Inhibition of Nanoceria's Catalytic Activity due to Ce3+ Site-Specific

Aug 1, 2014 - Nanoscience and Technology Center (NTSC), University of Central Florida (UCF), 4000 Central Florida Blvd, Orlando, Florida. 32816, Unite...
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Inhibition of Nanoceria’s Catalytic Activity due to Ce3+ Site-Specific Interaction with Phosphate Ions Rameech N. McCormack,†,∥ Priscilla Mendez,‡,∥ Swetha Barkam,§,∥ Craig J. Neal,‡ Soumen Das,∥,⊥ and Sudipta Seal*,§,∥,⊥ †

Department of Mechanical and Aerospace Engineering (MAE), ‡Department of Molecular Biology and Microbiology (M&M), Department of Material Science and Engineering (MSE), ∥Advanced Material Processing and Analysis Center (AMPAC), and ⊥ Nanoscience and Technology Center (NTSC), University of Central Florida (UCF), 4000 Central Florida Blvd, Orlando, Florida 32816, United States §

ABSTRACT: Cerium oxide nanoparticles (CNPs) exhibit superoxide dismutase (SOD) and catalase mimetic activities. Therefore, based on its catalytic activities, CNPs can potentially be used to treat diseases associated with oxidative stress. The potency of CNPs can be hindered by ion interaction due to chemical modifications. The issue is that phosphate ions are relatively ubiquitous in all biological relevance medium and body fluid. Our ventures in this study were to understand the phosphate ion interaction and fabricate CNPs that are biocompatible and simultaneously retain their catalytic properties in the presence of phosphate ions. CNPs were coated with polyethylene glycol and dextran in order to enhance biocompatibility. A series of experiments determined that maximizing the preserved catalytic responses were highly dependent on the Ce3+:Ce4+. Results have shown that the particles engineered with higher concentrations of Ce4+ on the surface are more robust and retain catalytic activity post buffer exposure.

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oxide radical scavenging,18 superoxide dismutase mimetic (SOD),19 and catalase mimetic activity.20 The catalytic performance of CNPs is highly dependent on its surface physicochemical properties. The correlation between surface Ce3+/Ce4+ oxidation state ratios and CNPs catalytic activity has been well established.21 CNPs with higher surface 3+ oxidation state are SOD mimetic, whereas surfaces with higher 4+ oxidation state are more catalase mimetic.4 Therefore, nanoparticles engineered for catalytic/antioxidant activity need to have the ability to retain their surface physiochemical properties for maximum efficiency. Several factors affect nanoparticles’ catalytic property in biological environment. Among these factors, ion interactions with the surface of nanoceria can affect the catalytic potency by neutralizing antioxidant mimetic reactive sites. This neutralizing mechanism could be due to ion adsorption or formation of a coordination compound (complex). Either of these mechanisms alter CNPs’ surface chemical states and hence its catalytic response. Cells are dependent on the acid−base homeostasis for continuous stabilization of pH. Therefore, all biological medium and body fluids have a buffering capacity. These buffer systems often determine cellular fluid uptake or release

INTRODUCTION In recent years, nanoparticles have become a viable therapeutic and diagnostic agent. Some prominent endeavors using nanoparticles include imaging, targeting, and systematic drug delivery. Cerium oxide nanoparticles (CNPs, nanoceria) are attracting researchers from fields of nanomedicine and pharmacology due to its unique antioxidant property. There are numerous applications of CNPs in biomedical research; whereby CNPs are used to reduce oxidative stress.1,2 In addition, CNPs have been shown to be effective in retinal protection,3,4 enhancing tissue regeneration,5−7 and treating neurodegenerative diseases.4,8,9 CNPs have also proven to exhibit properties such as ultraviolet shielding10 and radioprotection,11,12 and act as an analgesic to reduce inflammatory response of external stimulants.13 The redox activity of the CNPs is facilitated by the metal ions ability to switch between 3+ and 4+ oxidation states. The proposed switching mechanism between Ce3+ and Ce4+ is attained through manipulating the oxygen vacancy concentration in CNPs’ lattice.14,15 The catalytic property of the CNPs is a consequence of the ease at which oxygen atoms can be extracted from (or donated to) the lattice.16 Switching between oxidation states enables CNPs to exhibit unique antioxidant mimetic properties. The redox mechanism of CNPs has proven to protect cells against oxidative stress by scavenging excess reactive oxygen species (ROS).17 This redox mechanism of CNPs enables it to mimic potent antioxidants such as nitric © 2014 American Chemical Society

