HECKMAN ET AL.
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CXXXX American Chemical Society
Custom Cerium Oxide Nanoparticles
Autoimmune Degenerative Disease
in the Brain
Karin L. Heckman,†,r,*William DeCoteau,‡,rAna Estevez,†Kenneth J. Reed,§Wendi Costanzo,§
David Sanford,§James C. Leiter,^Jennifer Clauss,†Kylie Knapp,†Carlos Gomez,†Patrick Mullen,†
Elle Rathbun,†Kelly Prime,†Jessica Marini,†Jamie Patchefsky,†Arthur S. Patchefsky,)
and Joseph S. Erlichman†
Richard K. Hailstone,#
†Departments of Biology and‡Psychology, St. Lawrence University, Canton, New York 13617, United States,§Cerion Enterprises, LLC, Rochester, New York 14610,
United States,^Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire 03756, United States,
Department of Pathology, Fox Chase Medical Center, Temple Health, Philadelphia, Pennsylvania 19111,United States, and#Chester F.Carlson Center for Imaging
Science, Rochester Institute of Technology, Rochester, New York 14623, United States.rBoth authors contributed equally to this work.
ufacturing processes including catalysis,
fuel cells, polishing agents, and combustion
adjuvants. The ability of nanoceria to parti-
cipate in redox coupled reactions has made
these nanoparticles attractive candidates
for development in biological and medical
applications,1?6and recent studies have
focused on the biological outcomes of ad-
ministering cerium oxide nanoparticles to
experimental animals using disease models
in which free radicals are thought to play a
anocrystalline cerium oxide is widely
used in myriad industrial and man-
Cerium is a lanthanide element with a
crystalline structure that can exist in two
oxidation states (Ce3þor Ce4þ), which can
be interchanged depending on the redox
environment within biological systems.5,8,9
The catalytic properties of ceria have been
attributed to the presence of highly mobile
lattice oxygen present at the surface,10?13
nanoparticles in vitro has been studied
extensively.14?21The half-cell potential of
ceria nanoparticles falls between the redox
potential of superoxide, hydroxyl ion and
*Address correspondence to
Received for review June 4, 2013
and accepted November 22, 2013.
ABSTRACT Cerium oxide nanoparticles are potent antioxidants,
based on their ability to either donate or receive electrons as they
alternate between the þ3 and þ4 valence states. The dual
oxidation state of ceria has made it an ideal catalyst in industrial
applications, and more recently, nanoceria's efficacy in neutralizing
biologically generated free radicals has been explored in biological
applications. Here, we report the in vivo characteristics of custom-
synthesized cerium oxide nanoparticles (CeNPs) in an animal model
ofimmunological and free-radical mediated oxidative injury leading
toneurodegenerative disease. TheCeNPs are 2.9 nm indiameter, monodispersed and havea ?23.5 mV zetapotential when stabilized with citrate/EDTA.
This stabilizer coating resists being 'washed' off in physiological salt solutions, and the CeNPs remain monodispersed for long durations in high ionic
strength saline. The plasma half-life of the CeNPs is ∼4.0 h, far longer than previously described, stabilized ceria nanoparticles. When administered
also able to penetrate the brain, reduce reactive oxygen species levels, and alleviate clinical symptoms and motor deficits in mice with a murine model of
multiple sclerosis. Thus, CeNPs may be useful in mitigating tissue damage arising from free radical accumulation in biological systems.
KEYWORDS: nanoparticles.free radicals.experimental autoimmune encephalitis.tissue distribution.blood brain barrier
HECKMAN ET AL.
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peroxynitrite (all have lower half-potentials) and hy-
drogen peroxide (half-cell potential = ?1.7 V). Thus,
ceria is well positioned to oxidize biologically impor-
tant reactive oxygen and nitrogen species with lower
half-potentials, such as superoxide and peroxynitrite.
Further, nanoceria catalyze the same electron trans-
fer reactions as superoxide dismutase, catalase and
glutathione.22The regenerative oscillation between
valencestates suggeststhat nanoceriamayhavevalue
as a therapeutic agent in the treatment of diseases
related to the accumulation of reactive oxygen species
(ROS).3,4However, the majority of studies have used
reduced preparations to study cellular effects in vitro,
which, despite providing important insight into the
mechanisms of action of the particles, do not fully
The studies of ceria function in vivo used nano-
ceria ranging in size from 5 to 55 nm,1,2,7,28often with
negative zeta potentials (<?50 mV), administered
as dispersions or nominally stabilized with citrate or
polyethylene glycol.2,29In rodent models, these parti-
cles were prone to aggregate and accumulate in the
spleen and liver.1,29?31This pattern of tissue distribu-
tion is characteristic of 'bare' nanoparticles and nano-
particles that have not been adequately stabilized. In
either case, excess deposition in the liver may result in
toxicity at higher doses.32?34Nanoceria in the pre-
have promoted oxidative stress in some settings.32
Discrepancies among the biological effects of various
nanoceria formulations have been attributed to many
factors including synthetic routes, composition/purity,
particle size and surface charge.3,4,36Unfortunately,
none of these characteristics, a priori, have been
particularly helpful in evaluating biological outcomes,
which suggests that the interaction of the particles
with proteins in different cellular compartments may
provide more useful insight into biological differences
Clearance of nanoceria from the body is another
previousin vivo studies, administration ofunstabilized,
bare or citrate-stabilized ceria nanoparticles was asso-
ciated with high accumulation in the reticuloendothe-
lial organs, tissue toxicity and poor tissue clearance
rates,29,34all of which suggest that additional surface
functionalization/stabilization may be required before
ceria nanoparticles can be developed for therapeutic
applications. To this end, our group has synthesized
unique cerium oxide nanoparticles (CeNPs) with char-
acteristics distinct from commercially available parti-
cles and those used by other groups.
Our custom CeNPs were synthesized by a novel pro-
cess that generates smaller, highly uniform (2.9 nm)
particles with a less negative zeta potential, and the
particles were treated with a stabilizer comprised of
citrate and EDTA, which resists 'washing' off in physio-
logical solutions.37EDTA seems to alter the surface
by binding to the ceria. Citrate may act as an electron
transfer agent between the particle and reactive oxy-
gen species that reside in the oxygen vacancies on the
surface of the nanoceria crystal. Here, we describe the
biological effects of adminstering our unique CeNPs in
a murine model of multiple sclerosis, experimental
autoimmune encephalomyelitis (EAE). Oxidative stress
generated by immune cells plays a significant role in
this autoimmune and neurodegenerative disease,
which results in the loss of motor function and im-
paired limb movement in affected animals. Intra-
venous delivery of CeNPs reduced clinical disease
severity and improved motor function compared to
control animals when the particles were delivered in
both preventative and therapeutic treatment regi-
mens. CeNP-treated animals also exhibited reduced
ROS levels in the brain following drug administration,
demonstrating that the nanoparticles retained antiox-
idant properties and may be promising candidates for
further development in the treatment of oxidative
Characterization of Custom-Synthesized CeNPs. After syn-
yellow and displayed Tyndall scattering when illu-
minated with a low intensity laser, indicating that
the solution contained well-dispersed colloidal par-
ticles. Figure 1 shows transmission electron microscopy
(TEM) (a) and electron diffraction (ED) images (c) of the
CeNP material. The diffraction pattern is consistent
with a fluorite lattice structure of CeO2; the most
intense line (111) corresponds to 0.312 nm lattice
spacing as expected for the fluorite structure of CeO2.
Results of X-ray diffraction performed on dried parti-
cles corroborated the fluorite structure. The nanopar-
ticles consisted of single 2.4 nm crystallites of cerium
dioxide (Figure 1D). In water, the average hydrody-
namic diameter of the particles was 2.9 ( 0.3 nm
(Figure 1B) with a polydispersity of 0.19 ( 0.03, based
on dynamic light scattering (DLS) measurements
(Figure 1D). The zeta potential of the final particle
was ?23.5 ( 1.3 mV. The material did not change size
when stored at room temperature for up to two years
after synthesis, and repeated, high-speed centrifuga-
tion (100000g, 4 ?C for 3 h) failed to pellet the material
or significantly change its size as determined by DLS
CeNP Size in High Ionic Strength Solutions. To eval-
uate the likelihood of CeNP aggregation in high ionic
strength solutions that mimic biological solutions, our
custom synthesized CeNPs were placed in simulated
body fluid (SBF; 1:1 dilution by volume; ∼305 mOsm)
HECKMAN ET AL.
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for 48 h, and the size of the particles was measured
periodically using dynamic light scattering. Once the
of the CeNPs was unchanged relative to the stock
solution and remained monodispersed for up to 48 h
Plasma Half-Life and Clearance of CeNPs in Healthy
Animals. Next we evaluated the plasma clearance of
the CeNPs when delivered in vivo. The plasma half-life
of the CeNPs (10 mg/kg intravenous dose) was ∼3.7 h
with a mean residence time of 5.4 h in Sprague?
