Reactivity of nanoceria particles exposed to biologically relevant catechol-containing molecules

Abstract

The interaction of nanoceria particles with catechol-like molecules of physiological importance, including dopamine, norepinephrine, epinephrine, serotonin, 3,4 dihydroxyphenylaceticacid (DOPAC) and L-3-(3,4-dihydroxyphenylalanine) (L-DOPA) was studied to obtain predictive information of their behavior in biological systems. A suite of complementary techniques including UV-Vis spectroscopy, electrochemistry, dynamic light scattering (DLS), thermogravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FTIR) demonstrated alteration in the spectral, redox and surface properties of nanoceria exposed to these molecules in an aqueous environment. Binding of catechol to the surface of nanoceria diminished the oxidase-like activity of these particles against an organic dye, TMB (3,3,5,5-tetramethylbenzedine), but enhanced their ability to react with and inactivate reactive oxygen species. Therefore, the reactivity of these particles can be modulated by addition of catechol-like molecules. These findings can help develop predictive models of the behavior and potential effects of nanoceria particles in complex environments.

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Introduction

Cerium oxide (CeO₂) nanoparticles, or nanoceria, have been subject to significant scientific interest in recent years due to their catalytic and free radical scavenging properties,¹ attributed to the presence of a dual oxidation state (Ce³⁺/Ce⁴⁺) on the surface. These unique properties have been explored for a variety of applications in the biological field which include, but are not limited to, protection of hippocampus neuron cell line from oxidative stress,² decrease in progression of retinal degeneration,³ reduction of nitric oxide, superoxide and peroxynitrite formation in ischemic cardiomyopathy,⁴ hippocampal brain slices⁵ and ischemic brain.⁶ In addition, these particles have been used as a catalyst and an oxygen carrier in the construction of enzyme-based biosensors,⁷ and as a redox active colorimetric agent,⁸ catalytic amplifier and signal generating probe in bioanalytical assays.⁹ They were also found to act as oxidase,¹⁰ superoxide dismutase and catalase enzyme mimetics.¹¹ In addition to the many promising biological applications, nanoceria is extensively used as a polishing agent in the microelectronics industry and has been employed as a fuel additive to decrease emissions from diesel engines.¹² As a result of these applications, nanoceria particles can be found in a variety of biological systems, where they can interact with components of the environment and change their properties.¹³ In particular, nanoceria can participate in redox reactions, a property related to the co-existence of both Ce³⁺ and Ce⁴⁺ on their surface. Studies in model biological systems have shown potential neurotoxic effects and alteration in the level of serotonin in living systems exposed to nanoceria¹⁴ as well as adsorption of dopamine in human serum.¹⁵ While research is progressing, not much is known about the fundamental parameters driving the interaction of these particles with catecholic molecules, which limits the understanding of their reactivity and potential effects.

Several studies reported surface adsorption and complexation of phenol and catechol on metal oxide nanoparticles. For example, colloidal TiO₂ was found to form stable complexes with benzene derivatives including catechol by displacing the surface-bound hydroxyl groups with the catecholateligands.¹⁶ The surface adsorption significantly altered the interfacial electron transfer at colloidal TiO₂ particles. Surface modification of nanocrystalline TiO₂ with bidentate benzene derivative was reported to change their optical properties due to formation of charge transfer complexes. In another study, dopamine formed bidentate chelates with the metal center of Fe₂O₃ nanoparticles.¹⁷ Similar processes involving adsorption of catechol compounds were reported with MnO₂, Cr₂O₃ ¹⁸ and Al₂O₃.¹⁹ The interaction mechanism involving formation of bidentate and monodentate ligands depends on the type of metal oxide, the chemical structure of the benzene derivative, and the environmental conditions.²⁰ The interaction of nanoceria particles with members of the catechol family has not been studied systematically. Given the increasing use of these particles in many applications assessing this interaction is critical for understanding their reactivity and behaviour in complex environmental and biological media.²¹

Herein, we studied the interaction of nanoceria with a number of catechol-like neurotransmitters including dopamine, serotonin, L-DOPA, DOPAC, epinephrine, norepinephrine and phenylalanine, evaluated their reactivity in relation with the chemical structure and assessed changes in the surface properties of these particles as a result of this interaction. We further investigated whether the observed changes alter the fundamental redox properties of nanoceria that are central to many of their practical applications. This study contributes to further understanding of the interaction of catechol-containing molecules with nanoceria particles, and how this reactivity can be modulated for potential use in practical applications. These findings can also help develop predictive models of the behavior and potential effects of nanoceria particles in complex environments.

