Silica coating influences the corona and biokinetics of cerium oxide nanoparticles
Konduru et al. Particle and Fibre Toxicology
Silica coating influences the corona and biokinetics of cerium oxide nanoparticles
Nagarjun V. Konduru 0
Renato J. Jimenez 0
Archana Swami 0
Sherri Friend 2
Vincent Castranova 1
Philip Demokritou 0
Joseph D. Brain 0
Ramon M. Molina 0
0 Molecular and Integrative Physiological Sciences Program, Department of Environmental Health, Harvard T.H. Chan School of Public Health , 665 Huntington Avenue, Boston, MA 02115 , USA
1 Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University , P.O. Box 9530, Morgantown, WV 26506 , USA
2 National Institute for Occupational Safety and Health , Morgantown, WV , USA
Background: The physicochemical properties of nanoparticles (NPs) influence their biological outcomes. Methods: We assessed the effects of an amorphous silica coating on the pharmacokinetics and pulmonary effects of CeO2 NPs following intratracheal (IT) instillation, gavage and intravenous injection in rats. Uncoated and silica-coated CeO2 NPs were generated by flame spray pyrolysis and later neutron-activated. These radioactive NPs were IT-instilled, gavaged, or intravenously (IV) injected in rats. Animals were analyzed over 28 days post-IT, 7 days post-gavage and 2 days post-injection. Results: Our data indicate that silica coating caused more but transient lung inflammation compared to uncoated CeO2. The transient inflammation of silica-coated CeO2 was accompanied by its enhanced clearance. Then, from 7 to 28 days, clearance was similar although significantly more 141Ce from silica-coated (35 %) was cleared than from uncoated (19 %) 141CeO2 in 28 days. The protein coronas of the two NPs were significantly different when they were incubated with alveolar lining fluid. Despite more rapid clearance from the lungs, the extrapulmonary 141Ce from silica-coated 141CeO2 was still minimal (<1 %) although lower than from uncoated 141CeO2 NPs. Post-gavage, nearly 100 % of both NPs were excreted in the feces consistent with very low gut absorption. Both IV-injected 141CeO2 NP types were primarily retained in the liver and spleen. The silica coating significantly altered the plasma protein corona composition and enhanced retention of 141Ce in other organs except the liver. Conclusion: We conclude that silica coating of nanoceria alters the biodistribution of cerium likely due to modifications in protein corona formation after IT and IV administration.
Nanoceria; Bioavailability; Protein corona; Silica
With rapid growth in nanotechnology-enabled consumer
products, engineered nanomaterials (ENMs) are
increasingly common. At the same time, there are rising public
concerns about adverse effects of ENMs on human health
and the environment. Among the ENMs introduced into
the global nanotechnology market, nanoceria (CeO2) has
moved to the fore with a wide array of applications. The
ability of cerium to switch oxidation states between Ce
(III) and Ce (IV) is crucial for many nanobiomedical
applications [1–4]. Further, parameters such as the method
employed to synthesize CeO2, its particle size, and the
extent of doping with other agents may alter the cerium
oxidation state [3, 5].
The toxicity data on CeO2 from studies undertaken
during the last decade are mixed and report a range
of biological effects . A number of in vivo and in
vitro studies evaluating the biological effects of CeO2
have reported toxicity and oxidative stress [7–10].
However, recently there are also reports highlighting
putative antioxidant activity of CeO2 and its ability to
protect against oxidative stress-driven disorders [11–13].
Baer et al. have shed light on the influence of synthesis
method, particle size and aging of CeO2 on biological
outcomes . Many studies have underscored the need for
defining nanoparticle characteristics employed in
biological studies. There are conflicting data on CeO2 toxicity
and the consequences of different concentrations [14, 15].
© 2015 Konduru et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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It is important to consider the extent of particle
agglomeration in air and liquid media as a crucial factor
contributing to discrepancies between in vivo inhalation
versus instillation studies. Nanoparticle agglomeration
is primarily influenced by NP intrinsic properties such
as surface chemistry, charge, and primary particle size,
but also from properties of suspending medium such as
ionic strength [16–19].
Nanoparticle recognition by alveolar macrophages is a
determinant of effective lung clearance. There is
evidence that particle agglomeration aids in promotion of
effective phagocytosis in alveolar macrophages; smaller
(<100 nm) and more abundant structures may make
macrophage mediated “surveillance” less effective .
Scientists are creating nanoparticles with functional
surfaces designed to reduce inflammogenicity and lower
toxicity while improving useful physicochemical properties.
Developing strategies to mitigate toxicity of NPs without
altering their core properties (a safer-by-design approach)
is a vigorously pursued area of research [21–23]. In some
cases, surface encapsulation of nanomaterials with a
thin layer of amorphous silica renders them less cytotoxic
and reduces DNA damage. Coating nanoparticles with
amorphous silica can enhance nanoparticle stability in
colloidal suspensions and facilitate effective uptake by
professional phagocytes, stem cells, and other cell types
with reduced toxicity [24–26]. Unlike crystalline silica that
induces sustained inflammation and resultant fibrosis,
amorphous silica evokes a transient and reversible
inflammatory response .
We recently investigated the pulmonary clearance and
extrapulmonary translocation of radiolabled Ce after
intratracheal instillation of CeO2 . Our study showed
that only 12 % of the instilled Ce dose was cleared from
the rat lung during 28 days post-exposure. In another
investigation, we found that inhalation of CeO2 caused
more lung injury and inflammation than CeO2 coated
with amorphous silica after one day post-exposure .
Previous reports have proposed that the protein corona
formed on particles can influence biological effects .
