Direct monitoring of the interaction between ROS and cerium dioxide nanoparticles in living cells

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Shcherbakov, E. Vitukova, A. Yegorova, Y. Scripinets, I. Leonenko, A. Baranchikov, V. Antonovich and V.

Ivanov, RSC Adv., 2014, DOI: 10.1039/C4RA08292C.

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A method of direct monitoring of reactive oxygen species (ROS) interaction with nanoceria in

living cells is proposed, based on the use of a calcein-nanoceria complex

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View Article OnlineDOI: 10.1039/C4RA08292C


Direct monitoring of the ROS-nanoceria interaction in living cells

Journal: RSC Advances

Manuscript ID: RA-ART-08-2014-008292

Article Type: Paper

Date Submitted by the Author: 07-Aug-2014

Complete List of Authors: Zholobak, Nadezhda; Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Shcherbakov, Alexander; Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Vitukova, Ekaterina; Bogatsky Physicochemical Institute, National Academy of Sciences of Ukraine, Yegorova, Alla; Bogatsky Physicochemical Institute, National Academy of Sciences of Ukraine,

Scripinets, Yulia; Bogatsky Physicochemical Institute, National Academy of Sciences of Ukraine, Leonenko, Inna; Bogatsky Physicochemical Institute, National Academy of Sciences of Ukraine, Baranchikov, Alexander; Kurnakov Institute of general and inorganic chemistry RAS, Antonovich, Valeriy; Bogatsky Physicochemical Institute, National Academy of Sciences of Ukraine, Ivanov, Vladimir; Kurnakov Institute of general and inorganic chemistry RAS, ; National Research Tomsk State University,

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1

Direct monitoring of the ROS-nanoceria interaction

in living cells

Nadezhda M. Zholobak,†

Alexander B. Shcherbakov,†

Ekaterina O. Vitukova,‡

Alla V. Yegorova,‡

Yulia V. Scripinets,‡

Inna I. Leonenko,‡

Alexander Ye. Baranchikov,§

Valeriy P. Antonovich,‡

and

Vladimir K. Ivanov§,¶*

† Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine,

Kyiv D0368, Ukraine

‡Bogatsky Physicochemical Institute, National Academy of Sciences of Ukraine, Odessa,

Ukraine

§Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences,

Moscow 119991, Russia

¶National Research Tomsk State University, Tomsk 634050, Russia

KEYWORDS. Cerium dioxide, nanoparticles, calcein, luminescence, hydrogen peroxide, latex,

VSV-infection.

ABSTRACT


living cells is proposed, based on the use of a calcein-nanoceria complex. The calcein-nanoceria

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complex easily penetrates cell membranes and is decomposed readily by endogenous or

exogenous ROS, releasing brightly fluorescent calcein, which can be observed using

conventional luminescent microscopy. This complex is less cytotoxic than individual nanoceria

and provides effective protection from oxidative stress induced by either action of hydrogen

peroxide, or latex beads treatment, or infection by the vesicular stomatitis virus.

INTRODUCTION

Cerium is the most abundant of the rare earth elements. Its Clarke value in the Earth’s crust is

comparable or exceeds that of copper, cobalt, tin, and tungsten [1]. A wide range of cerium

compounds’ modern technological applications is largely determined by its unique electronic

structure, allowing switching to be achieved easily between two major oxidation states, namely

Ce3+

and Ce4+

. For instance, cerium dioxide (ceria) serves as a key redox-active component in

industrial and automotive catalysts and solid-oxide fuel cells [2, 3].

In recent years, nanocrystalline ceria has been shown to possess enormous biological activity,

which also originates from outstanding redox activity of this material, as well as its low toxicity,

making in vivo application of cerium dioxide preparations relatively safe [4-10]. Cerium dioxide

nanoparticles (CDN) are able to participate in the redox processes under biologically relevant

conditions, being excellent scavengers of reactive oxygen species (ROS) deleterious to living

cells [5, 8, 9]. CDN are expected to be useful in the therapy of aging-associated diseases

including various types of cancer [11-13], diabetes [4, 14], ischemic stroke [15, 16], Alzheimer's

disease [5, 17] and other neurological oxidative stress diseases [18-20], retinal degenerations

[21-25], among others.


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Hydrogen peroxide (H2O2) is one of the most important ROS in a living cell. In the presence of

some enzymes (catalase, glutathione peroxidase), hydrogen peroxide is decomposed into non-

toxic constituents (water and oxygen). These enzymes are the components of the primary

antioxidant system of the cell’s protection. Recently, nanoceria has been shown to exhibit

excellent catalase-mimetic activity and thus it is able to protect cells against ROS-induced

damage too [4, 26-27]. The mechanism of decomposition of the hydrogen peroxide by nanoceria

is not clear yet. Presumably, several consecutive processes take place when a catalase-like

scheme of nanoceria action is activated. The trivalent cerium ions on the surface of CDN are

oxidized by hydrogen peroxide to Се4+

, while H2O2 is simultaneously bonded to the surface of

CDN, forming cerium perhydroxide [28]; in turn, cerium perhydroxide further decomposes to

release oxygen. Earlier cerium perhydroxide formation was observed during the interaction of

cerium compounds: not only with H2O2, but also with other ROS (e.g. O2-radicals) [29]. Since

hydrogen peroxide is strongly adsorbed on the surface of cerium dioxide nanoparticles, it can

probably displace inorganic or organic ligands (stabilizers, dyes etc.).