Dawley rats (Figure 2A). To evaluate tissue clearance,
healthy mice (n = 10) were injected with a single,
intravenous dose of CeNPs (20 mg/kg), and ceria
content was measured at different time points. Tissues
were harvested 24 h later to determine the initial
loading dose of ceria in various organs. CeNPs were
detected by inductively coupled mass spectroscopy
(ICP-MS), and the highest tissue levels were measured
in the liver (Figure 2B) and spleen (Figure 2C), followed
by the kidney (Figure 2E) and the brain (Figure 2D).
Over time, the ceria levels in the liver and brain declined
continually, and low levels of ceria were retained even
5 months after administration (Figure 2B,D). CeNP
levels in the spleen and kidney initially rose after the
loading levels were achieved, peaked at 1?2 months
postadministration and then declined (Figure 2C,E). In
contrast to the other organs, the CeNP content in the
the level present at the loading stage. It is noteworthy
that measurable brain levels of ceria were detected
even in these healthy mice with intact BBBs.
Application of CeNPs to a Murine Model of Oxidative
Stress. We sought to observe whether the CeNPs
retained their antioxidant capabilities using an in vivo
disease model. Given the efficacy of the CeNPs in
distribution to the brain in vivo, a central nervous
in (B). Electron diffraction analysis of CeNPs (C) demonstrated the presence of ceria, while X-ray diffraction (D) identified the
crystalline structure. CeNPs were added to simulated body fluid, and their size was determined over a 48 h period using DLS
(E). High ionic strength solutions had little effect on agglomeration and size.
Figure 2. CeNP half-life and tissue clearance. (A) A total of
10 mg/kg of CeNPs was administered intravenously to
healthy Sprague?Dawley rats, and blood was collected
over a 24 h period. Ceria content was measured by ICP-
MS. (B?E) Healthy SJL/J mice were injected with one
intravenous 20 mg/kg CeNP dose, and various tissues were
harvested 24 h (Load) or 1, 2, 3, 4, or 5 months postadmi-
nistration. Liver (B), spleen (C), brain (D), and kidney (E)
as microgramsof Ce pergramof tissuewetweight(μg Ce/g
HECKMAN ET AL.
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system (CNS) disease involving oxidative damage was
selected for study. Multiple sclerosis (MS) is a chronic,
immunological and neurodegenerative disease char-
acterized by degradation of neuronal myelin sheaths
and, at times, neuronal death, resulting in impaired
nerve impulse conduction and, ultimately, cognitive
and motor dysfunction in affected patients.38The
cellular mediators of disease include macrophages
that invade the brain39and produce reactive oxygen
species.38Superoxide anions, peroxynitrite and hydro-
gen peroxide participate in disruption of the BBB
in MS40and also oxidize DNA, proteins and lipids,
damage that can induce genomic instability and the
death of neurons.41We studied a murine model of
MS, experimental autoimmune encephalomyelitis (EAE),
hypothesizing that administration of the CeNPs would
reduce levels of free radicals and alleviate disease
severity in this well-characterized animal model.
Chronic progressive EAE was induced in C57BL/6
mice using the MOG35?55peptide. A variety of CeNP
doses and dosing regimens were tested (Table 1), and
these results were compared with the effectiveness of
the immunomodulatory agent fingolimod, which is
currently used to treat patients with relapsing MS
and which we used to benchmark the effectiveness
of CeNP treatments.42Clinical scores, which take
into account limb mobility, were assigned as a metric
of disease severity. A typical example of EAE disease
progression is depicted in Figure 3A, and the mean
clinical scores of animals treated preventatively or
TABLE 1. Sample Size for Each CeNP Dose and Treatment Regimena
10 mg/kg20 mg/kg30 mg/kg
Preventative: 15 mg/kg the day before and day of induction, on day 3, followed by maintenance dose at right
Therapeutic 3 day delay: first dose day 3 after induction then maintenance dosing
Therapeutic 7 day delay: maintenance dosing only
Control: vehicle control injections
Fingolimod: provided continuously beginning day 7 after induction
n = 13
n = 15
n = 12
n = 17
n = 15
n = 15
n = 15
n = 15
n = 15
n = 15
aCeNPs and control solutions were delivered in 100 μL volume intravenously. Maintenance doses were given every 7 days after induction through day 35.
Figure 3. CeNP treatment delays disease onset and alleviates disease severity of EAE. (A) The temporal pattern of disease
regimen (see Table 1) and in the matched control group. Delay in disease onset (B) and disease severity (AUC expressed as a
percent of the value in control animals) (C) are shown as a function of CeNP dose (10, 20, or 30 mg/kg) and administration
regimen. (D) Mean disease severity during the last 4 days of treatment is shown as a function of the CeNP dose (10, 20, or
30 mg/kg) and administration regimen. For (B?D), black bars indicate preventative CeNP treatment regimens, while
white bars indicate therapeutic treatment regimens of either CeNPs or fingolimod. pre: preventative CeNP regimen.
d3: day 3 therapeutic CeNP regimen. d7: fingolimod or 30 mg/kg CeNP treatment started 7 days after induction of EAE.
fing: fingolimod. Asterisk (*) indicates p < 0.05 compared to control animals.
HECKMAN ET AL.
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therapeutically with 20 mg/kg CeNPs are compared
with those of control animals. Both of the CeNP dosing
regimens were associated with significantly reduced
disease severity compared to control treatment start-
ing approximately 19 days after EAE induction (p <
0.001). Average responses for all treatment groups are
summarized in Figure 3B?D. EAE onset was signifi-
cantly delayed by the preventative 30 mg/kg CeNP
dose and by fingolimod treatment (Figure 3B). In all
dosing paradigms except the 10 mg/kg therapeutic
3 day delay regimen, CeNP treatment significantly
decreased total disease severity (measured as the area
Fingolimod also significantly reduced total disease
severity, and the efficacy of preventative treatment
with 30 mg/kg CeNPs was similar to the efficacy of
fingolimod. Since this EAE model mimics a chronic
(nonrelapsing) form of MS, we examined the durability
of the treatment effects by assessing the average
clinical scores in the last four days (days 32?35) of
compared to control treatment (p e 0.045), including
CeNPs (Figure 3D). Further, the higher doses of CeNPs
were as effective as, and not significantly different from,
the late response to fingolimod treatment (p > 0.05).
Effect of CeNP Treatment on Motor Function.
Though the manifestations of EAE generally develop
in a caudal to rostral direction,43the clinical scoring
scale is dominated by caudal motor function. Since we
were concerned that subtle improvements in motor
metric, we performed specific tests of motor function
to assess the effects of CeNP treatment more comple-
tely: rotarod (mainly caudal function), hanging wire
(rostral function only), and balance beam (both caudal
and rostral function). Predisease performance did not
differ among treatment groups (p > 0.1), and with the
exception of the 10 mg/kg dose, preventative CeNP
administration yielded higher latencies to fall in the
rotarod and hanging wire tasks and improved balance
beam scores compared to the effects of the delayed,
therapeutic dosing regimen (Figure 4). This finding
could be either the result of differences in the timing
of drug delivery or the cumulative dose administered
(animals treated earlier also received a larger cum-
ulative dose of CeNPs). Within a dosing regimen
(i.e., preventative or therapeutic), a significant CeNP
rotarod test (calculated as AUC) in each animal is expressed as a function of the CeNP dose (10, 20, or 30 mg/kg) and
preventative (A) or 3 day therapeutic (B) administration regimen. Cumulative latency to fall on the hanging wire test
20, or 30 mg/kg) and administration regimen. Dashed lines indicate the mean (rotarod and hanging wire tests) or median
(balance beam test) scores of the matched control animals at each dose of CeNPs studied. sec: seconds. Other abbreviations
and symbols are as described in Figure 3.
HECKMAN ET AL.
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higher doses of CeNPs preserved motor function more
effectively in all tests (Figure 4).
CeNP Dose Response Characteristics. In general, cu-
mulative disease severity (measured as AUC of clinical
scores) was inversely related to the individual
CeNP dose regardless of the timing of administration
(Figure 5A). Expressing the reduction in disease sever-
demonstrated that, regardless of dosage timing, high-
er cumulative doses of CeNPs were significantly corre-
lated with the reduction in the AUC of disease severity
may be attributable to brain penetration of the CeNPs,
since tissue levels of CeNPs were well-correlated with
the cumulative dose of CeNPs delivered (r2= 0.83;
p = 0.03) (Figure 5C). No plateau in the relationships
between cumulative CeNP dose and either disease
severity reduction or brain CeNP levels was observed,
suggesting that the maximum therapeutic brain
concentration of CeNPs was not reached in these
Biodistribution of CeNPs in EAE Animals. ICP-MS
analysis of brain tissue indicated that multiple doses
of CeNPs accumulated in the CNS, particularly in the
cerebellum (Figure 6A), a region with significant dam-
age in EAE animals.44Moreover, cerebellar ceria levels
cord and the other brain regions (Figure 6A). Consis-
tent with previous in vitro findings22and our mass
spectroscopy data, TEM analysis of the cerebellum of
CeNP-treated animals revealed deposition of CeNPs
throughout the intracellular compartments in myeli-
nated processes, axons, dendrites and mitochondria
(Figure 6B): sites of ROS production and targets of free
radical damage. Though hepatic toxicity and particle
aggregation have been observed after administration
of other nanoparticle formulations in rodents,30,32?35
we detected relatively low levels of CeNPs in the liver
compared to the CNS (Figure 6A), and we found no
evidence of liver pathology in CeNP-treated animals
with cumulative doses as high as 130 mg/kg of CeNPs
in the preventative treatment regimen. Both control
and treatment groups showed mild focal portal lym-
was minimal, suggesting that these responses in the
liver may have been related to disease induction as
opposed to specific drug treatment effects.
