
(16), inflammation (17), and in many other pathological
conditions (18). In particular, ROS are able to oxidize many
biological macromolecules, affecting in particular the hy-
drocarbon chains of unsaturated fatty acids, aminoacid res-
idues in proteins, carbohydrates, and nitrogenous bases in
nucleic acids. This oxidation compromises structure and
function of the biological macromolecules, often resulting
in cell death (19).
Concerning neurological applications, NC has been
very recently proposed for the treatment of neurological
diseases (20), such as Alzheimers, Parkinsons, and
Gehrigs. As an example, Cimini et al. proposed polyeth-
ylene glycol (PEG)-coated and antibody-conjugated NC
for the selective targeting of amyloid-β142 aggregates,
that are the main responsible of Alzheimersdisease,
showing an improvement of neuronal survival, by mod-
ulating the brain-derived neurotrophic factor (BDNF)
signaling pathway (21).
Another study showed the neuroprotective mechanisms
of NC in a model of ischemia (22). The neuroprotective
effects resulted due to a modest reduction in reactive oxygen
species, and, more significantly, to a strong decrement of the
levels of ischemia-induced 3-nitrotyrosine, a modification of
tyrosine residues in proteins induced by the peroxynitrite
radical.
In this paper, we report on the effects of incubation of
PC12 neuron-like cells with increasing concentrations of a
commercial NC. After an extensive characterization of the
nanomaterial, we evaluated viability, proliferation and differ-
entiation of the cells in the presence of CeO
2
nanoparticles.
No evidences of toxicity were found; instead, an increment of
differentiation in terms of neurite length was found when cells
underwent treatment with NC. ROS production evaluation
confirmed anti-oxidant properties of NC also in this kind of
cells. Finally, and most interestingly, we analyzedthe effects of
NC on dopamine production, that is impaired in Parkinsons
disease, showing beneficial effects of nanoparticles in dopa-
mine secretion. Effects of NC on PC12 cells were also inves-
tigated at gene level. All these results corroborate the
need of further investigations of NC as a therapeutic
agent, suggesting special attention to neurodegenerative
processes.
MATERIALS AND METHODS
Nanoparticle Characterization
Cerium oxide nanoparticles (nanoceria, NC) were pur-
chased by Sigma (code 544841) and underwent extensive
characterization before biological testing.
Transmission electron microscopy was performed on a
Zeiss Libra 120 operating at 120 kV. The samples were
prepared by deposition of CeO
2
nanoparticles in physiolog-
ic saline solution (0.9% NaCl in H
2
O) on a 300 mesh
carbon coated copper grid.
Energy-dispersive X-ray spectroscopy (EDS) microanaly-
sis was performed using a scanning electron microscope
(SEM, EVO MA10 Zeiss) with NC deposited on silicon
wafers and gold-sputtered before analysis.
Cerium nanoparticles were characterized through X-
ray photoelectron spectroscopy (XPS) in order to quali-
tatively and quantitatively assess Ce
3+
and Ce
4+
content.
XPS measurements were performed using a Specs Lab2
electron spectrometer equipped with a monochromatic
X-raysource(setat1,253eV)andwithaPhoibos
analyzer Has 3500 (Emispherical Energy Analyzer). The
applied voltage of the Mg KαX-ray source was set at
13 kV and the applied current at 15 mA. The pressure
in the analysis chamber was approximately 7·10
9
mbar.
For Ce 3d
5/2
and Ce 3d
3/2
spectra acquisition, the
energy pass was 30 eV, the energy step 0.2 eV and the
scan number 40. The spectra were then analyzed using
CasaXPS software.
Nanoparticles were mixed with the appropriate culture
medium at a concentration of 1 mg/ml and sonicated for
2 h (with a Bransonic sonicator 2510) using an output power
of 20 W, just before their administration to cell cultures.
The obtained dispersion was then diluted at fixed concen-
trations (see next sections for details) for biological experi-
ments. For EDS analyses, nanoparticles were dispersed in
distilled water using the same procedure.
Particle size distribution and Z-potential of the disper-
sionswereanalyzedwithaNanoZ-Sizer90(Malvern
Instrument). For both analyses, each acquisition was
performed three times, using samples appropriately diluted
in complete cell culture medium.