To our knowledge this is the first study investigating the
influence of surface properties of cerium oxide
nanoparticles on protein corona formation, pulmonary effects,
and the translocation and distribution of nanoceria
after pulmonary and intravenous administration. We
employed amorphous silica coating as a model to test
the hypothesis that surface coating of CeO2 would alter
its protein corona and thus influence the biokinetics of
the core nanoceria. We chose nanoceria due to its slow
lung clearance and relatively low solubility [15, 30–32].
The aim of our study was to compare the clearance kinetics
and bioavailability of cerium after intratracheal, intragastric,
and intravenous administration of silica-coated versus
uncoated CeO2 in rats.
Synthesis and characterization of CeO2 and silica-coated
Uncoated and silica-coated CeO2 were made by flame
spray pyrolysis using the Versatile Engineered
Nanomaterial Generation System (VENGES) at Harvard
University . Detailed physicochemical and morphological
characterization of these NPs was reported earlier [21, 28].
In summary, the uncoated and silica-coated CeO2 had a
cubic fluorite-like structure (Fig. 1). A nanothin (2–4 nm)
amorphous silica layer hermetically encapsulated the
CeO2 core in a coating reactor after their initial synthesis
in an aerosol reactor  (Fig. 1b). The silica coating on
the surface was revealed as fine optically transparent film
surrounding the dark and opaque CeO2, as verified by
Xray diffraction (XRD) and electron microscopy analyses.
The average crystal size of the primary uncoated and
silica-coated NPs was 32.9 and 32.6 nm, respectively. Their
specific surface areas (SSA) were 28 m2/g (uncoated) and
27.8 m2/g (silica-coated) (Table 1). The extent of the
silica coating was assessed by X-ray photoelectron
spectroscopy and by photocatalytic methods. The persistence
of the silica coating in the lungs of rats was at least 3 days
after inhalation .
Assessments by dynamic light scattering (DLS) showed
that as an aqueous dispersion the particles essentially
behaved as “nanoagglomerates” of 136 ± 1.1 nm (uncoated)
and 208 ± 2.9 nm (silica-coated). The hydrodynamic
diameters of the two CeO2 types are shown in Table 1. The zeta
potential of NP suspensions was also evaluated in distilled
water. Uncoated CeO2 exhibited a positive zeta potential
(34.5 ± 3.1 mV) and the silica coating changed the zeta
potential to negative −26.8 ± 0.3 mV (Table 1). DLS analysis
was also performed on both nanoceria after in vitro
incubation with harvested rat bronchoalveolar lining (BAL) fluid
to determine if the lipoprotein corona alters agglomeration
size and zeta potential. We found that this corona
significantly increased the hydrodynamic diameter
(136 to 1463 nm) and changed the zeta potential
(34.5 to −20.8 mV) of uncoated CeO2. The effects of
the lipoprotein corona on silica-coated CeO2 were
more modest (Table 1). After incubation in rat plasma
and the formation of the protein corona, the hydrodynamic
diameters of both CeO2 NP types were significantly
increased and the surface charge of uncoated CeO2 was also
altered from positive to negative zeta potential (Table 1).
Similar to protein corona formed with BAL incubation, the
increase in DH with plasma protein corona formation was
more pronounced with uncoated CeO2 NPs.
Pulmonary responses to intratracheally instilled CeO2 and
We compared the pulmonary responses of rats to
uncoated versus silica-coated CeO2 at 1 and 5 days after
Fig. 1 Appearance of CeO2 NPs used in this study. a Electron micrograph of uncoated and b silica-coated CeO2 NPs. Arrow shows a thin silica coating
IT instillation in rats as described previously . This
experiment was performed to also determine the safe
dose for intratracheal instillation of CeO2 and
silicacoated CeO2 NPs where inflammation or injury was
minimal. Groups of 6 rats (272 ± 13 g body weight) were
instilled with 0.2, 1 or 5 mg/kg of each type of CeO2.
Control animals were instilled with an equivalent
volume of distilled water. We found that coated and
uncoated CeO2 NPs induced a dose-dependent injury and
inflammation as indicated by increased neutrophils
(Fig. 2a) in the BAL fluid at 24 h post-instillation. Both
NPs also increased the levels of myeloperoxidase
(MPO), albumin and lactate dehydrogenase (LDH)
(Fig. 2b). Interestingly, the numbers of lavaged
macrophages increased for uncoated and decreased for
silica-coated CeO2 with increasing dose (Fig. 2c). At
0.2 and 1 mg/kg doses, only the silica-coated CeO2
instilled rats showed elevated LDH, MPO, and
albumin levels. However, five days post-dosing with 1 mg/
kg of silica-coated CeO2 there were decreased PMN
counts (Fig. 2d). At this time, there were also
reductions in other inflammatory biomarkers such as MPO,
albumin and LDH (Fig. 2e). However, significant
increase in macrophage numbers was observed in
silicacoated CeO2 groups (Fig. 2f ).
Table 1 Physicochemical characterization of nanoparticles used
In vivo clearance and translocation of 141CeO2 and
silica-coated 141CeO2 after IT instillation in rats
The lung levels of 141Ce after a single IT instillation of
either radioactive uncoated CeO2 or silica-coated CeO2
were evaluated in rats for 28 days. Animals were sacrificed
at 5 min, and 2, 7 and 28 days post-instillation and various
organs were collected to determine the retained cerium
concentration. The lung clearance profiles for both
nanoparticle types showed no differences during the first two
days post-IT instillation. Interestingly, the lung clearance
was markedly different between day 2 and day 7 for the
two NPs (Fig. 3a). We observed that ~22 % of the 141Ce
from the silica-coated CeO2 and only ~8 % of the 141Ce
from the uncoated CeO2 dose disappeared from the lungs
during this period. Between day 7 and day 28 post-IT
instillation, the difference in the fraction of cleared NPs was
statistically significant but relatively small (8.1 % for uncoated
141CeO2 vs. 10.4 % for silica-coated CeO2). By 28 days
postinstillation, ~81 % of uncoated CeO2 still remained in the
lungs. Coating of CeO2 with amorphous silica enhanced
the overall clearance of CeO2 by an additional 16 %.
