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Accepted Article

Title: Cerium vanadate nanozyme with specific superoxide dismutase

activity regulates mitochondrial function and ATP synthesis in
neuronal cells

Authors: Namrata Singh, Somanathapura K NaveenKumar, Motika

Geethika, and Govindasamy Mugesh

This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.

To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202011711

Link to VoR: https://doi.org/10.1002/anie.202011711

Angewandte Chemie International Edition 10.1002/anie.202011711

RESEARCH ARTICLE
Cerium vanadate nanozyme with specific superoxide dismutase
activity regulates mitochondrial function and ATP synthesis in
neuronal cells
Namrata Singh, Somanathapura K. NaveenKumar, Motika Geethika, and Govindasamy Mugesh*
Abstract: Nanoparticles that functionally mimic the activity of metal- disease condition known as Amyotrophic Lateral Sclerosis
containing enzymes (metallonanozymes) are of therapeutic (ALS).[4g-i] Thus, the artificial enzymes with specific SOD-like
importance to treat various diseases. However, it is still not clear activity that can maintain the mitochondrial integrity and a steady
whether such nanozymes can completely substitute the function of redox stature of cells will be of great therapeutic importance in
natural enzymes in living cells. In this work, we show for the first time various neurological diseases.

Accepted Manuscript
that a cerium vanadate (CeVO4) nanozyme can substitute the
Recently, nanomaterials with enzyme-like characteristics,
function of superoxide dismutase 1 and 2 (SOD1 and SOD2) in the
termed nanozymes, have attracted significant attention as they
neuronal cells even when the natural enzyme is down-regulated by
can be used for various biomedical applications.[5] However,
specific gene silencing. The nanozyme prevents the mitochondrial
most of the nanozymes have been reported to exhibit oxidase or
damage in SOD1 and SOD2-depleted cells by regulating the
peroxidase activity and examples of nanomaterials that
superoxide levels and restores the physiological levels of the anti-
functionally mimic the redox modulatory enzymes such as SOD,
apoptotic Bcl-2 family proteins. Further, the nanozyme effectively
catalase (CAT), and glutathione peroxidase (GPx) are very
prevents the mitochondrial depolarization, leading to a significant
rare.[6] A few nanozymes with SOD-like activity have been
improvement in the cellular levels of ATP under oxidative stress.
studied for their ability to combat oxidative stress in various
cellular and animal models.[6] However, it is still not clear
Introduction whether a nanozyme can fully substitute the key functions of a
natural antioxidant enzyme when the enzyme is down-regulated
Superoxide (O2.−) is one of the major reactive oxygen species
in the living cells. In this paper, we report that cerium vanadate
(ROS) produced in the mammalian cells, and when generated in
(CeVO4) nanorods function as SOD and regulate the ATP levels
excess, it causes oxidative damage to various cellular
by restoring the mitochondrial function and integrity in neuronal
components.[1] Therefore, the level of superoxide in the cells is
cells. The nanozymes maintain the physiological levels of
tightly regulated by a metalloenzyme, superoxide dismutase
superoxide and restore the functions of the crucial anti-apoptotic
(SOD), which forms the first line of defense against oxidative
Bcl-2 family proteins under oxidative stress conditions.
stress.[2] The enzyme effectively converts superoxide radicals to
molecular oxygen (O2) and hydrogen peroxide (H2O2). The three Results and Discussion
isoforms of SOD in mammalian cells, i.e. cytosolic SOD (SOD1
The CeVO4 nanorods of different sizes were synthesized
or Cu-Zn-SOD), mitochondrial SOD (SOD2 or Mn-SOD) and
utilizing hydrothermal method. The size and morphology of the
extracellular SOD (SOD3 or Cu-Zn-SOD) have evolved for
obtained nanoparticles were ascertained using scanning
organelle-specific activity and redox signalling.[3a-c] The most
electron microscopy (SEM) and transmission electron
abundant isoform SOD1 is also localized in the inter-membrane
microscopy (TEM). The micrographs indicate the formation of
space and plays an important role in maintaining the
monodisperse, polycrystalline nanorods of different sizes
mitochondrial integrity along with SOD2.[3]
(CR1≈50 nm, CR2≈100 nm and CR3≈150 nm) (Figure 1a,c,e,
The SOD1 deficiency promotes carbonylation of proteins,
Figure S1 and S2). The high resolution TEM (HR-TEM) images
including the anti-apoptotic Bcl-2 family proteins that play key
display regular fringes corresponding to the [200] planes of the
roles in governing the mitochondrial membrane permeabilization
tetragonal CeVO4 nanorods (Figure 1b,d,f). The presence of
and apoptosis. The loss of mitochondrial membrane potential
cerium (Ce), vanadium (V) and oxygen (O) in the samples were
(Δψm) due to inefficiency or deficiency of SOD enzyme causes
confirmed by X-ray mapping (Figure S3). Further, the phase of
mitochondrial dysfunction, leading to cellular energy crisis.[4] As
the materials was determined by X-ray powder diffraction (XRD).
neurons have high demand of ATP even at resting stage due to
The diffraction peaks of different sized nanorods can be indexed
their higher metabolic rate, these cells are highly susceptible to
to the pure tetragonal (zircon-type) CeVO4 phase (JCPDS No-
oxidative damage by ROS. Therefore, the impairment of SOD
72-0282)[7a] (Figure S4). The absence of peaks corresponding to
activity is associated with various neurological disorders such as
other phases indicates high purity of the samples. The phase of
Alzheimer’s and Parkinson’s disease.[4b,4f] In addition, a mutation
the materials was further confirmed by Raman spectroscopy
in the SOD1 gene is also well-documented in a neurological
(Figure S5a)[7b-7d] and Fourier-transform infrared spectroscopy
(FT-IR) (Figure S5b).[8]