Cell Culture and Viability Assays
PC12 cells (ATCC CRL-1721) are derived from a trans-
plantable rat pheochromocytoma, and represent a valuable
2134 Ciofani et al.
model for neuronal differentiation, mimicking many fea-
tures of central dopaminergic neurons, including dopamine
production. Moreover, they can reversibly respond to the
administration of nerve growth factor (NGF), expressing
sympathetic neuronal phenotype (23). PC12 were cultured
in Dulbeccos modified Eagle medium (DMEM) with
10% horse serum (HS), 5% fetal bovine serum (FBS),
100 IU/ml penicillin, 100 μg/ml streptomycin and
2 mM L-glutamine. Differentiation was induced by ad-
ministration of a low-serum (1% FBS) medium
supplemented with 50 ng/ml of NGF.
For viability testing, PC12 cells were seeded in 96-well plates
(5,000 cells per well) and incubated with increasing concentra-
tions of NC (0, 10, 20, 50 and 100 μg/ml). At 24 and 72 h since
beginning of treatment, cell metabolism was assessed with
the WST-1 assay (2-(4-iodophenyl)-3-(4-nitophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazoilium monosodium salt, provided
in a pre-mix electro-coupling solution, BioVision). Cell cul-
tures were treated with 100 μl of culture medium added
with 10 μl of the pre-mix solution for 2 h and, finally,
absorbance was read at 450 nm with a microplate reader
(Victor3, Perkin Elmer).
Viability was further qualitatively investigated at 72 h
with the Live/Dead® viability/cytotoxicity Kit (Molecular
Probes). The kit contains calcein AM (4 mM in anhydrous
DMSO) and ethidium homodimer-1 (EthD-1, 2 mM in
DMSO/H
2
O 1:4 (v/v)), and allows for the discrimination
between live cells (stained in green by calcein) and dead cells
(stained in red by EthD-1). Cultures were rinsed with PBS,
treated for 10 min at 37°C with 2 μM calcein AM and 4 μM
EthD-1 in PBS, and finally observed with an inverted fluo-
rescence microscope (TE2000U, Nikon) equipped with a
cooled CCD camera (DS-5MC USB2, Nikon) and with
NIS Elements imaging software.
Differentiation capabilityinthepresenceofNCwas
evaluated providing cell cultures (30,000 cells in 24-well
plates) with differentiating medium doped with 0, 20 and
50 μg/ml of NC. After 3 days, differentiating status was
assessed through immunofluorescence analysis of specific
neuronal differentiation markers, β3-tubulin and
neurofilament-66, followed by ImageJ analysis (http://
rsb.info.nih.gov/ij/) of the neurite length (at least 100
neurites per sample were measured for statistical analysis
purpose).
Samples were rinsed with PBS and fixed in paraformalde-
hyde (4% in PBS) for 15 min. After rinsing with PBS, they
were incubated with sodium borohydryde (1 mg/ml in PBS)
for 10 min to reduce autofluorescence. Cellular membranes
were then permeabilized with 0.1% Triton X-100 in PBS for
15 min. Antibody aspecific binding sites were saturated with
10% goat serum in PBS for 1 h, and, subsequently, a primary
antibody (rabbit polyclonal IgG anti-tubulin, Sigma, diluted
1:75 in 10% goat serum, or rabbit monoclonal IgG anti-
neurofilmanent, Millipore, diluted 1:250 in 10% goat serum)
was added. After 30 min of incubation at 37°C, samples were
rinsed with 10% goat serum; then, a staining solution was
added, composed of a secondary antibody (fluorescent goat
anti-rabbit IgG, Invitrogen) diluted 1:250 in 10% goat serum
and of 1 μM DAPI for nucleus counterstaining. After 30 min
of incubation at room temperature, samples were rinsed with
0.45 M NaCl in PBS for 1 min to remove weakly bound
antibodies and, after rinsing in PBS, observed with the
inverted fluorescence microscope.
ROS Production Assessment
Intracellular ROS production was induced in cells cultured
in 24-well plates (30,000 cells per well) by a 50 μMH
2
O
2
treatment of 30 min in complete medium, after being ex-
posed for 72 h to 0, 20, and 50 μg/ml of NC. Controls on
cells non-treated with H
2
O
2
were also performed. ROS
production was quantified with the 20,70-dichlorfluorescein
diacetate (DCFH-DA) test. DCFH-DA is a membrane-
permeant compound that, once inside cells, is deacetylated
by endogenous esterases to form the non-fluorescent 20,70-
dichlorfluorescein (DCFH). DCFH is thereafter converted
to green fluorescent dichlorofluorescein (DCF) compound
by the action of cellular oxidants. Following ROS produc-
tion, green fluorescence is therefore emitted by the cells.