Durability of CeNPs’ Mechanism of Action. To test
whether the CeNPs detected in the cerebellum re-
tained their catalytic activity over time, ROS levels in
the brainswereassessed lateintheEAE diseasecourse
(day 42). Fresh cerebellar brain slices were harvested
from control, fingolimod or CeNP (preventative regi-
men 30 mg/kg dose) treated animals and stained with
the free radical indicator dye, H2CMDCFDA, to provide
an estimate of the total ROS load. ROS levels in tissues
from the fingolimod and control treated animals did
not differ significantly (Figure 7A; p = 0.619), though
yielded disease protection similar to that of the fingolimod treatment, regardless of when the CeNP treatment was started.
The decrease in cumulative disease severity compared to control animals (B) and deposition of CeNPs in the brain (C) are
expressed as a function of the cumulative dose of ceria given in each treatment group. Cerium content in the brain was
analyzed by mass spectroscopy in a subset of animals; thus, the cumulative dose of ceria (x-axis) is not equal in (B) and (C).
HECKMAN ET AL.
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ROS levels in tissues from CeNP treated mice were
reduced by ∼31% (p < 0.001 compared to either
control or fingolimod treated animals), indicating that
the CeNPs retained their catalytic, antioxidant activity.
The majority of the ROS signal was derived from cells
located in the granular/Purkinje cell layers of the
ROS signal reflected the intrinsic ROS generation rate
of different types of neurons rather than nonspecific
slice trauma). Moreover, immunohistochemical stain-
ing of the microvasculature (Figure 7B) showed an
entirely different pattern of staining compared to the
ROS staining, illustrating that intravenously adminis-
tered CeNPs were not captured solely by endothelial
cells, and that ROS were not being neutralized only in
the vascular compartment of the brain. CeNP deposi-
tion extended far beyond vessel endothelial cells and
Figure 6. CeNP deposition in CNS tissues. (A) Tissues from EAE mice treated with 20 mg/kg CeNPs in preventative or
therapeutic regimens were harvested from PBS-perfused animals and subjected to ICP-MS. Brain (without cerebellum) and
spinal cord samples were pooled (br þ cord) for analysis. (B) TEM images of the cerebellum of an EAE mouse treated with 30
mg/kg CeNPs (preventative regimen) are shown; note the dense black particles at the highest magnification. These particles
were particularly prominent in mitochondria. Arrows indicate putative particles. cereb: cerebellum. prevent: preventative
Figure 7. CeNPs decrease cerebellum ROS levels in EAE mice. (A) ROS levels were measured with a nonspecific free radical
fluorophore (CM-H2DCFDA) in brain slices prepared from 30 mg/kg CeNP treated (preventative regimen) or fingolimod
treated mice beginning on day 42 after induction of EAE. This time point fell at least 7 days after the last CeNP treatment. (B)
The cerebellum was stained with an antilaminin AlexaFluorreagent to illustrate the location of the detected ROS staining (C)
relative to vasculature in brain slices. (C) Representative images of ROS detected in brain slices from a CeNP treated animal
(left) and a control animal (right), which was injected with saline only. G/P: granular/Purkinje layer; M: molecular layer.
HECKMAN ET AL.
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penetrated into the actual brain tissue (Figure 7B),
in the TEM images (Figure 6B).
Lack of Effect of CeNPs on Immune Cell Distributions.
Diminished ROS levels in the brains of CeNP-treated
EAE animals implied that the ROS component of EAE
pathology was therapeutically altered by CeNP treat-
ment (Figure 7), which likely contributed to decreased
disease severity (Figures 3 and 4). This efficacy is
encouraging, given that the immune system might
recognize nanomaterials as foreign antigens and
mount an immune response against the CeNPs.45
It seems unlikely that the CeNP-dependent improve-
ments in clinical disease severity scores were derived
from changes in T helper cell or macrophage function
in the CNS, but we tested the possibility that CeNPs
may ameliorate EAE by modulating immune cell func-
tion. We harvested the brains of EAE mice treated with
10 mg/kg CeNPs in preventative or therapeutic 3 day
delay dosage regimens on day 45 (10 days after the
last CeNP treatment). CD4þT helper cells and macro-
phages (CD11bþ CD45þ cells) were quantified using
fluorescence-activated cell sorting (FACS). Levels of
each cell type were lower in CeNP-treated animals
was great variability within treatment groups, and the
differences among the control, preventative and ther-
apeutic groups were not statistically significant (CD4þ
the brains of treated animals, immune cell infiltration
into the brain was not different among the treatment
and control groups, and we have no evidence of
immunomodulatory effects of CeNPs in this model of
EAE, though we tested only the lowest dose of ceria
nanoparticles that we studied.
The CeNP formulation used in the current study
important respects including size (2.9 nm), surface
charge and the stabilizer bound to the surface of the
particle that remains monodispersed in physiological
solutions, resists agglomeration even when centri-
fuged, and has an extended plasma half-life approxi-
mately four times longer than previous ceria/citrate
formulations of similar size.
Delivery of nanoceria to an intact biological system
can yield changes to structure and function that are
distinct from in vitro properties. When weakly stabi-
lized or unmodified nanoceria enter the body, they
interact with proteins, lipids and cells in a complex
milieu with a high ionic strength. Interactions among
the constituents of this milieu seem to modify the
form (i.e., the Vroman Effect).46,47For many materials,
the complement of proteins bound to the particle's
surface results in significant deposition in the reticu-
loendothelial organs (i.e., liver and spleen) and little
opportunity for deposition in other tissues, in particu-
lar, the brain. Commercially available, unstabilized or
HECKMAN ET AL.
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citrate-stabilized cerium oxide nanoparticles were de-
for up to 90 days.1,29?32In the present study, tissue
levels of CeNPs in the liver and spleen were extremely
studies using 5 nm, citrate-stabilized nanoceria at
similar time points. Moreover, the CeNP deposition in
the liver of the animals with EAE (Figure 6A) was
considerably lower than that in healthy mice, suggest-
ing that disease state may influence nanoparticle
distribution in the reticuloendothelial system. These
deposition patternsareconsistent withthe notionthat
our CeNPs adsorbed fewer proteins that direct the
particles to the reticuloendothelial system, and for this
reason, our particles exhibited much longer circulation
times (3.7 vs 1 h). The combination of citrate/EDTA,
perhaps like PEG on other nanoparticles, seemed to
reduce the association of proteins to the surface of the
The ceria content of the organs measured in the
present study decreased below loading values, except
in the spleen. This contrasts with previous reports that
showed little decrease in tissue ceria content up to
3 months postinjection.29For example, using nonsta-
bilized, 3?5 nm particles, Hirst et al. showed that a
cumulative dose of 2.5 mg/kg delivered over a 5 week
period resulted in the deposition of >20000 μg/g of
ceria in the liver and >30000 μg/g in the spleen one
week after the final injection.27In rats, a single
85 mg/kg injection of citrate-stabilized ceria (5 nm)
resulted in 500 μg/g of ceria in the liver up to 90 days
citrate-stabilized ceria, Yokel et al. showed that a
in the liver and spleen across the 3 month study.29
Thus, nonstabilized nanoceria were retained in the
reticuloendothelial system to a much greater extent
than citrate-stabilized particles, regardless of particle
deposition compared to other stabilizer formulations.
