Cerium fluoride nanoparticles protect cells against oxidative stress

Cerium fluoride nanoparticles protect cells against oxidative stress

Materials Science and Engineering C 50 (2015) 151–159 Contents lists available at ScienceDirect Materials Science and Engineering C journal homepage...

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Materials Science and Engineering C 50 (2015) 151–159

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Cerium fluoride nanoparticles protect cells against oxidative stress Alexander B. Shcherbakov a, Nadezhda M. Zholobak a, Alexander E. Baranchikov b, Anastasia V. Ryabova c,d, Vladimir K. Ivanov b,e,⁎ a

Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv D0368, Ukraine Kurnakov Institute of General and Inorganic Chemistry of the Russian Academy of Sciences, Moscow 119991, Russia Prokhorov General Physics Institute of the Russian Academy of Sciences, Moscow 119991, Russia d National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Moscow 115409, Russia e National Research Tomsk State University, Tomsk 634050, Russia b c

a r t i c l e

i n f o

Article history: Received 12 September 2014 Received in revised form 12 January 2015 Accepted 30 January 2015 Available online 2 February 2015 Keywords: Cerium fluoride nanoparticles Terbium doped CeF3 Luminescence Living cells Virus Oxidative stress Toxicity

a b s t r a c t A novel facile method of non-doped and fluorescent terbium-doped cerium fluoride stable aqueous sols synthesis is proposed. Intense green luminescence of CeF3:Tb nanoparticles can be used to visualize these nanoparticles' accumulation in cells using confocal laser scanning microscopy. Cerium fluoride nanoparticles are shown for the first time to protect both organic molecules and living cells from the oxidative action of hydrogen peroxide. Both non-doped and terbium-doped CeF3 nanoparticles are shown to provide noteworthy protection to cells against the vesicular stomatitis virus. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Biomaterials engineering is one of today's most promising areas of materials science. Substantial progress is observed in this field due to the widespread use of artificial nanomaterials [1]. Particular success has been achieved in the development of nanoparticle-based theranostic systems, including drug delivery platforms, luminescent and magnetic biomarkers, and sensoric and diagnostic devices. [2,3]. In very recent years, nanocrystalline ceria has been shown to possess an enormous biological activity, which originates from its ability to participate readily in the redox processes under biologically relevant conditions, as well as its relatively low toxicity, making biological application of ceria-based materials relatively safe [3–6]. Ceria-based nanomaterials belong to a new class of artificial enzymes (nanozymes) [7] and are able to scavenge various reactive oxygen species (ROS) deleterious to living cells [8–10]. For instance, nano-ceria exhibits a superoxide dismutase-mimetic activity [8,9] and catalase-mimetic activity [10], protecting living cells against superoxide anion and hydrogen peroxide. Nano-ceria is expected to be useful in the therapy

⁎ Corresponding author at: Kurnakov Institute of General and Inorganic Chemistry of the Russian Academy of Sciences, Moscow 119991, Russia. E-mail address: [email protected] (V.K. Ivanov).

http://dx.doi.org/10.1016/j.msec.2015.01.094 0928-4931/© 2015 Elsevier B.V. All rights reserved.

of aging-related diseases, including various types of cancer, diabetes, ischemic stroke, and Alzheimer's disease [3–16]. From a chemical point of view, the mechanism of the nano-ceria protective action is not quite clear yet. According to a common opinion, ceria becomes strongly non-stoichiometric in a nanocrystalline state and thus can participate in various redox processes [1–14]. However, the role of cerium valence states in ROS inactivation is still under question. Celardo et al. [15] demonstrated that it is Ce3 + ions which determine the redox-dependent anti-apoptotic effect of cerium oxide nanoparticles. On the other hand, Das et al. [16] stated that the catalase mimetic property of nano-ceria (unlike the SOD mimetic one) depends essentially on the content of Ce4+ in ceria nanoparticles. We can assume that both valence states of cerium actually play an important role in the scavenging of hydrogen peroxide. While performing the cell protection function, Ce3+ is oxidized by hydrogen peroxide to the tetravalent state. Simultaneously, Н2О2 is bonded strongly to Се4+ ions on the surface of nano-ceria [17], forming cerium perhydroxide, which is further decomposed to form non-toxic water and oxygen. Until now, the question is open as to whether other ceriumcontaining nanomaterials can protect living cells from hydrogen peroxide or other reactive oxygen species when the cerium ion would be initially in a trivalent state only. A good candidate for such examination is cerium fluoride (CeF3). This compound would presumably be nontoxic, because the toxicity of fluoride-containing inorganic substances