After H
2
O
2
treatment, cells were incubated with 50 μM
DCFH-DA in DMEM without serum for 30 min at 37°C
and then observed at the fluorescence microscope or lysed
though three freezing/thawing cycles to quantitatively assess
fluorescence through the microplate reader (excitation
wavelength 485 nm, emission wavelength 535 nm).
Dopamine Secretion Assessment
Dopamine production was quantified collecting superna-
tants after 72 h of incubation of proliferating cells with 0,
20, and 50 μg/ml of NC (30,000 cells per well, in 24-plate
wells). A solid phase Enzyme-Linked Immunosorbent Assay
(ELISA), based on the sandwich principle, was carried out
with a Dopamine ELISA kit (GenWay Biotech Inc.), follow-
ing the manufacturers instructions. Briefly, 20 μlof
extracted samples were incubated for 2 h at room temper-
ature in wells coated with a goat anti-rabbit antibody, di-
rected towards a dopamine epitope. An enzyme-conjugated
secondary antibody, directed towards a different region of
the dopamine molecule, was thus added and incubated at
room temperature for 1 h. Appropriate substrate was there-
after provided to the enzyme-conjugated antibody and re-
action was allowed to occur for 40 min at room
temperature. Absorbance of the colored product was finally
measured at 405 nm on the microplate reader and com-
pared to a calibration curve (obtained with known amounts
Effects of Cerium Oxide Nanoparticles on PC12 Neuronal-Like Cells 2135
of dopamine) for neurotransmitter concentration determi-
nation. Obtained values were normalized to the cell num-
ber, evaluated by measuring total DNA content with the
PicoGreen® kit (Molecular Probes) following the manufac-
turers instruction. After cell lysis, the PicoGreen® dye binds
to ds-DNA and the resulting fluorescence intensity is directly
proportional to the ds-DNA concentration in solution and,
therefore, to the cell number. Fluorescence intensity was
measured with the microplate reader using an excitation
wavelength of 485 nm and an emission wavelength of
535 nm.
Quantitative RT-PCR
Transcription of specific genes of markers of neuronal cell
maturation (neurofilament-66, Nefl, and β3-tubulin, Tub), of
ROS mechanisms (glutathione peroxidase 1, Gpx1; glutathi-
one synthetase, Gss; thioredoxin reductase-1, Txnrd1), of
dopamine metabolism (tyrosine hydroxylase, Th;mono-
amine oxidase A, Maoa; catechol-O-methyltransferase,
Comt), and of dopamine transport (dopamine transporter,
Dat; vesicular monoamine transporter-2, Vmat2) were eval-
uated with quantitative real-time qRT-PCR at 3 days of
incubation with 0, 20, and 50 μg/ml of NC. Total RNA was
isolated from cell cultures using High Pure RNA Isolation
kit (Roche) according to the manufacturersprotocol.
Extracted RNA was diluted ten times in pure water
(MilliQ Millipore) and RNA concentration was measured
at 260 nm with a spectrophotometer (Lambda 45, Perkin
Elmer). RNA retrotranscription into cDNA was performed
with 400 ng of RNA in a total volume of 20 μl, including
4μl of iScriptTM Reverse Transcription Supermix (5X,
Bio-Rad). The synthesis program included an initial incu-
bation at 25°C for 5 min, followed by incubation at 42°C
for 45 min and at 48°C for 15 min. Reaction was
inactivated by heating at 85°C for 5 min, and the reaction
volume was finally increased up to 200 μl with pure water.
Quantitative RT-PCR wasperformedwithaCFX
ConnectReal-Time PCR Detection System (Bio-Rad)
to determine the transcription of different genes. Results
were normalized to the transcription levels of a selected
housekeeping gene, glyceraldehyde 3-phosphate dehydroge-
nase (Gapdh). The obtained cDNA (5 μl) was mixed with 1 μl
of specific forward and reverse primers (8 μM), 4 μlof
MilliQ and 10 μl of SsoAdvancedTM SYBR®Green
Supermix (Bio-rad). The thermal protocol was applied with
one cycle of 30 s at 98°C for enzyme activation, followed by
40 cycles at 98°C for 3 s and 60°C for 7 s. After the last
reaction cycle, the protocol included a temperature ramp
from 65°C to 95°C, with 0.5°C/s increments, to exclude
unspecific products with melting curve results. Each assay
included no templatesample and all tests were carried out
in triplicate. The cycle threshold (Ct) value relative of
control sample was adopted as reference for the calculation
of ΔΔCt (difference between ΔCt values deriving from dif-
ference between Ct of target and housekeeping gene) for the
subsequent samples. Primer sequences (forward and reverse)
of the investigated genes are reported in Table I.
