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

Abstract

A method for the direct monitoring of the interaction of reactive oxygen species (ROS) with cerium dioxide nanoparticles (CDN) in living cells is proposed based on the use of a complex of calcein and CDN. The CDN–calcein complex penetrates easily into a living cell and is decomposed readily by endogenous or exogenous ROS, releasing brightly fluorescent calcein, which can be observed using conventional fluorescent microscopy. This complex is less cytotoxic than individual cerium dioxide nanoparticles and provides effective protection from oxidative stress induced by the action of hydrogen peroxide, treatment with latex beads, or infection by the vesicular stomatitis virus.

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Introduction

Cerium is the most abundant of the rare earth elements. Its Clarke value in the Earth's crust is comparable to or exceeds that of copper, cobalt, tin, and tungsten.¹ The wide range of cerium compounds' modern technological applications is largely determined by its unique electronic structure, allowing switching to be achieved easily between its two major oxidation states, namely Ce³⁺ and Ce⁴⁺. For instance, cerium dioxide serves as a key redox-active component in industrial and automotive catalysts and solid-oxide fuel cells.^2,3

In recent years, cerium dioxide nanoparticles (CDNs) have been shown to possess considerable biological activity, which also originates from the high level of redox activity of this material, as well as its low toxicity, making in vivo application of cerium dioxide preparations relatively safe.^4–12 CDNs are able to participate in the redox processes under biologically relevant conditions since they are excellent scavengers of reactive oxygen species (ROS) deleterious to living cells.^5,8,9,13,14 CDNs are expected to be useful in the therapy of aging-associated diseases including various types of cancer,^15–17 diabetes,^4,18 ischemic stroke,^19,20 Alzheimer's disease^5,21 and other neurological oxidative stress diseases,^22–24 and retinal degenerations,^25–29 among others.

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 cell's protective primary antioxidant system. Recently, CDNs have been shown to exhibit excellent catalase-mimetic activity and are thus able to protect cells against ROS-induced damage as well.^4,30,31 The mechanism of decomposition of hydrogen peroxide by CDNs is not clear yet; presumably, several consecutive processes take place when a catalase-like scheme of CDN action is activated. Trivalent cerium ions are oxidized by hydrogen peroxide bonded to the CDN surface to form cerium(IV) perhydroxide;³² in turn, cerium perhydroxide further decomposes to release oxygen. Formation of cerium perhydroxide was previously observed during the interaction of cerium compounds not only with H₂O₂, but also with other ROS (e.g., O₂ radicals).³³ Since hydrogen peroxide is strongly adsorbed on the surface of the CDN, it probably has the capability to displace inorganic or organic ligands (such as stabilizers and dyes).

Calcein is a well-known low-toxicity fluorescent organic dye, λ_ex/λ_em ∼495 nm/∼520 nm (Fig. S1–S3†). Fluorescence of calcein is quenched by some transition metal ions, such as Co²⁺, Ni²⁺, and Cu²⁺. Recently, it was established that rare earth ions also quench calcein's fluorescence at the physiological pH range (∼6–8), the strongest effect being demonstrated by the Ce³⁺ ion.³⁴ Since the calcein 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 fluorescence. In turn, during interaction between cerium dioxide and stronger ligands, calcein would be desorbed from the oxide's surface. In this way, interaction of cerium dioxide with a ROS such as hydrogen peroxide would lead to the release of free calcein, accompanied by regeneration of its fluorescence. In this paper, we have focused our efforts on showing that it is possible to directly monitor the interaction between ROS and CDNs in living cells by detecting the fluorescence of calcein as it is being released from the CDN–calcein complex. We have also paid attention to determining the antioxidant properties of the CDN–calcein 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.^35,36 For additional details, see ESI,† section “materials and methods”.

Optical measurements

Fluorescent measurements of calcein (excitation and emission wavelengths of 485 and 508 nm, respectively) and cerium(III) ions (excitation and emission wavelengths of 250 and 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 the 200–900 nm wavelength range.

Cell culture

The reference diploid epithelial swine testicular cell line (ST-cells) from the collection of the Institute of Veterinary Medicine, Ukrainian Academy of Agrarian Sciences (UAAS), was used to study the cytotoxicity of the CDN and the CDN–calcein complex, and their protective effect against ROS of different origins. Synthetic nutrient medium 199 (Biotest Laboratory, Ukraine) supplemented with 5% (v/v) fetal bovine serum (Sigma, USA), 25 mM HEPES, 10 mM glutamine, 100 units per mL penicillin, and 100 μg mL⁻¹ streptomycin was used as the growth medium. Cultured cells were kept at 37 °C in a 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 the suspension containing 5 × 10⁵ cells per ml were placed in 96-well Costar plates and incubated at 37 °C for 24 h in a TC-80M-2 thermostat in humid air (98%) containing 5% CO₂. The cell-supporting medium consisted of nutrient medium 199, 1% fetal bovine serum, 25 mM HEPES, 10 mM glutamine, 100 units per mL penicillin, and 100 μg mL⁻¹ streptomycin. The cell monolayer was finally washed with medium 199, without fetal bovine serum.