Frequent and rapid agglomeration of other nano-
nanomaterial be delivered in water. For many typical
surface treatments used to condition nanoparticles
(citrate, lactate, etc.), high ionic strength solutions
seem to strip away the surface treatment, allowing
the particles to aggregate, which reduces their biolo-
gical activity. In contrast, the citrate/EDTA stabilized
CeNPs were resistant to stabilizer loss in high ionic
strength solutions, remained dispersed in solution and
retained their small size for relatively long periods of
time (Figure 1E). Following administration of nanopar-
ticle formulations used by other groups, both the
large size of the aggregates and the exposure of
the particle surface may accelerate association with
proteins in the body that promote deposition in the
reticuloendothelial system.48?50For example, the
binding of complement proteins to nonstabilized nano-
particles promotes uptake and clearance of nanoma-
terials by circulating and tissue resident macro-
phages.51,52The presence or lack of stabilizer also
correlates with plasma half-life: ∼ 7 min for nonstabi-
lized nanoceria and 1 h for citrate stabilized par-
ticles.30,31,53In contrast, the half-life of our CeNPs was
3.7 h, considerably longer than that of ceria stabilized
with citrate alone. Treatment with a combination of
citrate and EDTA during synthesis of the CeNPs
the CeNPs to evade active removal by cells of the
immune system. In the future, establishing bioequiva-
lence profiles among nanoparticle formulations would
be prudent given the potential differences in calcu-
used by different investigators (e.g., compartmental vs
noncompartmental). We used a noncompartmental
analysis since it appeared tofit the data well,hasfewer
assumptions than the compartmental models, and has
been used to evaluate the pharmacokinetics of other
The differences in nanoceria tissue clearance could
reflect either the physical or chemical features of the
a role in cellular uptake of liposomes, polymer nano-
particles, artificial viruses (DNA coated glycocluster
nanoparticles) and inorganic nanostructures.57?60
Once the material is inside the cell, cellular clearance
may be influenced by the type of cell the material is
sequestered in (i.e., resident tissue macrophages or
other organotypic cells), the subcellular location of the
nanoparticles, and natural degradation processes. We
have not yet examined these possibilities for these
custom CeNPs, but having a better understanding of
the cycle of cellular uptake and clearance is a critical
requirement for the therapeutic development of na-
nomaterials, especially since the patterns of tissue
deposition and clearance appear to vary significantly
among different species and different strains within
Previous studies examining nanoceria deposition
in the brain following intravenous administration
have been inconclusive: brain ceria content was very
low.31,32,35,53The brain deposition of the CeNPs in the
current study was dose-dependent and significantly
higher than that observed in other studies, even
when normalized to the nanoceria dose.27,29,31,32TEM
images from brains of animals with EAE indicate that
our CeNPs were widely distributed within cells of the
cerebellum and were not simply taken up by endothe-
lial cells (Figure 6B). Moreover, the cumulative CeNP
dose correlated well with biological action (Figure 5B),
which is consistent with regional pathological effects
of EAE within the brain. Similar brain ceria content was
measured in both the EAE and healthy mice treated
HECKMAN ET AL.
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with the same doses of CeNPs, which suggests that
our CeNP formulation may cross the normal BBB
(Figures 2D and 6A). Since the tight junctions of the
BBB exceed the size of our particles (4?20 nm), it is
possible that someofthe CeNPs mayhaveentered the
brain via paracellular or transcellular transport pro-
cesses. Alternatively, adsorption of plasma proteins
such as ApoE or albumin may facilitate movement of
CeNPs into tissues such as the brain, or the CeNPs may
enter the brain by selective transport mechanisms.
Resolving this question will require further study.
The amount of CeNPs deposited in the brain was
similar to other metal oxide nanomaterials (Au, Pt, and
Ti) that have either bypassed the BBB via intranasal
delivery or have been engineered to use endogenous,
selective BBB transport mechanisms to improve brain
deposition.61?64We hypothesize that the unique pro-
tein corona formed around these citrate-EDTA stabi-
lized CeNPs is responsible for their organ deposition
pattern, as such protein interactions are among the
We may be able to increase brain deposition of our
CeNPs by functionalizing the surface with peptide
moieties using approaches described by others.67
CeNPs are an extremely powerful superoxide dis-
mutase mimetic; 1 μg/mL of CeNPs is equivalent to
antioxidants currently under development, CeNPs are
unsurpassed in their ability to neutralize free radicals,
while their 'durability' and regenerative catalytic capa-
city allow infrequent dosing compared to other
drugs.70?74This type of antioxidant activity was pre-
served in vivo, as shown by the reduced ROS levels in
the cerebellum of CeNP-treated mice (Figure 7A). The
decreased ROS levels appear to be physiologically
relevant in that these animals with the MOG35?55-
induced, chronic progressive model of EAE also exhib-
ited decreased disease symptoms and preserved
motor function (Figures 3 and 4). In fact, the amount
of CeNPs delivered to the brain correlated with the
decrease in cumulative disease severity, represented
by the AUC analysis (Figure 5B).
The efficacy of CeNPs depended on the dose given
and the timing of treatment initiation. For example,
disease onset was significantly delayed only at higher
CeNP doses when the CeNPs were given before or
shortly after EAE induction, illustrating that higher
individual and cumulative doses and an earlier start
to treatment were needed to mitigate disease severity
preventative regimen) even demonstrated similar
efficacy as fingolimod therapy, though fingolimod
was capable of delaying disease onset even when
delivered 7 days after EAE induction. This comparison
for CeNPs and continuous in the drinking water for
that started later (day 3 or 7) also resulted in lower
cumulative doses of CeNPs. Therapeutic administra-
tion of greater CeNP doses starting at 7 days after
induction of EAE might have matched the benefit of
fingolimod started after EAE induction if higher cumu-
lative doses of CeNP had been given (and preliminary
data from our lab indicates that larger cumulative
doses of CeNP given after EAE induction can match
or exceed the efficacy of fingolimod in EAE).
CeNPs may have to be present at the time of inflam-
matory cell entry into the CNS at some threshold
concentration in order to reduce the levels of ROS in
this disease model. The graduated, serial dosing regi-
threshold level of activity until later in the experiment.
Consistent with such a hypothesis, clinical severity
during the latter phase of disease (days 32?35) was
significantlyreduced atmostCeNP dosescomparedto
control treatment (p e 0.045). Moreover, even starting
the 30 mg/kg dose 7 days after EAE induction reduced
disease severity (Figure 3), and in this late phase,
symptom severity was not different between preven-
tative treatment, fingolimod treatment and the 7 day
delay 30 mg/kg treatment groups (p > 0.05) (Figure 3D).
formation of the MOG35?55model of EAE. Therefore,
full expression of the disease model. To the extent this
is true, the response to CeNPs may be disease model
specific. The late beneficial effects of therapeutic CeNP
regimens argue against this line of reasoning since the
therapeutic dose schedules allowed full development
in the illness. Moreover, we conducted studies of a
relapsing model of EAE induced by immunization with
a different proteolipid protein (PLP139?151), and treat-
ment with CeNPs effectively mitigated symptoms in
this model as well.75Regardless of the timing of CeNP
administration relative to disease onset, the cumula-
tive damage was significantly reduced as the CeNPs
accumulated and the capacity for ROS neutralization
Specific tests of motor function were also better
demonstrating a similar dose-dependent effect as that
observed with clinical disease scores. In general, we
expected CeNPs to improve rostral motor function
more than caudal motor function, where the extent
of neuropathology is greater. There was a trend in this
direction: higher CeNP doses and earlier initiation of
treatment tended to preserve function on the hanging
wire test, while lower CeNP doses and delayed treat-
of EAE in our studies seemed to reflect more equal loss
of both caudal and rostral motor function. By the same
HECKMAN ET AL.
’ NO. XX
token, however, CeNPs improved both caudal and
rostral motor function, especially at higher doses and
when given at the time of EAE induction.
Fingolimod and ceria nanoparticles have distinct
mechanisms of action. Fingolimod interferes with im-
mune cell infiltration into the brain, induces signaling
events through sphingosine-1-phosphate receptors
and modulates glial reactivity.76,77Since migration of
activated T cells to their target sites typically takes
approximately 7 days, administration of fingolimod
within 7 days of EAE induction could still block the
majority of T cell infiltration and, thus, T cell mediated
pathology. In contrast, ROS levels were significantly
lower in cerebellar brain slices of CeNP-treated EAE
animals compared to brain slices in fingolimod treated
ties of the CeNPs were at least partially responsible for
the therapeutic effects of the CeNPs. The clinical dis-
ease severity was similar in the CeNP and fingolimod
treated animals. Therefore, the differences in ROS
levels between CeNP-treated animals and fingolimod
treated animals reflect the action of the CeNPs late in
the disease rather than any differences in disease
severity. Fingolimod may decrease ROS levels earlier
in the disease by reducing infiltration of inflamma-
tory cells and ROS production. The antioxidant effect
of CeNPs occurs mechanistically downstream from
the infiltration of inflammatory cells and may be
inadequate to neutralize the levels of ROS generated
by the early inflammatory events of EAE when either
CeNP therapy is started later or smaller doses of
CeNPs are given. In any event, low levels of ROS
measured in the late phase of disease highlight the
unique and durable mechanism of the CeNPs com-
pared to fingolimod.
CeNPs did not alter levels of T cells or macrophages
present in the brains of EAE animals (Figure 8), sug-
gesting that CeNPs do not impair immune cell entry
into the CNS. The immune cell infiltration studies
were conducted at the lowest tested dose of CeNPs
(10 mg/kg), which showed relatively modest protec-
tive effects. It is possible that some alteration in CNS
infiltration of immune cells could be induced by a
higher CeNP dose (i.e., 30 mg/kg); however, further
analysis of the kinetics of immune cell infiltration,
including phenotype and the polarity of T helper
cells, is needed before a more definitive conclusion
can be made about any immunomodulatory proper-
ties of CeNPs.
While naturally occurring and synthetic antioxidant
therapies have shown some efficacy in similar models
of EAE, decreasing clinical scores 30?70%,70?73the
efficacy of such antioxidant therapies is contingent
upon several factors.78First, the reagent must be able
to cross the BBB and localize not only to affected
tissues, but also individual cells with a high ROS load.