Calcein (CLC) is a well-known low-toxic luminescent organic dye, λex/λem ~495 nm/~520 nm

(Figures S1-S3). Fluorescence of calcein is quenched by some transition metal ions (Co2+

, Ni2+

,

Cu2+

etc.). Recently, it was established that rare earth ions also quench calcein’s luminescence at

the physiological pH (~6÷8), the strongest effect being demonstrated by the Ce3+

ion [30]. Since

the calcein’s molecule bears several anchoring carboxylic groups, it can presumably also be

bound to the surface of metal oxides, including cerium dioxide. It is anticipated that the

formation of a surface cerium-calcein complex would also result in quenching calcein’s

luminescence. In turn, during interaction between ceria and stronger ligands, calcein would be

desorbed from the oxide's surface. In this way, cerium dioxide interaction with ROS, e.g.


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hydrogen peroxide, would lead to the release of free calcein, accompanied with regeneration of

its luminescence. In this paper, we have focused our efforts on the demonstration of the

possibility of direct monitoring of ROS-nanoceria interaction in living cells, by detecting the

luminescence of calcein releasing from the calcein-CDN complex. We have also paid attention to

elucidation of antioxidant property of the calcein-CDN complex, as well as its antiviral activity.

MATERIALS AND METHODS

Starting materials. Cerium salts and calcein (Sigma-Aldrich) were used without additional

purification. Non-stabilized and citrate-stabilized cerium dioxide nanoparticles were synthesized

according to previously reported protocols [31, 32]. For additional details, see Supplementary

Information, section “Materials and Methods”.

Optical measurements. Luminescent measurements of calcein (excitation and emission

wavelengths of 485/508 nm, respectively) and cerium(III) ions (excitation and emission

wavelengths of 250/358 nm, respectively) were carried out on a Varian Cary Eclipse

spectrofluorimeter equipped with a xenon lamp (150 V). Spectrophotometric measurements were

carried out using a Shimadzu UV-2401PC spectrophotometer in a 200-900 nm range.

Cells culture. The reference diploid epithelial swine testicular cell line (ST-cells) from the

collection of the Institute of Veterinary Medicine UAAS was used to study cytotoxicity of CDN

and calcein-CDN complex and their protective effect against ROS of different origin. Synthetic

nutrient 199 medium (Biotest Laboratory, Ukraine) supplemented with 5% (v/v) fetal bovine

serum (Sigma, USA) 25 mM HEPES, 10 mM glutamine, 100 units/mL penicillin, and 100

µg/mL streptomycin was used as the growth medium. Cultured cells were kept at 37ºC in a


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humidified 5% CO2 incubator. Once the cells reached confluence, the culture medium was

removed and the cells were rinsed three times with sterile Hank's Balanced Salt Solution (HBSS,

Biotest Laboratory, Ukraine). The confluent cell monolayers were removed using EDTA (Gibco,

USA) and re-suspended in a culture medium. To form cell monolayers, aliquots (0.1 ml) of

suspension containing 5 *105 cells per ml were placed in 96-well Costar plates and incubated at

37oC for 24 h in a TC-80M-2 thermostat in humid air (98%) containing 5% CO2. The cell-

supporting medium consisted of nutrient 199 medium, 1% fetal bovine serum, 25 mM HEPES,

10 mM glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. The cell monolayer

was finally washed with 199 medium, without fetal bovine serum.

The cytotoxicity of the nanoparticles was analyzed using colorimetric MTT (3-(4,5-

dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, C18H16BrN5S) in vitro assay [33]. MTT

(M5655, Sigma-Aldrich) is a classic colorimetric metabolic activity indicator in cell viability

assays. MTT produces a yellowish solution converted to dark blue, water-insoluble MTT

formazan by mitochondrial dehydrogenases of living cells. The reduction of nitroblue

tetrazolium is a sign of the change in the energy potential of the cells and a decrease in the total

activity of mitochondrial dehydrogenases, and thus it is an indicator of the viability of cells in

culture. The test protocol for cytotoxicity evaluation was adopted from [34], taking into account

the reliability of the method [35].

Nanoparticles were suspended in distilled water by consecutive dilution across a 96-wells

plate (100 µL), and transferred to the cell culture media in a ratio of 1/10 (v/v). Nanoparticles

and cells were kept together for 24 h at 37ºC in humid air (98%) containing 5% CO2. Four hours

prior to the end of the exposure period, the culture medium was removed, and MTT solution in

PBS (0.1 mg/ml, 100 µL/well) was added. Upon the completion of the exposure period, the


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supernatant was removed, and lysing solution (DMSO and 0.1% SDS) was added. Plates were

shaken for 5 min, placed in a Thermo/LabSystems Multiskan MS Microplate Reader with a

vertical beam, and the absorbance of the blue crystals of formazan formed was then read

colorimetrically at 540 nm. Each experiment was repeated three times with four replications.

Protective effect of ceria nanoparticles against ROS in vitro. The protective action of

ceria nanoparticles or CDN-calcein complex against ROS in vitro was determined as described

earlier [36]. Briefly, a CDN-calcein complex or ceria sols of different concentrations (10 µM – 2

mM) were added to the cell monolayer 24 h before the hydrogen peroxide treatment. 4 h after the

application of hydrogen peroxide (the final concentration of H2O2 in each well was 0.5 mM), the

number of survived cells was determined by measuring the absorbance of cells stained with

crystal violet. After staining, the excess dye was removed and the stained monolayer was washed

with distilled water and dried. Absorbance of stained cells was measured at 540 nm using a

Thermo/LabSystems Multiskan MS Microplate Reader.

