The influence of agglomeration of nanoparticles on their superoxide dismutase-mimetic activity

Colloids and Surfaces A: Physicochem. Eng. Aspects 409 (2012) 176–182

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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa

The influence of agglomeration of nanoparticles on their superoxide dismutase-mimetic activity Vladimir K. Klochkov ∗ , Anna V. Grigorova, Olga O. Sedyh, Yuri V. Malyukin Institute for Scintillation Materials National Academy of Sciences of Ukraine, 60, Lenin ave., 61001 Kharkov, Ukraine

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

 Three types of nanoparticles CeO2 , GdYVO4 :Eu and CePO4 :Tb are compared in this work.  Colloidal systems are in a dynamic equilibrium of agglomerizationpeptization.  Superoxide dismutase mimetic activity of NP increases during the decay of agglomerates.

a r t i c l e

i n f o

Article history: Received 26 March 2012 Received in revised form 15 June 2012 Accepted 18 June 2012 Available online 26 June 2012 Keywords: Nanoparticles Colloidal solutions Coagulation Peptization Antioxidant Superoxide radical

a b s t r a c t Three types of colloidal systems based on CeO2−x , GdYVO4 :Eu3+ and CePO4 :Tb3+ are compared in this work. Nanoparticles with diameter 1.9 ± 0.3 nm were stabilized by sodium citrate. Ultramicroheterogeneous dispersion in water was found to be a typical hydrophobic colloidal system with negatively charged particles surface. CeO2−x colloidal solutions were chosen as a model system. Using coagulation by inorganic electrolytes, it was shown, that both individual nanoparticles and their agglomerates, which remain in a dynamic equilibrium of agglomerization-peptization, can be present in solutions. The reaction of the autoxidation of epinephrine to adrenochrome was used in order to determine the ability of nanoparticles to inactivate superoxide radicals. Superoxide dismutase (SOD) activity of CeO2−x nanoparticles increases during the decay of agglomerates in solution. Nanoparticles GdYVO4 : Eu3+ as well as CeO2−x exhibit SOD mimetic activity. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In recent years, there has been a dramatic increase in research, technology, and production of nanoparticles. Some of these nanoparticles are widely used in a diverse array of applications including material science and biology. Nanoparticles of metals, semiconductors and dielectric materials have useful properties such as fluorescence, optoelectronic and magnetic behavior [1]. Nanoparticles possess unique properties with potentially wideranging therapeutic applications [2]. The development of inorganic

∗ Corresponding author. Tel.: +380 57 341 03 89. E-mail address: [email protected] (V.K. Klochkov). 0927-7757/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfa.2012.06.019

luminescent nanoparticles offers a unique opportunity for biologists to use their high photostability for the dynamic tracking of individual species during in vitro or in vivo experiments [3]. These unusual properties depend on individual as well as collective properties of nanoparticles and their sizes and shapes [4–6]. On the one hand, nanoparticles can exhibit antibacterial and antioxidant properties; on the other hand, there is potential hazard of nanoparticles on human health [7–9]. It has become important to determine the influence of nanomaterial on biological objects. Results of toxic action studies for various nanoparticles seem to be contradictory. In particular, some sources indicate that the fullerene, cerium oxide and yttrium oxide nanoparticles have an antioxidant effect [10–14], according to other sources, the effect is prooxidant [16–18]. The ability of nanoparticles to protect or destroy cells depends not

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Fig. 1. TEM image of the NP:GdYVO4 :Eu3+ (A), CeO2−x (B), and CePO4 :Tb3+ (C).

only on their elemental composition, but also on their geometric parameters, the environment, the crystal structure and agglomeration of primary particles in the investigated systems [9,18–21]. The concentration of nanoparticles also affects the investigated biological systems. For example, in [10] was shown that OH• removing efficiency of hydrated C60 fullerene was in inverse correlation with fullerene concentration. It was conjectured that the antiradical action of fullerene in water medium is generally due to a “nonstoichiometric” mechanism, supposedly to a hydrated free radical recombination (self-neutralization), which is catalyzed by specific water structures ordered by fullerene. One of the factors for increased antioxidant activity together with a decrease in the concentration of nanoparticles may be the agglomeration of particles at higher concentrations [11]. The size growth of a nanoparticle can occur by aggregation and agglomeration. Small particles have larger surface area and higher particle number than bigger particles at the same mass concentration. Thus, extremely small nanoparticles (diameter <3 nm) could not be easily stabilized and aggregated rapidly [19,20]. A study of small nanoparticles’ aggregation and agglomeration influence on an experimental model is of particular importance for a correct interpretation of results of biological research. Recent studies have provided evidence that cerium oxide nanoparticles act as direct antioxidants to protect cells from various forms of lethal stress in vitro and exhibit SOD mimetic activity [14–17,22–24]. Previously, we have reported that spherical luminescent particles of GdYVO4 :Eu3+ were accumulated in the nuclei of liver cells [25]. In this paper, we carry out a comparative study of CeO2−x , GdYVO4 :Eu3+ and CePO4 :Tb3+ colloidal solutions with nanoparticles’ size <3 nm.