Received: January 23, 2014 Revised: July 24, 2014 Published: August 1, 2014 18992

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Figure 1. HRTEM images with inset SAED image of CNPs. (a) WB1 with SAED patterns and particle of 3−5 nm range. (b) PEG-CNPs sample with a particle size distribution ranging from 5 to 8 nm. (c) DEX-CNPs particles with a size variation from 3 to 5 nm. (A, B, C, D are the 111, 200, 220, and 311 lattice planes).

were then added to the CNPs in a 4:1 (buffer to particle) molar ratio in order to analyze the particle−ion interaction. The particles were incubated with the solution for a period of 24 h, and ions were removed by either dialysis or multiple centrifugation before analysis. Methods of Characterizing CNPs. The ζ-potential and electrolytic conductivity of the particles were analyzed through a process of dynamic light scattering (DLS) using a Zetasizer, Nano Series, Malvern Instrument. The UV−vis absorbance of the particles were analyzed using a Lambda 750S Spectrophotometer from PerkinElmer. The photoluminescence (PL) properties of the particles were analyzed using a F-7000 Fluorescence Spectrophotometer from Hitachi High Technologies America. High-resolution transmission electron microscopy (HRTEM) was conducted using FEI Technai F30 TEM, with a 0.2 nm resolution, at a potential of 300 kV. The TEM samples were prepared by dropped casting suspension onto the surfaces of holey carbon grids. The oxidation states of the particles were obtained using a 5400 ESCA system (X-ray photoelectron spectrophotometers, XPS) from Phi Electronics, which incorporated an unmonochromatized Mg Kα X-ray source (1253.6 eV). XPS samples were analyzed at high vacuum, with a maximum internal pressure of 5 × 10−8 Torr. Another XPS (Phi Electronics’ VersaProbe) with a monochromatic Al Kα X-ray source was used in order to characterize the surface chemistry of the coated particles after sputtering with C60 ion gun. Analysis of CNPs’ Catalytic Properties. The catalase mimetic activity was assessed in order to quantify CNPs’ ability to scavenge hydrogen peroxide (H2O2). The catalase experiments were conducted using an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Life Technology, cat no. A22188). SOD mimetic activity, the capability to scavenge superoxide free radical, was analyzed using an SOD assay kit (Sigma-Aldrich, kit no. 19160−1KTF).25 Deionized water was used as the control for both catalase and SOD experiments.

depending on ion concentrations. In our prior study, phosphate ions have been shown to alter the catalytic activity of cerium oxide nanoparticles.22 Phosphate ions are abundant in biological mediums, and oftentimes, phosphate buffered salines (PBS) are used for administering drugs. Therefore, we have selected PBS as our medium in order to monitor the interaction between the surface of nanoceria and phosphate ions. In this study, it has become apparent that CNPs have a means by which they can bypass the interaction with the anions without neutralizing its catalytic potency. The complexity of our endeavor lies within the fact that one has to circumvent CNPs−ion interaction and retain its nontoxic and antioxidant properties. Therefore, this study focuses on preventing the surface modification of CNPs due to phosphate anion interaction by engineering CNPs with enhanced biocompatibility and similar catalytic mimetic activity as bare CNPs. In this endeavor, CNPs’ surfaces were coated with dextran and polyethylene glycol (PEG), in order to enhance nanoparticle biocompatibility. The catalytic responses such as SOD and catalase mimetic activity were then analyzed in order to compare the effects of ion interaction on the particles.