Statistical Analysis
Analysis of the data was performed by analysis of variance
(ANOVA) followed by Tukeys post-test to test for signifi-
cance, which was set at 5%. Results are presented as mean
value ± standard deviation (n=6 for the WST-1 assays, n=3
for all other analysis).
RESULTS
NC Characterization
Transmission electron microscopy shows that the CeO
2
nanoparticles are quite dispersed in size, in a range going
from 5 nm to 80 nm. Powder electron diffraction patterns
takenonareasof10μm in diameter, that are rich of
nanoparticles, show sharp diffraction rings indicating that
the nanoparticles have a crystalline nature. The patterns can
be indexed with a cubic face centered lattice. A Le Bail fit
Table I Primer Sequences for qRT-PCR Analysis
Gene Sequence
Gapdh 5-AACCTGCCAAGTATGATGAC-3
5-GGAGTTGCTGTTGAAGTCA-3
Nefl 5-GAGAAGCAGAAGAGGAGGAG-3
5-TGGCATCTTCAGACTCATCC-3
Tub 5-ATTCTGGTGGACCTGGAG-3
5-CACTCTGACCGAAGATAAAGTT-3
Gpx1 5-CATTGTTTGAGAAGTGCGAGGTG-3
5-ACTGGGTGCTGGCAAGGC-3
Gss 5-CCAGCGTGCCATAGAGAAC-3
5-CCCTTTCAGAGACATCTTCAAATC-3
Txnrd1 5-TGGAAGAGCATGGTATCAAG-3
5-CACGGTCTCTAAGCCAATAG-3
Th 5-CTGTTCTCAACCTGCTCTTC-3
5-TGGCTTCAAATGTCTCAAATAC-3
Maoa 5-CAGACACACCAGACAACAC-3
5-TACGGACATAGGCACTGAG-3
Comt 5-CCTGACTTCCTGGCGTATG-3
5-TTCTCCAAGCCGTCTACAAC-3
Dat 5-TGGACTTCTACCGACTCTG-3
5-GGAGGTGGTGATGATTGC-3
Vmat2 5-AGCATCTTCTCTTACTATAACAAC-3
5-AAACAGCAGCCCAACTTG-3
2136 Ciofani et al.
(24) of the integrated pattern gives a unit cell parameter
of a=5.402±0.005 Å which is compatible with that one
of CeO
2
cubic ceria.
EDS analysis (Fig. 1b) performed on the sample con-
firmed its high purity, being composed by Ce ~30% and
O ~65% (atom content). A small amount of gold, deriv-
ing from the sputtering procedure, is also present (~4%);
impurities were found to be less of the 1% of atom
content.
Since oxidation state is a key component of NC catalytic
activity, it is mandatory to assess the presence of adequate
amounts of Ce
3+
and Ce
4+
on the nanoparticle surface.
Figure 1c shows the Ce 3d
5/2
and Ce 3d
3/2
XPS spectra
and the related Gaussian peak deconvolution acquired from
the NC powder. The peaks in the range 875895 eV belong
to the Ce 3d
5/2
, while the peaks in the range 895910 eV
correspond to the Ce 3d
3/2
levels. Basing on the shift
reported in the literature (25), we could assign peaks at
0
10
20
30
40
50
a
b
c
012345678910
keV
CPS
0.0E+00
5.0E+02
1.0E+03
1.5E+03
2.0E+03
2.5E+03
3.0E+03
870880890900910920930
Bindin
g
ener
gy
(
eV
)
CPS
O
Ce Au Ce
Ce
XPS signal
Fitting
Ce
4+
Ce
3+
Fig. 1 Cerium oxide
nanoparticle characterization:
TEM bright field imaging (left),
powder electron diffraction
pattern (bottom right), radially
integrated profile of the pattern
fitted with the Le Bail method (top
right) with the positions and the
indices of the diffracted peaks (a);
EDS analysis (b) and high
resolution XPS analysis of Ce 3d
5/
2
and Ce 3d
3/2
(c).