2. Materials and methods 2.1. Materials Lanthanide chlorides 99.9% and anhydrous sodium metavanadate (NaVO3 , 96%) (“Acros Organics” company) were all used without further purification. Sodium tripolyphosphate (Na5 P3 O10 , 98%), sodium citrate (Na3 C6 H5 O7 , 99%), hexamethylenetetramine ((CH2 )6 N4 , 99%), H2 O2 , 35%, NH4 OH, 25% from “Macrochem” Co. Ltd. were used. The inorganic salts and isopropanol (coagulants) were commercial products of reagent grade. Na3 VO4 solution with pH value of 13 was obtained by adding solution NaOH (1 mol/L) to NaVO3 water solution. Solution of epinephrine hydrotartrate 0.18% pharm. Borate buffer (pH 10.7).

stoichiometric formula Ce(0.8) Tb(0.2) PO4 have been synthesized following the method [28,29]. Solutions of nanoparticles CeO2−x were obtained by the method developed in this study. A typical synthesis: 100 ml of solution CeCl3 (0.002 mol/L) were mixed with 100 ml of solution hexamethylenetetramine (0.004 mol/L) and stirred by using a magnetic stirrer for 3 h at room temperature. After that, 1.8 ml NH4 OH and 0.6 ml of H2 O2 were added into the solution. Then, the solution was put in round-bottom flask and was refluxed for 5 h. As a result, transparent colorless solutions were obtained. The solution was evaporated in a rotary evaporator flask under vacuum at the bath temperature of 70 ◦ C to 30 ml. A solution of 2 M NaCl was added to the obtained solution until the resulting solution became turbid. Then the solid phase was precipitated by centrifugation. The precipitate was separated and a solution of sodium chloride was added again. The procedure of precipitate cleaning was repeated three times. After the last stage of centrifugation, a solution of sodium citrate with molar ratio CeO2 /NaCt as 1:1 was added to the precipitate. Thus, the impurity-free solution was found to be transparent in transmitted light at the concentration CeO2 > 1 mg/ml with a poorly yellow color. All colloidal suspensions (lanthanum orthophosphate, orthovanadate and cerium oxide) were stabilized by sodium citrate with molar ratio 1:1. The solutions were additionally dialyzed for 24 h against deionized water to remove the excess of ions and organics species. Dialysis membrane tubing with a molecular weight cutoff of “Cellu Sep H1” 3.5 KDa (pore size of less than 1.5 nm) was used, and water was renewed each 6 h (the water/colloid volume ratio is 40). All sols were transparent in transmitted light. Moreover, the sols passed through membrane filters with pores of 100 nm without loss. The concentration of working solutions was chosen to be 1–2 mg/ml. The process solutions have physiological value pH = 7.2–7.8 and, therefore, they can be used for biological testing. 2.2. Methods UV–vis absorption spectra of colloidal solutions were measured with a SPECORD 200 spectrometer (“Analytik Jena”). Photoluminescence spectra were recorded by a spectrofluorimeter on the base of a grating monochromator at room temperature under excitation by the helium-cadmium laser with  = 325 nm. Transmission electron micrographs of particles were taken by using TEM-125K (“SELMI”) electron microscope. The samples for microscopy were prepared by evaporating dilute solution droplets onto carbon coated copper discs. 3. Results and discussion

2.1.1. Nanoparticles (NPs) synthesis Solutions of nanoparticles with stoichiometric formula Gd(0.7) Y(0.2) Eu(0.1) VO4 have been synthesized following the method reported earlier [26,27]. Solutions of nanoparticles with

GdYVO4 :Eu3+ , CeO2−x and CePO4 :Tb3+ nanoparticles (NPs) with diameter of 1.9 ± 0.3 nm were stabilized by sodium citrate and had identical concentration of solid phase in water (Fig. 1A–C).