Second, the compound must accumulate in the brain
at a high enough concentration to be clinically effec-
tive in the treatment of disease. Last, the therapeutic
agent must have a half-life in tissue sufficient to inhibit
ROS levels long enough to have an appreciable effect
on disease progression/symptoms and must retain
sufficient catalytic activity to neutralize excessive
amounts of ROS produced as part of the disease
process. Given that the majority of patients with
MS eventually convert to the chronic, progressive
treatment is high, and a less frequent dosing regi-
men is desirable. In this study, the effectiveness of
the relatively infrequent dosing regimen was made
possible by the long half-life of cerium oxide, the
cumulative effect of repeated doses and the dur-
ability of the regenerative, catalytic, antioxidant
activity of CeNPs.
This is the first report to demonstrate efficacy of
cerium oxide nanoparticles in the treatment of a well-
characterized, autoimmune, neurodegenerative dis-
ease model in intact animals. Most of the therapeutic
potential of commercial cerium oxide nanoparticles
has been assessed using in vitro or cell culture
models6,24,26or in vivo in models with no clear clinical
correlate.27Nevertheless, CeNPs have reduced retinal
damage,1reduced the size of infarcts in a middle
cerebral artery model of ischemia in rodents,2and
improved cardiac function in a murine model of
effects of cerium oxide nanoparticles have been attrib-
uted to the antioxidant activity of the particles. In the
current study, CeNPs mitigated the severity of EAE in
mice, as indicated by reduced disease severity scores
and preserved motor function. Thus, CeNPs are novel
synthetic antioxidant catalysts with measurable CNS
penetrance, prolonged retention in the CNS, and high,
regenerative catalytic activity that hold great promise
for use in MS patients and, potentially, other ROS-
mediated disorders of the CNS.
Synthesis and Morphology of CeNPs. Aqueous 20?30% NH4OH
was added under high speed mixing to a heated water
(EDTA) and citric acid for stabilization. Aqueous solutions of Ce
H2O2solution was added to the vessel and mixed with a high
raised to 80 ?C and held at this temperature for 60 min. The
reaction was cooled to room temperature overnight and diafil-
tered to remove excess free ions with a Millipore, cellulose
HECKMAN ET AL.
’ NO. XX
regenerated column to a pH of 7.2 and conductivity less than
10 mS. Particle size and composition were confirmed via
electron diffraction (ED), X-ray diffraction (XRD), dynamic light
scattering (DLS) and transmission electron microscope (TEM).
The combination of EDTA and citrate prevented aggregation
only with EDTA did not exhibit significant neuroprotective
effects when tested in our in vitro brain slice assay.4
X-ray Diffraction (XRD) Analysis. The stock CeNP solutions were
concentrated, placed onto a zero background quartz disk,
allowed to dry under a heat lamp, and then dried in an oven
for four hours at 80 ?C in vacuum. The solid sample on quartz
was analyzed by XRD in a N2dry cell attachment. The sample
was analyzed for crystallite size in the CeO2(220) direction,
determined using the Scherrer technique.
Dynamic Light Scattering (DLS). The CeNP stock solution (73 mM)
was filtered (0.22 μm), and the particle size was determined
using a Brookhaven DLS Instrument.
Electron Diffraction. Selected-area diffraction patterns were
imaged with a 100 μm diameter aperture and a camera
length of 55 cm. The camera length was calibrated using an
Al polycrystalline standard. The radii of diffraction rings were
determined using the radial profile plug-in in Image J software.
fluid (SBF): 140 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 2.5 mM
CaCl2, 1.5 mM MgSO4, 10 mM Na-Hepes (pH: 7.25). DLS analysis
was used to assess particle size at various time points.
CeNP Half-Life. Three Sprague?Dawley rats (∼200 g body
weight) received a single, 10 mg/kg intravenous injection of
CeNPs, and blood was drawn periodically from the lateral tail
vein. Ceria content from the blood collected at each time point
was measured using ICP-MS. Plasma half-life was calculated
with a noncompartmental analysis model based on WinNonlin,
a pharmacokinetic analysis program.
Clearance of CeNPs from Healthy Mice. Female SJL/J mice were
purchased from Jackson Laboratories and used at 8?10 weeks of
age. CeNPs were delivered intravenously at 20 mg/kg on day 0.
Animals were euthanized and transcardially perfused with phos-
phate buffered saline (PBS) before organs were harvested at
the following time points: 24 h, 1 month, 2 months, 3 months,
Induction of EAE in Mice. All protocols involving animals were
approved by the St. Lawrence University Institutional Animal
Care and Use Committee (IACUC). Female C57BL/6 mice were
purchased from Jackson Laboratories and maintained in our
animal facility. Chronic progressive EAE was induced in 8?
10 week old mice on day 0 by subcutaneous injection of a
homogenized mixture of 200 μg MOG35?55peptide (Genscript,
Piscataway, NJ) in 50 μL of PBS with 50 μL of incomplete
Freund's adjuvant (Sigma Aldrich, St. Louis, MO) containing
4 mg/mL Mycobacterium tuberculosis H37Ra (BD Diagnostic
Systems, Sparks, MD). An intraperitoneal injection of 200 ng of
pertussis toxin (List Biological Laboratories, Campbell CA) in
100 μL of PBS was also delivered on days 0 and 2.
CeNP and Fingolimod Administration. CeNPs were administered
to the mice in a sterile, sodium citrate buffer (70 mM NaCl,
70 mM Na-citrate, 10 mM Na-Hepes, pH 7.4; 100 μL injection
volume), andthissolution alonewas usedforcontrol injections.
CeNPs were administered intravenously in one of three regi-
mens: preventative, therapeutic-3 day delay, or therapeutic-7
day delay. The preventative group received CeNPs starting one
day before disease induction, on the day of induction, on day 3
and then at set intervals after induction (see Table 1), whereas
the therapeutic groups received the drug starting either 3 or
7 days after induction of EAE. Fingolimod (Cayman Chemical,
Ann Arbor, MI) was prepared fresh daily and given in the
drinking water (2 μg/L) beginning on day 7.
Clinical Scoring. Disease severity was rated twice daily, inde-
pendently by two or more observers. Clinical scores were
assigned as follows: 0, normal tail tone and limb movement;
0.5, tail drags when walking, but can still curl around observer's
finger; 1, tail has no tone or curl and drags when animal walks;
2, impaired or clumsy gait; 2.5, partial paralysis of one or both
paralysis of both hind limbs; 4, partial or complete paralysis of
one or both front limbs; 5, moribund. Any mouse with a clinical
score g4 oraclinicalscore of3.5 andaninability togroom itself
for greater than 5 days was euthanized. Two or three investi-
gators were involved in the clinical scoring, and the diversity of
clinical scores among the investigators was less than 15%. As a
measure of cumulative disease severity, the area under the
curve (AUC) of daily clinical scores was calculated for each
animal. Clinicalscores waxandwaneinEAE,andthearea under
the curve is less sensitive to day-to-day variations in clinical
scores and provides a sensitive index of disease severity over
the entire time course of the disease.79The AUC of the clinical
score for each animal and the means between the treatment
groups were compared. The onset of illness was defined as the
the clinical scores increased by g20%. Estimates for disease
by averaging the clinical scores of the last four days of the
experiment (days 32?35).
Tests of Motor Function. For the rotarod test, each mouse was
placed onto a drum (Med Associates, St. Albans, VT) rotating at
28 rpm, and the latency to fall from the drum (300 s, maximum)
was turned upside down 60 cm above the counter top, and the
latency to fall was measured. The balance beam apparatus
consisted of a 28 mm square beam (100 cm in length and
elevated 50 cm above the counter top) that led to a 20 cm ?
20 cm dark enclosure. A 60 W light bulb illuminated the open
end of the beam and motivated the mice to traverse the beam
and reach the dark goal box. Each mouse was placed on the
illuminated end of the beam and given up to 60 s to reach
the dark, goal box. Balance and gait quality were scored using a
5 point scale (5 = normal gait to 0 = fallsoff beam immediately).
For all tasks, each animal was trained for 5 days before EAE
induction. After induction, tests were performed daily for the
duration of the experiment.
Biodistribution of CeNPs in EAE Mice. EAE mice were euthanized
by isoflurane overdose and transcardially perfused with PBS,
and organs were removed to analyze tissue-specific ceria con-
tent.Thedissection tools werecleaned betweeneachanimal to
decrease cross contamination with ceria. Tissues were frozen
and analyzed by ICP-MS at the Trace Metal andAnalytics facility
at Dartmouth College. Organ samples (50?100 mg) were im-
cool. After cooling was achieved, deionizedwater was added to
each sample tube to achieve a final acid content of 5%.
TEM Analysis. Tissue samples for TEM analysis were fixed in
glutaraldehyde and osmium tetraoxide, then treated with suc-
cessively higher ratios of ethanol/water and finally immersed in
100% acetone. These dehydrated samples were immersed in
3/1 acetone/Embed 812 for 12 h, 1/1 acetone/embed 812 for
4 h, 3/1 acetone/Embed 812 for 4 h, and finally 100% Embed
812 for 12 h. Last, each sample was cured at 60 ?C for 12 h. Thin
sections (70 nm) of the epoxy-embedded tissue were prepared
with an ultramicrotome and transferred to lacey-carbon cov-
ered, copper TEM grids. The sections were imaged at 100 kV
using a JEOL 100CX II transmission electron microscope.
after behavioral testing and drug therapy had ended (post-
induction day 42), mice exhibiting the most severe clinical
symptoms in each group were sacrificed by rapid decapitation.