Effects of Cerium Oxide Nanoparticles on PC12 Neuronal-Like Cells 2137
882.9, 889.8, 900.9, 908.6 and at 917.4 eV to Ce
4+
, while
peaks at 884.4 and 902.9 eV to Ce
3+
. From the ratio of
the integrated peak areas, we can deduce a Ce
3+
content
of ~23%, that has been demonstrated to be optimal for
NC redox activity (26).
In order to characterize NC assembling in cell culture
medium and to have a hint of the actual sizes of the
nanocomplexes in the cultures, dynamic light scattering
(DLS) and Z-potential analyses were performed in complete
cell culture medium. DLS revealed a 92.0% peak at a size of
about 230 nm, with a polydispersity index of 0.290.
Moreover, a Z-potential of about 15 mV (deriving from
an absorption of serum proteins on the nanoparticles) de-
noted a good stability of the obtained dispersion.
PC12 Cells Viability and Differentiation
in the Presence of NC
WST-1 provided an excellent metabolic activity in cells
treated up to 100 μg/ml of NC (Fig. 2a), with no statistically
significant decrement of viability, at all the tested concen-
trations, after both 24 and 72 h.
Viability was further investigated with the Live/Dead®
viability/cytotoxicity Kit. Also in this case, the compati-
bility of NC resulted optimal at each concentration
(Fig. 2b) after 72 h of incubation. No evidence of cell
membrane damage was present following treatment of
cells up to 100 μg/ml of NC, clearly demonstrating a
viability comparable to that of the control cultures (less
than 1% of red, i.e., necrotic, cells).
PC12 cells retained differentiation capability in the pres-
ence of NC, as depicted in Fig. 3. After 3 days since differ-
entiation induction, PC12 cells exhibited a well-developed
neurite network, with a strong expression of typical neuronal
marker such as β3-tubulin and neurofilament-66, as
suggested by the intense fluorescence signal of the images.
Interestingly, a quantitative analysis carried out on
neurite length showed a beneficial effect of NC on neuronal
differentiation. Figures 4acdepict length distribution of
neurites of cells treated with 0, 20 and 50 μg/ml of NC
during the 3-day differentiation. Control cultures exhibit a
median neurite length value of about 55 μm (min 25 μm,
average 60 μm, max 175 μm), that increases to about 65 μm
(min 30 μm, average 70 μm, max 140 μm) in the case of
cells treated with 20 μg/ml of NC, and up to 85 μm (min
35 μm, average 90 μm, max 180 μm) when the concentra-
tion of NC was 50 μg/ml.
An improved differentiation was confirmed by analysis at
gene level with qRT-PCR (Fig. 4d). In this case, a slight, but
statistically significant increment (p<0.05) of gene transcrip-
tion is appreciable just in cells treated with 50 μg/ml of NC,
that show a 1.5 fold increment of both β3-tubulin and
neurofilament-66 genes.
NC as ROS Scavenger in PC12 Cells
Intracellular antioxidant activity of nanoceria was tested
both on cultures stimulated and non-stimulated with
H
2
O
2
. Figure 5a shows fluorescence images of cells stimu-
lated with H
2
O
2
for 30 min after a 3-days incubation with
NC. It can be clearly seen that NC reduces production of
ROS in cells in a dose-dependent manner.
This result is confirmed by a quantitative evaluation of the
fluorescence due to DCF production. Figure 5b indicates the
variation percentage of fluorescence intensity with respect to a
control culture (cells not treated with NC and not stimulated
with H
2
O
2
). It can be seen that H
2
O
2
produces a ~25% of
ROS increment. ROS are efficiently scavenged by NC, and
their values drop to a ~5% with respect to the basal levels in
cells treated with 20 or 50 μg/ml of NC. No statistically
significant scavenging effects are appreciable in cultures incu-
bated with just 10 μg/ml. Most interestingly, NC induces a
decrement of ROS basal levels even in cells not stimulated
with H
2
O
2.
In this case, it can be again appreciated a slight,
but significant, decrement of ROS production in cultures
treated with 20 (~5%) and with 50 μg/ml of NC (~10%) with
respect to the basal levels.
In order to investigate the mechanism of basal ROS
decrement in NC-treated cells, qRT-PCR was carried
out. Among the selected genes indicative of the redox
status of the cells, a significant down-regulation of Gss
and Gpx1 induced by the 50 μg/ml NC 3-days treatment
could be appreciated; Gpx1 resulted down-regulated also
by an incubation with 20 μg/ml NC; instead, no signif-
icant effects on Txnrd1 were found at all the tested
concentrations (Fig. 5c).