Translocation of radioactive cerium from the lungs to
other organs was evaluated by measuring 141Ce in the
different collected tissues. Low detectable fractions of
radioactivity for Ce from both NP types were found in the liver,
−25.2 ± 2.8
−31.8 ± 2.7
CeO2 in DI water
Silica-coated CeO2 in DI water
CeO2 in BAL Silica-coated CeO2 in BAL
−26.8 ± 0.3
−20.8 ± 3.4
−15.4 ± 2.0
Fig. 2 Bronchoalveolar lavage analysis after IT instillation of uncoated or silica-coated CeO2 NPs. a Dose-dependent increases in lavaged neutrophils and
b lactate dehydrogenase levels in BAL at 24 h post-instillation. c Lavaged macrophages increased with uncoated but decreased with silica-coated CeO2 at
the highest NP dose. d Lavaged neutrophils and e lactate dehydrogenase, MPO and albumin (data not shown) returned to normal levels but
f macrophage recruitment was observed at 5 days post-instillation of 1 mg/kg CeO2. (* increased, # decreased, P < 0.05, MANOVA. Data are
mean ± SEM, n = 5/group)
bone/bone marrow, spleen and kidneys (<1 %) (Fig. 3b).
Estimated tissue cerium concentration in these organs were
higher for uncoated CeO2 (Table 2). The elimination of
141Ce from both particle types was mostly via the feces
(Fig. 4b) and to a much lesser extent via the urine (Fig. 4a).
Furthermore, we found that the total recovered 141Ce in
examined tissues, feces, and urine was significantly higher in
uncoated than silica-coated CeO2 (Figs. 3 and 4). In the case
of silica-coated CeO2, we speculate that the missing
radioactivity may have been in organs not examined such as
lymph nodes, adipose tissue, pancreas, adrenals, teeth, nails,
tendons, nasal tissues, and the rest of the head.
Biodistribution of CeO2 within the lungs and protein
Since the protein corona on NP surfaces may modulate
their cell interaction and overall biological effects, we
examined the composition of adsorbed proteins on the NP
surface when incubated with collected cell-free BAL fluid.
First, we found that incubation of NPs in concentrated
BAL fluid significantly altered their aggregate sizes (Table 1).
Compared with the suspension in deionized water, both
nanoceria types exhibited larger and more variable
hydrodynamic diameter. Uncoated nanoceria also formed larger
agglomerates than the coated NPs. In addition, we found
that the total amount of adsorbed protein was significantly
higher in silica-coated than uncoated CeO2 especially
albumin, C3, and transferrin (Fig. 5a, b). However, we found
no differences in the quantitative distribution of the two
NP types 24 h post-IT instillation among the three
measured compartments (Fig. 5c). The majority of 141Ce
activity was associated with the lavaged lungs. Additionally,
hyperspectral imaging analysis, to determine the extent of
NP uptake in BAL cells after 5 days post-IT instillation,
revealed a higher number of particle-containing cells in the
silica-coated than uncoated group (Fig. 6).
Table 2 Cerium concentration at 28 days post-instillation of
uncoated or silica-coated CeO2 NPs
95193.25 ± 1766.39 *
Fig. 3 Biokinetics of IT-instilled 141CeO2 nanoparticles. a Lung clearance
of both CeO2 NPs was slow. Although similar during the first
two days post-IT instillation, it was different between from day 2
to day 7. Approximately 22 % of silica-coated CeO2 and only ~8 % of
uncoated CeO2 total dose cleared the lungs during this period. By
28 days post-instillation, 81 % of uncoated and 66 % of silica-coated
CeO2 remained in the lungs. b Translocated 141Ce from the lungs
gradually accumulated in extrapulmonary organs. By 28 days, only
0.9 % of instilled 141Ce dose from uncoated and 0.7 % from
silica-coated CeO2 were retained in all extrapulmonary organs examined.
Data are mean ± SEM, n = 5/group
Biodistribution of uncoated and silica-coated CeO2 after
gavage administration in rats
At 5 min and 7 days post-gavage of uncoated CeO2 or
silica-coated CeO2 we measured absorption of 141Ce
from the gut. As expected, nearly 100 % of the dose was
recovered at 5 min in the stomach for both types of NPs
(Fig. 7a). The 141Ce levels in tissues other than the
gastrointestinal (GI) tract were extremely low (0.004 %
for uncoated, 0.002 % for silica-coated CeO2) by day 7
(Fig. 7b). Very low levels of 141Ce were excreted in the
urine (Fig. 7c) and nearly 99 % of both CeO2 NPs was
excreted in feces by day 7 (Fig. 7d). As there was very
low radioactivity detected in any of the collected organs
and in urine samples over a period of 7 days, we
conclude that both uncoated and silica-coated CeO2 do not
significantly translocate through the intestinal barrier.
Data are mean ± SE ng/g cerium concentration, n = 5/group
Ce concentration was estimated (ng/μCiNPs x μCi/gtissue)
*P < 0.05, CeO2 vs. silica-coated CeO2
Tissue distribution of 141CeO2 and silica-coated 141CeO2
NPs after intravenous injection
The distribution of intravenously injected NPs at 2 h
and 2 days post-injection is shown in Fig. 8a and b,
respectively. Radioactive 141Ce from both NP types was
predominantly retained in the liver, spleen, and bone,
organs that typically take up circulating particles by
macrophages with access to the blood. The silica coating led
to a redistribution of 141Ce over a period of 2 days from
the liver to the spleen and other organs (Fig. 8b). The
silica coating also enhanced the tissue concentration
of 141Ce in several organs but decreased in the liver
(Tables 4 and 5).