[*] Dr. Namrata Singh,[‡] Dr. S. K. NaveenKumar[‡], Dr. Motika
Geethika, and Prof. Dr. G. Mugesh
Department of Inorganic and Physical Chemistry
Indian Institute of Science, Bangalore 560012, India
E-mail: mugesh@iisc.ac.in
[‡] These authors contributed equally to this work.
Supporting information of this article can be found under:
https://doi.org/10.1002/anie.2019xxxx.
The surface property and oxidation state of materials was
investigated employing X-ray photoelectron spectroscopy (XPS)
(Figure S6). The peaks near 882.0 and 885.8 eV corresponds to
Ce3d5/2 transition and the peaks at around 900.2 and 904.3 eV
belongs to Ce3d3/2 confirming the presence of Ce in +3 oxidation
state in the nanomaterial (Figure S6a,c,e). The XPS data
indicates the presence of only a negligible amount of Ce in +4

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RESEARCH ARTICLE
directly proportional to the amount of superoxide generated in
the reaction mixture. The SOD-like activity of the nanorods was
quantified by measuring the inhibition of formazan formation.
The nanorods exhibit high SOD-like activity and the IC50 value
for the inhibition of formazan production by the rods of size ≈ 50
nm, 100 nm and 150 nm were determined to be 4.12 ± 0.19,
3.93 ± 0.20, 2.57 ± 0.07 ng µL−1, respectively (Figure 2b,c,d).
The activities of nanorods with different sizes were found to be
comparable and displayed SOD-like activity at much lower
concentrations. A relatively lower surface area of the CR1 as
compared to that of CR2 and CR3 does not affect the catalytic
activity, probably due to the larger pore size on the nanorods
surface that can provide larger active site for the catalysis
(Figure S8). The disproportionation of superoxide by SOD

Accepted Manuscript
generates H2O2 and oxygen. To understand the formation of
H2O2 in the presence of CeVO4 nanozymes, we carried out the
reaction in the presence of horseradish peroxidase (HRP) and
the peroxidase substrate 2,2’-azino-bis(3-ethylbenzthiazoline-6-
sulfonic acid) (ABTS). The H2O2-mediated oxidation of ABTS in
the presence of HRP was measured spectrophotometrically at
405 nm.[10b] The results indicate that all three nanorods mediate
the formation of H2O2 from superoxide (Figure 2e).

Figure 1. TEM and corresponding HRTEM images of CeVO4 nanorods of

different sizes: (a-b) 50 nm (CR1); (c-d) 100 nm (CR2); and (e-f) 150 nm
(CR3). Inset of figure a, c and e shows SAED pattern.

