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Silica supported ceria nanoparticles: a hybrid

nanostructure to increase stability and surface
Cite this: RSC Adv., 2014, 4, 8421
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reactivity of nano-crystalline ceria†

Prabhakaran Munusamy,* Shail Sanghavi, Tamas Varga
and Thevuthasan Suntharampillai

Received 14th August 2013
Accepted 18th November 2013
DOI: 10.1039/c3ra44345k
www.rsc.org/advances
The mixed oxidation state (3+/4+) of ceria nanoparticles of smaller sizes make them attractive materials for
their catalytic antioxidant biological properties. However the unmodified smaller ceria nanoparticles are
limited in their use due to particles agglomeration and reduced surface chemical reactivity in the
solutions used to disperse the nanoparticles. This work describes an effort to stabilize small ceria
nanoparticles, retaining their desired activity, on a larger stable silica support. The ceria nanoparticles
attached to silica was synthesized by a solution synthesis technique in which the surface functional
groups of silica nanoparticles were found to be essential for the formation of smaller ceria nanoparticles.
The surface chemical and vibrational spectroscopy analysis revealed cerium–silicate (Ce–O–Si) covalent
bond linkage between silica and cerium oxide nanoparticles. The colloidal properties (agglomerate
particle size and suspension stability) of ceria attached to silica was significantly improved due to
inherent physico–chemical characteristics of silica against random collision and gravitation settling as
opposed to unmodified ceria nanoparticles in solution. The bio-catalytic activity of ceria nanoparticles in
the 3+ oxidation state was not found to be limited by attachment to the silica support as measured by
free radical scavenging activity in different biological media conditions.

properties of ceria nanoparticles.8,9 An intrinsic instability of the

1. Introduction
Within the category of oxides, cerium oxide nanoparticles are
found to be useful for their redox active properties. For some
applications the useful properties of ceria appear when nano-
particles are partially reduced (mixed Ce 3+/Ce 4+ valence state).
The redox active properties (reversible oxidation state between
Ce 3+ and Ce 4+) appear to allow the ceria nanoparticles to act as
free radical scavengers to prevent oxidative stress related
cellular damage and this antioxidant property has made them
attractive to several research groups studying structure–prop-
erty relationships related to biomedical applications.1–4 Several
synthesis approaches have been used to produce such particles
and these include producing particles with grain sizes less than
5 nm (which stabilizes more Ce 3+), adding various dopants, or
by synthesis conditions.5–7
Many different factors including the synthesis approach,
environmental exposure, storage conditions, and processing or
testing (e.g. aggregation to form larger crystals) exposure have
been demonstrated to inuence the physico–chemical
Environmental Molecular Sciences Laboratory, Pacic Northwest National Laboratory,
Innovation Blvd, Richland, WA 99354, USA. E-mail: Prabhakaran.munusamy@pnnl.
gov; Tel: +1 509 371 7051
† Electronic supplementary information (ESI) available.
10.1039/c3ra44345k
nanoparticles in aqueous media conditions over a longer ageing
period has been demonstrated to have a direct inuence on the
fate and impact of nanoparticles used for biological applica-
tions.10–12 For example, ceria nanoparticles can remain as single
particles in aqueous media or aggregate to bigger sizes, thereby
reducing the desirable size dependent properties and
decreasing their stability in suspension, which are important
properties for biomedical applications. In addition to the
particle size, the chemical state of ceria particles in solution has
been observed to vary with time and environmental condi-
tions.13 Hence long term stability of particles in different envi-
ronmental conditions (pH, ionic strength, presence of
biomolecules, etc.) can be a barrier in the production of func-
tional particles. Therefore it is of importance to develop stabi-
lization methods by which the desired physical and chemical
properties of cerium oxide nanoparticles, oen with sizes below
10 nm, can be maintained for longer periods of time.
Stabilization of cerium oxide nanoparticles in aqueous
media conditions has been oen achieved by surface func-
tionalization or graing with polymer, surfactants or surface
ligands. Surface functionalization with organic moieties can
increase the surface charge density to provide enough steric or
electrostatic repulsive forces for particles to remain non-aggre-
See DOI:
gated and stable in the colloidal state.14 Surface modication of
nanoparticles with polymers or surfactants oen requires