Experimental section

Materials and reagents

Dopamine hydrochloride, L-phenylalanine; DL-norepinephrine hydrochloride, TMB (3,3,5,5-tetramethylbenzedine) and cerium oxide(IV), 20 wt% colloidal dispersion in 2.5% acetic acid were purchased from Sigma-Aldrich. 3,4-Dihydroxyphenylacetic acid and L-3-(3,4-dihydroxyphenylalanine) (L-Dopa) were from Acros. Serotonin hydrochloride 99% was obtained from Alfa Aesar. DL-Epinephrine was from MP Biomedicals. Stock solutions of these molecules were freshly prepared before the experiments.

Instrumentation

UV-Vis measurements were carried out with a Schimadzu P2041 spectrophotometer equipped with a 1 cm path length cell. Electrochemical experiments were performed with a CHI660 potentiostat (CH Instruments, Austin, TX) connected to a conventional three electrochemical cell. Screen printed carbon electrodes (DRP 110) from DropSens (Spain) were used for cyclic voltammetric (CV) studies. A Zeta PALS (Brookhaven Instruments) analyzer was used for zeta potential measurements and assessment of the particle diameter. Fourier Transform Infrared Spectroscopy (FTIR) spectra were recorded using a Mattson Galaxy 2020 spectrometer. The thermogravimetric analysis (TGA) was carried out using a Seiko Exstar 6200 TG/DTA analyzer.

Methodology

UV-Vis spectroscopy was used to monitor the optical properties of nanoceria upon interaction with the selected catechol-containing molecules. The spectra were run between 420 and 800 nm. The kinetic study was performed by incubating 1 mg mL⁻¹ nanoceria with 0.5 mM of reacting molecules. CV measurements were performed in the potential range of −0.2 to 0.7 V in the presence of 1.5 mM of test molecules. Particle size measurements were performed with 10 mg mL⁻¹ nanoceria exposed to 2 mM catechol-like molecules. FTIR spectroscopy was used to evaluate the binding and adsorption mechanism on the nanoceria surface. FTIR experiments were performed with 2 mg mL⁻¹ nanoceria particles reacted with 5 mM catechol molecules. Following incubation, the particles were filtered under vacuum using a 10 nm pore size polycarbonate membrane (Osmonics) and washed with DI water to eliminate the excess of weakly adsorbed reagents. The resulting powders were dried under vacuum for at least 48 h. TGA analysis was performed with nanoceria dispersions exposed to reacting molecules. Samples were washed three times with distilled water to remove weakly adsorbed reagents and dried before the TGA analysis.

Experiments to determine the change in oxidant/antioxidant properties of nanoceria functionalized with catechol-bearing molecules were performed using nanoceria particles (1 mg mL⁻¹) pre-treated with catechol or dopamine in the concentration range of 0.5 to 5 mM. The mixture was centrifuged for 30 min and the supernatant was removed to discard the unbound molecules. These experiments were performed with dopamine selected as example. The pellets were re-dispersed in distilled water. To perform experiments with TMB, 1 mL of re-dispersed treated particles were transferred to UV-vis cuvettes. TMB at a concentration of 0.05 mg mL⁻¹ was added to the solution to develop the blue color. The UV-vis spectra after addition of TMB was recorded in the wavelength range between 580 and 780 nm.

The superoxide dismutase (SOD) activity of nanoceria was determined by quantifying the ability of these particles, and their catechol-treated congeners to inactivate enzymatically generated superoxide radicals. The inactivation of superoxide radicals with and without particles was determined using a cytochrome c reduction based-colorimetric test as described previously.³³ In this study, 0.05 mM hypoxanthine (HX), 3.3 mU mL⁻¹ xanthine oxidase and 0.01 mM cytochrome c were used. The kinetics data were recorded using a UV-2401PC UV-Vis spectrophotometer (Shimadzu, Japan). All samples included 50 U mL⁻¹ catalase to eliminate residual H₂O₂ which may react with nanoceria and cytochrome c. The SOD activity assay was performed in 0.1 M PBS at pH 7.5.