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generally depends on their solubility in the water. Similar to cerium dioxide, cerium fluoride has low solubility in water and biological fluids [18]. For instance, the CeF 3 solubility product constant (Ksp = 8 ∗ 10− 16) is substantially lower than that of fluorspar CaF2 (K sp = 4 ∗ 10 − 11 ), which is classified as “not dangerous” [19]. Another good reason to study CeF3 is the prominent luminescence properties of Ce3 + species (unlike the Ce4 + ones), especially when doped with some other rare earth elements (terbium, europium etc. [20]). The luminescence of Ce3 + is strongly quenched when it is oxidized by oxygen or ROS to Ce4 + [21]. Thus the luminescence of cerium fluoride (or doped cerium fluoride) could allow simultaneous monitoring of CeF3 nanoparticles' behavior in cells and their redox state in a microenvironment. There are plenty of synthetic methods for obtaining cerium fluoride dispersions and sols: thermolysis of fluorine-containing precursors, co-precipitation from aqueous solutions, reversed micelle and micro-emulsion precipitation, hydrothermal synthesis, precipitation from non-aqueous solutions and solvothermal synthesis, and the sol–gel method, among others [22]. For example, monodisperse photoluminescent CeF3, CeF3:Tb3 +, and CeF3:Tb3 [email protected] core/shell nanoparticles of small size (b 15 nm) have been synthesized by thermolysis of rare earth oleate complexes in a high boiling mixture of oleic acid and 1-octadecene [23]. Cerium fluoride nanoparticles (15–30 nm) have been successfully prepared from water-in-oil microemulsions [24]. Re-dispersible CeF3 nanoparticles were synthesized by a polyol route, using ethylene glycol [25] or diethylene glycol [26] as a solvent. CeF3 nanocrystals with plate-like and perforated morphologies were synthesized via a hydrothermal route, using polyvinylpyrrolidone as a stabilizer [27]. Large-sized nanoparticles of cerium fluoride were prepared in water under sonication without any surfactants, using cerium nitrate and ammonium hydrofluoride as the starting materials [28]. A Triton X-100 surfactant was used for aqueous synthesis of [email protected] and [email protected]@ SiO2 complex nanostructures [29]. Unfortunately, only a small part of the methods listed above are suitable for biological applications. Toxic solvents (ethylene or diethylene glycols, octadecene) or toxic capping agents (oleylamine, ethoxylated phenols) are typically used in the syntheses, so the products demand thorough purification; in turn, surfactant-free syntheses in aqueous media (hydrothermal, microwave etc.) often lead to the coarse-grained and/or agglomerated specimens that can't be peptized to form stable transparent sols. Here we report on the synthesis of stable transparent aqueous sols containing nearly monodisperse luminescent CeF3 or CeF3:Tb nanoparticles by a novel facile surfactant-free low-temperature technique. These materials are shown to be non-toxic and capable of protecting organic molecules and living cells from the oxidation by hydrogen peroxide. Finally, the antiviral activity of CeF3 and CeF3:Tb nanoparticles against the vesicular stomatitis virus in vitro is demonstrated for the first time. 2. Materials and methods 2.1. Synthesis of CeF3 and Tb-doped CeF3 nanoparticles We have elaborated a novel method, allowing CeF3 and CeF3:Tb nanoparticles synthesis via facile precipitation in alcoholic media at room temperature. Briefly, 1.86 g of cerium(III) chloride heptahydrate (5 mmol) (Aldrich, #228931) was dissolved in 15 ml of distilled water and added to 150 ml of isopropyl alcohol (Aldrich, W292907). Hydrofluoric acid (20 mmol) (Sigma-Aldrich, #30107), dissolved in 50 ml of isopropyl alcohol, was added drop-wise to a cerium salt solution under vigorous stirring. The resulting white sediment was filtered and washed carefully by pure isopropyl alcohol, several times. Then the suspension was slightly dried to form a paste-like substance and dispersed in 110 ml of distilled water, using an ultrasonic bath. The resulting transparent colloid solution was boiled for 5 min to

remove residual alcohol. Tb-doped CeF3 nanoparticles were synthesized by the same protocol, using a mixture of 4.25 mmol of cerium chloride and 0.75 mmol (15%) of terbium chloride (Aldrich, #212903) as a starting material. 2.2. Synthesis of cerium oxide nanoparticles A non-stabilized ceria aqueous sol (ceria sol #1) was synthesized by hydrothermal-microwave treatment of the colloid solution, formed upon anionite treatment of a cerium(III) nitrate aqueous solution [30]. Briefly, ion-exchange resin Amberlite IRA 410 CL (Aldrich, #216569), preliminarily converted to the OH-form, was gradually added to a 0.01 M cerium(III) nitrate (Aldrich, #238538) solution until pH reached 10.0. Sols formed in this way were separated from the resin by filtering, immediately transferred to 100 ml polytetrafluoroethylene autoclaves (filled to 50%) and subjected to microwave-hydrothermal treatment in a Berghof Speedwave MWS-3 + setup at 190 °C for 3 h. Upon completion of the synthesis the autoclaves were withdrawn from the microwave oven and cooled down to room temperature in air. A citrate-stabilized ceria aqueous sol (ceria sol #2) was synthesized by the previously reported procedure [31]. Briefly, 2.0 g of citric acid (Sigma-Aldrich, #251275) was mixed with 25 ml of a 0.4 M aqueous cerium(III) chloride (Aldrich, #228931) solution. The resulting solution was rapidly poured under stirring into 100 ml of a 3 M ammonia (analytic grade, Chimmed, Russia) solution, and then exposed for 2 h at ambient conditions and further boiled for 4 h. Then the solution was cooled to room temperature and purified from precursors and by-products by sedimentation and further re-dispersion. 2.3. Characterization of nanoparticles Transmission electron microscopy was performed using a Leo 912 AB Omega electron microscope operating at 100 kV. Before the analysis sols were brought onto the copper grids using micropipette without any specific pretreatment and dried in ambient air. Particle size measurements by dynamic light scattering were carried out on a Malvern Zetasizer Nano ZS analyzer. The light source used was a helium-neon laser (the radiation wavelength was 632.8 nm). Local elemental analysis (with ca. 0.5 μm resolution) of CeF3 and CeF3:Tb nanoparticles pre-deposited on a conductive carbon tape was performed using a Carl Zeiss NVision 40 scanning electron microscope, equipped with an Oxford Instruments X-MAX energy-dispersive X-ray analyzer, operating at a 20 kV acceleration voltage. The overall Ce:Tb atomic ratio in the CeF 3:Tb sample was additionally checked by total reflection X-ray fluorescence (TXRF) spectrometry, using a Bruker PICOFOX S2 laboratory spectrometer equipped with an aircooled Mo-anode X-ray tube. UV–vis absorption spectra of CeF3 and CeF3:Tb colloid solutions were recorded using standard quartz cells for liquid samples, on an Agilent Technologies Cary 5000 UV–Vis spectrophotometer. Photoluminescence spectra of CeF3 and CeF3:Tb colloid solutions were recorded using a Perkin Elmer LS55 spectrometer at room temperature (resolution: 0.5 nm; slit width: 3–8 nm). The laser scanning microscopy investigations were carried out using a Carl Zeiss LSM-710-NLO microscope equipped with a pulse femtosecond Chameleon Ultra II laser system (Coherent Inc., USA), tunable in the 690–1060 nm range. The luminescence spectra of CeF3:Tb nanoparticles was registered by the 32 channel GaAsP detector under excitation by a 488 nm CW laser or 735 nm 80 MHz pulse laser, with a pulse width of 140 fs. Scanning was performed at the lowest speed (177 μs/pixel) because of the prolonged luminescence lifetime of Tb3+. 2.4. Hydrogen peroxide inactivation Indigoid dyes are easily decomposed by ROS [32,33], the discoloration of indigo by hydrogen peroxide being used in biochemical assays