Effects of NC on Dopamine Production
ELISA test confirmed a production of dopamine following
the 3-day incubation at all the tested NC concentrations.
Dopamine amount, normalized to cell number, amounted
to 2.5±2.3, 11.6±3.5, and 15.4±3.6 pg/ml/cell in the
supernatants of PC12 cells treated with 0, 20, and
50 μg/ml of NC, respectively (Fig. 6a). These values indi-
cate a strong effect of NC on dopamine secretion by the
cells, with significant increase also at the lowest NC concen-
tration tested (20 μg/ml).
At a gene level, a modulation induced by NC treatment
for Comt and Maoa (1.5 fold increment at 50 μg/ml) could be
noticed, while Th resulted unaffected (Fig. 6b). An interest-
ing phenomenon of up-regulation in gene coding for protein
involved in dopamine transport is highlighted: Dat transcrip-
tion was 2 fold higher in cells treated with 20 μg/ml of NC,
and about 3 fold higher for the 50 μg/ml dose (in both cases
p<0.05); instead, Vmat2 exhibited a 2 fold up-regulation
after cell treatment with 50 μg/ml of NC (Fig. 6c).
2138 Ciofani et al.
DISCUSSION
Nanoceria holds great promises for the treatment of all
those conditions where oxidative stress plays a key role.
Impressive results have been already achieved: it is worth
to mention the work by Rzigalinski and collaborators, that
verified as NC behaves as an extremely efficient anti-ageing
agent, increasing median lifespan in Drosophila melanogaster by
18 days (27).
Aiming at the exploitation of NC in the neurological
field, here we have proposed a study of interactions of
cerium oxide nanoparticles with PC12 cells. As already
proven with many other cell lines, NC did not adversely
affect viability and metabolic activity of PC12 cells, as
NC 0 µg/ml
NC 10 µg/ml NC 20 µg/ml
NC 20 µg/ml NC 50 µg/ml
200 µm
0
20
40
60
80
100
120
a
b
0 10 20 50 100
nanoparticle concentration (µg/ml)
WST-1 (% of control)
24 h 72 h
Fig. 2 WST-1 results at 24 and
72 h after incubation of PC12
cells with increasing
concentrations of NC (a); Live/
Dead® assay performed after
72 h (b).
Effects of Cerium Oxide Nanoparticles on PC12 Neuronal-Like Cells 2139
demonstrated by both the Live/Dead® and the WST-1
assay, up to 100 μg/ml. Assessment of cytocompatibilty
remains however a fundamental step before any other
consideration; there are in fact, in the literature, some
examples that show as NC could impair cell activities
(28,29). These discrepancies can be explained by con-
sidering nanoparticle preparation procedure, nanoparti-
cle size, internalization route, and cell line (30): it has
been proven, in fact, as NC could be selectively toxic
towards certain cancer cells, thus opening interesting
perspectives also in cancer therapy (31). Nanoceria also
did not impair neuronal differentiation of PC12 cells.
Indeed, NC treated cells demonstrated a significant
improved differentiation, in terms of a higher median
neurite length (50% higher in cells treated with
50 μg/ml of NC) and of higher gene transcription of
markers of neuronal differentiation, such as β3-tubulin
and neurofilament-66. β3-tubulin is an early marker of
neuronal differentiation (32), while neurofilament-66, also
known as α-internexin, plays a key role in neurite outgrowth
and regulates the expression of other neurofilaments (33), and
was found to be expressed in cell somas and in the proximal
segment of neurites.
Improvement of the differentiation process could be ascrib-
able to a decrement of stress induced by ROS accumulation in
the cells. As already proven for other cell models, NC is able to
scavenge intracellular ROS also in PC12. A dose of 20 μg/ml
was sufficient to reduce from 25% to 5% of the basal level
ROS produced after H
2
O
2
stimulation. Moreover, in cells not
stimulated with H
2
O
2
, NC further reduces the basal levels of
ROS up to 10% (at a dose of 50 μg/ml).
In order to investigate redox phenomena at gene level,
qRT-PCR was carried out on genes coding for enzyme
typically involved in ROS metabolism of PC12 cells (34).