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elaborate synthetic schemes and can be a cumbersome process.
Moreover surface modication with organic chemical groups
can also alter the nanoparticle surface interactions by changing
surface properties and limit their application for a desired
biological end use.15,16 The long term stability of surfactant or
polymer surface functionalized nanoparticles may be limited
due to instability of surface ligands under harsh environmental
conditions like highly acidic conditions, or enzymatic degra-
dation.17,18 Nanoparticles stabilized by electrostatic repulsive
functional groups can agglomerate to bigger sizes due to surface
charge neutralization upon contact with highly ionic biological
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media conditions. The extent of particles agglomeration can
affect the biological endpoints where cells uptake and behavior
or translocation within different organs can be different. Hence
the need to develop a ceria nanostructure with excellent
stability, controlled size, and re-dispersibility in biological
relevant media conditions.
The ceria attached to silica materials is attractive for chemical
mechanical polishing in the semiconductor industry and such
materials are utilized as abrasive elements due to their high
polishing rates and selective removal.19 However attempts to
directly coat ceria to the silica surface have proven to be a difficult
task.20–22 Most studies have focused on fabricating silica–ceria
composites and studied their properties as abrasives without any
detailed analysis of silica–ceria interactions or suspension
stability. Frequently the synthesis of ceria attached to silica
nanoparticles involved post heat treatment which lead to hard
aggregation and non-homogenous coatings.20 To the best of our
knowledge there is no reported literature on ceria–silica nano-
structure hybrids developed for biomedical applications with a
focus on optimizing the dispersibility of unstabilized cerium
oxide nanoparticles powder in biologically relevant media.
In this study novel hybrid nanostructures consisting of small
ceria particles attached to larger silica particles have been
designed and synthesized using a wet chemical synthesis
technique. The larger silica particles of size
50–60 nm
synthesized by sol–gel chemical process was selected to be used
as a guest template to grow small ceria nanoparticles. The silica
particles enable only limited diffusivity and interparticle inter-
actions in solution by their larger size and high surface charge
density which provides improved dispersibility and stability for
small ceria nanoparticles which are highly unstable in solution.
The particle size and structural characteristics of the silica–ceria
hybrid nanostructure were analyzed by dynamic light scattering
(DLS) X-ray diffraction (XRD), scanning electron microscopy
(SEM), and transmission electron microscopy (TEM) imaging
techniques including energy dispersive X-ray chemical analysis
(EDX). The nature of bonding between silica and ceria nano-
particles were characterized by X-ray photoelectron spectros-
copy (XPS) and Fourier transform infrared spectroscopy (FTIR)
analysis. The silica–ceria hybrid nanostructure solution
behavior was compared with unstabilized ceria by comparing
the particle size changes and settling velocity in different bio-
logical media conditions. The bio-catalytic activity of the silica–
ceria nanostructure was characterized by measuring the
superoxide dismutase (SOD) mimetic activity in different bio-
logical media conditions in comparison to a control reagent.
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2. Experimental section
2.1. Chemicals
The chemicals used in this work are tetraethylorthosilicate
(TEOS, Sigma-Aldrich), cerium(III) nitrate hexahydrate (99.99%,
Sigma-Aldrich), urea ($98%, Sigma-Aldrich), ethanol (200 proof,
Sigma-Aldrich), and ammonium hydroxide (28–30%). Unmodi-
ed ceria nanoparticles powders of mean size 8 nm were
purchased commercially from Melorium Technologies (product #
09817). The deionized (DI) water used in our experiments has
resistance greater than 18 Ohm at room temperature. All chem-
icals were used as received without any further modication.
2.2. Synthesis of the silica nanoparticles template
The silica nanoparticles of average size 50–60 nm were synthe-
sized by a modied Stöber synthesis process.23 The initial
reaction mixture consisted of 114 ml ethanol and 5.7 ml
ammonium hydroxide in a 250 ml conical ask. The above
mixture was stirred for 15 min followed by drop wise addition of
3.8 ml tetraethylorthosilicate; the nal mixture was allowed to
undergo vigorous stirring for 15 h at room temperature. The
nal silica nanoparticles sol obtained appeared milky white in
color and was used as prepared without further modication.
2.3. Synthesis of the silica–ceria hybrid nanostructure
The silica–ceria nanostructure was synthesized by a solution
chemical synthesis process previously reported for GaN/SiO2
nanocomposites.24 In common with this procedure a required
amount of aqueous urea (0.6 gm/3 ml DI water) was added to
15 ml of prepared silica nanoparticles solution and the mixture
was stirred (350 rpm) for 15 min at 85  C. The ceria nano-
particles precursor was prepared by dissolving 0.0938 gm of
cerium(III) nitrate hexahydrate in 1 ml DI water. This solution
was then added to the urea–silica mixture and continued to stir
for another 3 h at 85  C. The ratio of Ce–Si in the nal product
was calculated as 5 wt%. The resulting yellowish colored solu-
tion was centrifuged to isolate the particles obtained as
precipitates. The nal product was washed 3 times with DI
water and nally with anhydrous ethanol followed by drying at
room temperature for 24 h.
2.4. Preparation of the silica–ceria hybrid nanostructure
suspension in biological relevant media
An aqueous solution of silica–ceria was prepared in DI water at a
concentration of 40 mg ml1. The prepared suspension was
dispersed by sonication for 15 min in a water ultrasonicator
(Model: Branson 2510). The prepared nanoparticle suspension
was used as a stock solution for further dispersion in other
aqueous media.
2.5. Characterization of the silica–ceria hybrid
nanostructure
Dynamic light scattering (DLS) particle size measurements.
The average median or effective diameters (Z-average) of the
particles were measured by dynamic laser light scattering
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measurements. The instrument setup used for measurements
was Brookhaven Zeta PALS particle size analyzer with a xed He–