To determine the influence of silica coating on
NP-plasma protein interactions, we analyzed the
hydrodynamic diameters of NPs and characterized
the protein corona formed after incubation of NPs in
rat plasma in vitro. We found significant increases in
agglomerate sizes of both NP types compared to when
suspended in protein-free deionized water (Table 1). We
also found differences in the protein corona composition
between the 2 NP types (Fig. 9a, b). The fecal excretion of
141Ce post-injection of NPs during the first 24 h was far
Fig. 4 Elimination of 141Ce post-IT instillation. a. Only 0.03 % – 0.05 %
was excreted in the urine in 28 days. b However, 19 % of 141Ce from
uncoated and 12 % from silica-coated CeO2 was excreted in the feces.
Data are mean ± SEM, n = 5/group
lower than after IT instillation (0.05 % v. 3 %), suggesting
that some CeO2 NPs in the lungs may be removed by
mucociliary transport. It also suggests that absorbed
cerium is eliminated slowly from the body.
Progress in nanotechnology has produced a variety of
nanoparticle generation systems which synthesize
nanoparticles of desired size and properties. The in-house
VENGES system employed in this study enabled us to
control primary particle size and aerosol size
distribution. This platform also allowed for in-flight coating of
CeO2 with a nanothin layer of amorphous silica .
This flame-based silica-coating process has recently been
explored as a means of high yield scalable manufacturing
of silica-coated nanosized ENMs with cores of TiO2,
Fe2O3, or Ag .
In this study, we sought to examine the effect of
surface modification of CeO2 with amorphous silica on
acute pulmonary responses as well as on CeO2
pharmacokinetics after IT instillation, gavage, and IV injection.
We observed that exposure of rats to silica-coated CeO2
caused higher dose-dependent inflammatory responses
compared to uncoated particles and a vehicle-only
control group, as evidenced by increases in BAL parameters.
However, the inflammatory effects induced by
silicacoated CeO2 were transient and subsided by day 5
(Fig. 2d and e). This is consistent with our recent study
in which 1 mg/kg dose of silica-coated CeO2 NPs also
caused higher but transient inflammation . We note
that these findings are in contrast to our previously
published report on the toxic and inflammatory effects of
the same particles after inhalation exposure, where we
showed that inhaled silica-coated CeO2 induced less
toxicity and inflammation after exposure for 2 h per day for 4
consecutive days . This discordance may be explained
based on the higher doses used here and the different
exposure method (bolus IT instillation vs. inspired aerosols
over 8 h). Although IT instillation is a reliable method for
administering a precise dose to the lungs, it differs from
inhalation exposure in terms of particle distribution, dose
rate, the extent of NP agglomeration and ressulting
patterns of injury and clearance. Baisch et al. observed that
inflammatory responses following intratracheal instillation
were higher than those seen following whole body
inhalation for single and repeated exposures of titanium
dioxide NPs when deposited doses were comparable .
Fate of intratracheally-instilled nanoceria
The lung clearance of uncoated CeO2 observed in this
study was similar to our recent report on CeO2 NM-212.
NM-212 was synthesized by a precipitation method unlike
the CeO2 NPs used here which were flame-generated .
Our data are consistent with a study by He et al. where
63.9 ± 8.2 % of the intratracheally instilled dose still
remained in the lungs after 28 days . We found that
the extent of silica-coated CeO2 clearance from the lung
was significantly higher (~35 %) than uncoated CeO2
(~19 %). But an important finding was the significant
influence of the silica coating on the lung clearance of
CeO2 from days 2 to day 7. This period of more rapid
clearance coincided with the initial phase characterized by
greater inflammation and increased air-blood barrier
As the pulmonary surfactant lies at the outermost
aspect of the air-blood barrier, inhaled and deposited
NPs first encounter the biomolecules of the alveolar lining
layer. This fluid consists of an ultra-thin layer of aqueous
hypophase and a surface active lipoprotein mixture usually
known as the pulmonary surfactant layer . Pulmonary
surfactant is composed of 85-90 % w/w phospholipids and
Fig. 5 Analysis of nanoparticles after incubation with BAL fluid. a NP-bound rat BAL proteins were analyzed by 1D gel electrophoresis and Mass
Spectrometry. The molecular weights (kDa) of reference proteins are shown in lane MW. Five proteins identified by LC-MS are indicated on right.
b LC-MS profiles of the same five proteins show the influence of silica coating on the protein corona profile. c Compartmental distribution of
neutron activated uncoated and silica-coated CeO2 at 24 h post-instillation. No significant differences in distribution were observed between the
two CeO2 NPs. Data are mean ± SEM, n = 5/group
10 % w/w proteins . Adsorption of phospholipids and
proteins on the NP surface takes place rapidly .
Therefore, it is reasonable to assume that interactions of NPs
with lung cells occur mostly with the NP-lipoprotein
complex and not with bare NP surfaces . Importantly,
the adsorption of proteins and phospholipids on NPs may
modulate their overall biological effects [43, 44].
We examined the protein corona formed on the surface
of our test NPs as they encounter the lung lining fluid.