Three genes were selected: Txnrd1,Gss and Gpx1,which
respectively encode thioredoxin reductase-1, glutathione syn-
thetase, and glutathione peroxidase-1. The glutathione
disulfide/glutathione pair (GSSG/GSH) and the thioredoxin
reductase/thioredoxin system are in fact the major players in
maintaining a favorable intracellular redox environment
(35,36). After NC treatment, a down-regulation of Gss at
50 μg/ml and of Gpx1 at both 20 and 50 μg/ml of NC
concentration was noticed; on the contrary, no change in
Txnrd1 was detected. This result could be explained taking
into account a mechanism of negative regulation by the
cells: since NC acts as an exogenous anti-oxidant, natural
3-tubulin
0 µg/ml
100 µm
Neurofilament-66
20 µg/ml50 µg/ml
Fig. 3 Immunofluorescence
staining of β3-tubulin and
neurofilament-66 of PC12 cells
differentiated for 3 days in the
presence of increasing
concentrations of NC.
2140 Ciofani et al.
mechanisms of the cells are expressed in a lower extent,
because of the presence of a further ROS scavenger
represented by NC. The absence of oxidative stress even in
asituationofGss/Gpx1 down-regulationisinfactconfirmedby
absence of significant alteration of Txnrd1, usually up-regulated
following an oxidative insult (37). Nanoceria therefore acts
in parallel to enzymes that usually scavenge ROS in cells.
However, it should be carefully considered that it is also
important to maintain a specific ROS level in cells. There
are, in fact, some metabolic pathway were ROS play a
relevant role in order to appropriately sustain cell activities,
acting as useful signaling molecules that regulate physiolog-
ical processes. This is the case, for example, of muscle
tissue, where ROS have been shown to trigger many
relevant signaling pathways (38). An accurate evaluation of
NC effects on different cells and tissues is therefore man-
datory, as well as a systematic consideration of the dose
administered to the biological systems. If a systemic admin-
istration is envisioned, a specific functionalization of the
nanoparticles should also be considered, in order to focus
their ROS scavenger actions just on the desired
cells/tissues, thus preventing potential side effects where,
on the contrary, higher ROS levels are physiologically
needed.
As previously pointed out, PC12 cells mimic many features of
central dopaminergic neurons, including dopamine production.
In evaluating whether the production of dopamine is affected by
NC treatment, we highlighted an impressive, dose-dependent,
increment of dopamine secretion by cells treated with
nanoparticles, arriving at a 7-fold increment in cultures treated
with 50 μg/ml of NC with respect to the controls.
In order to depict a better picture of NC effects on
dopamine, we investigated transcription of Th,Maoa, and
Comt genes, that are coding for tyrosine hydroxylase, mono-
amine oxidase A, and catechol-O-methyltransferase, respec-
tively. Tyrosine hydroxylase is a key enzyme in dopamine
synthesis, while monoamine oxidase A and catechol-O-
methyltransferase are involved in dopamine metabolism
(39). Nanoceria did not exhibit significant effects in Th
trancription, but, at the highest tested concentration, an
up-regulation of Maoa and Comt was found. An increased
transcription of gene coding for enzyme involved in dopa-
mine metabolism can be easily explained in view of a com-
pensatory effect in cells induced by a higher dopamine
content. The absence of a direct influence on Th suggests
that beneficial effects of NC in dopamine production could
be ascribable to its role in ROS depletion, rather than to a
direct effect on the dopamine pathway.
The transcription of Dat and Vmat2, which encode for two
transporters (dopamine transporter and vesicular monoamine
transporter-2) was also affected by NC treatment. Dat,inpar-
ticular, showed a 2-fold and a 3-fold transcription increment in
cells respectively treated with 20 and 50 μg/ml of NC. Usually
expressed at low levels in PC12 (39), dopamine transporter is a
membrane protein involved in the re-uptake of dopamine,
while vesicular monoamine transporter-2 is an integral mem-
brane protein responsible of the transport of monoamines from
cellular cytosol into synaptic vesicles.Onceagain,theirup-
0.0
0.5
1.0
1.5
2.0
Nefl Tub
Normalized expression ( Cq)
0 µg/ml
20 µg/ml
50 µg/ml
0
5
10
15
20
25
30
35
40
45
a
b
c
d
0-20
20-40
40-60
60-80
80-100
100-120
120-140
140-160
160-180
180-200
>200
length (µm)
% neurites
0
5
10
15
20
25
30
35
0-20
20-40
40-60
60-80
80-100
100-120
120-140
140-160
160-180
180-200
>200
length (µm)
% neurites
0
5
10
15
20
25
30
35
0-20
20-40
40-60
60-80
80-100
100-120
120-140
140-160
160-180
180-200
>200
length (µm)
% neurites
**
Fig. 4 Quantitative evaluation of PC12 differentiation: neurite length
distribution of cells incubated in the presence of 0 (a), 20 (b) and 50 μg/
ml (c) of NC; qRT-PCR results of β3-tubulin and neurofilament-66 gene
transcription (c); * p<0.05.