Ne laser. All the measurements were performed at 90 scattering
angle. The number average particle size and/or Z-average were
derived from correlation function and cumulant size analysis
using particle sizing soware. The size analysis was conducted
except unless noted by dispersing the dry powder particles at a
concentration of not less than 10 mg ml1. All sample preparation
was made at room temperature. Before each measurement the
nanoparticles dispersed solution was sonicated for a few minutes
and vortexed for uniform dispersion. The nal volume of particle
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dispersion used for size measurements was
1 ml.
X-ray diffraction (XRD) analysis. A portion of the particles
was smeared uniformly over the double sided tape attached to
metal sample holder for X-ray diffraction (XRD) measurements
which were generated using a Cr Ka (2.2897 Å, 40 kV, 40 mA)
radiation source. The measurements were performed using a
0.3 mm collimator under the following measurement condi-
tions: Omega 25–45 deg., Phi 0–360 deg. The intensity data were
collected over a 2q range of 10–180 . The average crystallite size
was calculated using the Debye–Scherer equation. The standard
cubic indexation method was used to calculate the cell “a”
parameter value for the prominent ceria base peak (111).
Scanning electron microscopy (SEM)/energy dispersive X-ray
spectroscopy (EDS) measurements. The SEM and EDS
measurement was done using FEI Quanta (Dual FIB/SEM). For
image analysis, a portion of the particles was deposited
uniformly over double sided tape mounted on a metal stub.
Before imaging, a thin conducting layer of carbon lm was
deposited on the surface for better conductivity.
High resolution transmission electron microscopy (HRTEM)
analysis. A JEOL JEM 2010 TEM was used to acquire the electron
microscopy images at accelerating voltage of 200 keV. The
sample preparation included dispersing the sample in DI water
and sonication for 10 min followed by drop casting few drops of
as prepared dispersion on a lacey copper grid (200 mesh) coated
with carbon. The grids were allowed to dry at room temperature
for
24 h before the actual analysis.
X-ray photoelectron spectroscopy (XPS) analysis. XPS was
used to determine the chemical speciation and elemental
composition of silica–ceria hybrid nanostructures. The XPS
measurements were performed using a Phi 5000 Versa Probe.
This system consisted of a monochromatic focused Al Ka X-ray
(1486.7 eV) source and a hemispherical analyzer. A 25 W X-ray
beam with 100 mm in diameter was incident normal to the
sample surface and the photoelectrons were collected at 45
relative to the surface normal. Wide energy scan data were
collected using pass energy of 117.4 eV. High-resolution scans
were obtained using pass energy of 23.5 eV. Low energy elec-
trons at
1 eV, 40 mA and low energy Ar+ ions (5 eV) were used to
minimize the surface charging. The XPS spectra were calibrated
to an energy scale with binding energies for Cu 2p3/2 at
932.67  0.05 eV and Au 4f at 84.0  0.05 eV.
Fourier transform infrared spectroscopy (FTIR) analysis. For
FTIR measurements solid loose silica supported ceria powder
was made into a pellet by mixing with dried KBr in
10 : 1 ratio.
The measurements were performed using a Thermo Scientic
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FTIR spectrometer, model Nicolet iS10. All the measurements
were an average of 50 scans and recorded at resolution of 1 cm1.
2.6. Colloidal stability characteristics of the silica–ceria
hybrid nanostructure in aqueous biological media conditions
Agglomeration kinetics of the silica–ceria hybrid nano-
structure. The agglomeration kinetics of the unmodied and
silica–ceria hybrid nanostructure was monitored by dynamic light
scattering measurements over a period of 2 h. The nanoparticles
were diluted from stock solution to 10 mg ml1 concentration in
the respective solutions. The effect of pH on the stability of the
nanoparticles was tested at constant ionic strength solution of
0.02 M NaCl with the pH of the solution adjusted using either HCl
or NaOH. The particles suspension was sonicated for 3 min and
vortexed for 10 seconds before starting the rst measurement and
le undisturbed for subsequent measurements.
Characterization of the suspension stability by sedimenta-
tion kinetics. The unmodied ceria and silica–ceria hybrid
nanostructure settling velocity was evaluated by characterizing
time dependent suspension stability analysis. The sedimenta-
tion of the nanostructure was monitored by in situ absorbance
measurements over a period of 12 h with 30 min time interval
between each measurement. The stock nanoparticles suspen-
sion (40 mg ml1) was diluted to a concentration of 25 mg ml1
in the required testing media, DI water, phosphate buffer saline
(PBS) or complete cell culture media (CCM), RPMI (Roswell Park
Memorial Institute Medium) with 10 vol% fetal bovine serum
(FBS). The diluted nanoparticle suspension was vortex mixed in
a vortexer (Fisher Vortex, Gene 2) for 15–20 seconds. The
changes in maximum absorbance (lmax
300–320 nm) inten-
sity of ceria nanoparticles in different aqueous media was
recorded as a function of time in a 10 mm path length cuvette
using a UV-Vis spectrometer (Shimadzu UV-2501 PC). The
suspension stability index was calculated as % of initial absor-
bency at t ¼ 0 and other subsequent time points up to 12 h.
Analysis of SOD activity of silica–ceria hybrid nano-
structures. The bio-catalytic activity of the silica–ceria hybrid
nanostructure was analyzed by SOD mimetic assay. The proce-
dure for SOD mimetic activity was followed as prescribed in the
Trevigen's HT Superoxide Dismutase Assay Kit (Cat# 7501-500-
K). In general the superoxide radical generated from the
conversion of xanthine by xanthine oxidase converts WST-1 to
WST-1 formazan, a colored reagent, which absorbs light at
450 nm. In our experiment silica supported ceria nanoparticles
dispersed in different biological relevant media conditions was
assessed for reduction of superoxide ion concentrations in
competition to the SOD control reagent. The measure of silica
supported ceria nanoparticles SOD activity was represented by
the extent of reduction in the appearance of WST-1 formazan
measured using an absorbance plate reader.
3. Results and discussion
3.1. Particle characterization and structural evolution
A schematic illustration of the steps followed in the synthesis of
the silica–ceria hybrid nanostructure is shown in Scheme 1. The
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nano-crystalline ceria nanoparticles were synthesized using sol–