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EXPERIMENTAL SECTION Preparation of Nanoparticles and Coating. The method used to synthesize CNPs in polymeric media such as PEG (PEG-CNPs) is synonymous to that performed by A. Karakoti et al.23 The PEG-CNPs were coated with 300 Da molecular weight polyethylene glycol. Synthesis of dextran coated CNPs (DEX-CNPs) was completed in accordance to that described by L. Alili et al.2 The DEX-CNPs particles used in these experiments were coated with 1000 Da molecular weight dextran polymers. Bare nanoparticles are designated as Water Based 1 (WB1) and 2 (WB2) and were synthesized by two different methods. WB1 particles were prepared according to the synthesis procedure describe by Hirst et al.,24 and WB2 particles were synthesized by the addition ammonium hydroxide to an aqueous solution of cerium nitrate while the solution was stirred. Phosphate Ion Treatment. A 50 mM aqueous solution of disodium hydrogen phosphate (Na2HPO4) and a 50 mM aqueous solution of sodium dihydrogen phosphate (NaH2PO4) was made, prior to treating the CNPs. These solutions were combined in a 1:1 ratio to obtain a solution of phosphate ion pseudo buffer of pH 7. Equivalent quantities of buffer solution

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RESULTS AND DISCUSSION Size and Crystallinity Determination Using HRTEM Analysis. HRTEM was used to analyze size and morphology of the synthesized nanoparticles. In Figure 1a, the WB1 sample shows CNPs’ small crystallite sizes ranging from ∼3 to 6 nm. The selected areas electron diffraction (SAED) pattern obtained in the TEM analysis of the WB1 sample indicates the presence of 111, 200, 220, and 311 lattice planes. This SAED 18993

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Figure 2. UV−vis absorbance spectra of bare CNPs and polymer coated CNPs. (a) WB1 and treated WB1, with peaks at 251 and 300 nm, which correlates to Ce3+ and Ce4+, respectively. The 271 nm peak in the treated WB1 is not typical of CNPs. (b) PEG-CNPs and incubated PEG-CNPs with peaks positioned at 252 and 300 nm. (c) Treated and untreated WB2 particles with peak absorptions at 296 nm (Ce4+). (d) DEX-CNPs with peak absorption at 283 nm for the treated and untreated sample. (e) Cerium phosphate with peaks at 259 and 277 nm.

pattern confirms the cubic fluorite structure of CNPs. In Figure 1b, relatively larger crystal sizes of ∼5−8 nm were obtained in the TEM analysis of PEG-CNPs. In addition, PEG-CNPs’ prominent SAED pattern shows the 111, 200, 220, and 311 indicated rings, hence the formation of cerium oxide. Figure 1c also shows a similar SAED pattern to that of cerium oxide and the size distribution of the DEX-CNPs ranges from ∼3−5 nm. The size distribution of the WB2 CNPs synthesized varied from ∼8 to 10 nm. Absorption Characteristics Post Ion Interaction. The UV−vis absorbance spectra of WB1, PEG-CNPs, and DEXCNPs before and after phosphate ion incubation are shown in Figure 2.

Particles were treated with phosphate ions in a 4:1 molar ratio. Comparative spectra of WB1 versus treated WB1 can be observed in Figure 2a. Figure 2a shows that WB1 has peak absorptions at 251 and 300 nm which prior research has assigned to Ce3+ and Ce4+, respectively.26,27 Once WB1 particles were exposed to phosphate ion, their maximum absorbance was at peak 271 nm. The peak shoulders of Ce3+ and Ce4+ can still be observed (Figure 2a) in treated WB1 sample. Figure 2b shows the UV−vis absorption of PEG-CNPs and treated PEG-CNPs. The untreated sample showed similar absorption pattern to that of WB1, while treated PEG-CNPs samples exhibited the highest absorbance at 273 nm due to ion interaction. Figure 2c shows that the WB2 particles have an 18994

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Figure 3. PL spectra of bare CNPs and polymer coated CNPs samples at room temperature. (a) Treated and untreated WB1 samples show a prominent peak at 349 nm for their major emission. (b) PEG-CNPs treated and untreated with respective peaks emission at 344.4 and 349.2 nm. (c) WB2 CNPs treated and untreated show no emissions. (d) DEX-CNP treated and untreated samples are showing no emissions. (e) Cerium phosphate spectrum as reference with no PL property.