The incubation of NPs in BAL fluid significantly increased
their hydrodynamic sizes and changed the zeta potential
of CeO2 NPs likely due to their interactions with
phospholipids and proteins. Presumably, instilled NPs would
immediately acquire protein coronas in vivo changing
their surface charge and extent of aggregation unlike those
in water suspension and in dry aerosols. The type of
proteins comprising the corona may also impact NP
translocation . Aggregate size alterations could also
influence the pulmonary effects and translocation of the
core nanoceria. Notably, we found significantly more
protein adsorbed in the “hard corona” of silica-coated
compared to uncoated CeO2. The amounts of specific proteins
comprising the hard corona shown in Fig. 5b were based
on NP mass (μg/mg NPs). When expressed as amount of
protein per unit surface area (μg/m2) of NPs, silica-coated
CeO2 still bind more BAL proteins than uncoated NPs.
Significantly more albumin, SP-A, α-1 antitrypsin,
transferrin, and C3 proteins were present in the corona of
silica-coated CeO2. These belong to the class of proteins
that shuttle across the alveolar-epithelial barrier .
Receptor-mediated transport processes in the alveolar
epithelium have been reported for albumin and transferrin
. Translocation of intratracheally instilled 125I-albumin
from air spaces into the blood compartment has been
reported previously . Rapid translocation of synthetic
organic NPs comprised of human serum albumin and a
fluorophore has been demonstrated . Whether this
enhanced adsorption of albumin and transferrin onto
Fig. 6 Quantitative assessment of uptake of CeO2 by alveolar
macrophages at 24 h post-instillation. BAL cells were analyzed using
hyperspectral imaging. a The image shows uncoated and silica-coated
CeO2 mapped as bright pixels (pointed arrows) inside the cells. BAL cells
isolated at 24 h and 5 days after IT-instillation were scored. b Numbers of
macrophages with or without internalized CeO2 at 1 and 5 days
postinstillation. Significantly more cells with ingested silica-coated CeO2 were
seen at 5 days. Data are mean ± SEM, n = 3 rats/group, n = 3000 cells
scored/group. * P < 0.05, Student t test
coated nanoceria contribute to their small but higher
translocation through the lungs needs further investigation.
Studies have reported that some of the proteins
present in BAL exhibit immunological functions (e.g.,
C3 and SP-A) [49–51]. It has been shown that coating of
magnetite and TiO2 with SP-A improved their uptake in
macrophages . Our findings that the lipoprotein
corona changes the agglomerate size and zeta potential of
CeO2 also suggest that the corona can affect the manner
in which alveolar macrophages interact, recognize,
phagocytose, and process CeO2 NPs. Alveolar macrophages are
the primary phagocytic cells for ultrafine particles in the
lungs . Particles may adhere to the surfaces of type I
and type II epithelial cells as well, but lung parenchymal
cells are less capable of phagocytosis . AMs play a
critical role in NP-induced inflammation and oxidative stress.
Most of the deposited particles in the alveolar region are
phagocytosed within a 24 h period after particle
deposition, as long as the dose is not beyond the ingestion
capacity of AMs [55, 56]. Notably, functionalized NPs are
more effectively phagocytosed than non-functionalized
NPs [57–60]. Recognition and phagocytosis of
nanoparticles by AMs is a key component in nanoparticle
dissolution and clearance.
We examined whether silica coating affects the
distribution of CeO2 within the different lung compartments
after the first 24 h post-instillation. We found no
significant differences in the amount of radioactive CeO2 in
lavaged alveolar cells, in cell-free supernatant, or in
lavaged lungs. Furthermore, no significant difference was
found in the number of AMs with internalized CeO2
NPs assessed by hyperspectral imaging of lavaged AMs.
However, at 5 days post-instillation, significantly more
AMs were found to have internalized silica-coated than
uncoated CeO2. This enhanced uptake could be due to
different corona profile, altered aggregate size or abundant
recruitment of AMs observed with silica-coated CeO2. It
is possible that this enhanced uptake of silica-coated CeO2
by activated AMs and the higher inflammation could lead
to greater translocation of particles or particle-containing
cells into the lymphatic system. For the lung parenchyma,
clearance involves a slower phase, occurring in the alveoli.
It consists of phagocytosis of particles from the lung
surface by AMs and to a lesser extent by particles entering
the lymphatics and subsequent accumulation in the
regional lymph nodes.
We were unable to measure the lymphatic clearance
of CeO2 NPs since lymph nodes were not included in
this study. However, we have previously shown that 65Zn
from 65ZnO NPs was more significantly translocated to
tracheobronchial lymph nodes when coated similarly
with amorphous silica . Interestingly, despite the
greater clearance from the lungs, 141Ce from silica-coated
CeO2 was slightly lower in all the organs we examined
(0.73 vs. 0.93 %). The cerium concentration retained in
the liver, bone, kidneys, heart, and testes was lower.
Excretion in the feces was also lower (12 vs. 19 %).
Fate of ingested nanoceria
Data from animal and human studies show that inhaled
nanoparticles are subject to different site-dependent
clearance mechanisms . These mechanisms include a
fast clearance phase, which can be observed in the
tracheobronchial region and is attributed to the mucociliary
elimination with subsequent ingestion into the
gastrointestinal tract and excretion via the feces. Thus, the oral
exposure to nanoparticles is pertinent from an
environmental exposure perspective, such as the ultrafine fraction
of air pollution exposures. As a surrogate for entry of
particles into the GI tract from the lungs, we also investigated
the influence of silica coating on the bioavailability of
Fig. 7 Tissue distribution of 141Ce post-gavage. a Immediately post-gavage, nearly 100 % of both CeO2 were recovered in the stomach and to
much lesser extent in other organs. b At 7 days post-gavage, the total tissue 141Ce detected in all organs examined was negligible (0.003 ± 0.001 %).