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for a long time [34]. The ability of cerium fluoride nanoparticles to inactivate hydrogen peroxide was tested in the process of indigo carmine dye bleaching in alkaline media (Supplementary Information Figure S4). Briefly, 10 ml of indigo carmine (Aldrich, #57000) stock solution (1 mM) was diluted by distilled water to obtain an appropriate concentration (75 μM). pH was then adjusted to 10 by a 1 M NaOH solution. Hydrogen peroxide (non-stabilized 3% H2O2 preliminarily purified by distillation) was then added to make a 1 mM peroxide concentration; the solution was agitated and the absorption spectrum was recorded. Optical density at 611 nm was monitored in the absence and presence of different quantities of cerium fluoride.

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prior to the end of the exposure period the culture medium was removed, and MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide, Sigma-Aldrich, #M5655) solution in PBS (0.1 mg/ml, 100 μl/well) was added to the cells. After the completion of the exposure period, the supernatant was removed, and a lysis solution containing 0.1% SDS (Sigma-Aldrich, #L3771) solution in DMSO (Sigma-Aldrich, #D8418) was added. Plates were shaken for 5 min and placed on a Thermo/LabSystems Multiskan MS Microplate Reader, and the absorbance was read colorimetrically at 540 nm. Each experiment was repeated three times, with four replications. 2.8. The protective effect of nanoparticles against ROS in vitro

2.5. Cells preparation and analysis of viability Cell Culture. The effect of nanoparticles on the viability of cells was studied using a reference diploid epithelial swine testicular cell line (ST-cells) from the collection of the Institute of Veterinary Medicine UAAS. A synthetic nutrient 199 medium (Biotest Laboratory, Ukraine) supplemented with 5% (v/v) fetal bovine serum (Sigma-Aldrich, USA #F7524), 25 mM HEPES (4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid sodium salt, N-(2-Hydroxyethyl)piperazine-N′-(2ethanesulfonic acid) sodium salt (Sigma-Aldrich, USA, #H3784), 10 mM L-glutamine solution (Sigma-Aldrich, USA, #G7513), 100 units/ ml penicillin, and 100 μg/ml streptomycin as penicillin–streptomycin solution (Sigma-Aldrich, USA #P4333) 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 from the flask, 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 (Sigma-Aldrich, USA #E8008) and re-suspended in a culture medium. To form cell monolayers, aliquots (0.1 ml) of suspension containing 5 ∗ 10 5 cells per ml were placed in 96-well Costar plates (Sigma-Aldrich, USA #CLS3595) and incubated at 37 °C for 24 h in humid air (98%) containing 5% CO2. The cell-supporting medium consisted of a 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 washed with a 199 medium without fetal bovine serum. 2.6. Intracellular nanoparticles distribution Intracellular CeF3:Tb nanoparticle distribution was studied using an HeLa cell line. Cells were seeded in Petri dishes, preliminary treated with a 0.1% gelatin solution. The cells were then supplied with a DMEM medium containing 10% FBS and the CeF3:Tb colloid solution was added. The laser scanning microscopy investigations were carried out using a Carl Zeiss LSM-710-NLO microscope (see above). To discriminate between Tb3+ luminescence and cellular autofluorescence, “Online Fingerprint” mode was used. For this purpose, the luminescence spectra of CeF 3 :Tb sols and autofluorescence spectra of cells under excitation by 488 nm laser were preliminarily registered. The autofluorescence spectra contain a low-intensity broad peak in the range of 500–600 nm, and the Tb3+ ion spectra contain characteristic comb of narrow peaks thus allowing decomposition of the total fluorescence in each pixel into corresponding components. 2.7. The cytotoxicity of the nanoparticles The cytotoxicity of the nanoparticles was analyzed using the colorimetric MTT assay [35]. The test protocol for cytotoxicity evaluation was adopted from elsewhere [36,37]. Nanoparticles were suspended in distilled water, serially diluted across 96-well microtiter plates (100 μl), and transferred to the cultural medium to the cell monolayer in a ratio of 1/10 (v/v). Exposure time was 24 h at 37 °C in humid air (98%) containing 5% CO2. Four hours