The percentage of the cells absorbing crystal violet was determined according to the

following formula:

(Atest / Acontr)*100, (1)

Here, Atest is the optical density of the test cells; Acontr is the optical density of the intact (control)

cells.

Statistical treatment of data obtained was performed using BioStat 2009 Professional

5.8.1 software in accordance with standard recommendations. Data obtained from control cells

were taken as possessing 100% viability. Experimental data were presented as the median and


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the interquartile range Me (LQ-UQ), where Me – median (50% percentile), LQ – 25% percentile,

UQ – 75% percentile. In the entire series, the number of experiments conducted was five.

Protective effect of nanoparticles against virus infections in vitro.

The protective effect of cerium dioxide sols against vesicular stomatitis virus (VSV;

Rhabdoviridae family, Vesiculovirus genus serotype Indiana) infections was studied using the

same ST cell line. Ceria sols of different concentrations (16–2000 µM) were added to cell

monolayers according to the above-described protocol, 24 h before VSV-infection. At the end of

the exposure period, the culture medium was removed, and the cells were infected with VSV at a

concentration of 100 TCID50/ml. 40 minutes later, the unabsorbed virus was removed by

washing with 199 medium. A supporting medium (0.1 mL/well) was then added, and the cells

were incubated at 37oC for 24 h. The viability of cells was estimated 24 h after infection by

VSV.

For control intact cells (positive control) the cell monolayer remained intact (0% of cytopathic

effect, CPE), while for control cells infected by VSV (negative control) the cell monolayer was

completely destroyed (100% CPE).

The percentage of viable cells was determined according to the following formula:

(Atest – Avsv)/(Acontr – Avsv)*100 (2)

Here, Acontr is the optical density of intact cells stained with crystal violet; Avsv is the optical

density of VSV-infected cells stained with crystal violet; Atest is the optical density of stained

cells treated with nanoparticles and VSV. Statistical treatment of data obtained was performed as

described above.


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Microscopic visualization of the ROS-nanoceria interaction in living cells.

Microscopic visualization of the ROS-nanoceria interaction in living cells was performed using a

LOMO Mikmed-2 optical microscope equipped with a Canon Digital IXUS 9515 camera. The

cells treated with calcein or CDN-calcein complex were investigated under UV illumination

using a НВО-100W/2 mercury lamp with a “Green-2” filter as a light source (excitation and

emission wavelengths of 450-480/520-560 nm, respectively).

RESULTS AND DISCUSSION

500 525 550 575 600 625 650

0

100

200

300

400

500

600

700

1*10-3 M CDN

0 M CDN

Wavelength, nm

I, a

.u.

0 2 4 6 8 10

0

100

200

300

400

500

600

700

CCDN

, x10-4 M

I, a

.u.

Figure 1. Luminescence spectra of calcein (СCLC= 1×10-6

M, рН 7.2) in the presence of various

concentrations of a CDN sol.


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Calcein luminescence is strongly quenched by both cerium ions (Figures S4, S5) and cerium

dioxide nanoparticles (Figures 1, S8). When quenching is induced by calcein interaction with

Ce3+

ions, the intensity of luminescence is inversely proportional to the cerium concentration. In

turn, luminescence quenching by CeO2 nanoparticles proceeds through calcein interaction with

surface-located cerium ions only. For CDN nanoparticles of ~6 nm size, total quenching of

calcein luminescence is achieved when the molar concentrations ratio of calcein and cerium is

approx. 1:50...100 (Figures S6, S7).

Excitation of CDN by UV-irradiation at 250 nm (which corresponds to the characteristic

absorption band for cerium ions (III)) does not lead to emission at 358 nm, which is

characteristic for cerium(III) ions luminescence (Figure. S9). We assume that suppression of the

calcein luminescence by CDN is mainly caused by cerium(IV) ions, which are prevailing in the

cerium dioxide nanoparticles lattice. However, as we have demonstrated, both cerium(III) and

cerium(IV) ions were proved to be quenchers of the calcein luminescence (Figure 2), and the

efficiency of calcein luminescence quenching by cerium ions only slightly depends on the

oxidation state of cerium. Thus we can conclude that quenching of calcein luminescence by ceria

nanoparticles would not actually depend on their oxidation state, too.

Hydrogen peroxide slightly decreases the luminescence intensity of pure calcein (Figure S10).

The injection of hydrogen peroxide into the solution of non-luminescent cerium(IV)-calcein

complex leads to calcein release and restoration of its luminescence (Figure 3). When hydrogen

peroxide reacts with a cerium(III)-calcein complex, calcein luminescence is not restored but is

even additionally quenched (Figures S12, S13). The most probable reason of this effect is that

calcein is decomposed by peroxide radicals, which are formed by Fenton’s reaction in the


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presence of cerium(III) ions and hydrogen peroxide. Since during Fenton’s reaction the oxidation

of cerium(III) takes place, the intensity of its luminescence falls (Figures S14, S15):

Ce3+

+ H2O2 → Ce4+

+ HO• + OH

- (3a)

Calcein + HO•→ oxidized Calcein (non-fluorescent) (3b)

Figure 2. Luminescence spectra of CLC solution (1) and the same solution upon introduction of

cerium species (2 – Ce(III) ions; 3 – Ce(IV) ions; 4 – CDN) (СCLC= 1×10-6

M; СCDN= 5×10-4

M;

СCe(III) = 1×10-4

M; СCe(IV) = 5×10-5

M; рН=7.2).