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been described earlier [25–29]. The agglomeration of small colloidal particles is accompanied by the change in optical properties of a disperse system, for example, in absorption spectra [30]. These changes are related to the electrodynamics’ interaction between particles spaced at small distances in agglomerates. With increasing particles’ sizes and their agglomeration, a shift of max to the long-wave region is observed. The luminescence spectra of sols under 325 nm excitation by helium–cadmium laser are shown in Fig. 3A and B. The nanoparticles based on GdYVO4 :Eu3+ , CePO4 :Tb3+ possess a bright luminescence, and can be used as biological luminescent probes. Cerium oxide Ce4+ does not luminesce. It is known that changes in crystal lattice parameters occur when the size of cerium oxide nanoparticles decreases or crystal contains dopants [31–33]. The variation of the unit cell parameter is caused by gradual decrease of the cerium effective oxidation number as a result of removal of some oxygen atoms located on the particle surface from their crystallographic sites to give oxygen vacancies. According to some calculations, the particle size corresponding to the complete Ce4+ → Ce3+ transition is than less 1.9 nm [31,32,34]. The proposed model of partial reduction of cerium includes protonation of surface oxygen atoms followed by elimination of water to give oxygen vacancies. Therefore, in the investigated system, the nanocrystals of cerium oxide are in the form of CeO2−x .

3.2. The coagulation points We have used inorganic salts as coagulating agents. The systems behave as typically hydrophobic ones, colloidal particles have a negative charge on the surface, due to potential-determining citrate ions adsorbed on the their surface. After reaching a proper ‘threshold’ concentration of the coagulating agent, the opalescence becomes visible by the naked eye. The appearance of evident turbidity in 1–2 min after addition of an electrolyte was chosen as a criterion of fast coagulation. The typical examples of behavior of the systems at electrolyte concentrations below and above the critical concentration are shown in Fig. 4. As a result of turbidity increase, the light absorption increases at first but then decreases due to deposition of the coagulated nanoparticles. The value of the coagulation point (or the critical coagulation concentration) Y was calculated in the standard way [35]: Y = 0.5(Cx + Cx−1 )

Fig. 2. Absorption spectra of NP:GdYVO4 :Eu3+ (A), CeO2−x (B), and CePO4 :Tb3+ (C).

Nanoparticles of size less than 3 nm possess high surface activity and diffuse mobility that defines their aspiration to aggregation. Only stabilization of such particles (by surface-active agents) can be used to preserve the stability of sols [13]. The hydrosols which were obtained under our conditions and stored in sealed ampules have remained stable for more than 6 months. 3.1. Optical properties The absorption spectra of sols are shown in Fig. 2A–C. The spectra correspond with literary data for similar systems which have

(1)

where Cx is the concentration of fast coagulation, while Cx−1 is the nearest concentration of the electrolyte at which this phenomenon does not yet occur. Fig. 4 provides an example. The value of Y is calculated as an average value of the coagulant concentrations (mol/L) equal to 0.0010 (Fig. 4A) and 0.0011 (Fig. 4B) respectively. At the concentration of 0.0012 mol/L (Fig. 4C), the fast coagulation and immediate turbidity of the solution occur. This value does not meet the criterion of visual assessment of the coagulation point. Then Y = 0.5 (0.0011 + 0.0010) = 0.00105. The Y values determined at room temperature are presented in Table 1. As shown in Table 1, the ratio Y (NaCl)/Y (salt) for cations with different charges corresponds to the “threshold” character of coagulation. The ratio of Y values for inorganic salts is in accordance with the semi-empirical Schulze–Hardy rule and (DLVO) theory for Derjaguin–Landau–Verwey–Overbeek hydrophobic ‘negative’ sols [36–38]. These data reflect a substantial role of the universal mechanism of condensation of the

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Fig. 3. Luminescence spectra of NP: GdYVO4 :Eu3+ (A) and CePO4 :Tb3+ (B) (excitation 325 nm).