The protective action of nanoparticles against ROS in vitro was determined as described earlier [38]. Briefly, sols of different concentrations (10 μM–2 mM) were introduced to the cell monolayers, and 24 h later the hydrogen peroxide was added to cells (the concentration of H2O2 in each well was 0.5 mM). The cells were incubated in hydrogen peroxide for 4 h and subsequently washed and stained with crystal violet. The excess dye was then 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 optical density of stained cells corresponds to their viability. The percentage of the cells absorbing crystal violet was determined according to the following formula (Dex / Dcontr) ∗ 100, where Dex is an optical density of the experimental wells, and Dcontr is an optical density of the intact (control) wells. Statistical treatment of data obtained was performed using BioStat 2009 Professional 5.8.1 software, in accordance with standard recommendations. Experimental data are presented as the median and interquartile range Me (LQ–UQ), where Me = median (50% percentiles), LQ = 25% percentiles, and UQ = 75% percentiles. In the entire series, the number of experiments conducted was five. 2.9. The protective effect of nanoparticles against viral infection in vitro The protective effect of ceria sols against the vesicular stomatitis virus (VSV) infection 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 cells were infected with VSV at a concentration of 100 TCID50/ml. The unabsorbed virus was removed in 40 min by washing with a 199 medium. The cell supporting medium (0.1 ml per well) was added, and the cells were incubated at 37 °C for 24 h. The viability of cells was estimated 24 h after infection by VSV, by measuring the optical density of cells stained with crystal violet. The positive control consisted of staining intact cells; the negative control consisted of staining the VSV-infected cells. The optical density of cells stained with crystal violet was measured using a microwell plate reader (Thermo/LabSystems Multiskan MS Microplate Reader) at 540 nm. Each experiment was performed in triplicate. The percentage of viable cells was determined by the formula (Atest − Avsv) / (Acontr − Avsv) ∗ 100, where Acontr is the optical density of stained intact cells (positive control); Avsv is the optical density of stained VSV-infected cells (negative control); and Atest is the optical density of stained test cells treated with CeF3, CeF3:Tb or CeO2 nanoparticles. The statistical treatment of data obtained was performed as described above. 3. Results and discussion 3.1. Microstructure and luminescent property of the nanoparticles Transmission electron microscopy (TEM) data indicate that obtained CeF3 and CeF3:Tb samples consist of nearly monodisperse

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Fig. 1. TEM images and SAED patterns (top) and particle size distributions according to the DLS (bottom) of CeF3 (A) and CeF3:Tb (B) samples.

well-crystallized nanoparticles with an average size of 15–20 nm (Fig. 1A, B, top). Selected area electron diffraction (SAED) patterns correspond to CeF 3 crystal structure (PDF2 8-45, space group P63/mcm) and do not contain any reflections from impurity phases. The presence of circular rings in the SAED patterns indicates polycrystalline nature of CeF3 samples. No signs of orientational ordering or oriented attachment of the nanoparticles are observed [39]. According to DLS data, the mean hydrodynamic diameter is 18 nm for CeF3 nanoparticles and 24 nm for CeF3:Tb nanoparticles (Fig. 1A, B, bottom).

The coincidence of the mean particle size values measured by TEM and DLS indicates a very low (if any) degree of particle agglomeration in colloid solutions. Local energy dispersive X-ray spectroscopy data for CeF 3 and CeF3:Tb samples confirm the presence of Ce, Tb and F (Supplementary Information Figure S1). Ce/Tb atomic ratios are in the range from 0.87/0.13 to 0.85/0.15, which is very close to the set value. The same value (0.852/0.148) was obtained using total reflection X-ray fluorescence analysis.

Fig. 2. Excitation (λem = 543 nm) and emission (λex = 277 nm, 488 nm or 735 nm) spectra of the CeF3:Tb sol.