oxidation state on the nanomaterial surface (Table S1). The XPS

spectra corresponding to vanadium presents two strong peaks
at 517 and 524.7 eV that can be attributed to V2p 3/2 and V2p1/2
affirming the presence of vanadium in the +5 oxidation state
(Figure S6b,d,f). Furthermore, the O (1s) spectra show peaks at
around 529.9, 531.8 and 533.4 eV correlating to the lattice
oxygen in the tetragonal CeVO4, the oxygen of the hydroxide
ions and a small number of water molecules adsorbed on the
surface of CeVO4, respectively (Figure S6b,d,f). The binding
energy values of all the samples were in agreement with the
previously reported values.[9] The zeta potential values (ζ)
indicating the surface charge on the nanorods were measured to
be 19.7 ± 3.9, 33.3 ± 7.4, 27.6 ± 5.9 mV for CR1, CR2, and CR3,
respectively (Figure S7). To gain insight into the specific surface
area and pore-size distribution of the CeVO4 nanorods of
variable sizes, Brunauer-Emmett-Teller (BET) and Barrett-
Joyner-Halenda (BJH) measurements were performed. The
CeVO4 nanorods of different sizes show a distinct hysteresis
indicating the presence of mesopores on the surface. The BET
surface area of the CR1, CR2 and CR3 nanorods were found to
be 110.1, 157.5 and 149.8 m2 g−1 respectively. The
corresponding pore sizes obtained from BJH analysis for
Figure 2. (a) The SOD-like activity of CeVO4 nanorods, producing H2O2 and
nanorods were 19.5, 16.1 and 13.5 respectively (Figure S8). The oxygen; (b-d) Plots showing the SOD-like activity of CR1, CR2, and CR3,
pore size distribution was uniform and in the range of respectively; (e) The plot indicating formation of H2O2 during the SOD-like
mesopores resulting in higher surface areas, thus providing reaction as determined by ABTS/HRP assay; (f) A comparison of the SOD-like
activity of CR1, CR2 and CR3 with other related metal oxide nanoparticles.
large active sites for the catalytic activity.
The data is represented as mean of s.e.m. n=6.
The ability of CeVO4 nanorods to functionally mimic the SOD
enzyme was studied using a colorimetric assay (Figure 2a).[10a]
Briefly, the superoxide generated by one-electron reduction of A comparison of the activities of the nanorods with that of
oxygen by xanthine and xanthine oxidase can convert the water- other related nanomaterials indicate that the SOD-like activity of
soluble WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo the nanorods (CR1, CR2 and CR3) is much higher than that of
phenyl)-2H-tetrazolium) to spectrophotometrically detectable NiO,[6o] MnO2,[6c] V2O5,[5h] CeO2,[6m] but it is comparable to that of
formazan at 440 nm. Thus, the amount of formazan produced is previously reported CeVO4 nanocubes (Ch) (Figure 2f).[6g]

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RESEARCH ARTICLE
Interestingly, the nanorods (CR1, CR2 and CR3) do not exhibit nanorods do not mediate the oxidation of TMB (3, 3’,5,5’-
any cytochrome c oxidase (CcO) activity, which has been tetramethylbenzidine), indicating the absence of oxidase and
observed earlier for the nanocubes (Ch) (Figure S9a-c).[6g] A peroxidase activities (Figure S14-S15).
detailed analysis of the surface of the rods and cube indicates
To determine the antioxidant potential of CeVO 4 nanozymes
that the presence of mixed Ce4+/Ce3+ ions on the surface of the
in cellular environment, cells of neuroblastoma origin, SH-SY5Y,
nanocube is responsible for its dual SOD and CcO activities and
were utilized. As the cytoprotective effect of the nanomaterials
a specific SOD activity can be obtained by lowering the amount
relies on their cellular uptake and relative toxicity, we evaluated
of Ce4+ on the surface. In contrast, the CcO activity can be
the internalization of CeVO4 nanorods in the cells utilizing
enhanced by increasing the amount of Ce4+ ions on the
inductively coupled plasma optical emission spectroscopy (ICP-
surface.[6g] In agreement with our previous investigation, the
OES). We observed an effective uptake of these nanozymes in
nanorods having relatively less amount of surface Ce4+ exhibit
the SH-SY5Y cells (Figure 3a). The relative uptake at 1h and
much less CcO activity as compared to that of nanocubes (Ch)
24h follows the same order of CR1>CR3>CR2 and the surface
(Figure S9d and Table S1). These observations suggest that a
charge and size of the nanoparticles appears to play key roles in
nanozyme having predominant SOD activity could be obtained
the cellular uptake. It has been reported that an increase in the