Results and discussion

Selection of catechol-containing molecules

Fig. 1 shows the chemical structures of the molecules investigated in this study. Five of these compounds have been selected as representative catechol-containing molecules of biological significance: dopamine, norepinephrine, epinephrine, DOPAC and L-DOPA. In addition to the catechol ring, these compounds differ by their substituents to the benzene ring and include COOH or NH₂. Catechol is known to have high affinity for metal oxide surfaces²² via complexation of the hydroxyl (OH) groups with metal atoms.²³ Despite its high pK_a values, catechol can form strong coordination bonds with metals both in acidic and basic pH solutions.²⁴ Phenylalanine possessing a benzyl ring but lacking OH groups was also tested to determine the role of catechol chemistry. For comparison, serotonin that contains an indole nucleus with a OH group in the 5 position and a primary amine was also investigated. In addition to these molecules a larger class of monoaromatic benzoic compounds such as phenol, catechol, bisphenol A and hydroquinone, as well as more complex polynuclear aromatic compounds such as naphthol, naphtyl phosphate and ochratoxin A, were also investigated to determine the effect of geometry, steric orientation and the contribution of additional substituents such as phosphate to the binding and reactivity mechanism. The structure of these compounds and a summary of their stability and physicochemical properties are listed in Table S1 in the (ESI†). We have used commercially available nanoceria particle dispersion in acetic acid with a particle size of 17.5(±1.1) nm and a zeta potential value of +34.5(±3.4) mV that was characterized by high reactivity in our previous work.¹⁵

Fig. 1 Structure of primary catechol-bearing molecules selected as model compounds to study nanoceria reactivity.

Reactivity of nanoceria against catechol-containing molecules

To investigate the reactivity of nanoceria against catechol-like molecules and aromatic benzoic compounds with varying degrees of substitution we first characterized the optical properties of these particles in the absence and presence of these molecules using UV-vis spectroscopy. The incubation of nanoceria with catecholic molecules resulted in an immediate color change of the nanoceria dispersion from colorless to dark brown. It is known that nanoceria shows a distinctive peak with a maximum at ∼300 nm corresponding to the band gap of CeO₂ due to the charge transfer between O_2p and Ce_4f in O₂⁻ and Ce⁴⁺.²⁵ Addition of molecules with a catechol moiety to the nanoceria dispersion increased the maximum absorption peak at 300 nm. Simultaneously, strong peaks in the visible range were observed at ∼470–550 nm with variable intensities depending on the substituents in the catechol ring. The color intensity varied in the order: DOPAC > L-DOPA > epinephrine > norepinephrine > dopamine. These molecules contain a catechol moiety but have different p-substituents. The incubation of nanoceria with serotonin did not show an immediate color change. For this compound, a brownish color of lower intensity was observed following 60 min incubation. No change in the spectra of nanoceria was observed upon exposure to phenylalanine, which lacks OH groups on the benzyl ring, suggesting that these groups may be involved in the reactivity and binding mechanism of this class of compounds. The observed spectral changes are similar with those reported previously in studies of TiO₂ with catechol-type derivatives that suggested formation of charge transfer complexes involving the catechol moiety.²⁶ Fig. 2 shows comparative absorption spectra of nanoceria in the absence and presence of catecholic molecules. The spectral changes indicate alteration in the surface properties of these particles induced by these molecules.

Fig. 2 (A) Changes in the optical properties of nanoceria particles (5 mg mL⁻¹) upon incubation with 0.1 mM, (a) only ceria, (b) phenylalanine, (c) serotonin, (d) dopamine, (e) L-DOPA, (f) DOPAC, (g) epinephrine, (h) norepinephrine; (B) corresponding UV-Vis spectra.