Table 1 The coagulation points of hydrosols by various electrolytes. Salt

z

NaCl CaCl2 LaCl3

1 2 3

CePO4 :Tb3+

CeO2−x

GdYVO4 :Eu3+

Y (mol/L)±(5–15%)

Y (NaCl)/Y (salt)

Y (mol/L)±(5–15%)

Y (NaCl)/Y (salt)

Y (mol/L)±(5–15%)

Y (NaCl)/Y (salt)

0.354 0.0025 0.00105

1 140 340

0.165 0.001225 0.00065

1 135 254

0.125 0.002625 0.000275

1 48 454

The concentrations of nanoparticles were 1.0 mg/ml (CeO2−x , 0.0058 mol/L; CePO4 :Tb3+ , 0.0042 mol/L; GdYVO4 :Eu3+ , 0.0039 mol/L); z, charge of the cation.

diffuse part of the double electric layer. From Hamaker’s the following well-known relation can be obtained [35,36]: Yz6 = const

(2)

where z is the charge of the counterion, i.e. the ion, charged oppositely to the surface. But the Y−1 ratios of 1:64:729 for z = 1, 2 and 3 which are in exact agreement with Eq. (2) have seldom been observed. Deviations from the 6th power law are obviously due to the increased role of the specific adsorption of multicharged cations on the surface of nanoparticles stabilized by sodium citrate and a possibility of coagulation both into the ‘primary’ (close) and ‘secondary’ (distant) minima [37–40].

Fig. 4. Dynamics of CeO2−x hydrosol (1 mg/ml) coagulation in the presence of the coagulating electrolyte LaCl3 ; coagulant concentration (mol/L): 0.001 (A), 0.0011 (B), 0.0012 (C); Y = 0.00105; (vis = 500 nm).

The dependence of Y on the concentration of nanoparticles is of particular interest (Fig. 5). It is known that colloids concentration decrease can result in either an increase or a decrease of Y [35]. As shown in Fig. 5A the Y value for cerium oxide in a concentration range from 0.02 to 0.85 mg/ml increases with decrease of concentration in the case of coagulation with NaCl, and decreases in the case of coagulation with LaCl3 (Fig. 5B). The character of the dependence of this type agrees well with the Burton–Bishop rule [35]. According to the theory developed by Muller [41] increase of coagulation points with concentration decrease indicates the presence of agglomerates (secondary associates) in the system in which the primary particles are held by weak van der Waals forces. Typically, when agglomerated nanoparticle samples are added to a liquid they can be separated by overcoming the weaker attractive forces by several methodologies [21]. Agglomerates can be formed by interaction of the electrical double layers of primary particles in secondary minima, and in a state of dynamic equilibrium of coagulation–peptization. Dilution of sols in distilled water leads to decrease of ionic strength, increase of zeta potential of primary particles and destruction of the agglomerates. Adding of electrolytes leads to ionic strength increase which results in the electrical double layer compression. Hence, although the particle surface charge may be unchanged since coagulant ions do not interact with the particle surface, the zeta potential decreases with increasing of ionic strength, which leads to agglomeration and coagulation. In our case, at sodium chloride coagulation, a nonlinear increase in the values of Y has been observed. Moreover, there are three ranges of threshold concentrations. Different slopes of linear ranges of the curve (Fig. 5A, a and c) correspond to the agglomerates of two types in the solution: large (a) and small (c). Range (b) corresponds to transition state between two types of associates. Measurements of Y values for coagulation with NaCl was carried out in 1 and 24 h after dilution of the concentrated sol CeO2−x (C = 1.7 mg/ml). As shown in Fig. 5A Y values at low concentrations of CeO2−x are much higher for sol diluted 24 h before the measurement than for the same sol diluted 1 h before the measurement. Clearly, such a

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Fig. 5. Dependence of Y value on the nanoparticles concentration: CeO2−x with NaCl (A) and LaCl3 (B) coagulant; GdYVO4 :Eu3+ with NaCl coagulant (C); CePO4 :Tb3+ with NaCl coagulant (D).

difference is due to peptization of agglomerates, initially present in the concentrated hydrosols. Moreover, agglomerates are stable for quite a long time. Hydrosol CeO2−x is a system consisting of both individual particles and their agglomerates. Moreover, the processes of coagulation and peptization are in dynamic equilibrium. For diluted solution equilibrium is shifted toward lower quantity of agglomerates in the system and, conversely, with increasing concentration of solids in solution (e.g. evaporation of a dilute solution on a rotary evaporator) the content of agglomerates increases. This statement is also confirmed by the absorption peak bathochromic shift (∼3 nm) at concentration increase. Additionally, this effect can be observed for coagulation of CeO2−x sol with NaCl. Coagulation with sodium chloride is accompanied by an increase in light scattering in solution. Also if the precipitate is separated by centrifugation and then re-dissolved in water – a transparent solution with an initial value of the absorption maximum (max = 287 nm) will be obtained again. It is obviously that coagulation with NaCl occurs by the concentration mechanism due to compression of the diffuse electrical double layer and decrease of the  potential of the particles. Coagulation structures form in the secondary (distant) minima and the system is reversible. In the case of coagulation of hydrosols with LaCl3 (Fig. 5B) decrease of coagulation points with decrease of CeO2−x mass concentration is observed. Obviously, in this case, the role of cation adsorption (z = 3) is significant and coagulation takes place mainly by neutralization mechanism. Coagulum is not redissolved in water, i.e. the system is irreversible.