Brains were quickly removed and placed in a chilled, choline-
Bregma) using a vibratome.22The samples were allowed to
recover for 1 h in control, artificial cerebral spinal fluid (aCSF)
and bubbled with 5% CO2, 95% O2gas (pH 7.4, 300 mOsm). For
each ROS measurement, two sets of anatomically matched
brain slices were taken from EAE animals, one from a control
animal. The CeNp and fingolimod treated animals were also
matched on their peak clinical scores.
Measurement of Reactive Oxygen/Nitrogen Species in Brain Slices.
Brain slices were loaded with 20 μM 5-chloromethyl-20,70-
dichlorodihydro-fluorescein diacetate (CM-H2DCFDA; Invitrogen
HECKMAN ET AL.
’ NO. XX
Grand Island, NY) dissolved in DMSO and added to the aCSF
Hibernate A low fluorescence solution (Brain Bits, LLC, Spring-
field, IL). After a washing step, the sections were mounted on a
coverslip with Hibernate A and visualized with a Nikon TE
tial images were collected in a randomized order using a 4?
Plan Fluor objective (Nikon Instruments) under identical
conditions (i.e., light intensity, exposure time, camera acqui-
sition settings). Fluorescence was measured by briefly
(∼300 ms) exciting the tissue at 480 ( 40 nm. Emitted fluo-
rescence (535 ( 50 nm) from the probe was filtered using a
505 nm, long-pass, dichroic mirror (Chroma Technology
Bennington, VT), intensified and measured with a cooled
CCD gain EM camera (Hamamatsu CCD EM C9100; Bridge-
water, NJ). These exposure times did not induce photo-
oxidation in the sections even after repeated image capture.
SimplePCI 6.0 software (C Imaging Systems Cranberry Town-
ship, PA). Four images were captured, the total gray level for
each was calculated, and the light intensities were summed
to determine a total gray level for the section. Results were
expressed as the ratio of the fluorescence in each test
condition to fluorescence in the matched control slice im-
aged at the same time point within the experimental
Microvasculature Staining and Visualization. Alternatively, live
brain sections (prepared as described above) were stained with
mouse anti-laminin conjugated to Alexa Fluor 488 (EMD Milli-
pore; 1:100) for 1 h in aCSF. Tissue was washed twice in low
fluorescence Hibernate A at room temperature (15 min each
wash; Brain Bits, Springfield, IL). Sections were mounted on a
coverslip with a drop of Hibernate A, and microvasculature was
visualized using epifluorescence microscopy as described for
CNS Cellular Infiltration. On day 45, 10 days after clinical test-
ing and drug therapy had ended, mice that were treated with
overdose prior to transcardial perfusion with PBS. Brain and
spinal cord tissues were harvested and homogenized, and
lymphocytes were isolated using Percoll gradient separation
(GE Healthcare, Pittsburgh, PA). Any red blood cells remaining
among the isolated lymphocytes were lysed by exposure to
hypotonic conditions. The remaining lymphocytes were
stained with anti-CD4 PE (eBioscience, San Diego CA), anti-
CD11b PerCp (BioLegend, San Diego CA), and anti-CD45 PE
(BioLegend) antibodies and fixed with 4% paraformaldehyde
for flow cytometry analysis. Samples were analyzed at the
Wistar Institute (Philadelphia, PA), and data analysis was
performed using the FCS Express program (De Novo Soft-
ware, Los Angeles, CA).
Statistical Analysis. For statistical analysis, the area under the
curve was calculated for clinical scores and for motor tests by
summing the daily scores to generate cumulative disease
severity measurements. For ordinal data (clinical scores and
balance beam), Freidman's test for nonparametric, multiple
comparisons was used to evaluate main effects, and Dunn's
post-hoc test (one-tailed) was used to compare treatment
groups to control. Day-by-day comparisons and other compar-
hanging wire data were analyzed using a repeated measures
ANOVAto testformaineffects,andDunn's test(one-tailed)was
used for post-hoc tests when the ANOVA indicated that sig-
nificant differences existed among treatment groups. A re-
peated measures design to test for main effects was used to
assess dose?responses for clinical scores and motor testing
used, and the Holm-Sidak method was used for post-hoc
analysis. For statistical analysis of sections labeled with the
fluorescent, free radical probe, raw fluorescence values were
compared using a factorial ANOVA and Dunnett's post-hoc
Conflict of Interest: The authors declare the following com-
peting financial interest(s): Drs. Ana Estevez, William DeCoteau,
Joseph Erlichman, and Kenneth Reed have equity ownership that
exceeds 5% in Cerion NRx which funded a portion of this work.
REFERENCES AND NOTES
McGinnis, J. F. Nanoceria Extend Photoreceptor Cell Life-
span in Tubby Mice by Modulation of Apoptosis/Survival
Signaling Pathways. Neurobiol. Dis. 2011, 42, 514–523.
H.; Yang, H. S.; Kim, J. Y.; Park, H. K.; et al. Ceria Nanopar-
ticles That Can Protect against Ischemic Stroke. Angew.
Chem., Int. Ed. 2012, 51, 11039–11043.
3. Andreescu, A.; Ornatska, M.; Erlichman, J. S.; Estevez, A. Y.;
Leiter, J. C. Biomedical Applications of Metal Oxide Nano-
particles. In Fine Particles in Medicine and Pharmacy;
Matijevi? c, E., Ed.; Springer ScienceþBusiness Media, LLC:
New York, 2012; pp 57?100.
4. Estevez, A. Y.; Erlichman, J. S. Cerium Oxide Nanoparticles
for the Treatment of Neurological Oxidative Stress Dis-
eases. In Oxidative Stress: Diagnostics, Prevention, and
Therapy; Andreescu, S., Ed.; American Chemical Society:
Washington, DC, 2011; pp 255?288.
5. Tarnuzzer, R. W.; Colon, J.; Patil, S.; Seal, S. Vacancy
Engineered Ceria Nanostructures for Protection from
Radiation-Induced Cellular Damage. Nano Lett. 2005, 5,
6. Schubert, D.; Dargusch, R.; Raitano, J.; Chan, S.-W. Cerium
and Yttrium Oxide Nanoparticles Are Neuroprotective.
Biochem. Biophys. Res. Commun. 2006, 342, 86–91.
7. Colon, J.; Herrera, L.; Smith, J.; Patil, S.; Komanski, C.;
Kupelian, P.; Seal, S.; Jenkins, D. W.; Baker, C. H. Protection
from Radiation-Induced Pneumonitis Using Cerium Oxide
Nanoparticles. Nanomedicine 2009, 5, 225–231.
8. Campbell, C. T.; Peden, C. H. Oxygen Vacancies and
Catalysis on Ceria Surfaces. Science 2005, 309, 713–714.
9. Zhang, H. Z.; Chen, B.; Ren, Y.; Waychunas, G. A.; Banfield,
J. F. Response of Nanoparticle Structure to Different Types
of Surface Environments: Wide-Angle X-Ray Scattering
and Molecular Dynamics Simulations. Phys. Rev. B 2010,
10. Trovarelli,A.Structural andOxygenStorage/ReleaseProp-
erties of CeO2-Based Solid Solutions. Comments Inorg.
Chem. 1999, 20, 263–284.
11. Schalow, T.; Laurin, M.; Brandt, B.; Schauermann, S.; Gui-
mond, S.; Kuhlenbeck, H.; Starr, D. E.; Shaikhutdinov, S. K.;
Interface of Catalyst Nanoparticles. Angew. Chem., Int. Ed.
2005, 44, 7601–7605.
12. Dutta, P.; Pal, S.; Seehra, M. S.; Shi, Y.; Eyring, E. M.; Ernst,
R. D. Concentration of Ce3þand Oxygen Vacancies in
Cerium Oxide Nanoparticles. Chem. Mater. 2006, 18,
13. Sayle, T. X.; Molinari, M.; Das, S.; Bhatta, U. M.; Mobus, G.;
Parker, S. C.; Seal, S.; Sayle, D. C. Environment-Mediated
Structure, Surface Redox Activity and Reactivity of Ceria
Nanoparticles. Nanoscale 2013, 5, 6063–6073.
14. Chen, J.; Patil, S.; Seal, S.; McGinnis, J. F. Rare Earth Nano-
particles Prevent Retinal Degeneration Induced by Intra-
cellular Peroxides. Nat. Nanotechnol. 2006, 1, 142–150.
15. Wong, L. L.; Hirst, S. M.; Pye, Q. N.; Reilly, C. M.; Seal, S.;
McGinnis, J. F. Catalytic Nanoceria Are Preferentially Re-
tained in the Rat Retina and Are Not Cytotoxic after
Intravitreal Injection. PLoS One 2013, 8, e58431.
16. Das, S.; Singh, S.; Dowding, J. M.; Oommen, S.; Kumar, A.;
Sayle, T. X.; Saraf, S.; Patra, C. R.; Vlahakis, N. E.; Sayle, D. C.;
et al. The Induction of Angiogenesis by Cerium Oxide
Nanoparticles through the Modulation of Oxygen in In-
tracellular Environments. Biomaterials 2012, 33, 7746–
17. Dowding, J. M.; Dosani, T.; Kumar, A.; Seal, S.; Self, W. T.
(NO). Chem. Commun. 2012, 48, 4896–4898.