gel derived silica nanoparticles as a guest matrix. The surface
functional groups of silica mediated the growth of the nano-
crystalline ceria nanoparticles. The as synthesized silica–ceria
hybrid was isolated and washed by centrifugation, dried at
room temperature and processed as a loose powder for further
characterization. The formation of cerium oxide was indicated
by color change of the reaction solution from colorless to pale
yellow (oxide). The particle size, crystallinity, and morphology of
the silica supported ceria nanoparticles was characterized DLS
particle size measurements, XRD and SEM/EDX analysis as
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described in the later sections.

3.2. Particle size characterization

Fig. 1 shows the particle size characterization for the silica–ceria
hybrid nanostructure by DLS size analysis. The silica particles
size was measured as an average median size of 68.7 nm and
aer the attachment of ceria showed broad size distribution
with an increase in the average median size to 71.1 nm.
However the control unmodied ceria nanoparticles agglom-
erated to average median size of 300 nm (Fig. 1). The small
increase in average median particle size (
2 to 3 nm) and
broader size distribution can be ascribed to a change in
hydrodynamic size of the silica particles due to the attachment
of ceria. This observation is supported by the polydispersity
index (PDI) values derived from cumulant analysis based on
particle size distribution width. The PDI for bare silica particles
was measured as 0.018 representing the monodisperse silica
particles. Aer ceria attachment the PDI for silica particles
increased to 0.31 consistent with broad particle size distribu-
tion where homogenous particle size is disturbed by ceria
particles attachment and may be due to agglomeration of non-
attached ceria particles as seen by the size distribution of silica
tailing on the lower side. Similarly the PDI for bare ceria
particles was measured as 0.384 due to the intense agglomera-
tion of small ceria particles present in the suspension. The silica
nanoparticles were observed to be more stable in the dispersion
medium due to the strong electrostatic stabilization which
renders them dispersible in aqueous media. This allowed for
the facile dispersion of small ceria nanoparticles attached to
silica without any signicant agglomeration. However unstabi-
lized small ceria nanoparticles without any surface stabilizing
agent agglomerated intensively due to the weak repulsive forces
between particles which is related to the diffusivity of the
particles in solution and extends to favorable conditions for an
increase in collision efficiency. In the following section we have
Fig. 1 DLS particle size analysis for silica template, silica–ceria hybrid
and for comparison of unmodified ceria particles of similar size range.
The silica particle size distribution was measured using the as
synthesized silica sol without any further dilution. For the silica–ceria
hybrid and unmodified ceria particle size measurements, DI water was
used as the solvent for dispersion.
further characterized the “ageing solution stability” of silica–
ceria hybrids against agglomeration and settling in different
biological media conditions as compared to unmodied ceria
nanoparticles.
The XRD analysis was performed to characterize the phase
purity and crystallinity of the pure silica, unsupported ceria
(control), and silica–ceria hybrid. The XRD patterns for the
above mentioned samples are shown in Fig. 2. The pure silica
nanoparticles had no diffraction peak except a broad hump
approximately between 25 and 40 indicating the amorphous
properties of silica. The silica supported ceria samples had
main diffraction patterns at 2q ¼ 45 , 50 , 72 , 88 , 94 , 113
corresponding to (111), (200), (220), (311), (222) and (400) ceria
reections conrming the formation of cubic crystalline ceria
nanoparticles.
The silica supported ceria diffraction peak was broadened
compared to unsupported ceria samples indicating the small
crystallite size of the as formed ceria. The crystallite size was
calculated from the peak broadening using the Debye–Scherrer
equation.25 The average ceria crystallite size for unsupported

Scheme 1 Schematic representation of the synthesis of the silica–ceria hybrid nanostructure by a solution chemical synthesis process. The
larger silica nanoparticles (
50–60 nm) were used as a support matrix to grow and stabilize small cerium oxide nanoparticles.