c By 7 days post-gavage, less than 0.0004 % of dose was excreted in the urine. d Elimination of 141Ce via the feces was nearly 100 % from uncoated
and 94 % from silica-coated CeO2. Data are mean ± SEM, n = 5/group
Table 3 Cerium concentration in different tissues at 7 days after
gavage administration of uncoated or silica-coated CeO2 NPs
Data are mean ± SE ng/g cerium concentration, n = 5/group
Ce concentration was estimated (ng/μCiNPs x μCi/gtissue)
No significant difference was observed between the two group
CeO2 after gavage. Our data showed a rapid clearance of
both types of CeO2. We found that nearly 100 % of the
uncoated CeO2 and ~95 % of silica-coated CeO2 were
eliminated in the feces within 7 days post-gavage. Despite the
higher dose we used for gavage, there was negligible
radioactivity in any organ or in urine samples collected over a
period of 7 days. As has been demonstrated previously,
neither CeO2 NP type cross the intestinal barrier nor is there
dissolution followed by absorption [15, 32, 61].
Fate of intravenously injected nanoceria
Due to increasing interest in CeO2 for potential
nanomedical applications, we also investigated whether silica
coating would affect the tissue distributions of IV-injected
CeO2. Consistent with our earlier study , both CeO2
types were immediately taken up in organs rich in
mononuclear phagocytes with direct access to the circulating
blood, such as those in the liver (87 %), spleen (4 %), and
bone (0.5 %). At 2 h, the total recovered 141Ce in all
organs examined were 92.6 % (uncoated) and 92.2 %
(silica-coated CeO2) of the total injected dose. Despite
the significantly higher agglomerate size of uncoated
nanoceria after interaction with plasma proteins, their
liver uptake measured at 2 h was not different from
silica-coated NPs. However, the silica coating enhanced
the overall amount of cerium in some other organs. We
found that binding of plasma proteins to the CeO2 surface
Table 4 Cerium concentration in different tissues at 2 hours after
intravenous injection of uncoated or silica-coated CeO2 NPs
Fig. 8 Tissue distribution of 141Ce post-IV injection of CeO2 NPs. a At
2 h post-injection, 87 % of 141Ce dose was recovered in the liver,
and lower percentages in blood, spleen, bone, and bone marrow
from both CeO2 group. b Over a period of 2 days, 141Ce levels in the
liver decreased from 87 % to 80 % in the silica-coated group
with accompanying increases in the spleen, bone and bone
marrow. * P <0.05, MANOVA. Data are mean ± SEM, n = 5/group
was altered by the silica coating. Notably, bound albumin
and α-2 hs glycoprotein were higher in silica-coated CeO2.
A recent study showed that albumin-coated liposomes
were taken up more efficiently than uncoated liposomes
by murine macrophages . The silica coating in our
study also caused a significant reduction (6 %) in the liver
retention of 141Ce with concomitant increases in the
spleen and bone two days post-exposure. This likely
reflects either enhanced dissolution of Kupffer cell-ingested
silica-coated CeO2 or the release of intact NPs into the
blood likely due to their smaller aggregate size (Table 1).
Very small amounts of 141Ce (3.8-5.8 %) were cleared
from the body two days post-exposure, indicating that
absorbed cerium is biopersistent, as reported in other
studies [32, 64].
In summary, we found that silica coating of CeO2 caused
a higher but transient lung inflammation and a higher
Data are mean ± SE ng/g cerium concentration, n = 5/group
Ce concentration was estimated (ng/μCiNPs x μCi/gtissue)
*P < 0.05, CeO2 vs. silica-coated CeO2
lung clearance. It also altered the biodistribution of
cerium when CeO2 were injected intravenously. These
effects correlated with enhanced adsorption of proteins in
lung lining fluid and plasma onto the silica coating. As
surface chemistry greatly influences the formation of
the nanoparticle corona, our future studies will focus
on understanding nano-bio interactions with lung and
plasma lipoproteins and their influence on toxicity
and biokinetics of NPs.
Synthesis of CeO2 and silica-coated CeO2 nanoparticles
Detailed procedures of generating these nanoparticles
have been reported [21, 28, 33]. Uncoated and
SiO2coated CeO2 nanoparticles were synthesized by flame
spray pyrolysis (FSP) of cerium (III) ethylhexanoate
(0.05 M) dissolved in xylene and cerium (III)
ethylhexanoate (0.04 M) dissolved in xylene: EHA (3:1), respectively.
The precursor solutions were fed through a stainless steel
capillary at 5 ml/min, dispersed by 5 L/min O2 (Airgas,
purity >99 %, pressure drop at nozzle tip: ρdrop = 2 bar)
and combusted to form the desired nanoparticles. A
remixed stoichiometric methane-oxygen (1.5, 3.2 L/min)
supporting flame was used in conjunction with 40 L/min
O2 sheath gas. In the case of the synthesis of uncoated
Table 5 Cerium concentration in different tissues at 2 days after
intravenous injection of uncoated or silica-coated CeO2 NPs
Data are mean ± SE ng/g cerium concentration, n = 5/group
Ce concentration was estimated (ng/μCiNPs x μCi/gtissue)
*P < 0.05, CeO2 vs. silica-coated CeO2
CeO2, 16 L/min of pure N2 was injected into the reactor
through a torus ring with 16 equispaced and equisized
(dinner = 0.6 mm) jets at an injection height of 200 mm
above the FSP burner. In the case of SiO2-coated CeO2,
16 L/min N2 carrying hexamethyldisiloxane (HMDSO,
Sigma–Aldrich, St. Louis, MO, USA) vapor was fed
through the same torus ring at an injection height of
300 mm. HMDSO vapor was obtained by bubbling
0.11 L/min gas through liquid HMDSO (300 ml)
maintained at 11.3 °C using a temperature-controlled water
bath. At saturation conditions, this corresponds to an
HMDSO injection mass of 0.85 g/h into the reactor. In
both cases, the reactor was enclosed above and below the
torus ring by two quartz tubes (dinner = 45 mm). Uncoated
and silica-coated CeO2 NPs were collected on a
watercooled glass fiber filter (Whatman) located 80 cm above
the reactor and stored in glass vials prior to experiments.