The dependence of Y on concentration of nanoparticles of hydrosol GdYVO4 :Eu3+ (coagulant NaCl) is similar to the one obtained for CeO2−x hydrosol under the same conditions (Fig. 5C), but the differences in Y values measured in 1 and 24 h after dilution are not so considerable as for CeO2−x sol. It is probable that orthovanadate hydrosol at high concentrations also contains agglomerates. Otherwise looks the same dependence for CePO4 :Tb3+ hydrosol (Fig. 5D). Decrease of Y values with sol concentration decrease indicates an absence of unstable agglomerates in the system. At concentrations of CePO4 :Tb3+ hydrosol less than 0.26 mg/ml there is no clear threshold critical coagulation concentration, so the determination of Y is difficult. Determination of the Y concentration dependence for the coagulation of GdYVO4 :Eu3+ and CePO4 :Tb3+ sols with LaCl3 coagulant are also difficult due to the fast clarification of the sol after coagulation. This is probably due to the phenomenon of “irregular series” [41] due to the adsorption of triply charged cations and charged particles. 3.3. SOD mimetic activity of nanoparticles Recently discovered antioxidant properties of nanoparticles have caused a discussion about the mechanisms of their antioxidant activity [10,11,15–17,22,42]. Much attention is paid to the ability of cerium oxide nanoparticles to protect cells against oxidative stress in both cell culture and animal models. The nanocrystals of cerium dioxide exhibit superoxide dismutase (SOD) activity [11,15–17,22,42,43]. It is assumed that Ce3+ ↔ Ce4+ transfer on the

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Fig. 6. Inhibition of O2− • in the presence of nanoparticles at the epinephrine autoxidation.

surface of nanoparticle is a cause of the CeO2−x antioxidant activity. However, the nanoparticles such as fullerenes [10] or yttrium oxide [16] which do not contain ions of variable valence also possess antioxidant activity. To study the antioxidant properties of CeO2−x , GdYVO4 :Eu3+ and CePO4 :Tb3+ nanoparticles we used the method developed by Scherbakov at al. [43]. To determine the ability of nanoparticles to carry out a role of SOD mimetic, epinephrine–adrenochrome autoxidation reaction (which is accompanied by the formation of superoxide radicals) [44,45] was used. In the presence of SOD, which inactivates superoxide radicals, the adrenochrome content decreases. The nanocrystalline cerium dioxide, containing on the surface as Ce4+ , so Ce3+ ions possesses a similar effect [11,15–17]. The reaction has gone in the borate buffer (pH = 10.7). The test sols were intaken in the buffer solution so the final concentration of nanoparticles was from 0.02 to 100 mg/L. The concentration of epinephrine in the sample was 270 ␮M. The measurements were performed in 1 and 24 h after dilution the concentrated sols (2 mg/ml) to the studied concentrations. Control measurements were performed in the absence of nanoparticles, but in the presence of sodium citrate. The formation of adrenochrome was revealed by intensity changing of the 347 nm absorption band in 40 min from the start of the reaction. Fig. 6 shows the changes in the relative values of optical density at max = 347 nm depending on the concentration of sols. Relative values in percents were calculated as: A × 100 A0

(3)

where A is the optical density for the investigated sample, A0 is the optical density for the control sample. Changes of relative values of adrenochrome content correspond to relative change in O2−• content in the sample. It was found a significant difference between O2−• inactivation ability for nanoparticles of different nature (Fig. 6). Since orthophosphate NPs show a very weak antioxidant activity, orthovanadate NPs and cerium oxide ones have a pronounced ability to inactivate superoxide radicals with increasing concentrations of nanoparticles in solution. Moreover, the form of the curve for orthovanadates remained unchanged when measured both in 1 h and in 24 h after dilution of the concentrated solution. For the cerium oxide a considerable dependence on the time after the dilution of solutions was observed. When measured in 1 h after dilution of the concentrated sol, inactivation of superoxide radicals of cerium oxide NPs has maximum activity at a concentration of NPs – 0.005 mg/ml. When measured in 24 h after dilution of the concentrated sol increase