The cytotoxicity of the nanoparticles

The cytotoxicity of the nanoparticles was analyzed using a colorimetric MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, C₁₈H₁₆BrN₅S) in vitro assay.³⁷ MTT (M5655, Sigma-Aldrich) is a classic colorimetric metabolic activity indicator in cell viability assays. It produces a yellowish solution that is converted to a 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 evaluating cytotoxicity was adapted from that reported previously,³⁸ taking into account the reliability of the method.³⁹

Nanoparticles were suspended in distilled water by consecutive dilution across a 96-well 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 37 °C 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⁻¹, 100 μL per well) was added. Upon the completion of the exposure period, the 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 CDN against ROS in vitro

The protective action of the CDN or the CDN–calcein complex against ROS in vitro was determined as described earlier.⁴⁰ Briefly, CDNs or CDN–calcein complexes of different concentrations (10 μM–2 mM) were added to the cell monolayer 24 h before the hydrogen peroxide treatment. Four hours after the application of hydrogen peroxide (the final concentration of H₂O₂ in each well was 0.5 mM), the number of cells that survived 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:

(A_test/A_contr) × 100 (1)

here, A_test is the optical density of the test cells; A_contr 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. Control cells were considered to have 100% viability. Experimental data were presented as the median and 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 CDNs against vesicular stomatitis virus (VSV; Rhabdoviridae family, Vesiculovirus genus serotype Indiana) infections was studied using the same ST cell line. CDNs 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 (50% tissue culture infection dose) per ml. Forty minutes later, the unabsorbed virus was removed by washing with medium 199. A supporting medium (0.1 mL per well) was then added, and the cells were incubated at 37 °C 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), while for control cells infected with VSV (negative control) the cell monolayer was completely destroyed (100% of cytopathic effect).

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

(A_test − A_vsv)/(A_contr − A_vsv) × 100 (2)

here, A_contr is the optical density of intact cells stained with crystal violet; A_vsv is the optical density of VSV-infected cells stained with crystal violet; A_test is the optical density of stained cells treated with nanoparticles and VSV. Statistical treatment of data obtained was performed as described above.

Microscopic visualization of the ROS–CDN interaction in living cells

Microscopic visualization of the ROS–CDN 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 the CDN–calcein complex were investigated under UV illumination using a HBO-100W/2 mercury lamp with a “Green-2” filter as a light source (excitation and emission wavelengths of 450–480 and 520–560 nm, respectively).

Results and discussion

Fluorescence of calcein is strongly quenched by both cerium ions (Fig. S4 and S5†) and cerium dioxide nanoparticles (Fig. 1 and S8†). When this quenching was induced by Ce³⁺ ions, the intensity of the fluorescence was observed to be inversely proportional to the cerium concentration. Fluorescence quenching by CeO₂ nanoparticles proceeds through the interaction of calcein with cerium ions located only on the surface. For CDN nanoparticles ∼6 nm in size, total quenching of calcein fluorescence was achieved when the ratio of molar concentrations of calcein and cerium was between 1 : 50 and 1 : 100 (Fig. S6 and S7†).

Fig. 1 Fluorescence spectra of calcein (CLC) (C_CLC = 1 × 10⁻⁶ M, pH 7.2) in the presence of various CDN concentrations.

Excitation of CDNs by UV-irradiation at 250 nm (which corresponds to the characteristic absorption band for cerium(III) ions) does not lead to emission at 358 nm, which is the characteristic wavelength for cerium(III) ions fluorescence (Fig. S9†). We assume that suppression of the calcein fluorescence by CDNs is mainly caused by cerium(IV) ions, which prevail in the CDN lattice. However, as we have demonstrated, both cerium(III) and cerium(IV) ions were proved to be quenchers of the calcein fluorescence (Fig. 2), and the efficiency of calcein fluorescence quenching by cerium ions depends on the oxidation state of cerium.

Fig. 2 Fluorescence spectra of calcein (CLC) solution (1) and the same solution upon introduction of cerium species (2 – Ce(III) ions; 3 – Ce(IV) ions; 4 – CDN) (C_CLC = 1 × 10⁻⁶ M; C_CDN = 5 × 10⁻⁴ M; C_Ce(III) = 1 × 10⁻⁴ M; C_Ce(IV) = 5 × 10⁻⁵ M; pH = 7.2).