absorption peak position at 296 nm for both treated and untreated WB2 samples. Figure 2d shows the absorption spectra of DEX-CNPs. In Figure 2d, one can observe the absorption peak positioned at 283 nm for both treated and untreated dextran nanoparticles; the spectra is similar to that of treated and untreated WB2 samples. Figure 2e shows absorbance spectra of a colloidal suspension of cerium phosphate (cerium(III) phosphate, 99% min from Alfa Aesar)

in water. The cerium phosphate was not nanostructured in nature and showed peaks absorption at 277 and 255 nm. Interpretation of UV−vis Absorption Spectra. The peak at 271 nm in Figure 2a, after treatment of WB1, is indicative of the modification of the electronic structure of the particles. The emergence of the 271 nm peak after treatment proves the ion interaction with CNPs. The retention of the absorbance at ∼251 and 300 nm is an indication that quantities of Ce3+ and Ce4+ still exist. These results clearly indicate that 18995

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Figure 4. (a) The ζ-potential of PEG-CNPs and DEX-CNPs at pH 7. After treatment of the particles with phosphate ions, PEG-CNPs shows a significant change in ζ-potential as the ion concentration increases. (b) The electrolytic conductivity of PEG-CNPs and DEX-CNPs suspension with increase in phosphate ion concentration. (The shaded region is the standard deviation or error band of the respective plots).

Figure 5. Selective high-resolution XPS spectra analysis of cerium phosphate (CePO4). (a) Cerium’s 3d orbital spectrum with prominent Ce3+ peaks at 880.0, 885.0, 899.2, and 903.4 eV. (b) P 2p3/2 spectrum with a peak position at 133.5 eV. (c) O 1s spectrum with numerous oxygen species. The combined regions show the typical spectra of cerium phosphate.

It has been established that the absorption peak shifts of nanoparticles are often associated with variations in the particle size due to the electron energy transitions linked to quantum confinement. This very principle is attributed to the cerium phosphate sample absorbing at a much higher wavelength of

CNPs’ surface is being partially modified into cerium phosphate. Figure 2b also exhibits the same behavior as treated and untreated WB1 particles, showing that the PEG coating is not preventing phosphate ions’ interaction. In Figure 2c, the WB2 sample shows a lower absorption at the Ce3+ wavelength. 18996

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Figure 6. X-ray photoelectron spectroscopy of the WB2 pre and post ion incubation. The O 1s spectrum of (a) treated and (b) untreated WB2. There exists a significant shift in the asymmetry in the O 1s peak upon treating the WB2 sample. (c) Ce 3d spectral emission from treated WB2 samples. The emission envelope of the treated WB2 sample is not archetypal of CNPs. (d) The Ce 3d spectrum for the untreated WB2.

Emission Characteristics Post Ion Interaction. The excitation wavelength used in the PL analysis for all the nanoparticles in the study was 250 nm. In Figure 3a, the comparative spectra of WB1 treated and untreated particles are shown. Figure 3a shows that WB1’s major peak emissions is 349.8 nm, but upon treatment, the emission is quenched. In Figure 3b, the spectra of treated and untreated PEG-CNPs’ emissions are similar to that of treated and untreated WB1 samples, respectively. Untreated PEG-CNPs’ emission is most intense at 349.2 nm, while treated PEG-CNP experiences a hypsochromic (blue) shift (from 349.2 to 344.4 nm) and a quenched emission. In Figure 3c, the spectra of WB2 treated and untreated show no photoluminescent emission. Similarly, DEX-CNP treated and untreated spectra show no emission peak (Figure 3d). On the other hand, cerium phosphate also had no PL property (Figure 3e). Elucidating PL Emissions Spectrum Characteristics. PL of ceria is predominately due to charge transitions between Ce4+-O2− and interaction of Ce3+ ions’ with ligands.28,29 Therefore, the shifts observed in the spectra are solely due to changes in ligand interaction with cerium atoms. The quenching of the emission in Figure 3a at 349 nm, subsequent to ion incubation, is evidence that ions are in fact chemically reacting with the Ce3+ atoms. Figure 3b shows that PEG-CNPs have a similar emission as that of WB1 particles, where after the introduction of phosphate ions, a slight hypsochromic (blue) shift and quenching at 344.4 nm occurs due to ion interaction. It can be observed in Figure 3c, treated and untreated WB2 show no observable emissions. In Figure 3d, DEX-CNP, similar to WB2, shows no observable emission peaks prior and post treatment. This is attributed to the fact that DEX-CNP and WB2 have a greater concentration of Ce4+, which minimizes phosphate ion interaction. Particle Interaction by ζ-Potential and Electrolytic Conductivity. The ζ-potential of PEG-CNPs and DEX-CNPs were investigated in order to understand the effect of ion interaction on particles. In Figure 4a, the ζ-potential of PEGCNPs is initially positive, but when exposed to higher concentrations of phosphate ions the ζ-potential changes to a