18. Zhou, X. H.; Wong, L. L.; Karakoti, A. S.; Seal, S.; McGinnis,
J. F. Nanoceria Inhibit the Development and Promote the
Vldlr Knockout Mouse. PLoS One 2011, 6, e16733.
19. Cimini, A.; D'Angelo, B.; Das, S.; Gentile, R.; Benedetti,
E.; Singh, V.; Monaco, A. M.; Santucci, S.; Seal, S.
HECKMAN ET AL.
’ NO. XX
Antibody-Conjugated Pegylated Cerium Oxide Nanopar-
20. Karakoti, A.; Singh, S.; Dowding, J. M.; Seal, S.; Self, W. T.
Redox-Active Radical Scavenging Nanomaterials. Chem.
Soc. Rev. 2010, 39, 4422–4432.
21. Bhargava, N.; Das, M.; Karakoti, A. S.; Patil, S.; Kang, J. F.;
Stancescu, M.; Kindy, M. S.; Seal, S.; Hickman, J. J. Regen-
eration of Adult Mice Motoneurons Utilizing a Defined
System and Anti-Oxidant Nanoparticle. J. Nanoneurosci.
2009, 1, 130–143.
Lucky, J. J.; Ludington, J. S.; Chatani, P.; Mosenthal, W. P.;
Nanoparticles in a Mouse Hippocampal Brain Slice Model
of Ischemia. Free Radical Biol. Med. 2011, 51, 1155–1163.
23. Cohen, C. A.; Kurnick, M. D.; Rzigalinski, B. A. Cerium Oxide
Nanoparticles Extend Lifespan and Protect Drosophila
Melanogaster from Paraquat (Pq)-Induced Oxidative
Stress (Os). Free Radical Biol. Med. 2006, 41, S20–S20.
Cohen, C. A. Radical Nanomedicine. Nanomedicine (London,
U.K.) 2006, 1, 399–412.
25. Silva, G. A. Seeing the Benefits of Ceria. Nat. Nanotechnol.
2006, 1, 92–94.
26. Das, M.; Patil, S.; Bhargava, N.; Kang, J.-F.; Riedel, L. M.; Seal,
S.; Hickman, J. J. Auto-Catalytic Ceria Nanoparticles Offer
Neuroprotection to Adult Rat Spinal Cord Neurons. Bio-
materials 2007, 28, 1918–1925.
S.; Reilly, C. M. Bio-Distribution and in Vivo Antioxidant
Effects of Cerium Oxide Nanoparticles in Mice. Environ.
Toxicol. 2011, 28, 107–118.
28. Niu, J.; Azfer, A.; Rogers, L. M.; Wang, X.; Kolattukudy, P. E.
Cardioprotective Effects of Cerium Oxide Nanoparticles in
a Transgenic Murine Model of Cardiomyopathy. Cardio-
vasc. Res. 2007, 73, 549–559.
29. Yokel, R. A.; Au, T. C.; MacPhail, R.; Hardas, S. S.; Butterfield,
D. A.; Sultana, R.; Goodman, M.; Tseng, M. T.; Dan, M.;
Haghnazar, H.; et al. Distribution, Elimination, and Bio-
Ceria-Engineered Nanomaterial in Rats. Toxicol. Sci. 2012,
30. Yokel, R. A.; Florence, R. L.; Unrine, J. M.; Tseng, M. T.;
Graham, U.M.;Wu,P.Y.K.;Grulke, E.A.;Sultana,R.;Hardas,
S. S.; Butterfield, D. A. Biodistribution and Oxidative Stress
Effects of a Systematically-Introduced Commercial Ceria
Engineered Nanomaterial. Nanotoxicology 2009, 3, 234–
M.; Florence, R. L.; Unrine, J. M.; Graham, U. M.; Wu, P.;
Grulke, E. A.; et al. Brain Distribution and Toxicological
Evaluation of a Systemically Delivered Engineered Nano-
scale Ceria. Toxicol. Sci. 2010, 116, 562–576.
32. Hardas, S. S.;Sultana, R.;Warrier, G.; Dan, M.;Florence, R.L.;
Wu, P.; Grulke, E. A.; Tseng, M. T.; Unrine, J. M.; Graham,
U. M.; et al. Rat Brain Pro-Oxidant Effects of Peripherally
Administered 5 nm Ceria 30 Days after Exposure. Neuro-
toxicology 2012, 33, 1147–1155.
33. Nalabotu, S. K.; Kolli, M. B.; Triest, W. E.; Ma, J. Y.; Manne,
N. D.; Katta, A.; Addagarla, H. S.; Rice, K. M.; Blough, E. R.
Intratracheal Instillation of Cerium Oxide Nanoparticles
J. Nanomed. 2011, 6, 2327–2335.
Unrine, J. M.; Graham, U.; Butterfield, D. A.; Grulke, E. A.;
et al. Alteration of Hepatic Structure and Oxidative Stress
Induced by Intravenous Nanoceria. Toxicol. Appl. Pharma-
col. 2012, 260, 173–182.
35. Sousa, F.; Mandal, S.; Garrovo, C.; Astolfo, A.; Bonifacio, A.;
Multimodal Microscopic Brain Distribution Study. Nano-
scale 2010, 2, 2826–2834.
36. Dowding, J. M.; Das, S.; Kumar, A.; Dosani, T.; McCormack,
R.; Gupta, A.; Sayle, T. X.; Sayle, D. C.; von Kalm, L.; Seal, S.;
et al. Cellular Interaction and Toxicity Depend on Physi-
cochemical Properties and Surface Modificationof Redox-
Active Nanomaterials. ACS Nano 2013, 7, 4855–4868.
37. DiFrancesco, A. G.; Hailstone, R. K.; Langner, A.; Reed, K. J.
Method of Preparing Cerium Dioxide Nanoparticles. U.S.
Patent Application Publication 2011/0056123.
38. Hemmer, B.;Nessler,S.;Zhou,D.;Kieseier,B.;Hartung,H. P.
Immunopathogenesis and Immunotherapy of Multiple
Sclerosis. Nat. Clin. Pract. Neurol. 2006, 2, 201–211.
39. Raivich, G.; Banati, R. Brain Microglia and Blood-Derived
Macrophages: Molecular Profiles and Functional Roles in
Multiple Sclerosis and Animal Models of Autoimmune
Demyelinating Disease. Brain Res. Brain Res. Rev. 2004,
40. Huppert, J.; Closhen, D.; Croxford, A.; White, R.; Kulig, P.;
Pietrowski, E.; Bechmann, I.; Becher, B.; Luhmann, H. J.;
Waisman, A.; et al. Cellular Mechanisms of Il-17-Induced
Blood-Brain Barrier Disruption. FASEB J. 2010, 24, 1023–
41. van Horssen, J.; Witte, M. E.; Schreibelt, G.; de Vries, H. E.
Radical Changes in Multiple Sclerosis Pathogenesis. Bio-
chim. Biophys. Acta 2011, 1812, 141–150.
42. Cohen, J. A.; Barkhof, F.; Comi, G.; Hartung, H. P.; Khatri,
B. O.; Montalban, X.; Pelletier, J.; Capra, R.; Gallo, P.;
Izquierdo, G.; et al. Oral Fingolimod or Intramuscular
Interferon for Relapsing Multiple Sclerosis. N. Engl. J.
Med. 2010, 362, 402–415.
43. Ledeboer, A.; Wierinckx, A.; Bol, J. G.; Floris, S.; Renardel de
Lavalette, C.; De Vries, H. E.; van den Berg, T. K.; Dijkstra,
C. D.; Tilders, F. J.; van dam, A. M. Regional and Temporal
Expression Patterns of Interleukin-10, Interleukin-10 Re-
ceptor and Adhesion Molecules in the Rat Spinal Cord
During Chronic Relapsing EAE. J. Neuroimmunol. 2003,
of Tremor in Multiple Sclerosis. Brain 2001, 124, 720–730.
45. Dobrovolskaia, M. A.; McNeil, S. E. Immunological Proper-
46. Vroman, L.; Adams, A. L.; Fischer, G. C.; Munoz, P. C.
Interaction of High Molecular Weight Kininogen, Factor
XII, andFibrinogen inPlasma atInterfaces. Blood 1980,55,
47. Walkey, C. D.; Chan, W. C. Understanding and Controlling
the Interaction of Nanomaterials with Proteins in a Phy-
siological Environment. Chem. Soc. Rev. 2012, 41, 2780–
48. Fischer, H. C.; Chan, W. C. Nanotoxicity: The Growing Need
49. Aggarwal, P.; Hall, J. B.; McLeland, C. B.; Dobrovolskaia,
M. A.; McNeil, S. E. Nanoparticle Interaction with Plasma
Proteins as It Relates to Particle Biodistribution, Biocom-
patibility and Therapeutic Efficacy. Adv. Drug Delivery Rev.
2009, 61, 428–437.