Accepted Manuscript
by fine-tuning the Ce3+/Ce4+ ratio on the surface of the
positive zeta potential can enhance the cellular uptake due to a
nanomaterial. This is in agreement with the previous reports that
favorable interaction between the positively charged
the CeO2 nanomaterials can mimic the function of two different
nanomaterial and the anionic plasma cell membrane. [17]
enzymes, SOD or catalase, depending on the Ce 3+/Ce4+ ratio.[11]
However, the lower uptake of CR2 having maximum positive
Furthermore, the dissociation of H2O2 produced during the SOD-
surface charge as compared to that of CR1 and CR3 indicates
like activity may alter the reduction potential of the system,
that there is no clear correlation between the zeta potential and
which can drive the reduction of Ce4+ to Ce3+.[12]
cellular uptake. This discrepancy can be ascribed to the
To confirm the surface activity of the nanozymes and rule adsorption of serum proteins present in the media on the highly
out the possible involvement of leached ions in the SOD-like positive nanoparticle surface. The formation of protein corona is
activity, the rod-shaped nanoparticles were incubated in the known to decrease the interaction of nanoparticles with plasma
buffer solution for 1, 12 and 24h. The supernatant solution cell membrane and subsequently decrease the internalization as
obtained after incubation was used for the determination of observed for CR2.[18]
nanozyme activity. As shown in Figure S10, the leached ions did
To understand the possible toxicity of the nanomaterials, we
not exhibit any significant SOD-like activity, confirming the
studied the cytotoxicity of CR1, CR2 and CR3 at various time
surface reaction of the nanozymes. Further, the morphology and
points by using MTT assay. The cell viability was not affected in
phase of the nanorods remain unaltered after performing the
the presence of nanorods up to 24h, suggesting that the
dismutation reactions, indicating the stability of the materials in
nanozymes are not cytotoxic even at higher concentrations
an oxidizing environment (Figure S11 and S12). The vanadium
(Figure S16). Prior to evaluating the intracellular antioxidant
center in CeVO4 nanozymes appears to provide the required
potential, the effect of CeVO4 nanorods on the basal ROS levels
stability to the nanomaterial and prevents the auto-oxidation of
[13a] was analysed flow cytometrically using the superoxide specific
cerium center in oxidizing conditions. In the cellular
probe, dihydroethidium (DHE) and H2O2 specific dye, Amplex
environment, SOD works in coordination with other antioxidant
enzymes i.e. CAT and GPx to maintain
the basal cellular ROS levels. While CAT
effectively converts H2O2 to water and
oxygen, GPx converts H2O2 to water
using cellular glutathione as a cofactor.
Thus, the CeVO4 nanorods were
assessed for their ability to mimic other
antioxidant enzymes. The CAT and GPx-
like activities were measured
spectrophotometrically at 240 and 340
nm, respectively, using standard assay
methods.[13b-c] However, CR1, CR2 and
CR3 exhibited negligible CAT and GPx-
like activity, indicating their high
specificity towards superoxide radicals
(Figure S13). This is interesting
particularly when CeO2 and V2O5
nanozymes has been shown individually
to exhibit CAT and GPx activities, Figure 3. (a) Cellular uptake of CeVO4 nanorods in SH-SY5Y cells after 1 h and 24 h respectively as
respectively.[14] In addition, as cerium and determined by ICP-OES analysis. The plot is represented as the amount of Ce uptake per million cells
vanadium-based nanoparticles are also with respect to the untreated controls (UT); (b) Protective effect of CeVO4 nanorods in SH-SY5Y cells
upon inhibition of SOD1 by DDC. The cell viability was determined using calcein-AM dye; (c) The SOD-
known to exhibit oxidase and peroxidase
like activity of rod-shaped CeVO4 nanoparticles in the presence DDC as evaluated by flow cytometry
[15]
activities, we used spectroscopic using superoxide specific DHE; (d) FACS analysis of mitochondrial membrane potential utilizing JC-1.
methods to study the CeVO4 nanorods for Data is represented as relative change in folds of superoxide compared to UT. The data is presented as
their ability to mimic these enzymes.[16] mean ± SEM (n=5) and analysed using one-way ANOVA followed by Bonferroni post hoc test, p*/# <
0.05, p**/## < 0.01, p***/### < 0.001; *significant compared to untreated cells; #significant compared to
These experiments revealed that the
DDC alone treated cells.