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of activity in the range of concentrations 0.1–0.005 mg/ml was observed. At concentrations of nanoparticles less than 0.005 mg/ml decrease in solutions activity proportional to the concentration NPs have been observed. Obviously, that the nanoparticles in concentrated sols are mainly in the aggregated form. At sol dilution large agglomerates disintegrate to smaller ones and individual particles, thus increasing the overall surface area and, hence, the superoxide radical inactivation ability. However, for approach of dispersed equilibrium some time interval after dilution of sols is required. It should be noted that we have found that the antioxidant activity of orthovanadate NPs does not depend on the presence of particles in the crystal lattice of Eu3+ ions. Experimentally, we have not found out any difference in the SOD mimetic activity for GdYVO4 :Eu3+ and GdYVO4 nanoparticles. Thus, the antioxidant activity of small nanoparticles with identical sizes and environment depends on the nature of particles and equilibrium state of the dispersions of nanoparticles and their agglomerates. 4. Conclusion This work is devoted to a comparative analysis of colloidal solutions, with the varied nature of a solid phase (CeO2−x , GdYVO4 :Eu3+ and CePO4 :Tb3+ ). The nanoparticles of the diameter 1.9 ± 0.3 nm stabilized by sodium citrate were used. The ultramicroheterogeneous dispersion in water was found to be a typical hydrophobic colloidal system with negatively charged particles surface. The coagulation by inorganic electrolytes occurs according to the DLVO theory. It was found that nanoparticles of the small sizes tend to agglomerate in solutions. The particles of sols can consist of both individual nanoparticles and their agglomerates. Agglomerates can collapse at dilution of the solutions. Generally, sols with small nanoparticles can be considered as polydisperse system consisting of individual structures and their agglomerates, which are in “coagulation–peptization” dynamic equilibrium. The disperse structure can change, at dilution or concentration of solutions, and also in the presence of coagulants. Therefore, at biological testing on the nanoparticles, which play the part of the model systems, in “in vitro” and “in vivo” experiments appearance of effects connected with agglomeration of the particles in solutions is probable. On the example of the SOD mimetic activity model for nanoparticles we have shown, that antioxidant properties of nanoparticles CeO2−x depend on the time after dilution of the concentrated solution. We have found out that nanoparticles based on GdYVO4 :Eu3+ at particle concentration more than 0.0005 mg/ml, as well as CeO2−x nanoparticles inactivate superoxide radicals in a model system. NPs based on CePO4 :Tb3+ practically do not show SOD mimetic activity. References [1] M. Ferrari, Nat. Rev. Cancer 5 (2005) 161. [2] X. Gao, Y. Cui, R.M. Levenson, L.W.K. Chung, S. Nie, Nat. Biotechnol. 22 (2004) 969. [3] X. Michalet, F.F. Pinaud, L.A. Bentolila, J.M. Tsay, S. Doose, J.J. Li, G. Sundaresan, A.M. Wu, Science 307 (2005) 538. [4] S. Link, M.A. El-Sayed, J. Phys. Chem. B 103 (1999) 8410. [5] J.J. Storhoff, A.A. Lazarides, C.A. Mirkin, R.L. Letsinger, R.C. Mucic, G.C. Schatz, J. Am. Chem. Soc. 122 (2000) 4640. [6] M. El-Sayed, Acc. Chem. Res. 34 (1999) 257. [7] A. Nel, T. Xia, L. Madler, N. Li, Science 311 (2006) 622. [8] A.D. Maynard, R.J. Aitken, T. Butz, V. Colvin, K. Donaldson, G. Oberdorster, Nature 444 (2006) 267. [9] P.J. Borm, D. Robbins, S. Haubold, T. Kuhlbusch, H. Fissan, K. Donaldson, Part. Fibre Toxicol. 3 (2006) 11. [10] G.V. Andrievsky, V.I. Bruskov, A.A. Tykhomyrov, S.V. Gudkov, Free Radic. Biol. Med. 47 (2009) 786. [11] S. Babu, A. Velez, K. Wozniak, J. Szydlowska, S. Seal, Chem. Phys. Lett. 442 (2007) 405.

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