Mechanistically, these spectral changes can be attributed to a cumulative impact from deprotonation of the OH groups along with formation of charge transfer complexes on the nanoceria surface.^8,14,15 Since nanoceria is a redox active material with a dual oxidation state (Ce³⁺/Ce⁴⁺), an oxidative reaction in which the catechols are first oxidized by nanoceria to their respective quinones is also possible. Such oxidative pathways involve reactive phenoxy radicals that can further react with and rapidly bind to the nanoceria surface. This binding along with the oxidation process are responsible for the strong color change. These results also indicate that catechol-like molecules can be used to functionalize nanoceria particles and modulate their optical properties.

In order to differentiate the contribution of the oxidation process and identify the effect of the OH/catechol groups to the overall reactivity, we investigated the interaction of nanoceria with a broader range of OH-containing molecules that lack a catechol ring and/or are electrochemically stable, including phenylalanine, okadaic acid, p-nitrophenylphosphate, ascorbic acid–phosphate and 1-naphthyl phosphate (Table S1†). When these compounds were reacted with nanoceria, there was no change in the optical properties. These results indicate that the presence of OH groups or a simple deprotonation process is not solely responsible for the observed optical changes. We further investigated the electrochemical behavior of these compounds and correlated the spectral changes with the oxidation potential. Fig. S1 in the ESI† section shows CVs representative of the electrochemical oxidation of the molecules that displayed the highest reactivity against nanoceria. The chemical structures, relative oxidation potential and the color intensity upon reaction with nanoceria are summarized in Table S1.† A correlation between the oxidation potential and the color intensity changes of the nanoceria dispersion was observed. Molecules with a lower potential generated a rapid and more intense change in the optical properties of the particles, as compared to those with higher potential. L-DOPA, DOPAC, epinephrine, norepinephrine and dopamine induced the strongest color intensity change. For these compounds, the reaction was almost instantaneous. Fig. S2 in ESI† shows the correlation between the extinction coefficient and the oxidation potential of these compounds. The extinction coefficient decreases as the oxidation potential increases which can be explained by a faster oxidation rate and complex formation between nanoceria and the more reactive and easily oxidizible compounds. We have further investigated the effect of pH and buffer conditions on this interaction using acetate and phosphate at varying pH values, with L-DOPA as a model molecule (Fig. S3†). Results showed that the reaction takes place in both acetate and phosphate buffer conditions but with varying levels of sensitivity. Higher reactivity was observed in distilled water as compared to acetate buffer. It is known that both acetate and phosphate can bind to nanoceria further affecting surface reactions. Nevertheless, these results indicate that reactivity is conserved in the presence of acetate and phosphate over a wide range of pH values. Further experiments to test nanoceria reactivity against catechol-containing molecules were performed in distilled water, which provided strong reactivity.

Fig. 3 shows the kinetic behavior of the reaction of nanoceria with catecholic molecules. High absorbance values are observed within seconds with a maximum absorbance achieved after 3 min, with the exception of serotonin which was characterized by slow kinetics, with a maximum absorbance being reached after 60 min. No change in absorbance was seen for phenylalanine even after an incubation period of 60 min, indicating non-reactive behaviour of this molecule against nanoceria. Easily oxidizible compounds such as catechol, ascorbic acid, 1-naphthol and hydroquinone generated very intense color changes upon reaction with nanoceria. On the other hand, molecules with higher oxidation potentials such as phenol, bisphenol a and ochratoxin a showed smaller intensity changes. The difference between catechol and phenol clearly indicates higher reactivity of nanoceria for catechol as compared to phenol-bearing molecules. The enhanced color change can also be related to formation of bidentate (for catechol) as compared to monodentate (for phenol) complexes. Molecules lacking a catechol or phenol moiety did not produce any change in the optical properties of nanoceria. Examples of non-reactive molecules include: phenylalanine, okadaic acid, p-nitro-phenyl phosphate, ascorbic acid–phosphate and 1-naphthyl phosphate.

Fig. 3 (A) Kinetic study of the nanoceria interaction (1 mg mL⁻¹) with 0.5 mM, (a) phenylalanine, (b) serotonin, (c) dopamine, (d) norepinephrine, (e) epinephrine, (f) L-DOPA, (g) DOPAC. (B) Illustration of surface binding of nanoceria with L-DOPA, dopamine, DOPAC, epinephrine and norepinephrine (from top to bottom in that order).