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Figure 3. Top panel: Luminescence spectra of CDN-calcein complex solutions in the presence of

H2O2 in various concentrations (Chydrogen peroxide = 5×10-5

M – 5×10-3

M; СCLC = 1×10-6

M; СCDN =

5×10-4

M; рН = 7.2). Bottom panel: Images taken under visible-light illumination (A, B) and

UV-illumination (A1, B1): 1, 2 – Samples of a CDN-calcein complex prepared from non-

stabilized and citrate-stabilized ceria aqueous sols synthesized according to protocols [31] and

[32], respectively; 3 – pure calcein (control). A, A1 – before H2O2 injection; B, B1 – after H2O2

injection.


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Stability of the CDN-calcein complex is high enough; taking into account that both Ce3+

and

Ce4+

ions interact with calcein, it should not depend on the effective oxidation state of cerium in

CDN. Our data indicate that the CDN-calcein complex is stable in the presence of a number of

extraneous ions (nitrates, sulphates, chlorides, carbonates, etc. – except for phosphates) and is

destroyed upon chemisorption of Н2О2 only. Since cerium dioxide nanoparticles have been

shown to be excellent scavengers of ROS (including H2O2) [4-10, 26, 27], we can suppose that

the CDN-calcein complex could also interact with H2O2 in a living cell, and this interaction

would presumably result in the release of free calcein and the appearance of calcein-specific

luminescence (see Figure 4). Thus, treatment of cell culture with a non-luminescent CDN-calcein

complex would allow observation of the process of hydrogen peroxide formation and

inactivation by ceria in vitro.

Figure 4. A non-fluorescent CDN-calcein complex interacts with ROS (hydrogen peroxide) in a

living cell with release of the fluorescent dye.


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Figure 5. Top panel: Toxicity of CDN and CDN-calcein complex to ST-cells upon 24-hour

exposure, as determined by MTT assay. Bottom panel: Protection of ST-cells against oxidative

stress caused by introduction of 0.5 mM of hydrogen peroxide.

Legend: control cells – intact ST-cells, control peroxide – ST-cells treated with H2O2 only.

1 – cells treated with H2O2 and non-stabilized nanoceria sols synthesized according to protocol

[31], 1a – cells treated with H2O2 and a CDN-calcein complex prepared from non-stabilized


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nanoceria sol synthesized according to protocol [31], 2 – cells treated with H2O2 and a citrate-

stabilized nanoceria sol, synthesized according to protocol [32], 2a – cells treated with H2O2 and

a CDN-calcein complex prepared from a citrate-stabilized nanoceria sol, synthesized according

to protocol [32].

Abscissa axis – cerium dioxide concentration, mM, ordinate axis – cells viability, %% to the

control cells.

It is worth noting that water-soluble cerium(III) and cerium(IV) compounds cannot be used by

themselves for the latter purpose because they are quite toxic (Ce(III) ions can participate in

Fenton reaction forming ROS, and Ce(IV) ions can cause oxidative stress by themselves). On the

contrary, because of the high antioxidant activity of CDN, a CDN-calcein complex could even

act as an ROS scavenger, protecting cells from oxidative stress caused by hydrogen peroxide,

and our experimental data prove this supposition (Figure 5).

According to our data, both CDN and CDN-calcein complexes actually have low toxicity to the

cell culture; the influence of calcein is practically negligible. In turn, the protection rates of ceria

sols against oxidative stress caused by hydrogen peroxide are different: citrate-stabilized CeO2

nanoparticles protect cells effectively in the 0.062–1.0 mM concentration range (see Figure 5),

while non-stabilized ceria nanoparticles even increase the H2O2 toxicity in high concentrations.

However, addition of calcein into ceria sols eliminates this difference, so both preparations of the

CDN-calcein complex protect cells in the concentration range 0.062–1.0 mM (see Figure 5.)

Highly polar hydrophilic calcein molecules cannot penetrate cell membranes. When cell culture

is treated with pure calcein the fluorescence is observed in intercellular space only, but not inside

the cells (see Figure 6, 1a-c). Washing of the cells with a buffer solution eliminates dye from the


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cells monolayer and the luminescence disappears. CDN-calcein complex is more hydrophobic

than pure calcein, so it quickly (in 3–5 min) penetrates into living cells. Since CDN-calcein

complex is not fluorescent, the UV-excited cell monolayer is dark both before (see Figure 6, 2a-

c) and after (see Figure 6, 3a-c) washing with buffer solution. Treatment by Н2О2 leads to the

destroying of non-fluorescent CDN-calcein complex, and the bright luminescence in a cytoplasm

appears (see Figures 4 and 6, 4a-c).