Effects of Cerium Oxide Nanoparticles on PC12 Neuronal-Like Cells 2141
regulation is a consequence of a compensation mechanism due
to an enhanced dopamine production by the cells.
Taken together, all the collected data indicate bene-
ficial effects of NC on PC12 cells, both on neuronal
differentiation and on dopamine production. These ef-
fects could be explained, in view of the performed
analyses, on a more favorable intracellular environment
due to lower ROS level, but direct effects on other
0.0
0.5
1.0
1.5
Gpx1 Gss Txnrd1
Normalized expression (ΔΔCq)
0 µg/ml
20 µg/ml
50 µg/ml
-15
-10
-5
0
5
10
15
20
25
30
35
a
b
c
0102050
Nanoparticle concentration (µg/ml)
Fluorescence variation (%)
control H2O2 treated
H2O2H2O2+ NC 10 µg/ml
H2O2+ NC 20 µg/ml H2O2+ NC 50 µg/ml
200 µm
*
*
*
*
*
*
*
Fig. 5 Fluorescence ROS
production detection in PC12
cells treated with increasing NC
concentrations and, thereafter,
stimulated with H
2
O
2
50 μM(a);
quantitative evaluation of ROS
level in PC12 cells treated with
increasing NC concentrations,
with and without H
2
O
2
stimulation (b); qRT-PCR
evaluation of transcription of
genes involved in cell redox
mechanisms following NC
treatment (c); * p<0.05.
2142 Ciofani et al.
metabolic pathway cannot be totally excluded. Future
studies towards clinical translation will be devoted to
the functionalization of cerium oxide nanoparticles in
order to allow their targeting towards desired tissues, as
well as their intracellular tracking. For in vivo neurolog-
ical applications, the problem of blood brain barrier
crossing will be addressed through functionalization
with specific monoclonal antibodies (i.e.,Ox26(40)).
Furthermore, NC exploitation following other therapeu-
tic approaches, like direct implantation of constructs
aiming at replacing the function of impaired dopami-
nergic neurons, will be explored. This is the case, for
example, of the transplant ofencapsulatedPC12cells
(41): NC could represent a promising physical cue to
be integrated into polymeric scaffolds in order to im-
prove dopamine secretion (42).
0
4
8
12
16
20
a
b
c
02050
nanoparticle concentration (µg/ml)
dopamine concentration (pg/ml/cell)
0.0
0.5
1.0
1.5
2.0
Comt Maoa Th
Normalized expression ( Cq)
0 µg/ml
20 µg/ml
50 µg/ml
0.0
1.0
2.0
3.0
4.0
Dat Vmat2
Normalized expression ( Cq)
0 µg/ml
20 µg/ml
50 µg/ml *
*
*
*
*
*
*
Fig. 6 Evaluation of dopamine
production by PC12 cells treated
with increasing NC concentrations
(b); qRT-PCR evaluation of
transcription of genes involved in
dopamine metabolism (b)and
transport following NC treatment
(c); * p<0.05.
Effects of Cerium Oxide Nanoparticles on PC12 Neuronal-Like Cells 2143
CONCLUSIONS
Applications of cerium oxide nanoparticles in the biomedi-
cal field have been exponentially grown during the latest
years (43). Their impressive catalytic properties make this
nanomaterial an extremely interesting inorganic antioxidant
agent, that could find application against a huge number of
pathological conditions (44). Our results on PC12 cells con-
firm the possibility of the exploitation of NC as a smart
material for modulating cell metabolism and functions.
Moreover, the highlighted increment of dopamine produc-
tion following NC treatment highlights its high potential as
countermeasure in neurodegenerative disorders. Of course,
deeper examinations will be necessary in order to better
clarify the mechanisms at the base of the documented ef-
fects; nonetheless, this study strongly encourages further in-
vestigations in this direction.
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Effects of Cerium Oxide Nanoparticles on PC12 Neuronal-Like Cells 2145
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