Neutron activation of CeO2 nanoparticles
Both nanoparticle powders were neutron activated at the
MIT Nuclear Reactor Laboratory (Cambridge, MA) with a
thermal neutron flux of 5 x 1013 n/cm2/s for 24 h. The
process generated the radioisotope 141Ce, which decays
with a half-life of 32.5 days and emits gamma rays with an
energy of 145.4 KeV. The specific activity was 2.7 μCi 141Ce
per mg CeO2 and 3.4 μCi 141Ce per mg silica-coated CeO2.
The protocols used in this study were approved by the
Harvard Medical Area Animal Care and Use Committee.
Male Wistar Han rats (8 weeks old) were obtained from
Charles River Laboratories (Wilmington, MA) and were
housed in standard microisolator cages under controlled
conditions of temperature, humidity, and light at the
Harvard Center for Comparative Medicine. They were fed
commercial chow (PicoLab Rodent Diet 5053, Framingham,
MA) and were provided with reverse-osmosis purified
water ad libitum. The animals were acclimatized in the
facility for at least 7 days before the start of experiments.
Fig. 9 Analysis of nanoparticle protein corona after incubation in plasma. a Analysis of NP-bound rat plasma proteins by 1D gel electrophoresis.
The molecular weights (kDa) of reference proteins are shown in lane MW. Twelve proteins identified by LC-MS are indicated on right. b LC-MS
profiles of the same twelve proteins and influence of silica coating on the corona profile
Preparation of CeO2 nanoparticle suspensions for animal
Particle suspensions at specified concentrations were
prepared in sterile distilled water in conical polyethylene
tubes. A critical dispersion sonication energy (DSEcr) to
achieve the smallest particle agglomerate size was used,
as previously reported . The suspensions were
sonicated at 242 J/ml (20 min/ml at 0.2 watt power output)
in a cup sonicator fitted on Sonifier S-450A (Branson
Ultrasonics, Danbury, CT, USA). The sample tubes were
immersed in running cold water to minimize heating of
the particles during sonication. The hydrodynamic
diameter (DH), polydispersity index (PdI), and zeta potential
(ζ) of each suspension were measured by dynamic light
scattering using a Zetasizer Nano-ZS (Malvern
Instruments, Worcestershire, UK).
Assessment of pulmonary effects of CeO2 nanoparticles –
Bronchoalveolar lavage and analyses
This experiment was performed to determine the influence
of an amorphous silica coating on CeO2 pulmonary effects
and also to identify a safe dose for pharmacokinetic studies
on instilled materials. Thirty five rats (wt. = 267 ± 15 g)
were instilled intratracheally with either uncoated or coated
CeO2 NP suspensions at 0.2, 1.0, and 5 mg/kg (n = 5 rats/
group). Another group of rats were instilled with an
equivalent volume of distilled water and served as controls.
The particle suspensions were delivered to the lungs
through the trachea, as described earlier . Twenty-four
hours later, rats were anesthetized and then euthanized via
exsanguination, with a cut in the abdominal aorta. The
trachea was exposed and cannulated. The lungs were then
lavaged 12 times with 3 mL of Ca++- and Mg++-free 0.9 %
sterile PBS. The cells from all washes were separated from
the supernatant by centrifugation (350 x g at 4 °C for
10 min). Total cell count and hemoglobin measurements
were made from the cell pellets. A dilute cell suspension
was cytocentrifuged, the cytospin was stained, and
differential cell counting was performed. The supernatant from the
first two washes was clarified via centrifugation (14,500 x g
at 4 °C for 30 min), and used for standard
spectrophotometric assays for LDH, MPO, and albumin .
Pharmacokinetics of intratracheally-instilled, gavaged and
intravenously injected 141CeO2 nanoparticles
The nanoparticle dose used for both NPs was 1 mg/kg
for IT instillation, 1 mg/kg for IV injection, and 5 mg/kg
for gavage administration. Neutron-activated 141CeO2
NPs were suspended in sterile distilled water at 0.67 mg/
ml for IT instillation (1.5 ml/kg body weight) at 1 mg/ml
for IV injection (1 ml/kg) or at 5 mg/ml for gavage
administration (1 ml/kg) and sonicated as described above.
The radioactivity in multiple aliquots of each suspension
was measured in a WIZARD Gamma Counter
(PerkinElmer, Inc., Waltham, MA).
Each rat was anesthetized with isoflurane (Piramal
Healthcare, Bethlehem, PA). The 141CeO2 NP suspension
was delivered to the lungs through the trachea, into the
bloodstream via the penile vein, or into the stomach via
the esophagus. Each rat was then placed in a metabolic
cage with food and water ad libitum for fecal and urine
sample collection. Five rats from the IT group were
humanely sacrificed at 5 m, 2 d, 7 d and 28 d post-dosing.
The same number of rats were analyzed at 5 m and 7 d
post-gavage, and at 2 h and 2 d post-IV injection.