Figure 6. Optical and luminescent micrographs of ST-cells (a – bright-field images, b –

luminescent images, c – merged). 1 – Cells treated with calcein solution (without further

washing). The intercellular space is bright since calcein doesn’t penetrate into cells. 2 – Cells


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treated with CDN-calcein complex (without further washing). A weak luminescence is observed

in the intercellular space, cell bodies are dark. 3 – Cells treated with CDN-calcein complex with

further washing with buffer solution. The excess of the CDN-calcein complex has been removed,

the luminescence is absent. 4 – Cells treated with CDN-calcein complex with further washing

and treatment with excess of Н2О2. Under oxidative stress conditions, bright luminescence is

observed in the cytoplasm.

Presumably, endogenous ROS can also cause a break-up of the CDN-calcein complex and thus

can be also monitored in the cells culture. To prove this hypothesis, we have tried to use one

well-known method to form oxidative stress in the living cells, namely their treatment by latex

beads, resulting in oxygen radical production [37, 38]. According to our data, latex particles

(dark agglomerates on Figure 7, 1a) do cause luminescence of ST-cells treated with a CDN-

calcein complex (Figure 7, 1b).

Viral infections can also cause oxidative stress, playing the key role in damaging infected cells

and surrounding tissues [39]. One good example of such an infection is vesicular stomatitis virus

(VSV) infection, which is an acute viral disease of a wide range of mammals, including equine,

cattle and swine. VSV infection of humans usually either is asymptomatic or causes a mild

influenza-like illness [40]. Earlier, we have reported that CDN can significantly reduce cells’

mortality caused by viruses [41]. Our present results indicate that treatment of cell culture by

CDN-calcein complex not only increases cells’ viability (Figure 8) but also allows the

monitoring of the development of the VSV infection process in vitro (Figure 7).


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Figure 7. Visualization of the ROS-nanoceria interaction in the living cells. Micrographs of ST-

cells treated with CDN-calcein complex and further treated by latex beads (1) or VSV (2, 3). 1 –

cells treated with latex beads, 2 mg/ml; 2 – VSV-infected ST-cells 12 h after infection; calcein is

distributed all over the cell, while in the nucleus its concentration is higher; 3 – VSV-infected

ST-cells 18 h after infection; calcein accumulates mainly in the nucleus, calcein luminescence in

the nucleus is higher. a – bright-field image, b – calcein luminescence image, c – merged

(overlay image).


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Figure 8. Antiviral effect of the CDN-calcein complex. Control cell – intact culture, control

virus – cells treated with VSV, 1 – cells treated with a CDN-calcein complex prepared from non-

stabilized ceria nanoparticles synthesized according to protocol [31] and VSV, 2 – cells treated

with a CDN-calcein complex prepared from citrate-stabilized ceria nanoparticles synthesized

according to protocol [32] and VSV. Abscissa axis – cerium dioxide concentration, mM, ordinate

axis – cells viability, %% to the control cells.

Interestingly, upon H2O2-induced oxidative stress conditions, CDN-calcein preparations

synthesized from non-stabilized [31] and citrate-stabilized [32] ceria sols demonstrate the same

antioxidant activity, while their antiviral action is rather different. A CDN-calcein complex

prepared from citrate-stabilized ceria nanoparticles is notably more active than its counterpart

prepared from non-stabilized ceria, and provides effective protection of the cells in a wider range


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of concentrations (see Figure 8). In our opinion, such a difference is presumably due to different

colloidal stability of surface-modified ceria nanoparticles in the biological environment.

CONCLUSIONS

The technique of oxidative stress visualization proposed here is based on the ability of ceria

nanoparticles to interact with calcein molecules, forming a non-fluorescent complex, simplifying

the dye penetration into the cell. Under oxidative stress conditions, the CDN-calcein complex is

decomposed and free calcein is released, allowing the visualizing of ROS in the cytosol. Our

data indicate that CDN-calcein complex can be used for visualization of either ROS directly

introduced in the cell (e.g., hydrogen peroxide) or ROS generated by the action of external

factors (e.g., latex beads treatment or viral infection). This technique allows the monitoring of

the ROS formation at different stages of oxidative stress. For instance, when oxidative stress is

induced by VSV infection, the luminescence of calcein is initially observed in the entire cell. At

the final stages of the viral process the dye is accumulated in the nucleus of the infected cell. It is

worth noting that the technique proposed has allowed us to monitor cerium dioxide

nanoparticles’ uptake by the cells. We have demonstrated that this is quite a fast process, taking

only ca. 5 minutes.

ACKNOWLEDGMENTS

This work was partly supported by the Russian Scientific Foundation (data concerning

bioactivity of nano-ceria were obtained under RSF project 14-13-01373).


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Luminescence spectra of calcein (СCLC= 1×10-6 M, рН 7.2) in the presence of various concentrations of a

CDN sol.

62x24mm (600 x 600 DPI)


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Luminescence spectra of CLC solution (1) and the same solution upon introduction of cerium species (2 –

Ce(III) ions; 3 – Ce(IV) ions; 4 – CDN) (СCLC= 1×10-6 M; СCDN= 5×10-4 M; СCe(III) = 1×10-4 M; СCe(IV) =

5×10-5 M; рН=7.2).

78x57mm (600 x 600 DPI)


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Top panel: Luminescence spectra of CDN-calcein complex solutions in the presence of H2O2 in various concentrations (Chydrogen peroxide = 5×10-5 M – 5×10-3 M; СCLC = 1×10-6 M; СCDN = 5×10-4 M; рН = 7.2).