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The CeF3 sol has a weak adsorption in the UV-region, and Tb-doping barely influences the position and intensity of the absorption bands of nanocrystals at all (Supplemetary Information Figure S1). The CeF3:Tb sol shows an intense luminescence in the green region of the visible spectrum under UV irradiation (277 nm), which is due to the electronic transitions of the Tb3 + ions [40–43]. The excitation and emission spectra of CeF3:(15% Tb3 +) are shown in Fig. 2. The sharp emission peaks originate from the 4f–4f transitions of Tb3 + ions: 5D4–7F6 (487.5 nm), 5D4–7F5 (543 nm), 5D4–7F4 (582 nm) and 5D4–7F3 (617 nm). The excitation spectrum for the 5D4–7F5 transition of Tb3+ consists of only one intense band, which corresponds to the 4f–5d transition of Ce3 +. The same band is observed in the excitation spectrum of undoped CeF3 nanoparticles emitting in the UV region (290–400 nm) only. Upon excitation at 277 nm the intensity of the nanoparticles' luminescence is the highest. This type of excitation can't be realized in confocal laser scanning microscopy and thus it can't be adopted for biomedical applications. Moreover, due to the harmful effect of UV radiation on living organisms, the biological experiments require usually an excitation at lower energy of irradiation. However, we have shown that CeF3:Tb nanoparticles can emit light upon excitation at higher wavelengths, e.g., at 488 nm or even at 735 nm (Fig. 2, Supplementary Information Figure S2). The 488 nm line of the continuous wave (CW) argon ion laser has previously been proved to be a convenient source for direct excitation of 7F6 → 5D4 transition of the Tb3 + ion in various coordination environments [44–46]. In our experiments, this source of excitation (CW 488 nm laser operating at 25 mW) is also shown to be suitable for terbium ion excitation in a cerium fluoride matrix. The terbium ion can be also excited by multi-photon absorption [47–51]. Two-photon excitation of the terbium ion was observed earlier in Cs2NaTbHal6 elpasolite [47], yttrium aluminum oxide [37], and fluoride crystals (CaF2:Tb, LaF3:Tb, pure TbF3) [49,50]. Rakov et al. [51] have registered three-photon excitation of terbium ion luminescence in terbium-doped lutetium silicate powders. Our results indicate clearly that both 488 nm CW laser and 735 nm pulse laser of the confocal laser scanning microscope are suitable excitation sources for multi-photon terbium ion excitation in a matrix of cerium fluoride. Green luminescence of CeF3:Tb nanoparticles can be used to visualize nanoparticles' accumulation in cells, using confocal laser scanning microscopy. We have obtained several spectrally resolved luminescent images of HeLa cells treated with CeF3:Tb nanoparticles at 488 nm excitation (Fig. 3). Fig. 3A shows that terbium-doped cerium fluoride nanoparticles' accumulation in living cells can be observed due to their luminescence. It is worth to note that nanoparticles are accumulated in the cytoplasm but not in the nucleus (Fig. 3A, C and D). Data presented in Figs. 3 and S3 clearly demonstrate the possibility of CeF3:Tb nanoparticles' spatial localization in vitro by using a wellestablished technique of confocal laser scanning microscopy. 3.2. Toxicity of cerium fluoride (non-doped and terbium-doped) nanoparticles Cerium fluoride (non-doped and terbium-doped) nanoparticles are quickly and strongly absorbed by the living cells, even when the cell monolayer was treated by CeF3 sols for less than 5 min. This fact was evidenced by the UV-luminescence of the cell monolayer exposed with CeF3 :Tb nanoparticles after thorough washing with Hank's Balanced Salt Solution (Supplementary Information Figure S3). The toxicity of cerium fluoride (non-doped and terbium-doped) nanoparticles was studied in a 0.0156–10.0 mM concentration range and compared with ceria sols #1 and #2. Ceria sol #1 (non-stabilized) was toxic for ST cells in a 0.3–2.0 mM concentration range, while cerium fluoride (non-doped and terbium-doped) nanoparticles decreased the viability of ST cells (by 10–15% to the control) only in the maximal studied concentration of 10 mM (Fig. 4). Therefore, cerium fluoride

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Fig. 3. Confocal laser scanning microscopy image (spectral imaging combined with linear unmixing) of HeLa cell treated with terbium doped cerium fluoride nanoparticles. The separation into individual channels after linear unmixing of spectral components (see insets): A — CeF3:Tb nanoparticles' luminescence, green pseudo-color; B — autofluorescence of the cell, red pseudo-color; C — bright-field image of the cell taken under visible light; D — merged image (A + B + C).

(non-doped and terbium-doped) nanoparticles, like ceria sol #2 (stabilized by citrate), are nontoxic for cells in the micromolar concentrations. 3.3. Hydrogen peroxide decomposition in the presence of cerium fluoride nanoparticles: CeF3–H2O2 interaction The main absorption band in cerium fluoride (non-doped and terbium-doped) spectra is located at ca. 250 nm (see Fig. 5, Figure S1). According to literature data, UV absorption spectra of Ce3 + ions in solution have a maximum at 253.6 nm with a molar extinction coefficient of 685 M−1 cm−1 [52], while the UV absorption spectra of Ce4 + ions have a maximum at ca. 320 nm with a molar extinction coefficient of 5580 M−1 cm−1 [53]. When cerium fluoride nanoparticles react with hydrogen peroxide at neutral (biological) and alkaline pH, an orange-colored sol is formed (see inset in Fig. 5), however the UV-adsorption band of the Ce4 + ion (290–310 nm) doesn't appear upon such interaction. The intensity of luminescence of the CeF3:Tb colloid solution decreases but still remains high enough. We can make an assumption that cerium(III) peroxo-compounds are formed on the surface of particles. According to literature data, interaction of Ce3 + ions with hydrogen peroxide leads to the formation of cerium hydroperoxide, having a broad absorption band at 350 nm with a shoulder at 500 nm; heating of cerium hydroperoxides leads to their decomposition and formation of Ce4+ species [54]. When the mixture of a cerium fluoride sol with hydrogen peroxide is left to stand for a long time or boiled, cerium peroxo-complexes are decomposed too, and Ce3 + ions are oxidized to Ce4 +. This leads to quenching of the colloid solution's luminescence (Fig. 5, inset). 3.4. CeF3 protects organic dye against oxidation by hydrogen peroxide Fig. 6 shows that CeF3 nanoparticles protect indigo carmine dye from oxidative discoloration caused by hydrogen peroxide in a dosedependent manner. The degree of protection depends proportionally