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Fig. 2 XRD diffraction analysis for (a) silica template, (b) silica–ceria
hybrid and (c) unstabilized ceria. The primary crystallite size of ceria
nanoparticles attached to silica was
3–5 nm. The silica particles
before ceria attachment only showed a broad peak representing the
amorphous nature of the particles.
ceria and silica supported ceria was calculated be 8 nm and 5
nm respectively. Absence of any other noticeable trace peaks for
the formation of additional complexes was evident from the
XRD analysis.
Formation mechanism of the silica–ceria hybrid nano-
structure. The nucleation and growth of nano-crystalline ceria
particles on the surface of amorphous silica template particles
were further investigated by pre-treatment of silica nano-
particles. To elucidate the mechanism of ceria growth on the
silica surface via the active functional groups (–OH and Si–O–
H), the silica sol nanoparticles were isolated by centrifugation
from the sol–gel synthesized liquid and were pre-treated by
heating @
60–80  C for 7 h and drying at 35  C overnight. The
synthesis of silica–ceria hybrid nanostructure procedure was
followed as mentioned in the experimental section using the
pre-treated silica as templates for growth of nano-crystalline
ceria. The XRD analysis of the resultant product showed no
distinguishable diffraction peaks for crystalline cerium oxide,
and that the material is apparently X-ray amorphous (Fig. 3(a)).
It is possible that the concentration or type of silanol groups
(non-hydrogen bonded or H-bonded) in silica particles was
decreased or modied with thermal treatment due to the
condensation of silanol functional groups to form a siloxane
bond.26 However utilizing the as prepared silica sol, without any
thermal treatment, at neutral or acidic pH provides negatively
surface charged Si–OH groups which acts as active binding sites
for increased local concentration of Ce 3+ cations directly on the
silica surface. Furthermore the addition of urea acts as a base
catalyst which upon mild heat treatment at 85  C decomposes
to ammonia which increases the base acidity of the silica sol
and the nucleation threshold for growth of Ce 3+ ceria cations
directly on the silica surface. The formation of cerium oxide
might have occurred with complexion with –OH groups on the
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silica surface to form intermediate hydroxide and to the nal
oxide phase. Our TEM image analysis further conrms our
assumption of the role of surface silica functional groups on the
growth of nano-crystalline ceria. Fig. 3(b) shows the electron
microscopy image of same product analyzed by XRD (Fig. 3(a)).
Several regions of image showed particles of irregular
morphology and size exceeding 100 nm. From the TEM
measurements it can be interpreted as nucleation and growth of
many small numbers of crystallites of ceria within a nano-
crystalline material which lacks long range order. This obser-
vation further conrms our assumption that with the reduction
of silica surface functional groups by thermal treatment, the
ability of the ceria precursor to form a chemical bond linkage
with the silica surface is reduced. This is due to the decompo-
sition of urea to ammonia and carbonate species in DI water
above 80  C which reacts with cerium nitrate metal salts to form
the particle like features of irregular size and shape within the
silica matrix.27 Our XPS surface analysis discussed in the later
sections further strengthens the proposed mechanism of silica–
ceria bond formation.
3.3. Morphology and structure analysis
The morphology and structure of unsupported ceria, silica and
the silica–ceria hybrid nanostructure were analyzed by electron
microscopy analysis. For TEM analysis the unsupported ceria
and silica–ceria dry powder was dispersed in DI water and
applied to TEM grids as described in the materials and methods
section. The unsupported ceria nanoparticles (control) looked
very irregular in shape and size (Fig. S1†). The surface
morphology of silica particles was evaluated before and aer
ceria coating. Fig. 4(a) and (b) shows the TEM image of amor-
phous silica particles which are spherical and monodisperse
and of median size
55 to 60 nm consistent with DLS size
analysis measurements. Fig. 4(c) shows an image of silica
nanoparticles aer ceria nanoparticles coating. The image
reveals darker regions representing ceria within the silica
spheres which appear lighter in contrast. The ceria nano-
particles were distributed all over the silica nanoparticles
surface in a more regular pattern than those obtained by
calcination at higher temperature which leads only to non-
homogenous coating and aggregation of ceria on the silica
surface.20 Fig. 4(d) shows a high magnication image of a single
silica particle aer attaching ceria. The image reveals a dense
surface coverage of ceria nanoparticles on the silica surface. The
lattice fringes seen in the TEM images can be attributed to
nano-crystalline ceria particles distributed on the surface of
amorphous silica particles (Fig. 4(d) inset). The measurement of
the lattice fringes distance substantiates the formation of cubic
crystalline ceria nanoparticles on the silica surface. The size
distribution of ceria nanoparticles attached to the silica surface
was measured in the size range of 3–5 nm consistent with XRD
crystallite size analysis. While most of the ceria particles are
bound to the surface of silica, the presence of unbound ceria
nanoparticles was also observed in small proportions possibly
due to nucleation and growth of isolated ceria crystallites in
solution. Fig. 4(e) and (f) show the SEM image/EDX analysis of
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Fig. 3 XRD diffraction analysis for silica–ceria hybrid control samples synthesized via thermal pre-treatment of the silica template which appears
to be (a) apparently X-ray amorphous, and (b) non-homogenous in size and shape.
silica–ceria which appears nearly spherical in shape and size.
The EDX analysis on the marked regions of SEM images showed
characteristic X-ray energies for O La1, Si Ka1, and Ce La1
conrming the presence of ceria within the silica matrix. All the
above observations suggest that by utilizing sol–gel derived
silica as a guest template it is possible to synthesize cerium
oxide particles with a high surface area to volume ratio. Simi-
larly Khalil et al. reported the synthesis of Fe2O3/SiO2 nano-
composites by hydrolysis of iron(III) nitrate in sols of silica
particles and our results are in agreement with their ndings
where sol–gel derived silica particles sol with surface active
functional groups can serve as anchor sites for nucleation and
growth of oxide nanoparticles.28
3.4. Nature of the bonding between silica and ceria
The chemical state of ceria nanoparticles and chemical bonding
information between silica and ceria was characterized by XPS, a
surface sensitive technique. Fig. 5(a) shows the XPS 3d spectral
region for the silica–ceria hybrid nanostructure. The spectral
features of silica supported ceria are consistent with the unsup-
ported ceria samples control which substantiates the formation
of cerium oxide. Assignment of the XPS peaks indicates the
presence of ceria in mixed oxidation states (3+ and 4+). The ceria
particles in the nano-size range, on an average less than 5 nm,
exist in a mixed oxidation state. The features observed at
880.60 eV, 885.80 eV and 903.9 eV can be attributed to ceria in the
3+ oxidation state. The presence of 3+ states can be ascribed to
oxygen vacancies in the atomic crystal structure.5 We further
probed the chemical bonding characteristics between silica and
ceria particles. The high resolution O 1s and Si 2p spectra for bare
silica and ceria attached silica are shown in Fig. 5(a) right and (b).
Comparing Si 2p and O 1s spectra for silica particles before and
aer attaching ceria, three main characteristic changes were
observed: (i) the shi in the binding energy of both Si 2p and O 1s
towards lower binding energy of 0.2 eV, (ii) the difference in full
width at half maximum (broadening) of the peaks was calculated
to be 1.7–2 eV (charge reference was made based on C 1s), (iii)
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compared to uncoated silica a shoulder peak at
530 eV was
observed aer ceria coating.
The shi in binding energy and peak broadening for silica–
ceria represents the modied local surface chemical environ-
ment of silica nanoparticles. The negative shi in the binding
energy of Si 2p and O 1s aer ceria coating represents an
increase in electron density on the surface of Si and
surrounding O, which infers that this region of Si was covered
by Ce. Furthermore the O 1s peak at
533 eV belongs to silica
nanoparticles and a shoulder at
530 eV for ceria attached silica
nanostructures represents Ce 3+ resulting from the formation
of Si–O–Ce bond on the surface of silica particles.
To characterize the dependence of ceria nanoparticles
formation on the surface silanol groups of silica, we performed
solid state FTIR spectroscopic analysis of the silica particles
template prior to and aer ceria deposition (Fig. 6). Prior to
ceria deposition, the structural Si–O–Si and Si–OH bands for
silica were observed. The broad absorption band observed
between at 3730 to 2550 cm1 and 1630 cm1 can be assigned to
the stretching (n) and bending (d) modes of adsorbed water
molecules and H-bonded silanols.29 The structural silica bands
nas (Si–O–Si) are observed at 1200 and 1090 cm1. The band at
950 cm1 corresponds to n (Si–OH) and SiO groups.30 Similarly
the band at 800 cm1 and 470 cm1 can be assigned to ns (Si–O–
Si) and d (Si–O–Si) mode.29,30 The silica–ceria nanostructure has
similar groups of vibrational bands with few denite charac-
teristic changes in the silanol bonds of silica. The Ce–O
stretching bands are expected to be ca. 400 to 450 cm1 and not
clearly distinguishable from silica stretching modes (Si–O–Si)
which occur in the same region.31 As such the absorption bands
at 950 cm1 due to dangling bands of oxygen atoms are reduced
in intensity and vibration modes of the Si–O–Si siloxane bonds
at 1090 cm1 and 1200 cm1 are shied. This can be attributed
to attachment of electropositive cerium ions to highly electro-
negative oxygen of silica nanoparticles.32
All the above observations indicate that ceria deposition on
the silica nanoparticle surface takes place via growth of ceria on
the silica nanoparticle surface. The proposed mechanism of
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Fig. 4 (a, b) TEM image of silica nanoparticle templates. (c) Silica nanoparticle after ceria coating marked by arrows (inset: silica nanoparticle
appears bright due to low atomic number covered by dark spots of ceria). (d) Image of single silica particle after ceria coating. Ceria particles are
of uniform size and morphology with visible lattice fringes confirming the formation of nano-crystalline ceria. SEM image and EDX chemical
analysis of the silica–ceria hybrid nanostructure (e and f).