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RESEARCH ARTICLE
red (Figure S17a-b). The relative fluorescence suggested that
the cells treated with different doses of CeVO4 nanorods did not
alter the basal ROS levels. In addition, the mitochondrial
membrane potential and the cellular GSH levels remain
unaffected upon treatment with the nanorods (Figure S17c-d).
Superoxide radicals are the parent ROS produced in the cellular
environment that mediate a variety of biological responses such
as cell proliferation, differentiation, and migration. [2a,19] They also
cause severe oxidative damage either by direct action or by
acting as progenitor for other deleterious ROS.[1,20a] Thus, we
investigated the ability of the CeVO4 nanorods to control the
enhanced levels of superoxide in the presence of
pharmacological inhibitor of SOD1,
diethyldithiocarbamate (DDC).[20b] The SH-
superoxide levels and mitochondrial membrane potential after
transfection with siSOD1 and siScr at various time points (24 h,
36 h and 48 h). We observed a substantial time-dependent
increase in the superoxide levels (Figure 4b) and decrease in
mitochondrial membrane potential (Figure 4c) in siSOD1
transfected cells. However, no significant cell death was
observed even after 36 h and 48 h of transfection with siSOD1
indicating presence of viable cells having SOD1 depletion
(Figure S20). As there was a significant alteration in the
superoxide and mitochondrial membrane potential in the SOD1
depleted cells after 48 h of transfection, we studied the effect of
CeVO4 nanorods under similar experimental conditions. We

Accepted Manuscript
SY5Y cells were treated with variable
concentration of DDC. The metabolic
activity was evaluated using MTT assay,
the superoxide levels were determined
using DHE dye and the mitochondrial
membrane potential was measured using
JC-1 dye. The flow cytometric analysis
revealed an increase in the superoxide
and decrease in the mitochondrial
membrane potential along with a
substantial cell death within 2 h of DDC
treatment (Figure S18). Interestingly, the
co-treatment of cells with CeVO4 nanorods
efficiently controlled the superoxide levels
(Figure 3c and Figure S19). In addition,
the rod-shaped CeVO4 nanoparticles
effectively restored the cell viability and
mitochondrial membrane potential and
upon inhibition of SOD1 (Figure 3b,d).

To explore the ability of CeVO4

nanorods to fully substitute the role of
SOD1,[21] we down-regulated SOD1 in SH-
SY5Y cells by transfecting the cells with
SOD1-specific siRNA. A scrambled siRNA
(siScr) that does not target any human
gene product was used as a negative
control. The respective immunoblot
analysis indicated a significant depletion
(up to 75%) of SOD1 protein at 36 h and
48 h after transfection (Figure 4a). To
understand whether the gene silencing of
SOD1 affects the mitochondrial Mn-SOD
(SOD2), we tested the relative expression
levels of SOD2 in SH-SY5Y cells. The
immunoblot analysis indicated that the
SOD2 expression was unaffected upon Figure 4. Down-regulation of SOD1 and protection by CeVO4 nanorods. (a) SH-SY5Y cells were
transfection of the cells with SOD1 specific transfected with SOD1 siRNA (siSOD1) or with Scr-siRNA (siScr). After transfection, cell lysates were
collected at various time points (24 h, 36 h and 48 h) and 40 µg of total protein extract was used for
siRNA (Figure 4a). The major sources of
immunoblotting analysis. Specific antibodies against SOD1 and SOD2 were used to evaluate the
superoxide production are mitochondrial expression levels. β-Tubulin was used as a loading control. (b) Flow cytometry analysis of superoxide
electron transport chain (ETC), plasma using DHE (5µM) and (c) Mitochondrial membrane potential using JC-1, after transfection with siSOD1 or
membrane-associated NADPH oxidase siScr at various time intervals. SH-SY5Y cells were transfected with SOD1-siRNA or with Scr-siRNA up
to 48 h for depletion of SOD1 (up to 70%). After 24 h of transfection, 50 ng µL−1 of nanomaterials (CeVO4
complex (NOX) and cytosolic xanthine
nanorods, NiO, MnO2, CeO2 and V2O5) were incubated for another 24 h, followed by treating the cells
oxidase (XO).[1a,22] Further, the depletion of with DHE (5µM), Calcein AM (1µM) and JC-1 (5µM) for (d) superoxide levels, (e) cell viability and (f)
SOD1 results in an increase in the mitochondrial membrane potential respectively. (g) Immunoblot analysis of SOD1 depletion control in the
superoxide that accumulates in the experimental condition. (h) Effect of CeVO4 nanorods (50 ng µL−1) on SOD1 expression under SOD1
depletion condition. β-Tubulin was used as loading control. The data is presented as mean ± SEM (n=5)
mitochondria along with intracellular space,
and analysed using one-way ANOVA followed by Bonferroni post hoc test, p*/# < 0.05, p**/## < 0.01,
leading to superoxide-mediated damages. p***/### < 0.001; *significant compared to untreated cells; #significant compared to siSOD1 alone
Therefore, we evaluated the cell viability, treated cells.