Bottom panel: Images taken under visible-light illumination (A, B) and UV-illumination (A1, B1): 1, 2 –

Samples of a CDN-calcein complex prepared from non-stabilized and citrate-stabilized ceria aqueous sols synthesized according to protocols [31] and [32], respectively; 3 – pure calcein (control). A, A1 – before

H2O2 injection; B, B1 – after H2O2 injection. 132x183mm (600 x 600 DPI)


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A non-fluorescent CDN-calcein complex interacts with ROS (hydrogen peroxide) in a living cell with release of the fluorescent dye.

118x56mm (300 x 300 DPI)


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Top panel: Toxicity of CDN and CDN-calcein complex to ST-cells upon 24-hour exposure, as determined by MTT assay. Bottom panel: Protection of ST-cells against oxidative stress caused by introduction of 0.5 mM of

hydrogen peroxide.

Legend: control cells – intact ST-cells, control peroxide – ST-cells treated with H2O2 only. 1 – cells treated with H2O2 and non-stabilized nanoceria sols synthesized according to protocol [31], 1a – cells treated with H2O2 and a CDN-calcein complex prepared from non-stabilized nanoceria sol synthesized according to protocol [31], 2 – cells treated with H2O2 and a citrate-stabilized nanoceria sol, synthesized

according to protocol [32], 2a – cells treated with H2O2 and a CDN-calcein complex prepared from a citrate-stabilized nanoceria sol, synthesized according to protocol [32].

Abscissa axis – cerium dioxide concentration, mM, ordinate axis – cells viability, %% to the control cells. 165x186mm (600 x 600 DPI)


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Optical and luminescent micrographs of ST-cells (a – bright-field images, b –luminescent images, c – merged). 1 – Cells treated with calcein solution (without further washing). The intercellular space is bright

since calcein doesn’t penetrate into cells. 2 – Cells treated with CDN-calcein complex (without further

washing). A weak luminescence is observed in the intercellular space, cell bodies are dark. 3 – Cells treated with CDN-calcein complex with further washing with buffer solution. The excess of the CDN-calcein complex

has been removed, the luminescence is absent. 4 – Cells treated with CDN-calcein complex with further washing and treatment with excess of Н2О2. Under oxidative stress conditions, bright luminescence is

observed in the cytoplasm. 299x313mm (72 x 72 DPI)


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Visualization of the ROS-nanoceria interaction in the living cells. Micrographs of ST-cells treated with CDN-calcein complex and further treated by latex beads (1) or VSV (2, 3). 1 – cells treated with latex beads, 2 mg/ml; 2 – VSV-infected ST-cells 12 h after infection; calcein is distributed all over the cell, while in the

nucleus its concentration is higher; 3 – VSV-infected ST-cells 18 h after infection; calcein accumulates mainly in the nucleus, calcein luminescence in the nucleus is higher. a – bright-field image, b – calcein

luminescence image, c – merged (overlay image). 400x307mm (72 x 72 DPI)


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Antiviral effect of the CDN-calcein complex. Control cell – intact culture, control virus – cells treated with VSV, 1 – cells treated with a CDN-calcein complex prepared from non-stabilized ceria nanoparticles

synthesized according to protocol [31] and VSV, 2 – cells treated with a CDN-calcein complex prepared from citrate-stabilized ceria nanoparticles synthesized according to protocol [32] and VSV. Abscissa axis – cerium

dioxide concentration, mM, ordinate axis – cells viability, %% to the control cells. 114x72mm (600 x 600 DPI)


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living cells is proposed, based on the use of a calcein-nanoceria complex


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Supporting Information

Direct monitoring of ROS-nanoceria interaction in

living cells

Nadezhda M. Zholobak,† Alexander B. Shcherbakov,

† Ekaterina O. Vitukova,

‡ Alla V. Yegorova,

‡

Yulia V. Scripinets,‡ Inna I. Leonenko,

‡ Alexander Ye. Baranchikov,

§ Valeriy P. Antonovich,

‡ and

Vladimir K. Ivanov§,¶*

† Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine,

Kyiv D0368, Ukraine

‡Bogatsky Physicochemical Institute, National Academy of Sciences of Ukraine, Odessa,

Ukraine

§Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences,

Moscow 119991, Russia

¶National Research Tomsk State University, Tomsk 634050, Russia

Materials and methods

The following solutions were used as starting materials: 0.01 M СeCl3 solution in water, 0.01 M

Сe(SO4)2 solution in 0,5 M H2SO4, 0.01 M non-stabilized aqueous ceria sol synthesized

according to previously reported protocol [1], 0.001 M calcein solution in water-DMSO (9:1)

mixture.

Luminescent measurements were carried out on a Varian Cary Eclipse spectrofluorimeter

equipped with a xenon lamp (150 V). Spectrophotometric measurements were performed on a

Shimadzu UV-2401PC spectrophotometer. рН value of the media was adjusted to 7.2, using a

Tris-buffer solution (Sigma-Aldrich).


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UV-vis spectroscopy of starting solutions

UV-vis absorption spectra of calcein (CLC), cerium(III) chloride, cerium(IV) sulfate) aqueous

solutions and CDN aqueous sol are shown in Figure S1.

Calcein is a fluorescent dye with excitation and emission wavelengths of 495/515 nm,

respectively (see Figure S2).

Figure S1. UV-Vis absorption spectra: 1 - 1×10-5

M calcein, 2 - 1×10-4

M СeCl3, 3 - 1×10-4

M

Сe(SO4)2, 4 – 1×10-4

M non-stabilized CDN sol.