cerium oxide nanocrystals growth could have occurred with the
formation of covalent bond linkage between ceria and silica
forming cerium–silicate bond. Our proposed mechanism is
supported by the experimental observation of color change of
the stirring reaction solution from colorless to murky white
(hydroxide) to yellowish (oxide).
3.5. Agglomeration kinetics and sedimentation stability of
silica–ceria nanoparticles in biological media conditions
For biomedical applications nanoparticles have to be dispersed
in aqueous biological media conditions and stability of nano-
particles, surface modied or not, is most oen affected by
constituents and media conditions. Hence we examined the
stability of silica–ceria hybrid by measuring the change in mean
particle size in different biological media conditions (Fig. 7). The
particle size measurements were started immediately following
dispersion of particles in different media suspension. We found
that silica–ceria hybrid particles remained stable in all of the
media with minimal agglomeration indicated by a minor
increase in particle size. In contrast unsupported ceria (control)
showed much poorer dispersion in all of the tested media. The
particle size varied based on type of medium, ionic strength of
media, where particles agglomerated to form small to very large
aggregates in the micron size range. The increase in aggregate
size of unstabilized ceria particles in tested dispersion media can
be explained based on the Brownian agglomeration mechanism
where intrinsic surface charge of small ceria particles in high
ionic strength media, like PBS, will be neutralized which leads to
a reduced surface charge barrier and the chances of particle
random collision and sticking together is increased. By attach-
ment of small particles like ceria to larger silica particles the