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Fig. 4. Relative viability of ST cells treated with cerium dioxide and cerium fluoride nanoparticles in comparison to intact cells (control cells). 1 — ceria sol #1; 2 — ceria sol #2; 3 — CeF3 sol; 4 — CeF3:Tb sol,.

on the cerium fluoride concentration; in all cases, the reaction obeys a first order rate law. Thus revealed CeF3 behavior greatly resembles the protective ability of CeO2 nanoparticles, which possess a pronouncing catalase-mimetic property [10,16]. The well-known mechanism of hydrogen peroxide decomposition by catalase includes the following reactions [55]: E-Fe3+ + H2O2 → E(*)-Fe4+ = O + H2O, E(*)-Fe4+ = O + H2O2 → E-Fe3+ + H2O + O2.

The commonly accepted mechanism of ceria nanoparticle (NP) catalase-like activity includes the following reactions [12]: NP-Ce3+ + H2O2 → NP-Ce4+ = O + H2O, NP-Ce4+ = O + H2O2 → NP-Ce3+ + H2O + O2. In our opinion, this scheme is not fully correct, because the process of hydrogen peroxide decomposition in the presence of cerium-containing nanoparticles could be more complex and actually can proceed in

Fig. 5. UV-absorption spectra of 0.5 mM CeF3 sol in the course of interaction with H2O2: 1 — initial sol; 2 — 0.005 mM H2O2 added; 3 — 0.05 mM H2O2 added; 4 — 0.1 mM H2O2 added; 5 — 0.5 mM H2O2 added; 6 — 5 mM H2O2 added; 7 — the same sol (6) stored for a week. Inset: 1 — CeF3:Tb sol, 2 — CeF3:Tb sol with H2O2. 3 — CeF3:Tb sol with H2O2 added and left to stand for 2 weeks. Top — pictures taken under visible light illumination; bottom — pictures taken under UV illumination.

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Fig. 6. Kinetics of indigo carmine dye (35 μM) discoloration by hydrogen peroxide (0.5 mM) in alkali media in the absence and presence of cerium fluoride nanoparticles, as measured by the change of optical density at 611 nm: 1 — control (without CeF3); 2 — 5 μM CeF3 added; 3 — 10 μM CeF3 added; 4 — 20 μM CeF3 added; 5 — 30 μM CeF3 added; 6 — 40 μM CeF3 added.

ligands (stabilizers, dyes, etc.) [17]. This phenomenon determines the ability of direct monitoring of the interaction between ROS and cerium dioxide nanoparticles in living cells. The mechanism of perhydroxide decomposition on a surface of ceria nanoparticles is not clear yet. In the case of aqueous solutions of cerium salts the formation of hydroxyl and superoxide (hydroperoxyl) radicals as intermediates is assumed [57,58]; it was established that the decomposition of hydrogen peroxide by Ce3 + ions occurs via Fenton/Haber–Weiss mechanisms [59]. However, in the case of cerium fluoride nanoparticles (just as ceria nanoparticles) these free radicals either cannot be formed or are inactivated just after their formation, and do not destroy the organic substrate. The ability to decompose H2O2 without releasing the aforementioned harmful oxygen radicals is a specific feature of cerium dioxide nanoparticles that distinguishes them from both cerium salts and most other metal oxides (including nanocrystalline ones). Our current study indicates that cerium fluoride nanoparticles can also decompose hydrogen peroxide without the formation of reactive oxygen radicals, so CeF3 sols can protect organic molecules against oxidation by H2O2. Such protective properties of cerium fluoride nanoparticles indicate a significant contribution of Ce3 + ions into the redox mechanism of H2O2 decomposition by cerium-containing nanoparticles, probably including cerium dioxide ones.

several stages [11,56]. At the first stage Ce3 + ions on the surface of nanoparticle are oxidized by hydrogen peroxide to Се4+:

3.5. CeF3 protects living cells against oxidative stress caused by hydrogen peroxide

NP-Ce3+ + H2O2 → NP-Ce4+(OH) + OH−.

As cerium fluoride is capable of protecting the organic molecules from ROS, we can assume that it would protect living systems also. Our data indicate that CeF3 do actually protect cells against oxidative stress caused by hydrogen peroxide (Fig. 7). Cerium fluoride (non-doped and terbium-doped) nanoparticles protect cells utterly against oxidative stress in a 0.08–2.5 mM concentration range. It is worth noting that terbium-doped cerium fluoride nanoparticles protect living cells from oxidative stress induced by hydrogen peroxide, even when applied in sub-toxic concentrations (e.g. 10 mM), while non-doped cerium fluoride nanoparticles taken in the same concentration don't provide any protection of the cells. However, in the lowest concentrations, non-doped cerium fluoride nanoparticles show a credibly higher protective effect. Non-stabilized nano-ceria aqueous sols (ceria sol #1) do not protect cells against oxidative

Simultaneously, Н2О2 is bonded to the surface of ceria nanoparticles, forming cerium perhydroxide: NP-Ce4+(OH) + H2O2 → NP-Ce4+(OOH) + H2O. In turn, cerium perhydroxide further decomposes with oxygen formation: NP-Ce4+(OOH) → NP-Ce4+(OH) + ½O2. Actually, hydrogen peroxide is shown to be strongly adsorbed on the surface of cerium dioxide nanoparticles, displacing inorganic or organic

Fig. 7. Protection of ST cells by cerium-containing nanomaterials against oxidative stress caused by the introduction of 0.5 mM of hydrogen peroxide. Legend: control cells — intact cells, control peroxide — cells treated with H2O2, 1 — cells treated with H2O2 and CeF3, 2 — cells treated with H2O2 and CeF3:Tb, 3 — cells treated with H2O2 and ceria sol #1, 4 — cells treated with H2O2 and ceria sol #2.