Figure 7. XPS spectral line of P 2p3/2 region of WB2. (a) Treated and (b) untreated WB2. Treated WB2 shows weak emissions from the region, which indicates the presence of negligible amount of phosphorus.

277 nm in Figure 2e, than absorbing at 271 nm, as observed in WB1 and PEG-CNPs after phosphate treatment. Treatment of the WB2 particles, interestingly, does not change the absorption peak position of WB2 after ion incubation (Figure 2c). These particles are uncoated, and no other form of shielding mechanism is evidently occurring in order to protect the particles’ surface. The phosphate ions are significantly interacting with the more abundant Ce3+ surface site of WB1 and PEG-CNPs. This reduced ion interaction might be due to the insufficient coordination number required to energetically stabilize the formation of cerium phosphate with Ce4+ surface sites. Therefore, the ratios of oxidation states on the surface of the nanoparticles are critical to the generation of cerium phosphate. 18997

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Figure 8. X-ray photoelectron spectroscopy of the WB1. The O 1s orbital spectrum of (a) treated and (b) untreated WB1. A significant shift in the O 1s peak can be observed upon treating the WB1 particles. (c) Ce 3d spectral emission from treated WB1 sample. (d) Ce 3d spectrum for the untreated WB1.

concentration of ions increased to 20 mM. In the case of DEX-CNPs’ colloidal suspension, the initial conductivity of the solution was 0.86 mS/cm which further rises as a relatively linear function as the ion concentration increased. PEG-CNPs, on the contrary, exhibited an initial nonlinear behavior in the conductivity measurement with the increase in ion concentration. ζ-Potential Changes and Their Correlation to Anion− Particle Interaction. Derjaguin−Landau−Verwey−Overbeek theory states that in order for particles to stabilized in a suspension there needs to be a process of ion adhesion− desorption from the surface of the particles in order to bring the system to a state of electrostatic equilibrium. This process of disassociation of ions, in order to stabilize the suspension, could also occur from either or both the polymer and the surface of the CNPs in order to stabilize the particle−medium electrostatic charge. Under the experimental conditions, such as neutral pH, temperature, and ion concentration, the pKa (disassociation constant) values of both dextran and PEG indicate that the polymer will not disassociate. Cerium oxide is also stable at this pH; therefore, the ions of the buffer will need to interact with both the polymers and surfaces of CNPs in order to generate the required interfacial electrokinetic potential. The ionic concentration of the solvent has a direct effect on the nanoparticle ζ-potential by modifying the outer Helmholtz plane and compressing the double layer as the ions concentration increases. In Figure 4a, the ζ-potential of PEGCNPs drastically changes with the increase in the concentration of phosphate ions in the suspension to the extent to which an isoelectric point could be observed. It can be deduced that PEG-CNPs are selectively adsorbing phosphate anion onto its surface due to chemical interaction. This same principle has been observed in other systems, where the chemical interaction with ions has reversed the signs (negative to positive or vice versa) of the ζ-potential.30 The ζ-potential of DEX-CNPs showed an initial decrease in value upon the introduction of ions, which then stabilizes to ∼30 mV as the ion concentration increases to 2.5 mM. As the ion concentration increases even further, the latter variations in ζ-potential of DEX-CNPs seem

Figure 9. XPS emission envelope of P 2p3/2 region of treated WB1. (a) Treated and (b) untreated WB1. Treated WB1 shows intense emissions in the P 2p region, which indicates a significant amount of phosphate on the surface of WB1.