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200 250 300 350 400 450 500 550 600 6500

100

200

300

400

500

600

2

Wavelength, nm

I, a

rb.u

nits

1

Figure S2. Calcein molecule and its excitation (1) and emission (2) spectra (СCLC= 1×10-5

M).

Preparations of calcein calibrating solutions

In a set of 10.0 ml calibrated flasks 0.1, 0.3, 0.5, 0.7, 1.0, 2.0, or 5.0 ml of calcein solution (СCLC

= 1×10-5

M) and 1.0 ml of the Tris-buffer solution (рН=7.2) were added. Flasks were filled up to

a level with distilled water, solutions were mixed for 5-10 minutes, and the intensity of

luminescence of calcein solutions at λemiss = 508 nm (λexcit = 485 nm) was measured. Data

obtained were used for the calibration curve preparation (see Figure S3).


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Figure S3. Luminescent spectra of CLC (above) and corresponding calibration curve (below),

pH=7.2.

In the presence of cerium(III) ions, cerium(IV) ions, or CDN sol, the suppression of intensity of

calcein luminescence was observed (see the following sections).


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Preparation of solutions for study of the influence of cerium(III) ions on the luminescence

of calcein

In a set of 10.0 ml calibrated flasks 1.0 ml of calcein solution (СCLC = 1×10-5

M), 1.0 ml of the

Tris-buffer solution (рН=7.2) and 0.1, 0.3, 0.5, 0.7, 1.0 or 5.0 ml of cerium chloride solution

(СCe(III)= 1×10-3

M) were added. Flasks were filled up to a level with distilled water, solutions

were mixed for 5-10 minutes, and the intensity of luminescence of calcein solutions at λemiss =

508 nm (λexcit = 485 nm) was measured (see Figure S4).

Figure S4. Intensity of calcein solutions luminescence (СCLC = 1×10-6

M, рН=7.2) in the

presence of various concentrations of cerium(III) ions.


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Preparation of solutions for study of the influence of cerium(IV) ions on the luminescence

of calcein

In a set of 10.0 ml calibrated flasks, 1.0 ml of calcein solution (СCLC = 1×10-5

M), 1.0 ml of the

Tris-buffer solution (рН=7.2) and 0.0, 0.5 or 1.0 ml of 1×10-5

M cerium (IV) sulphate solution or

0.05, 0.1, 0.3 or 0.5 ml of 1×10-3

M cerium (IV) sulphate solution were added. Flasks were filled

up to a level with distilled water, solutions were mixed for 5-10 minutes, and the intensity of

luminescence of calcein solutions at λemiss = 508 nm (λexcit = 485 nm) was measured (see Figure

S5).

Figure S5. Intensity of the calcein solutions (СCLC = 1×10-6

M, рН=7.2) luminescence in the

presence of various concentration of cerium (IV) ions.


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Grafting the density of calcein on the surface of CDN

When calcein is in planar orientation on the CDN surface, it occupies a site containing ca. 20-25

atoms of cerium (see Figure S6).

Figure S6. Top – Cerium dioxide nanocluster with a grafted calcein molecule (covalent radii).

Bottom – the same structure plotted using van der Waals radii.


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Figure S7. The calculated number of surface and bulk cerium atoms in CeO2 nanoparticles, as a

function of the particle size.

The quantity of atoms on the surface and in the bulk of the ceria nanoparticle depends on its size,

as shown in Figure S7 (data for calculations taken from [2]). For the particle of ~6 nm size there

are approximately (500/1200 *100) ≈ 40% of cerium atoms on the surface. For the particle of ~8

nm size there are approximately (850/2600 *100) ≈ 30% of cerium atoms on the surface. In case

of dense packing, all the calcein molecules are bound to the CeO2 surface when the molar ratio

of calcein to cerium is 1: (60…80). Taking into account that the surface of the particles is non-

ideal, this molar ratio can be estimated as 1:50...100.

Preparation of solutions for study of the influence of cerium dioxide nanoparticles on the

luminescence of calcein

In a set of 10.0 ml calibrated flasks, 1.0 ml calcein solution (СCLC= 1×10-5

M), 1.0 ml of the Tris-

buffer solution pH=7.2 and 0.0 or 0.5 ml of 1×10-5

M CDN sol, or 0.05, 0.1, 0.3, 0.5, 0.7, or 1.0

ml of 1×10-3

M of CDN sol were added. Flasks were filled up to a level with distilled water,

solutions were mixed for 5-10 minutes, and the intensity of luminescence of calcein solutions at

λemiss = 508 nm (λexcit = 485 nm) was measured (see Figure S8).

Results obtained indicate clearly that CDN also quenches the luminescence of calcein. In Stern-

Folmer coordinates, the linear dependence between the intensity of the luminescence (I0/I) and


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the concentration of cerium is observed in a narrow interval of concentrations (5·10-7

- 1·10-4

M)

(see Figure S8).

0 2 4 6 8 100

5

10

15

20

CCDN

, 10-4 M

I 0/I

Figure S8. Dependence of the intensity of calcein luminescence on CDN concentration.