To further investigate whether the color formed is a result of the reaction of nanoceria with the product of the oxidation reaction, rather than intermediate oxidative products, we exposed the particles to a model quinone, benzoquinone. It is known that the products of the oxidation reaction of phenolic and catechol compounds are quinones. No change in the optical properties of nanoceria was observed upon exposure to benzoquinone, indicating that both oxidation by nanoceria and surface attachment of the oxidized products are responsible for the observed color changes (Fig. S4†). To further verify formation of an organic layer on the surface of nanoceria and demonstrate that the color change is indeed related to surface attachment, we have performed a series of electrochemical and spectroscopic experiments to assess the presence of these molecules to the particle surface. When solutions of catechol molecules were incubated with nanoceria, the particles formed a highly stable and visible brown-colored dispersion, as shown in Fig. 2. When the reaction mixture was sonicated for 20 min and centrifuged for 30 min, the colored particles precipitated at the bottom of the vial, as shown in Fig. 4. The residual color of the supernatant is due to smaller suspended particles that are difficult to settle by centrifugation. By comparison, nanoceria particles alone and particles exposed to phenylalanine showed no color change. The results suggest that the reaction is occurring on the particle surface leading to coverage of the particle surface.

Fig. 4 Visual characterization showing precipitation of nanoceria particles (1 mg mL⁻¹) in the presence of 5 mM of: (a) DOPAC, (b) norepinephrine, (c) dopamine, (d) L-DOPA, (e) epinephrine, (f) serotonin and (g) phenylalanine.

Additional evidence was obtained by performing electrochemical studies of these molecules before and after exposure to nanoceria. Fig. 5 shows CVs of the six most reacting catecholic compounds in the absence and presence of nanoceria. The voltammograms clearly show changes in the oxidation and reduction waves of these compounds after exposure to particles. For all compounds, the oxidation peaks decreased significantly. In the same time a new broader peak appeared at a lower potential together with an increase in the reduction peak. The absorption spectra of L-DOPA exposed to nanoceria showed no change before and after application of the electrical potential which indicates that the observed changes are a result of a chemical process (Fig. S5†). These changes suggest a rapid nanoceria-induced oxidation and conversion of these compounds into oxidative metabolites which could attach to the particle surface and alter their reactivity.

Fig. 5 CVs obtained in the presence (grey line) and absence (black line) of nanoceria particles for (A) dopamine, (B) L-DOPA, (C) serotonin, (D) DOPAC, (E) epinephrine and (F) norepinephrine. All experiments were performed with 1 mM of reacting molecules and 2 mg mL⁻¹ nanoceria.

The presence of an organic layer to the nanoceria surface was further characterized by measuring changes in the particle size before and after incubation with the catechol-bearing molecules. The bare water-dispersed nanoceria particles have an average size of 17.5(±1.1) nm. The particles exposed to the most reactive molecules showed an increase in the particle size (Fig. 6), with the highest increase for DOPAC, L-DOPA and epinephrine. A lower increase was observed for compounds which showed smaller optical changes in the UV-Vis measurements. A small increase was also observed upon incubation with phenylalanine due to physical adsorption. The increase in the particle size demonstrates binding of these molecules to the nanoceria surface.

Fig. 6 Changes of particle size of the nanoceria (10 mg mL⁻¹) upon incubation with catechol based molecules (2 mM).

The surface attachment was further confirmed by TGA analysis (Fig. 7). Standard samples of the selected molecules exhibit a first sharp decomposition event in the 200–300 °C range, corresponding to the thermal decomposition of these compounds, and a second broad weight loss at 380 °C due to the combustion of the remaining organic matter. The negligible weight loss in the TGA curve of nanoceria corresponds to the elimination of physisorbed water. The weight loss between 200 and 300 °C observed for particles exposed to catecholic compounds after washing and drying demonstrates presence and strong attachment of these molecules. A negligible weight loss was observed upon incubation with phenylalanine which may be due to physical adsorption rather than chemical complexation. Zeta potential experiments of coated particles showed a slight decrease in the zeta potential value from +34.5(±3.4) to +28 and +30 in the presence of dopamine and catechol respectively. These experiments provide additional evidence confirming formation of an organic corona of catechol derivatives at the nanoceria particle surface.