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Fig. 5 Comparison of XPS high resolution spectra between control
ceria, silica template, and silica–ceria hybrid nanostructure (a) Ce 3d
and O 1s spectra for control ceria and silica–ceria. (b) O 1s and Si 2p
spectra of silica and silica–ceria nanostructure.
diffusivity of the particles will be less by which random collision
between particles will be possibly reduced. Moreover the intrinsic
surface property of silica, negative surface charge, provides
enough surface screening, electrostatic repulsion, by which
particles remain in solution and dispersion properties are
improved. Though the concentration of ceria nanoparticles may
be different between unsupported and silica–ceria the high
agglomeration of unsupported ceria particles was observed even
at very low concentrations indicating the instability of these
systems under all of the measurement conditions.
The agglomeration of nanoparticles in solution can affect the
“life time” stability of nanoparticles in solution which in turn can
affect the dosimetry for in vitro biological cell uptake experiments.
To further evaluate the long term colloidal stability of silica sup-
ported ceria nanoparticles we performed time resolved DLS
particle size measurements and characterized the suspension
stability of silica–ceria nanoparticles by sedimentation stability
8428 | RSC Adv., 2014, 4, 8421–8430
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Paper
Fig. 6 FTIR spectra of the silica nanoparticles template before and
after attachment of ceria nanoparticles.
Fig. 7 Average particle size of silica–ceria hybrid nanostructures in
different pH conditions, PBS and CCM.
analysis (Fig. 8(a) and (b)). The agglomeration kinetic studies
indicate no change in average agglomerate particles size for the
silica–ceria hybrid over a period of 2 h. However based on type of
media used little change in initial particle size was observed and
steady state was maintained throughout the end of the experi-
ment. By comparison the unstabilized small ceria nanoparticles
steeply increased in size purposefully in PBS media. This obser-
vation further conrms our Brownian agglomeration mechanism
for smaller sized ceria nanoparticles due to surface charge
neutralization in high ionic strength solutions. Our proposed
hybrid nanostructure was successfully shown to overcome the
higher diffusivity of small particles by attaching to the bigger
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support matrix by which rapid continuous movement of nanosize
particles can be greatly reduced. Fig. 8(b) shows the change in the
concentration of particles, “life time stability” measured by UV-Vis
spectroscopic analysis. The cerium oxide nanoparticles have
optical absorbance in the UV region centered on 300 and 325 nm.
The settling rate was calculated by monitoring changes in absor-
bance intensity of the ceria nanoparticles suspension as a func-
tion of time. In agreement with dispersion results the silica
supported ceria nanoparticles exhibited a high level of stability
without any sign of rapid settling in all of the tested suspension
media over the measurement time period. However aer a period
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the surface of oxide nanoparticles can act as a reactive site that
mimics SOD catalyst activity. However for biological testing,
nanoparticles have to be dispersed in variety of cell cultures and
physiological buffer conditions which contain small molecules,
proteins, peptides, enzymes etc. which can limit the activity of
small nanoparticles like ceria by surface screening effect or
agglomeration. Hence we tested the superoxide radical scav-
enging ability of ceria which exists in 3+ oxidation state due to
higher oxygen and electron vacancy as attached to silica
templates. The silica–ceria nanostructures were incubated in
the selected biological media DI water, CCM, and aqueous