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RESEARCH ARTICLE
observed a significant decrease in the superoxide levels (Figure
4d), improvement of the mitochondrial membrane potential
(Figure 4f) and a complete restoration of cell viability (Figure 4e).
Although some of the previously reported nanozymes such as
CeO2, NiO and MnO2 also exhibited SOD-like activity, the
CeVO4 nanorods (CR1, CR2 and CR3) were more efficient in
bringing the superoxide to the control level (Figure 4d and
Figure S21). In addition, the GPx-mimetic nanozyme, V2O5, had
no effect in restoring the superoxide levels, mitochondrial
membrane potential and cell viability (Figure 4d-f and Figure
S21). Strikingly, at lower concentrations (i.e. 1, 5, 10 and 25 ng
µL-1, CR2 exhibited much lower activity as compared to that of
CR1 and CR3, which can be ascribed to the relatively poor
cellular uptake of CR2 (Figure S22 and S23). Thus, to make a
The SOD1 inefficiency results in oxidative damage to
mitochondria, which is implicated in the pathogenesis of motor
neuropathy.[4d-4f] The depletion of SOD1 is known to significantly
alter the mitochondrial membrane potential. This in turn alters
the pro-survival B-cell lymphoma 2 (Bcl-2) family proteins, which
are the key regulators of apoptotic mitochondrial pathways
(Figure 5a).[24] The Bcl-2 family members contain both anti-
apoptotic (Bcl-2, Bcl-xL, Mcl-1, etc.) and pro-apoptotic pore
forming (Bax, Bak) proteins. The relative affinity and abundance
of these proteins dictates their interactions, which in turn
regulates mitochondrial outer membrane permeabilization.[25]
Thus, Bcl-2 proteins control the mitochondrial potential,
mitochondrial morphology dynamics and the balance between
survival and death in several cell types including neurons.[4b,22]

Accepted Manuscript
reasonable comparison of activities and cytoprotective potential Recent animal studies on familial amyotrophic lateral sclerosis
among the nanorods of variable sizes, we used 50 ng µL-1 in all (FALS) showed that Bcl-2 overexpression delayed onset of
the experiments. At this concentration, all three nanorods motor neuron disease.[26]
displayed similar SOD-activity in the cells although a difference
When we analysed the expression of major Bcl-2 family
in their cellular uptake was observed. Further, the major
proteins containing both anti-apoptotic (Bcl-2, Bcl-xL and Mcl-1)
contribution of the CeVO4 nanorods in the cytoprotection of
SOD1-depleted cells was
confirmed by studying the SOD1
expression in the presence or
absence of nanorods by
immunoblotting. It was found that
the expression of SOD1 remains
unaltered in the presence of
nanozymes (Figure 4h). It is
known that the cells use an
alternate defense line to combat
oxidative stress when one or
more enzymes in the antioxidant
machinery are affected. One such
strategy is to increase the levels
of cellular glutathione (GSH).[23]
As expected, a significant
increase in the cellular GSH
levels was observed in the
SOD1-depleted cells as
compared to that of the control
cells. However, when the
oxidative stress is caused mainly
by superoxide, an increase in the
GSH level alone is not sufficient
to restore the cellular redox
homeostasis. When the SOD-1-
depleted cells were treated with
nanorods, the cellular GSH levels
remained unaffected (Figure S24),
indicating that the nanozymes
substitute the function of SOD1
without affecting the cellular
antioxidant machinery.

The SOD1 present in the
inter-membrane space
mitochondria prevents
collapse of membrane potential
under oxidative stress
conditions.[4b] In neurons, the
presence of SOD1 in the inter-
membrane space is essential for
the maintenance of motor axon.
Figure 5. Down-regulation of pro-survival Bcl-2 proteins induced by SOD1 deficiency and restoration by CeVO4
of nanorods. (a) Schematic representation of pro-survival and pro-apoptotic Bcl-2 family proteins alignment on the
the mitochondrial membrane. (b,c) Expression levels of Bcl-2, Bax, Bak, Mcl-1 and Bcl-xL in SOD1 depleted SH-
SY5Y cells and densitometric analysis after normalization with the respective loading controls. (d) Immunoblots
of Bcl-2 and (e) Bcl-xL expression in SOD1 depleted cells in the presence or absence of CeVO4 nanorods (10-
50 ng μL−1) along with its densitometric analyses after normalization with the respective loading controls. (f)
Schematic representation of effect of SOD1 depletion on ATP levels. (g) Estimation of cellular ATP level after
48h of transfection with siSOD1 in the presence or absence of CeVO4 nanorods using bioluminescence
detection kit. (h) Immunoblot analysis of SOD1 depletion control. The data is presented as mean ± SEM (n=5)
and analysed using one-way ANOVA followed by Bonferroni post hoc test, p*/# < 0.05, p**/## < 0.01, p***/### <
0.001; *significant compared to untreated cells; #significant compared to siSOD1 treated cells.