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stress caused by hydrogen peroxide. The protective ability of citratestabilized nano-ceria aqueous sol (ceria sol #2) matches the protective ability of cerium fluoride (non-doped and terbium-doped) nanoparticles: a maximal cell survival rate (90–100% of the living cells in comparison to control intact cells) was found for the 0.08–2.5 mM CeO2 concentration range. Notable difference in the protection activity of ceria sols #1 and #2 probably arises from different contents of Ce3 + in corresponding nanoparticles. Our recent studies of the chemical composition of ceria nanoparticles in these sols have shown that sol #1 contains only trace amounts of Ce 3 + ions, while sol #2 contains ca. 15% of Ce 3 + [60]. Thus the present study clearly demonstrates that Ce3 + plays the predominant role in the protective effect of Ce-containing nanoparticles.

3.6. Antiviral activity of cerium fluoride nanoparticles The presented data (Fig. 8) indicate that cerium fluoride nanoparticles can protect cells against viral infection too. For cerium fluoride (non-doped and terbium-doped) sols, the greatest protective effect against cytopathic effect (CPE) of vesicular stomatitis virus (VSV) is observed at 0.312 mM concentration, which is four times lower than for citrate-stabilized nano-ceria aqueous sol (1.25 mM). For non-doped cerium fluoride sols, the range of concentrations providing protection against viral СРЕ for more than 50% of the cells is 0.312–1.25 mM. Terbium-doped cerium fluoride nanoparticles provide the same protection, even at lower concentrations (0.15–0.625 mM). The protective antiviral effect of non-stabilized nano-ceria aqueous sols is minimal and is observed in the 0.031–0.125 mM concentration range; protection of 50% of the cells is observed for 0.062 mM concentration only. Previously, we have reported that cerium dioxide nanoparticles (ceria sol #2, 3–5 nm, citrate-stabilized, zeta potential of − 20 mV) provide virus resistance in L929 and RF cells in both prophylactic and therapeutic (1 h after infecting) schemes. Nano-ceria also demonstrates a significant virucidal effect, reducing the titer of an RNA virus (vesicular stomatitis, VSV) and a DNA virus (herpes simplex type 1) by 2.6–4.8 lg [61,62]. The selectivity index (the ratio of the effective antiviral and cytotoxic doses) is more than 16.0, which is typical for active antiviral drugs. Our current results demonstrate that cerium fluoride nanoparticles, as well as doped ones, protect cells against oxidative stress caused by VSV infection more effectively than nano-ceria aqueous sols.

In our opinion, this effect (just as the protective effect against hydrogen peroxide) is dictated by different Ce3+ contents in nanoparticles. The possible mechanisms of ceria nanoparticles' protective action against viral infection depend mostly on the redox-properties of nanoceria. It is well known that oxidative stress plays an important role in a number of viral infections [63,64], and cerium species can dramatically decrease this pathogenic factor [62]. Cell death in VSV-infected cells occurs mainly by oxidative stress-induced apoptosis, which is accompanied by morphological changes in the nuclei, DNA fragmentation and activation of caspase-3 in different systems [65–67]. The ST cell line is highly sensitive to VSV, which causes an in vitro characteristic cytopathic effect. Previously, we have shown that VSV causes oxidative stress due to the formation of ROS in the infected ST-cells, and nano-ceria scavenges these oxygen species by means of strong binding [17]. Another possible mechanism of antiviral action of ceriumcontaining nanoparticles is based on their influence on cellular antiviral pathways. For their own replication, viruses use the signaling pathways and transcription factors of living cells. They activate IKK kinase; phosphorylation of the protein-inhibitor IκB causes the activation of the nuclear factor NF-κB; and NF-κB activation increases the expression of viral genetic material. It is worth noting that conventional antiviral drugs do not affect IKK cascades. It has been recently shown that cerium dioxide nanoparticles exhibit not only oxidoreductase-like activity, but also phosphatase-like one [70–72] and inhibit phosphorylation of IκBα, thus reducing the activation of NF-κB [73]. The phosphatase-like activity strictly depends on Ce3+ content in nanoparticles; pre-oxidation of nano-ceria by hydrogen peroxide leads to the decrease in the concentration of Ce3+ and also to the drastic decrease of the dephosphorylation reaction rates [70]. The higher concentration of Ce3 + in cerium fluoride nanoparticles is probably responsible for their higher phosphatase-mimetic activity in comparison with nano-ceria. In our opinion, the development of effective drugs, inhibiting the phosphorylation of IκB, and their introduction into clinical practice, would open a new perspective in the treatment of viral infections, thus nanosized cerium-containing species have a great potential in the field [68,69]. 4. Conclusions A novel facile method of synthesis of non-doped and terbium-doped cerium fluoride nanoparticles is proposed, allowing the production of stable CeF3 and CeF 3 :Tb aqueous sols. We have shown for the

Fig. 8. Protection of ST cells by cerium-containing nanomaterials against the cytopathic effect of vesicular stomatitis virus (VSV). Legend: control cells — intact culture, control virus — cells treated with VSV, 1 — cells treated with VSV and CeF3, 2 — cells treated with VSV and CeF3:Tb, 3 — cells treated with VSV and ceria sol #1, and 4 — cells treated with VSV and ceria sol #2.

