Titanium doping reduces superoxide dismutase activity, but not oxidase activity, of catalytic CeO₂ nanoparticles

Abstract

In this paper we report the enzymatic properties of Ti-doped CeO₂ nanoparticles. The superoxide dismutase activity of Ti-doped nanoparticles is reduced in comparison to undoped nanoceria. In contrast, the oxidase activity of these nanoparticles was unchanged. The change in enzymatic activity was accompanied by a dramatic change in shape to a spherical nanostructure. In addition to reporting a new type of enzymatically-active nanoparticle, Ti-doped cerium oxide nanoparticles are well suited for biological applications.

Although enzymatic activity is traditionally thought of as a biochemical function of proteins, recent studies have shown that several inorganic nanoparticles display enzymatic activities. Among these enzymatic activities are peroxidase, oxidase, catalase, and superoxide dismutase [1-5]. Indeed, complex biomimetic characteristics such as bacterial-like motility are exhibited when certain nanoparticles catalyze chemical reactions [6]. For example, cerium oxide nanoparticles (nanoceria) exhibit superoxide dismutase (SOD) mimetic activity [2, 3]. Asati et al. [5] have reported that nanoceria also possess an intrinsic oxidase-like activity at acidic pH, as they quickly oxidize several organic substrates without oxidizing agents. As tissue damage in diseases including macular degeneration, diabetic retinopathy, Alzheimer's disease, amyotropic lateral sclerosis, and many others is believed to be mediated by superoxide anions and other reactive oxygen species (ROS), there is a rapidly growing interest in using inorganic nanoparticles as therapeutic agents to deflect tissue damage [7]. Although ROS damage tissues at supraphysiological concentrations, under normal conditions they are required for cellular signaling pathways and the regulation of cell functions [8-12]. As nanoceria exhibit an SOD activity level much greater than that of the native biological enzyme SOD [2], the nanoparticles may be harmful to cells. Therefore, we sought nanoceria with reduced superoxide dismutase activity to more closely parallel the biological enzyme. One potential strategy to influence the enzymatic activity of nanoceria is to include dopants. We now show that nanoceria doped with Ti exhibit reduced SOD activity.

CeO₂, Ce_0.95Ti_0.05O₂, Ce_0.90Ti_0.1O₂, and Ce_0.85Ti_0.15O₂ nanoparticles, lot numbers 6SB232A2, 6SB254A, 6SB257B, and 6SB257D, respectively, were purchased from Nanocerox, Inc. (Ann Arbor, MI). Titanium oxide nanoparticles (size 10-25 nm) were purchased from US Research Nanomaterials Inc. WST-1 was obtained from Dojindo Molecular Tech. (Rockville, MD). All other chemicals were purchased from Sigma Chem. Co. (St. Louis, MO). Pt nanoparticles were synthesized as previously described by others [13].

The SOD assay was performed using the method of Ukeda et al. [14], with some modifications. Briefly, 240 μl of carbonate buffer (0.1M, pH 9.2) with 3 mM of EDTA were added into a 1.5 ml centrifuge tube, followed by the addition of 40 μl of a nanoparticle dispersion or buffer solution, 40 μl of 3 mM xanthine, 40 μl of 1.0 mM WST-1 and 40 μl of xanthine oxidase (0.05U/ml). These mixtures were vortexed, incubated for 25 minutes at room temperature in the dark, and then centrifuged at 2000 rpm for 5 min. The clear supernatant was pipetted into a 96-well plate (Costar 3626, Corning). The absorbance at 440 nm was measured with a plate reader (Molecular Devices, Sunnyvale, CA). Each sample was measured in triplicate (3 wells, 100 μL each). Sample readings were corrected by subtracting the value of the control without xanthine oxidase from all sample readings.

The oxidase assay was performed in a 96-well plate using N, N-diethyl-p-phenylenediamine sulfate salt (DPA) as the chromogenic substrate [5]. Briefly, 320 μl of citrate buffer (0.1M, pH 4.0) were pipetted into a centrifuge tube followed by the addition of 40 μl of nanoparticle dispersion and 40 μl of freshly prepared DPA (5 mM in citrate buffer) solution. These mixtures were incubated for 15 min. at room temperature in the dark and then the nanoparticles were removed by centrifugation. Absorbances at 550 nm were measured with a plate reader (Molecular Devices, Sunnyvale, CA). Experiments were performed in triplicate. All sample readings were corrected for non-specific absorbance by subtracting the absorbance measured for tests in which nanoparticles were omitted.

The nanoparticles were characterized using a 200 kV JEOL 2010F transmission electron microscope, as described [15]. Fig. 1A shows a TEM image of CeO₂ nanoparticles. A size range of 14×13 nm to 150×140 nm was observed for these nanoparticles. However, Ce_0.95Ti_0.05O₂, Ce_0.90Ti_0.1O₂, and Ce_0.85Ti_0.15O₂ nanoparticles (Fig. 1B-D) were characterized by the appearance of spherical nanoparticles (spherical diameter range 15 to 150nm; non-spherical range 16×15 nm to 120×110 nm). Thus, Ti-doping promotes a transition from the cubic crystal phase of CeO₂ to a spherical nanostructure.