Hydrogen peroxide slightly decreases the fluorescence intensity of pure calcein (Fig. S10†). The injection of hydrogen peroxide into the solution of the non-fluorescent cerium(IV)–calcein complex was observed to lead to calcein release and restoration of its fluorescence (Fig. 3). Upon reaction of hydrogen peroxide with a cerium(III)–calcein complex, calcein fluorescence was not restored but was observed to be even additionally quenched (Fig. S12 and S13†). The most probable reason for this effect is that calcein is decomposed by peroxide radicals, which are formed by Fenton's reaction in the presence of cerium(III) ions and hydrogen peroxide. Since the oxidation of cerium(III) takes place during Fenton's reaction, the intensity of its fluorescence falls (Fig. S14 and S15†):

Ce³⁺ + H₂O₂ → Ce⁴⁺ + HO˙ + OH⁻ (3a)

calcein + HO˙ → oxidized calcein (non-fluorescent) (3b)

Fig. 3 Top panel: fluorescence spectra of solutions of the CDN–calcein complex in the presence of H₂O₂ at various concentrations (C_{hydrogen peroxide} = 5 × 10⁻⁵ M − 5 × 10⁻³ M; C_CLC = 1 × 10⁻⁶ M; C_CDN = 5 × 10⁻⁴ M; pH = 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 CDN aqueous sols synthesized according to protocols reported in ref. 35 and 36, respectively; 3 – pure calcein (control). A, A1 – before H₂O₂ injection; B, B1 – after H₂O₂ injection.

The stability of the CDN–calcein complex is relatively high; taking into account that both Ce³⁺ and Ce⁴⁺ ions interact with calcein, this stability should not strictly depend on the effective oxidation state of cerium in CDNs. Our data indicate that the CDN–calcein complex is stable in the presence of inorganic ions such as nitrates, sulphates, chlorides, and carbonates (but not in phosphates) and is destroyed only upon chemisorption of H₂O₂. Since cerium dioxide nanoparticles have been shown to be excellent scavengers of ROS (including H₂O₂),^4–10,30,31 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 fluorescence (see Fig. 4). Thus, treatment of cell culture with a non-fluorescent CDN–calcein complex would allow observation of the process of hydrogen peroxide formation and inactivation by the CDN in vitro.

Fig. 4 The reaction of a non-fluorescent CDN–calcein complex with ROS (hydrogen peroxide) in a living cell releases the fluorescent dye.

It is worth noting that water-soluble cerium(III) and cerium(IV) compounds cannot be used by themselves for visualization of oxidative stress because they are quite toxic (Ce(III) ions can participate in the Fenton reaction to form ROS, and Ce(IV) ions can cause oxidative stress by themselves). In contrast, because of the high antioxidant activity of CDNs, 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 (Fig. 5).

Fig. 5 Top panel: toxicity of CDNs and the CDN–calcein complex to ST-cells upon 24 hour exposure, as determined by a MTT assay. Bottom panel: protection of ST-cells against oxidative stress caused by introduction of 0.5 mM of hydrogen peroxide. Abscissa axis – cerium dioxide concentration, mM, ordinate axis – cells viability. Control cells viability is taken as 100%. Legend: control cells – intact ST-cells, control peroxide – ST-cells treated only with H₂O₂. 1 – cells treated with H₂O₂ and non-stabilized CDN sols synthesized according to the protocol in ref. 35, 1a – cells treated with H₂O₂ and a CDN–calcein complex prepared from non-stabilized CDN sol synthesized according to the protocol in ref. 35, 2 – cells treated with H₂O₂ and a citrate-stabilized CDN sol, synthesized according to the protocol in ref. 36, 2a – cells treated with H₂O₂ and a CDN–calcein complex prepared from a citrate-stabilized CDN sol, synthesized according to the protocol in ref. 36.

According to our data, both CDNs and CDN–calcein complex actually have low toxicity to the cell culture; the influence of calcein is practically negligible. In turn, the protection rates of CDNs against oxidative stress caused by hydrogen peroxide are different: citrate-stabilized CeO₂ nanoparticles were observed to protect cells effectively in the 0.062–1.0 mM concentration range (see Fig. 5), while non-stabilized cerium nanoparticles even increased the H₂O₂ toxicity at high concentrations in our experiments. However, addition of calcein into cerium sols eliminated this difference, so both preparations of the CDN–calcein complex were observed to protect cells in the 0.062–1.0 mM concentration range (see Fig. 5).