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first time that cerium fluoride nanoparticles are able to protect organic molecules (e.g. indigo carmine dye) from the oxidizing action of hydrogen peroxide. In vitro studies demonstrate nontoxicity of both non-doped and terbium-doped CeF3 nanoparticles. Moreover, we have shown that these materials, in much the same way as CeO 2 nanoparticles, exhibit a well-pronounced protective action against hydrogen peroxide-induced oxidative stress in cell cultures. Both non-doped and terbium-doped CeF3 nanoparticles have been shown for the first time to provide noteworthy protection of cells against the cytopathic effect of vesicular stomatitis virus. Acknowledgments Nano-ceria biological activity studies were supported by the Russian Scientific Foundation (project 14-13-01373). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.01.094. References [1] C.R. Lowe, in: C.M. Niemeyer, C.A. Mirkin (Eds.), Nanobiotechnology: Concepts, Applications and Perspectives, Wiley-VCH, Weinheim, 2004, p. 1717. [2] J. Xie, S. Lee, X. Chen, Adv. Drug Deliv. Rev. 62 (2010) 1064. [3] A.A. Semenova, E.A. Goodilin, N.A. Brazhe, V.K. Ivanov, A.E. Baranchikov, A.E. Goldt, O.V. Sosnovtseva, S.V. Savilov, A.V. Egorov, A.R. Brazhe, E.Y. Parshina, O.G. Luneva, G.V. Maksimov, Y.D. Tretyakov, J. Mater. Chem. 22 (2012) 24530. [4] A.S. Karakoti, N.A. Monteiro-Riviere, R. Aggarwal, J.P. Davis, R.J. Narayan, W.T. Self, J. McGinnis, S. Seal, JOM 60 (2008) 33. [5] R.W. Tarnuzzer, J. Colon, S. Patil, S. Seal, Nano Lett. 5 (2005) 2573. [6] T. Naganuma, E. Traversa, Biomaterials 35 (2014) 4441. [7] H. Wei, E. Wang, Chem. Soc. Rev. 42 (2013) 6060. [8] C. Korsvik, S. Patil, S. Seal, W.T. Self, Chem. Commun. 10 (2007) 1056. [9] E.G. Heckert, A.S. Karakoti, S. Seal, W.T. Self, Biomaterials 29 (2008) 2705. [10] T. Pirmohamed, J.M. Dowding, S. Singh, B. Wasserman, E. Heckert, A.S. Karakoti, J.E.S. King, S. Seal, W.T. Self, Chem. Commun. 46 (2010) 2736. [11] V.K. Ivanov, A.B. Shcherbakov, A.V. Usatenko, Russ. Chem. Rev. 78 (2009) 855. [12] A.B. Shcherbakov, V.K. Ivanov, N.M. Zholobak, O.S. Ivanova, E.Yu. Krysanov, A.E. Baranchikov, N.Ya. Spivak, Yu.D. Tretyakov, Biophysics 56 (2011) 987. [13] A. Karakoti, S. Singh, J.M. Dowding, S. Seal, W.T. Self, Chem. Soc. Rev. 39 (2010) 4422. [14] M. Perullini, S.A.A. Bilmes, M. Jobbágy, in: R. Brayner, F. Fiévet, T. Coradin (Eds.), Nanomaterials: A Danger or a Promise? Springer, London, 2013, p. 307. [15] I. Celardo, M. de Nicola, C. Mandoli, J.Z. Pedersen, E. Traversa, L. Ghibelli, ACS Nano 5 (2011) 4537. [16] S. Das, J.M. Dowding, K.E. Klump, J.F. McGinnis, W. Self, S. Seal, Nanomedicine 8 (2013) 1483. [17] N.M. Zholobak, A.B. Shcherbakov, E.O. Vitukova, A.V. Yegorova, Y.V. Scripinets, I.I. Leonenko, V.K. Ivanov, RSC Adv. 4 (2014) 51703. [18] A.A. Migdisov, A.E. Williams-Jones, T. Wagner, Geochim. Cosmochim. Acta 73 (2009) 7087. [19] J. Aigueperse, P. Mollard, D. Devilliers, M. Chemla, R. Faron, R. Romano, J.P. Cuer, Ullmann's Encyclopedia of Industrial Chemistry, Wiley-VCH, Weinheim, 2005, p. 389. [20] C. Bouzigues, T. Gacoin, A. Alexandrou, ACS Nano 5 (2011) 8488. [21] X. Wang, D. Zhang, Y. Li, D. Tang, Y. Xiao, Y. Liu, Q. Huo, RSC Adv. 3 (2013) 3623. [22] P.P. Fedorov, A.A. Luginina, S.V. Kuznetsov, V.V. Osiko, J. Fluor. Chem. 132 (2011) 1012. [23] S. Gai, P. Yang, X. Li, C. Li, D. Wang, Y. Dai, J. Lin, J. Mater. Chem. 21 (2011) 14610. [24] S. Qiu, J. Dong, G. Chen, Powder Technol. 113 (2000) 9.

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