Fig. 1.

TEM images of nanoparticles. Micrographs of CeO₂, Ce_0.95Ti_0.05O₂, Ce_0.90Ti_0.1O₂, and Ce_0.85Ti_0.15O₂ nanoparticles are shown in panels A-D, respectively. It should be noted that Ti-doping promotes the acquisition of a spherical shape. (Bar=100nm)

As shown in Fig. 2A, nanoceria exhibited SOD activity in a nanoparticle concentration dependent fashion. For comparison, Pt nanoparticles were also tested as they have been reported to exhibit SOD mimetic activity [11-13]. The enzymatic activity of Pt nanoparticles shows a linear relationship with nanopaticle concentration (Fig. 2B), whereas the activity of nanoceria is exponentially dependent upon concentration (Fig. 2A).

Fig. 2.

SOD activity of nanoparticles. (A) The non-linear relationship between the production of WST-1 formazan at 440 nm and the CeO₂ nanoparticle concentration is shown. (B) The linear relationship between the production of WST-1 formazan at 440 nm and the abundance of Pt nanoparticles as judged by the concentration of Pt. Error bars were omitted when they were smaller than the symbol given in the figure. The R² values obtained for the datasets are shown in each panel.

To ascertain the effect of nanoparticle composition on SOD activity, we tested a panel of Ti-doped CeO₂ nanoparticles. Fig. 3 shows that 5% Ti doping in CeO₂ nanoparticles effectively reduces their SOD mimetic activity (12.6% residual superoxide for CeO₂ versus 65.6% for Ce_0.95Ti_0.05O₂). Further increases in Ti content from 5% to 15% had only a small effect on enzyme activity. In contrast, TiO₂ nanoparticles had no SOD activity. Thus, Ti-doping is accompanied by a significant reduction in SOD activity of nanoparticles.

Fig. 3.

Comparison of the residual superoxide levels of CeO₂, Ti-doped CeO₂, and TiO₂ nanoparticles. The nanoparticle concentration was 2 mg/ml. The statistical significance was evaluated by calculating p-values, which are shown at the right hand side of the illustration.

Nanoceria have also been reported to possess oxidase activity. We therefore examined the oxidase activity of Ti-doped CeO₂ nanoparticles. As Fig. 4A indicates, the oxidase activity of nanoparticles shows a linear relationship with particle concentration. In contrast to our results for SOD mimetic activity, the oxidase activity of nanoceria was not reduced by Ti doping; in fact, a slight increase in oxidase activity was noted (Fig. 4B). For comparison, TiO₂ nanoparticles had no oxidase activity.

Fig. 4.

Oxidase activity of nanoparticles. (A) The linear relationship between the production of the colored product N-ethyl-N-(4-iminocyclohexa-2,5-dien-1-ylidene)ethanaminium and CeO₂ nanoparticle concentration is shown. (B) Comparison of the oxidase mimetic activity of CeO₂, Ti-doped CeO₂ and TiO₂ nanoparticles is shown. To evaluate the statistical significance, p-values were calculated for the comparisons shown.

Our data show that nanoceria and Ti-doped nanoceria, but not TiO₂ nanoparticles, exhibit enzymatic activities. As hypothesized, Ti-doping blunted the SOD-like enzymatic activity of nanoceria. As the detailed mechanism of CeO₂ nanoparticle-mediated SOD catalysis is not certain, the mechanism responsible for its reduction by Ti is also uncertain. Although nanoparticle size does not appear to explain the functional differences noted between CeO₂ and Ti-doped CeO₂, a dramatic difference in shape was noted (Fig. 1). Previous studies have reported that changes in nanoparticle shape can alter nanoparticle catalysis [16, 17]. Thus, nanoparticle shape is a potential contributor to the observed changes in SOD activity. To the best of our knowledge, this is the first demonstration of a means to regulate metal oxide nanoparticle catalytic activity.

In contrast to our observations concerning SOD activity, we found that the oxidase-like activity of nanoceria was not diminished by Ti doping; indeed, the oxidase activity was somewhat enhanced (Fig. 4B). The chemical mechanisms of these two enzymatic activities of nanoceria appear to be fundamentally different. This is supported by the facts that, as mentioned above, Ti doping has dramatically different effects on SOD and oxidase activity. Furthermore, the concentration-dependent differences in SOD activity versus oxidase activity (Fig. 2A and 4A) of undoped nanoceria dramatically differ. Finally, the differences between these two catalytic activities are underscored by the fact that the SOD-activity appears to be sensitive to shape, while the oxidase activity does not.

The reduction in SOD-like activity of Ti-doped nanoceria is important because it provides a means of tailoring the enzymatic activities of inorganic nanoparticles to perform better within complex biological systems. It may be possible to further improve the enzymatic properties of these nanoparticles by altering Ti content or by changing the dopant. Another key advantage of Ti doping is that the nanostructure becomes spherical in shape, which is physically more compatible with a sensitive biological environment. The medical uses of nanoparticles are likely to continue to rise as they become tailored to specific applications.

Abbreviations

SOD

superoxide dismutase

ROS

reactive oxygen species

DPA

N, N-diethyl-p-phenylenediamine sulfate salt