Fig. 7 TGA analysis of: (a) nanoceria particles, (b) standard sample of the test catechol-based molecules, and (c) nanoceria exposed to the catechol-containing molecule.

FTIR spectroscopy was further used to determine the nature of the functional groups involved in the binding of these molecules with nanoceria. L-DOPA was selected as a representative molecule. L-DOPA contains two OH groups bonded to the adjacent carbon atoms of the aromatic ring as well as a carboxylic and an amino group on the side chain. These functional groups are typically associated with the adsorption or complex formation of molecules on surfaces. Fig. 8 shows the FTIR spectra (650–1900 cm⁻¹) for nanoceria particles, L-DOPA, and nanoceria exposed to L-DOPA. The FTIR spectrum of L-DOPA was run on L-DOPA powder used as received. The spectra of the colloidal nanoceria dispersion and the mixture of L-DOPA with nanoceria were run on washed and dried colloid samples. The peaks observed between 650 cm⁻¹ and 700 cm⁻¹ are assigned to the stretching vibration of Ce–O in ceria. The band at 870 cm⁻¹ corresponds to the out-of-plan wagging modes of hydrogen atoms of the OH groups in the catechol ring of L-DOPA. The disappearance of this band in the spectra of the L-DOPA modified nanoceria is direct evidence that OH groups of the catechol ring are involved in the surface attachment through a bidentate bridging. Similarly, the region between 1150 cm⁻¹ to 1170 cm⁻¹ and the band at 1250 cm⁻¹ could be assigned to the stretching vibration of HO–C–C–OH. The absence of these stretching modes in the L-DOPA modified nanoceria indicates that these groups are conjugated to the particles surface. The band at 1798 cm⁻¹ in L-DOPA is assigned to C–O stretching in the carboxylate group. The band at 1446 cm⁻¹ is associated to the rocking vibration of NH₂ group. The presence of these modes in the spectra of L-DOPA modified nanoceria suggests that attachment takes place though the OH groups of the catechol ring, and not by the other functional groups.

Fig. 8 FTIR spectra for: (a) L-DOPA, (b) nanoceria particles and (c) L-DOPA modified nanoceria particles.

Effect of surface complexation on nanoceria reactivity

These results support the hypothesis of an oxidation followed by surface chelation of catechol-based molecules with nanoceria. These changes potentially alter the functional surface properties and the reactivity of these particles, which are used in a large number of applications. Therefore, in the next set of experiments, we evaluated the reactivity change of the catechol-functionalized nanoceria, as compared to nanoceria alone, using two systems of interest for the practical implementation of these particles. In the first set of experiments, we studied whether surface modification of nanoceria with catechol ligands altered the oxidation ability of nanoceria against an organic dye, TMB, a mechanism that is used in the development of nanoceria based-sensors.²⁷ In the second set of experiments, we studied changes in the superoxide dismutase activity of these particles, a property that is explored to design nanoceria-based therapeutics for the treatment of oxidative stress related disease.^5,6,28 Particles exposed to catechol and dopamine were used in these tests to determine surface reactivity changes.

Evaluation of the oxidase-like activity against TMB

TMB was used to determine changes in the oxidizing ability of the nanoceria particles upon binding of catechol and dopamine (Fig. 9). The oxidation of TMB by nanoceria is an established reaction known to yield a blue colored product.¹⁰ The nanoceria particles used in this work showed strong oxidation ability against TMB. This can be seen by an increased blue color intensity of TMB upon addition of these particles with a maximum absorbance at 620 nm. In the same set of experiments, the use of nanoceria exposed to catechol or dopamine decreased the blue color intensity in a concentration dependent manner, as compared to bare nanoceria. This suggests that surface coating by catechol molecules blocks the redox active sites of these particles and diminishes their oxidation ability against TMB. Such blocking effect was also proposed previously using DNA on nanoceria.^29,30 These experiments demonstrate that coating of nanoceria affects the reactivity and surface properties of these particles. This property could be used to design sensing schemes based on reactivity changes at the surface of these particles, as we have demonstrated in our recent work for detection of dopamine³¹ and phosphatase activity³² or, for label free detection of clinical and food analytes when used in conjunction with biomolecular recognition elements.^27b,33 As an example, this assay could be used to detect dopamine and catechol, by measuring the decrease of TMB oxidation with varying concentrations, as shown in Fig. 9B and D. These results demonstrate that it is possible to modulate nanoceria reactivity, to either activate, or inactivate chemical reactions at the surface. For example, pre-coating nanoceria can prevent reactivity and possible adverse responses of these particle in biological environments.