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Angewandte Chemie International Edition 10.1002/anie.202011711

RESEARCH ARTICLE
and pro-apoptotic (Bax and Bak) proteins in the SOD1 depleted
cells, we observed a significant decrease in the expression
levels of anti-apoptotic proteins i.e. Bcl-2 and Bcl-xL and there
was a slight decrease in the Mcl-1 protein levels. However, the
expression of pro-apoptotic proteins remained unaltered,
suggesting that the depletion of SOD1 primarily affects the pro-
survival Bcl-2 family proteins (Figure 5a-c). The expression
levels of Bcl-2, Bcl-xL and Mcl-1 was restored by the CeVO4
nanorods in a dose-dependent manner (Figure 5d,e and Figure
S25). As the maintenance of appropriate mitochondrial
membrane potential (-135 to -140 mV) is crucial for the ATP
production via F1-F0 ATP synthase,[27] we studied the effect of
nanozymes on mitochondrial dysfunction and ATP production in
SOD1-depleted cells (Figure 5f). The ATP levels in siSOD1-
levels. Crucially, the ATP levels in the presence of CR1 and
CR3 were found to be almost identical to that of the control cells
(Figure S27b). It has been reported that the ATP production is a
highly efficient mechanism and even small changes in ATP
production by mitochondrial deformation can affect the
bioenergetics of the cells.[27d]
To determine the efficiency of the CeVO4 nanorods in
controlling the mitochondrial damage in the absence of SOD1,
the cells were extrinsically treated with ETC complex I inhibitor,
rotenone.[4b,4f,28] The SOD1 depleted cells treated with rotenone
showed a drastic increase in the superoxide levels (Figure 6a),
confirming the importance of mitochondrial SOD1 in maintaining
the amount of superoxide. In addition, we observed a significant
decrease in the mitochondrial membrane potential (Figure 6c)

Accepted Manuscript
transfected cells were measured using luminometric method at
and cell viability (Figure 6b). These results indicate the
various time points. Interestingly, a significant decrease in the
importance of SOD1 in protecting the cells from superoxide-
ATP production was observed after 36 h and 48 h of transfection
mediated mitochondrial damage and suggest that the CeVO4
(Figure S26), and treatment with CR1 and CR3 fully restored the
nanorods can effectively substitute SOD1 not only in controlling
ATP levels (Figure 5g). The nanorods do not show any
the superoxide levels, but also in restoring the mitochondrial
interference with the ATP assay as shown in Figure S28.
membrane potential and cell viability (Figure 6a-c). Further, we
However, CR2 was found be relatively less effective in restoring
observed a significant mitochondrial cardiolipin oxidation (Figure
the levels of ATP under SOD1-depleted conditions, especially at
6d) and increased cytosolic pro-apoptotic Bax level (Figure 6e)
lower concentrations (Figure 5g and Figure S27a). A
in the SOD1-depleted cells treated with rotenone. These results
comparison with the other SOD-mimetic nanozymes indicates
indicate that the cell viability is lost due to mitochondria-
that the CeVO4 is more efficient in restoring the cellular ATP
mediated cell death pathways. Treatment with CeVO4 nanorods
prevented the cardiolipin oxidation and up-
regulated the expression of pro-apoptotic
Bax level. As the depletion of SOD1 affects
the mitochondrial integrity and ATP levels
that can lead to pathological conditions in
neuronal cells, the CeVO4 nanozyme with
SOD-like activity can be considered as a
potential candidate for the treatment of
neurological conditions where the SOD1
activity is severely impaired.