Luminescence of Ce (III) solutions

300 350 400 4500

200

400

600

800

1000

4,5,6

3

2

I, a

rb.u

nit

s

Wavelength, nm

1

Figure S9. Luminescence spectra of cerium(III) solutions (1 - СCe(III) = 1×10-4

M; 2 - СCe(III) =

1×10-5

M; 3 - СCe(III) = 1×10-6

M) and solutions of CDN (4 - СCDN = 1×10-4

M; 5 – СCDN = 1×10-5

M, 6 – СCDN = 1×10-6

M). Excitation and emission wavelengths are 250/358 nm, respectively.


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Influence of hydrogen peroxide (Н2О2) on the intensity of luminescence of calcein and

complexes of calcein with CDN, cerium(III) ions, or cerium(IV) ions.

Hydrogen peroxide, in a wide range of concentrations (5×10-7

– 1×10-3

M), causes only

insignificant suppression of the luminescence of the calcein (not more than ~20 %) (Figure S10).

Figure S10. Luminescence spectra of CLC solutions (СCLC = 1×10-6

M; рН 7.2) in the presence

of various concentrations of Н2О2.

Preparation of solutions for study of the influence of hydrogen peroxide on calcein

luminescence in the presence of cerium(IV) and Ce(III) ions

In a set of 10.0 ml calibrated flasks, 1.0 ml of calcein solution (СCLC = 1×10-5

M), 1.0 ml of the

Tris-buffer solution (рН=7.2), 0.5 ml of cerium(IV) solution (СCe(IV)= 1×10-3

M) and 0.01, 0.1 or

1.0 ml of 1×10-3

M H2O2 solution, or 0.5, 1.0, 2.0 or 5.0 ml of 1×10-2

M H2O2 solution were

added. Flasks were filled up to a level with distilled water, solutions were mixed for 5-10

minutes, and the intensity of luminescence of calcein solutions at λemiss = 508 nm (λexcit = 485


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nm) was measured. The same procedure was used for the preparation of Ce (III)-containing

solutions.

In the Ce(IV)-H2O2-calcein system, upon increase of hydrogen peroxide concentration from

1×10-6

to 5×10-3

M, the luminescence intensity increases in ca. 10 times. However, this

dependence is non-linear (see Figure S11).

Figure S11. Luminescence spectra of CLC solutions in the presence of cerium (IV) ions and

various concentrations of hydrogen peroxide (1×10-6

- 5×10-3

). СCLC = 1×10-6

M; СCe(IV) = 5×10-5

M; рН 7.2.


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Introduction of hydrogen peroxide into a solution of CLC-Ce (III) complex gives a completely

different result, namely, additional quenching of the calcein’s luminescence (Fig. S12, S13).

Figure S12. Luminescence spectra of calcein in the presence of cerium(III) ions and various

concentrations of hydrogen peroxide (1×10-6

- 5×10-3

). СCLC = 1×10-6

M; СCe(III) = 1×10-4

M;

рН=7.2.

0,0 0 ,5 1,0 1,5 2 ,0

0

50

100

150

200

250

300

350

400

CH2 O2

, *10-3

M

I, a

rb.u

nit

s

Figure S13. Dependence of the intensity of calcein luminescence in the presence of Ce(III), upon

increase in hydrogen peroxide concentration from 1×10-6

M to 2×10-3

M.


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The luminescence of cerium(III) ions in the presence of calcein is quenched, and introduction of

hydrogen peroxide leads to additional suppression of the Ce(III) luminescence (Fig. S15, S16).

Figure S14. Luminescence spectra of cerium(III) ions in the presence of calcein and various

concentrations of hydrogen peroxide (1×10-6

- 5×10-3

). СCLC = 1×10-6

M; СCe(III) = 1×10-4

M;

рН=7.2; λех = 250 nm.


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0,0 0,2 0,4 0,6 0,8 1,00,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

5,0

I 0/I

CH2O2

, x10-3

M

Figure S15. Intensity of cerium(III) ions luminescence in the presence of calcein and various

hydrogen peroxide concentrations (1×10-6

- 2×10-3

) in Stern-Folmer coordinates. СCLC = 1×10-6

M; СCe(III) = 1×10-4

M; рН=7.2; λех = 250 nm.

It is well known that the intensity of Ce(III) luminescence decreases upon complexation.

Symbate Ce(III) ions luminescence quenching by CLC and H2O2 could be possibly explained by

the formation of calcein and peroxide complexes. However, it is more likely that calcein is

decomposed by peroxide radicals, which are formed at Fenton’s reaction in the presence of

cerium (III) ions and hydrogen peroxide [3], similarly to fluorescein decomposition [4].

Ce3+

+ H2O2 → Ce4+

+ HO• + OH

–

Calceine + HO• → oxidized Calceine (non-fluorescent)


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1. V.K. Ivanov, O.S. Polezhaeva, A.B. Shcherbakov and Y.D. Tret’yakov, Russian Journal

of Inorganic Chemistry, 2010, 55, 1.

2. C. Sun and D. Xue, Physical Chemistry Chemical Physics, 2013, 15, 14414.

3. E. G. Heckert, S. Seal and W. T. Self, Environmental science & technology, 2008, 42,

5014.

4. B. Ou, M. Hampsch-Woodill, J. Flanagan, E. K. Deemer, R. L. Prior and D. Huang,

Journal of Agricultural and Food Chemistry, 2002, 50, 2772.


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Direct monitoring of the interaction between ROS and cerium dioxide nanoparticles in living cells

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Transcript of Direct monitoring of the interaction between ROS and cerium dioxide nanoparticles in living cells