Fig. 9 The effect of nanoceria particle interaction with catechol (A and B) and dopamine (C and D) on the oxidation of TMB. (A and C) Spectra of nanoceria samples and controls showing oxidation of TMB by (a) catechol/dopamine (5 mM), (b) nanoceria and (c) nanoceria pre-treated with catechol/dopamine (5 mM). (B) Concentration dependent effect on TMB oxidation in presence of: (a) catechol/dopamine (5 mM) in absence of particles (b) nanoceria particles alone; and nanoceria exposed to varying concentrations of catechol/dopamine of (c) 0.5 mM (d) 1 mM, (e) 2.5 mM and (f) 5 mM. TMB concentration in all tests was 0.05 mg mL⁻¹. Nanoceria concentration was 1 mg mL⁻¹.

Evaluation of the superoxide-scavenging properties

Previous work showed that nanoceria particles are capable of scavenging free radicals including nitric oxide and superoxide and protect against free radical mediated stress in a variety of disease models.^5,6,34,35 Here we investigated whether chelation of the nanoceria surface by catechol-bearing molecules alters the ability of these particles to inactivate superoxide radicals. A spectroscopic SOD-mimetic activity assay was used to demonstrate the superoxide scavenging activity. Spectroscopic measurements of cytochrome c reduction by enzymatically generated superoxide were used to indirectly quantify the amount of superoxide before and after exposure to nanoceria. Fig. 10 shows a decrease in the absorbance intensity value in presence of nanoceria, indicating a decrease in the enzymatically produced superoxide, which demonstrates that these particles inactivate superoxide, equivalent to a SOD-mimetic activity. In the same set of experiments, addition of the same amount of nanoceria particles that have been exposed to catechol or dopamine induced an even stronger decrease in absorbance, suggesting enhanced scavenging activity. The enhanced SOD mimetic activity of nanoceria was observed only in the presence of surface-bound catechol and dopamine. These experiments demonstrate that functionalization of nanoceria with catechol-bearing molecules enhanced the ability of these particles to react with and inactivate superoxide, making them more powerful antioxidants. This finding could be of great interest in the biomedical field for scientists who are interested in exploring these particles for the treatment of oxidative stress-related diseases. Additionally, since dopamine and other catechol-containing molecules are present in biological systems, these results also show that the interaction of these molecules to nanoceria particles can change their scavenging activity, a process that could potentially impact the therapeutic efficacy of these particles.

Fig. 10 Superoxide scavenging activity of nanoceria particles exposed to 1 mM catechol (A) or 5 mM dopamine (B) as compared to the same amount of nanoceria. Control refers to enzymatically generated superoxide in the absence of nanoceria and catechol/dopamine.

Conclusions

In summary, we have studied the interaction of nanoceria particles with biologically relevant catechol-bearing molecules, with a primary focus on catecholamine neurotransmitters. Understanding these interactions is of great practical importance, as the oxidant/antioxidant properties of these materials are explored in a broad range of biomedical, sensing and industrial applications. Spectroscopic and electrochemical investigations showed that catechol-containing molecules altered the surface properties and reactivity of nanoceria. These molecules attach to the particle surface, forming an organic corona through the catechol's hydroxyl groups. This knowledge and the structure–activity relationship could be used to predict the behavior and transformation of these particles in complex environments. Changes in surface properties altered the ability of these particles to react with an organic dye, TMB and enhanced their superoxide scavenging activity. This work also demonstrates that catechol-like molecules can be used to functionalize nanoceria particles with organic ligands, and modulate the optical properties of these particles. The study demonstrates the need to carefully evaluate changes in nanoceria reactivity upon exposure to complex environments containing such molecules.

Acknowledgements

This work was supported by NSF # 0954919 grant. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the funding agency.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra07279h

‡ These authors have equal contribution.

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