As the ICP-OES data indicated the

internalization of the nanorods in cytosol as
well as mitochondria (Figure S29), it was
thought worthwhile to check whether the
CeVO4 nanorods can functionally mimic the
mitochondria residing SOD2 isoform as well.
We down-regulated the SOD2 by
transfecting the SH-SY5Y cells with SOD2-
specific siRNA for 48 h. This resulted in a
significant increase in the mitochondrial
superoxide generation, decrease in the
mitochondrial membrane potential and
subsequent lowering of the ATP levels
without affecting the cell viability (Figure 7b).
Interestingly, the treatment of the cells with
CeVO4 nanorods decreased the
mitochondrial superoxide levels (Figure 7a
and Figure S30) and increased the
Figure 6. Protective effect of CeVO4 nanorods on rotenone induced toxicity in SOD1 depleted SH-
mitochondrial membrane potential (Figure
SY5Y cells. After 24 h of transfection, siSOD and siScr cells were treated with 0.5 μM rotenone for
another 24 h in the presence or absence of CeVO4 nanorods (50 ng μL−1) and analysed for (a) 7c) with a significant improvement in the
superoxide level using DHE, (b) cell viability using calcein AM, (c) mitochondrial membrane potential ATP levels (Figure 7d). These observations
using JC-1 and (d) mitochondrial cardiolipin peroxidation using 10-Nonyl acridine orange (NAO). (e) suggest that CeVO4 not only mimic the
Immunoblots of pro-apoptotic Bax protein in presence of rotenone (0.5 μM), co-treated with CeVO4
cytosolic SOD1 function, but also enter the
nanorods in SOD-1 depleted SH-SY5Y cells. β-actin was used as loading control. (f) Schematic
representation of rotenone induced mitochondrial dysfunction, (g) Immunoblot analysis of SOD1 mitochondria and substitute the function of
depletion control. The data is presented as mean ± SEM (n=5) and analysed using one-way ANOVA SOD2. Therefore, CeVO4 represents the
followed by Bonferroni post hoc test, p*/# < 0.05, p**/## < 0.01, p***/### < 0.001; *significant first example of a nanozyme that functionally
compared to untreated cells; #significant compared to siSOD1+Rotenone alone treated cells.
mimics both SOD isoforms in mammalian

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Angewandte Chemie International Edition 10.1002/anie.202011711

RESEARCH ARTICLE
cells. The other nanozymes such as CeO2, NiO, MnO2 and V2O5
exhibited significantly lower activity in decreasing the superoxide
levels and improving the ATP concentration. Further, the
restoration of ATP levels by CeVO4 nanozymes upon depletion
of SOD1 is more pronounced as compared to that of SOD2
depletion, corroborating with the higher localization of the
nanozymes in the cytosol as compared to that of mitochondria
(Figure S29 and Figure S31).
biomarker for oxidative stress-induced apoptosis. The CeVO4
nanomaterial represents the first example of a nanozyme that
can improve the ATP levels in neuronal cells even when a key
antioxidant enzyme is down-regulated. The nanozyme reported
here may serve as a good therapeutic candidate for diseases
associated with mitochondrial dysfunction.
Acknowledgements

In summary, we report a CeVO4 nanozyme that can regulate
the ATP levels in neuronal cells under oxidative stress
conditions. The nanozyme catalytically converts superoxide to
hydrogen peroxide and oxygen under physiologically relevant
conditions and can effectively substitute the function of SOD1
and SOD2 enzyme in living cells without affecting the cellular
This study was supported by the Science and Engineering
Research Board (SERB, EMR/IISc-01/2016) and DST
Nanomission (SR/NM/NS-1380/2014), New Delhi. G. M.
acknowledges the DST for the J. C. Bose National Fellowship
(SB/S2/JCB-067/2015). N. S., M. G. and S. K. N. acknowledge

antioxidant machinery. The treatment of SOD1 and SOD2-
depleted cells with the nanozyme prevents the oxidative damage
to mitochondria, restores the mitochondrial integrity, and
improves the levels of pro-survival Bcl-2 family proteins. The
nanozyme also prevents the cardiolipin oxidation, which is a
Accepted Manuscript
the IISc and SERB, respectively, for a research fellowship. The
authors thank Mrs. Mary Sarkar for culturing and maintaining
SH-SY5Y cells, the Bio-imaging Facility, Division of Biological
Sciences, and CeNSE, IISc, for the microscopic studies.
Conflict of Interest

The authors declare no conflict of interest.

Keywords: ATP synthesis • cerium

vanadate • nanozymes • oxidative stress •
superoxide dismutase

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Angewandte Chemie International Edition 10.1002/anie.202011711

RESEARCH ARTICLE
RESEARCH ARTICLE
This paper describes the first
example of a nanozyme that Namrata Singh,
can fully substitute the function Somanathapura K.
of SOD1 and SOD2 in NaveenKumar, Motika
Geethika, and Govindasamy
neuronal cells and regulate the
Mugesh*
synthesis of ATP under
oxidative stress conditions. The Page No. – Page No.
nanozyme prevents the
mitochondrial dysfunction and Cerium vanadate nanozyme
improves the levels of pro- with specific superoxide
survival Bcl-2 family proteins. dismutase activity regulates
mitochondrial function and
ATP synthesis in neuronal

Accepted Manuscript
cells

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