50. Karmali, P. P.; Simberg, D. Interactions of Nanoparticles
with Plasma Proteins: Implication on Clearance and Toxi-
city of Drug Delivery Systems. Expert Opin. Drug Delivery
2011, 8, 343–357.
51. Walkey, C. D.; Olsen, J. B.; Guo, H.; Emili, A.; Chan, W. C.
Nanoparticle Size and Surface Chemistry Determine Ser-
um Protein Adsorption and Macrophage Uptake. J. Am.
Chem. Soc. 2012, 134, 2139–2147.
52. Rodriguez, P. L.; Harada, T.; Christian, D. A.; Pantano, D. A.;
Phagocytic Clearance and Enhance Delivery of Nanopar-
ticles. Science 2013, 339, 971–975.
53. Dan, M.; Tseng, M. T.; Wu, P.; Unrine, J. M.; Grulke, E. A.;
Yokel, R. A. Brain Microvascular Endothelial Cell Associa-
tion and Distribution of a 5 nm Ceria Engineered Nano-
material. Int. J. Nanomed. 2012, 7, 4023–4036.
54. Shahnaz, G.; Iqbal, J.; Rahmat, D.; Perera, G.; Laffleur, F.;
Rossi, D.; Bernkop-Schnurch, A. Development and in Vivo
Characterization of a Novel Peptide Drug Delivery System
HECKMAN ET AL. Download full-text
’ NO. XX
Providing Extended Plasma Half Life. J. Controlled Release
2012, 157, 375–382.
55. Arvizo, R. R.; Miranda, O. R.; Moyano, D. F.; Walden, C. A.;
Giri, K.; Bhattacharya, R.; Robertson, J. D.; Rotello, V. M.;
Reid, J. M.; Mukherjee, P. Modulating Pharmacokinetics,
Tumor Uptake and Biodistribution by Engineered Nano-
particles. PLoS One 2011, 6, e24374.
56. Chu, K. S.; Hasan, W.; Rawal, S.; Walsh, M. D.; Enlow, E. M.;
Luft, J. C.; Bridges, A. S.; Kuijer, J. L.; Napier, M. E.; Zamboni,
W. C.; et al. Plasma, Tumor and Tissue Pharmacokinetics
of Docetaxel Delivered via Nanoparticles of Different
Sizes and Shapes in Mice Bearing Skov-3 Human Ovar-
ian Carcinoma Xenograft. Nanomedicine 2013, 9, 686–
57. Rensen, P. C.; Sliedregt, L. A.; Ferns, M.; Kieviet, E.; van
Rossenberg, S. M.; van Leeuwen, S. H.; van Berkel, T. J.;
Biessen, E. A. Determination of the Upper Size Limit for
Uptake and Processing of Ligands by the Asialoglycopro-
tein Receptor on Hepatocytes in Vitro and in Vivo. J. Biol.
Chem. 2001, 276, 37577–37584.
58. Win, K. Y.; Feng, S. S. Effects of Particle Size and Surface
Coating on Cellular Uptake of Polymeric Nanoparticles for
Oral Delivery of Anticancer Drugs. Biomaterials 2005, 26,
Size and Shape Dependence of Gold Nanoparticle Uptake
into Mammalian Cells. Nano Lett. 2006, 6, 662–668.
60. Chithrani, B. D.; Chan, W. C. Elucidating the Mechanism of
Cellular Uptake and Removal of Protein-Coated Gold
Nanoparticles of Different Sizes and Shapes. Nano Lett.
2007, 7, 1542–1550.
61. Wang, J.; Liu, Y.; Jiao, F.; Lao, F.; Li, W.; Gu, Y.; Li, Y.; Ge, C.;
Zhou, G.; Li, B.; et al. Time-Dependent Translocation and
Potential Impairment on Central Nervous System by In-
tranasally Instilled TiO2Nanoparticles. Toxicology 2008,
62. Gan, C. W.; Feng, S. S. Transferrin-Conjugated Nanoparti-
cles of Poly(lactide)-D-alpha-tocopheryl Polyethylene Gly-
col Succinate Diblock Copolymer for Targeted Drug
Delivery across the Blood-Brain Barrier. Biomaterials
2010, 31, 7748–7757.
63. Lasagna-Reeves, C.; Gonzalez-Romero, D.; Barria, M. A.;
A.; Vergara, L.; Kogan, M. J.; Soto, C. Bioaccumulation and
Toxicity of Gold Nanoparticles after Repeated Administra-
tion in Mice. Biochem. Biophys. Res. Commun. 2010, 393,
64. Takamiya, M.; Miyamoto, Y.; Yamashita, T.; Deguchi, K.;
Ohta, Y.; Ikeda, Y.; Matsuura, T.; Abe, K. Neurological and
Pathological Improvements of Cerebral Infarction in Mice
with Platinum Nanoparticles. J. Neurosci. Res. 2011, 89,
65. Dutta, D.; Sundaram, S. K.; Teeguarden, J. G.; Riley, B. J.;
Fifield, L. S.; Jacobs, J. M.; Addleman, S. R.; Kaysen, G. A.;
Moudgil, B. M.; Weber, T. J. Adsorbed Proteins Influence
the Biological Activity and Molecular Targeting of Nano-
materials. Toxicol. Sci. 2007, 100, 303–315.
66. Luck, M.; Paulke, B. R.; Schroder, W.; Blunk, T.; Muller, R. H.
Analysis of Plasma Protein Adsorption on Polymeric
Nanoparticles with Different Surface Characteristics.
J. Biomed. Mater. Res., Part A 1998, 39, 478–485.
67. Malhotra, M.; Prakash, S. Targeted Drug Delivery across
Blood-Brain-Barrier Using Cell Penetrating Peptides
Tagged Nanoparticles. Curr. Nanosci. 2011, 7, 81–93.
68. Ganesana, M.; Erlichman, J. S.; Andreescu, S. Real-Time
Monitoring of Superoxide Accumulation and Antioxidant
Activity in a Brain Slice Model Using an Electrochemical
Cytochrome C Biosensor. Free Radical Biol. Med. 2012, 53,
69. Heckert, E. G.; Karakoti, A. S.; Seal, S.; Self, W. T. The Role of
Cerium Oxide Redox State in the SOD Mimetic Activity of
Nanoceria. Biomaterials 2008, 29, 2705–2709.
70. Aktas, O.; Waiczies, S.; Smorodchenko, A.; Dorr, J.; Seeger,
B.; Prozorovski, T.; Sallach, S.; Endres, M.; Brocke, S.; Nitsch,
R.; et al. Treatment of Relapsing Paralysis in Experimental
Encephalomyelitis by Targeting Th1 Cells through Ator-
vastatin. J. Exp. Med. 2003, 197, 725–733.
71. Hendriks, J. J.; Alblas, J.; van der Pol, S. M.; van Tol, E. A.;
Dijkstra, C. D.; de Vries, H. E. Flavonoids Influence Mono-
cytic GTPase Activity and Are Protective in Experimental
Allergic Encephalitis. J. Exp. Med. 2004, 200, 1667–1672.
M.; Kumanogoh, A.; Kusunoki, S.; Sakoda, S. Edaravone, a
Free Radical Scavenger, Ameliorates Experimental Auto-
73. Stanislaus, R.; Gilg, A. G.; Singh, A. K.; Singh, I. N-Acetyl-L-
cysteine Ameliorates the Inflammatory Disease Process in
Experimental Autoimmune Encephalomyelitis in Lewis
Rats. J. Autoimmune Dis. 2005, 2, 4.
74. Korsvik, C.; Patil, S.; Seal, S.; Self, W. T. Superoxide Dismu-
tase Mimetic Properties Exhibited by Vacancy Engineered
Ceria Nanoparticles. Chem. Commun. (Cambridge, U.K.)
Nanoparticles Reduce Disease Severity in a Mouse Model
of Multiple Sclerosis. Nanotechnol. 2012 2012, 3, 265–268.
76. Brinkmann, V.; Davis, M. D.; Heise, C. E.; Albert, R.; Cottens,
S.; Hof, R.; Bruns, C.; Prieschl, E.; Baumruker, T.; Hiestand, P.;
et al. The Immune Modulator FTY720 Targets Sphingosine
1-Phosphate Receptors. J. Biol. Chem. 2002, 277, 21453–
77. Choi, J. W.; Gardell, S. E.; Herr, D. R.; Rivera, R.; Lee, C. W.;
Noguchi, K.; Teo, S. T.; Yung, Y. C.; Lu, M.; Kennedy, G.; et al.
FTY720 (Fingolimod) Efficacy in an Animal Model of Multi-
pleSclerosis Requires Astrocyte Sphingosine1-Phosphate
Receptor 1 (S1p1) Modulation. Proc. Natl. Acad. Sci. U.S.A.
2011, 108, 751–756.
78. Schreibelt, G.; van Horssen, J.; van Rossum, S.; Dijkstra,
C. D.;Drukarch, B.; de Vries,H. E.TherapeuticPotentialand
Biological Role of Endogenous Antioxidant Enzymes in
79. Fleming, K. K.; Bovaird, J. A.; Mosier, M. C.; Emerson, M. R.;
Studies on Experimental Autoimmune Encephalomyelitis.
J. Neuroimmunol. 2005, 170, 71–84.