of 6 h the settling rate of the silica–ceria hybrid was found to acidic and basic buffers (Fig. 9). For the free radical scavenging
increase with the nal concentration of particles measured to be activity test, the silica–ceria nanostructure dispersed in biolog-

80%. This is understandable considering the gravitation sedi- ical media was tested along with cytochrome C control reagent.
mentation which is size dependent. However the control unsup- Experimentally the xanthine reagent gets converted to xanthine
ported ceria particles sediment out of the solution (PBS) at a much oxidase with free radical release which reacts with WST-1
faster rate due to the high degree of agglomeration with more reagent to form a colored product (formazan) which gives
than 60% of the particles sedimenting out within the rst 3 h of absorbance @ 450 nm. In comparison to the control reagent,
measurement duration. Considering the rate of settling and our
previous time dependent agglomeration studies it is possible that
the smaller unstabilized particles are predominantly dominated
by both Brownian motion and gravitational agglomeration while
as shown in Fig. 8(a) within few minutes (<2 min) the initial
agglomerate size of particles was
500 nm. This size was found to
increase followed by a correlative decrease in suspension stability
that indicates that the small ceria particles are dominated initially
by Brownian agglomeration and further by a gravitational colli-
sion mechanism. All of our observations indicated that similar
sized ceria nanoparticles upon surface stabilization with larger
silica nanoparticles exhibit higher colloidal stability by staying in
suspension over a longer ageing period by overcoming random
collision movement and gravitational agglomeration.

3.6. Analysis of surface reactivity of silica supported ceria

nanoparticles in various media conditions by SOD mimetic
assay
Recently the redox properties of nano-ceria have made them
useful candidates as free radical scavengers and redox cycling Fig. 9 Analysis of bio-catalytic activity of silica supported ceria
antioxidants. The ceria atoms trivalent oxidation state (3+) at nanoparticles in biological relevant media conditions.

Fig. 8 (a) Agglomeration kinetics of the silica–ceria hybrid in a variety of biological media commonly used for the dispersion of nanoparticles (b)
suspension stability of silica–ceria hybrid measured as function of time in DI water, PBS and CCM for a period of 12 h.

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the increase in absorbance was slow with inclusion of silica–
ceria hybrids dispersed in different medias which indicated that
the rate of catalytic activity or reaction of released free radical
with WST-1 reagent is reduced in the case of silica–ceria
inclusion as compared to the control reagent. This mechanism
was consistently observed in all of the media in which silica–
ceria was dispersed. Hence it is safe to say that compared to
cytochrome C control the ceria nanoparticles reactive oxidation
state (3+) is not limited by attachment to silica templates.
Furthermore the silica–ceria hybrid nanostructure scavenging
activity is as efficient as in any of the biological media condi-
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tions in which the catalytic activity was measured, and so is
unlikely to be inhibited by the constituents present in solution.
4. Conclusion
The colloidal stability of unmodied cerium oxide nanoparticles
of smaller sizes (<10 nm) can be improved by attaching to a
bigger support matrix like silica nanoparticles. The surface sila-
nol functional group of sol–gel derived silica nanoparticles was
found to be essential in promoting the growth of ceria on the
surface of amorphous silica. The silica–ceria hybrid nano-
structures showed excellent stability against agglomeration and
were characterized to be stable in suspension over longer ageing
periods in different biological media conditions. The synthesized
ceria nanoparticles which exist in 3+ oxidation states showed
remarkable SOD mimetic activity in DI water, acidic/basic pH and
culture media showing resistance to adverse biological media
conditions. All the above mentioned characteristics of the silica–
ceria hybrid nanostructure makes it a promising candidate for
further in vitro and in vivo studies as a bio-catalytic material for
oxidative stress related and other biomedical applications.
Acknowledgements
A portion of the research reported here was performed in EMSL,
a DOE user facility supported by the Office of Biological and
Environmental Research and located at Pacic Northwest
National Laboratory. We thank Dr Don Baer for his critical
review and useful insights of the manuscript. We acknowledge
Dr Chongmin Wang for assistance with TEM and Dr Nachi-
muthu Ponnusamy for XPS characterization and analysis.
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