Highly polar hydrophilic calcein molecules cannot penetrate cell membranes. When cell culture is treated with pure calcein, the fluorescence was observed only in the intercellular space, and not inside the cells (see Fig. 6 and 1a–c). Washing of the cells with a buffer solution apparently removed dye from the cell monolayer, as the fluorescence was observed to disappear. The CDN–calcein complex is more hydrophobic than pure calcein, so it quickly (in 3–5 min) penetrated into living cells. The mechanism of penetration has not been investigated yet, however, and the most probable route includes adsorption of low-polarity CDN–calcein nanoparticles on the cell's membrane and their following uptake via endocytosis, which takes just about one minute. Since the CDN–calcein complex is not fluorescent, the UV-excited cell monolayer remained dark both before (see Fig. 6 and 2a–c) and after (see Fig. 6 and 3a–c) washing with buffer solution. Treatment by H₂O₂ was observed to lead to the destruction of the non-fluorescent CDN–calcein complexes, and bright fluorescence in a cytoplasm appeared (see Fig. 4a–c and 6).

Fig. 6 Optical and fluorescent micrographs of ST-cells (a – bright-field images, b –fluorescent images, c – merged). 1 – Cells treated with calcein solution (without further washing). The intercellular space is bright since calcein does not penetrate into cells. 2 – Cells treated with the CDN–calcein complex (without further washing). A weak fluorescence can be seen in the intercellular space, cell bodies are dark. 3 – Cells treated with the CDN–calcein complex with further washing with buffer solution. When excess CDN–calcein complex was removed, the fluorescence stopped. 4 – Cells treated with CDN–calcein complexes with further washing and treatment with excess of H₂O₂. Under oxidative stress conditions, bright fluorescence can be seen in the cytoplasm.

Presumably, endogenous ROS can also cause a break-up of the CDN–calcein complex and thus be monitored in the cell culture. To prove this hypothesis, we tried to use a well-known method to create oxidative stress in the living cells, namely by treatment them with latex beads, which results in the production of oxygen radicals.^41,42 According to our data, latex particles (dark agglomerates on Fig. 7 and 1a) do cause fluorescence of ST-cells treated with a CDN–calcein complex (Fig. 7 and 1b).

Fig. 7 Visualization of the ROS–CDN interaction in living cells. Micrographs of ST-cells treated with the CDN–calcein complex and further treated with latex beads (1) or VSV (2, 3). 1 – cells treated with latex beads, 2 mg ml⁻¹; 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 fluorescence in the nucleus is higher. a – bright-field image, b – calcein fluorescence image, c – merged (overlay image).

Viral infections can also cause oxidative stress, playing the key role in damaging infected cells and surrounding tissues.⁴³ 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 equines, cattle and swine. VSV infection of humans usually either is asymptomatic or causes a mild influenza-like illness.⁴⁴ We previously reported that CDNs can significantly reduce levels of cell mortality caused by viruses.⁴⁵ Our present results indicate that treatment of cell culture by CDN–calcein complex not only increases cell viability (Fig. 8) but also allows the monitoring of the development of the VSV infection process in vitro (Fig. 7).

Fig. 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 CDN synthesized according to the protocol reported in ref. 35 and with VSV, 2 – cells treated with a CDN–calcein complex prepared from citrate-stabilized CDN synthesized according to the protocol reported in ref. 36 and with VSV. Abscissa axis – cerium dioxide concentration, mM; ordinate axis – cell viability, %% to the control cells viability.

Interestingly, upon H₂O₂-induced oxidative stress conditions, CDN–calcein preparations synthesized from non-stabilized³⁵ and citrate-stabilized³⁶ CDN demonstrated the same antioxidant activity, but with different antiviral actions. A CDN–calcein complex prepared from citrate-stabilized CDNs was observed to provide effective protection of the cells in a wider range of concentrations (see Fig. 8). In our opinion, such a difference is presumably due to different colloidal stabilities of the surface-modified CDNs in the biological environment.

Conclusions

The oxidative stress visualization technique proposed here is based on the ability of CDNs to interact with calcein molecules, forming a non-fluorescent complex, and simplifying the penetration of dye into the cell. Under oxidative stress conditions, the CDN–calcein complex is decomposed and free calcein is released, allowing the visualization of ROS in the cytosol. Our data indicate that the 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 bead treatment or viral infection). This technique allows the monitoring of the formation of ROS at different stages of oxidative stress. For instance, when oxidative stress is induced by VSV infection, the fluorescence 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. We have demonstrated that uptake of the CDN–calcein complex by the cells is quite a fast process, taking less than 5 minutes.

Acknowledgements

This work was partly supported by the Russian Scientific Foundation (data concerning bioactivity of CDNs were obtained under RSF project 14-13-01373).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra08292c

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