Analysis of rats at 5 min post-IT instillation and post-gavage
was performed to obtain an accurate measure of the
initial deposited dose. Since we anticipated that clearance
from the gastrointestinal tract would be relatively fast,
the gavage experiment spanned only 7 days. Twenty
four-hour samples of feces and urine were collected at
selected time points (0–24 h, 2–3 days, 6–7 days, 9–10
days, 13–14 days, 20–21 days, and 27–28 days post-IT
instillation; 0–24 h, 2–3 days, and 6–7 days post-gavage;
and 0–24 h post-IV injection).
At each endpoint, rats were anesthetized and as much
blood as possible was collected from the abdominal
aorta. Plasma and red blood cells were separated by
centrifugation at 3000 x g for 10 min at 4 °C. After
euthanasia, the whole lungs, brain, heart, spleen, kidney,
gastrointestinal tract, testes, liver, two femoral bones, and
multiple samples of skeletal muscle, bone marrow, and
skin were collected and placed in pre-weighed tubes. Each
sample weight was recorded. Radioactivity was measured
in a WIZARD Gamma Counter (PerkinElmer, Inc.,
Waltham, MA). Disintegrations per minute were
calculated from the measured counts per minute (minus
background) and the counter efficiency. Data were expressed
as μCi/g and as a percentage of the administered dose
retained in each organ. All radioactivity data were adjusted
for physical decay over the entire observation period. The
radioactivity in organs and tissues not measured in their
entirety was estimated from measured aliquots as a
percentage of total body weight as follows: skeletal muscle,
40 %; bone marrow, 3.2 %; peripheral blood, 7 %; skin,
19 %; and bone, 6 % [66, 67].
Pulmonary distribution of 141CeO2 nanoparticles
To determine the pulmonary distribution of instilled
141CeO2 NPs within the lungs at 1 d post-instillation, a
separate cohort of rats were IT-instilled with 1 mg/kg of
either 141CeO2 or silica-coated 141CeO2. Twenty-four
hours later, the lungs were lavaged as described above.
The BAL fluid was centrifuged at 350 x g for 10 min
at 4 °C to separate lavaged cells from the supernatant.
The cell pellets were resuspended in 0.5 ml PBS. The
lavaged lungs, BAL supernatants and cell pellets were
analyzed for 141Ce. The total radioactivity in each of
the three lung compartments was expressed as a
percentage of the total radioactivity recovered in the
Data were analyzed using multivariate analysis of variance
(MANOVA) followed by Bonferroni (Dunn) post hoc tests
using SAS Statistical Analysis Software (SAS Institute,
Cary, NC). CytoViva data were analyzed by Student t test.
Characterization of protein corona formation on CeO2
and silica-coated CeO2 nanoparticles in lung lining fluid
Nanoparticles (1 mg/mL) were incubated in 4 mL rat
plasma for 30 min at 37 °C. Then, the suspension was
centrifuged for 10 min at 14,500 x g. The resulting pellet
was washed in DI water three times. After the final
washing step, the NP pellet containing ‘hard corona’ was
suspended in 20 μL of DI water to which 10 μL of 4x
Laemmli sample buffer was added and vortexed. The
sample was then heated to 95 °C for 7 min. After cooling
to room temperature, 60 μL of mixed solution (57 μL
Laemmli and 3 μL βME) was added to 18 μL of the
sample. The samples were then loaded onto a gel and proteins
were visualized by 1D SDS-PAGE in combination with
Coomassie staining. Gel bands were excised and subjected
to a modified in-gel trypsin digestion procedure .
Peptides were later extracted and then dried in a speed-vac
(~1 h). The samples were then stored at 4 °C until
analysis. On the day of analysis, the samples were
reconstituted in 5–10 μL of HPLC solvent A (2.5 % acetonitrile,
0.1 % formic acid). A gradient was formed and peptides
were eluted with increasing concentrations of solvent B
(97.5 % acetonitrile, 0.1 % formic acid) . Eluted
peptides were subjected to electrospray ionization and then
analyzed in an LTQ Orbitrap Velos Pro ion-trap mass
spectrometer (Thermo Fisher Scientific, San Jose, CA).
Peptides were detected, isolated, and fragmented to
produce a tandem mass spectrum of specific fragment ions
for each peptide. Peptide sequences (and protein identity)
were determined by matching protein databases with the
acquired fragmentation pattern by the software program,
Sequest (ThermoFisher, San Jose, CA).
Assessment of alveolar macrophage uptake of nanoceria
Non-radioactive CeO2 and silica-coated CeO2 NPs were
instilled in a separate cohort of rats at the same dose
and concentration (1 mg/kg, 0.67 mg/ml). At 1 or 5 days
post-instillation, rats were sacrificed and their lungs
lavaged as described above. BAL cells were
cytocentrifuged and fixed on microscope slides. Uptake of nanoceria
by cells was analyzed in an Olympus BX-41 microscope
(CytoViva®, Auburn, AL) hyperspectral image analysis
software. Each macrophage was scored for the presence of
RMM, NVK, RJ, PD, and JDB designed, performed and evaluated the
experimental results. AS performed protein corona evaluation and SF
performed CytoViva imaging. This manuscript was written by RMM and NVK
and revised by JDB, PD, VC, NVK and RMM. All authors read, corrected, and
approved the manuscript.
We kindly acknowledge the financial support from the National Science
Foundation (grant no. 1235806), NIH (P30ES000002) and from BASF,
Ludwigshafen, Germany. This work was performed in part at the Harvard
Center for Nanoscale Systems (CNS), a member of the National Nanotechnology
Infrastructure Network (NNIN), which is supported by the National
Science Foundation under NSF award no. ECS-0335765. The authors also
gratefully acknowledge the technical help of Dr. Ross Tomaino with
mass spectrometry, Thomas Donaghey for statistical analyses and Melissa
Curran for editorial advice.
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