negative value. The ζ-potential of PEG-CNPs decreased further with the increase in ion concentrations. DEX-CNPs were negatively charged, and the increase in ion concentration forced the ζ-potential to a greater negative value after the addition of 2.5 mM solution of buffer. After this initial change in ζ-potential, DEX-CNPs’ ζ-potential was constant (∼30 mV) as the concentration of ions increased from 2.5 to 20 mM. Contrary to its counterpart PEG-CNPs, DEX-CNPs did not exhibit an isoelectric point within the ion concentrations administered in the experiment. In Figure 4b, the electrolytic conductivity of deionized water, PEG-CNPs’ suspension, and DEX-CNPs’ suspension were measured versus ion concentration. Figure 4b depicts a linear change in conductivity of the deionized water starting from 0 mS/cm and rises to approximately 3.9 mS/cm as the 18998

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Figure 10. XPS Ce 3d spectra of treated DEX-CNPs after consecutive sputtering cycles. (a) Ce 3d emission spectra of DEX-CNP after ion incubation. (b) Treated DEX-CNPs’ Ce 3d emission after the first sputtering cycle. (c) Treated DEX-CNPs’ Ce 3d emission after second sputtering cycle. (d) Treated DEX-CNPs’ P 2p emission after of ion incubation (e) Treated DEX-CNPs’ P 2p emission after the first sputtering cycle. (f) Treated DEX-CNPs’ P 2p emission after 2nd sputtering cycle. The dextran coating attenuates the electron emission, but nevertheless, after 2 cycles of sputtering the emission peak became evident for cerium and phosphorus.

Figure 11. Deconvoluted XPS emission envelopes of WB1 and WB2 spectra of Ce 3d. (a) Deconvoluted Ce 3d emission of WB1. (b) Ce 3d deconvolution of WB2. The quantified concentration of Ce3+ on the surface of WB1 particles was 59%. The WB2 samples had a higher concentration of Ce4+, showing a 21% concentration of Ce3+.

order to observe the changes in binding energy of surface Ce3+ and Ce4+ because of ion interaction. The spectrum observed in Figure 5 is used as a reference for cerium phosphate emission. Figure 6 shows the change in the surface chemistry of cerium and oxygen in CNPs before and after phosphate ion incubation. All XPS spectra have been referenced to adventitious carbon (C 1s spectral line) peak positioned at 284.5 eV in order to compensate for charging.31 The cerium spectrum in Figure 5a is due to both the cerium 3d5/2 and 3d3/2 electron emission and its associated satellite peaks. In Figure 5b, the spectral emission of the P 2p3/2 can be observed. In Figure 5c, the O 1s spectral envelope of cerium phosphate is asymmetric in nature due to adsorbed water and/or adsorbed hydroxyls groups, which are indicated by the higher binding energy shoulder of the emission envelope.32 The spectra of the O 1s of treated and untreated samples are shown in Figure 6a and 6b, respectively. The Ce 3d emission envelope of treated and untreated WB2 samples is shown in Figure 6c and 6d, respectively. It can be clearly observed in the spectra of WB2 that the O 1s peak structure is similar to that of untreated WB2. The absence of a major peak shift in the O 1s spectral region indicates that the O-bond configuration is not changing significantly because of the difference in binding energy of the P-O-Ce bond at approximately 531.8 eV. The P 2p spectral emission of treated and untreated WB2 samples is shown in Figure 7a and 7b, respectively. A negligible amount of phosphorus was detected

minimal in comparison to that of the PEG-CNPs. This decrease in the ζ-potential of DEX-CNPs after introduction of 10 and 20 mM of ions was purely electrostatic or ion adsorption into polymer matrix but not due to a chemical interaction.30 This minor drop in magnitude of the ζ-potential of the DEX-CNPs is indicative of the compression of the double layer. Electrolytic Conductivity of Suspensions Colloidal Suspension. Conductivity of a solution is dependent on the concentration of nanoparticles in a colloidal suspension, the cation/anion concentration, and their independent charge. In addition, there exists a linear correlation between ion concentration and electrolytic conductivity. In Figure 4b, a linear relationship between the conductivity and the concentration of ions exists, in the case of the DEX-CNPs. However, in PEG-CNPs, there exists an initial a nonlinear relationship in the conductivity as the ion concentration increases. This nonlinear relationship is attributed to the consumption of phosphate ions from the solution onto the surface of nanoceria by the generation of cerium phosphate. The linear region in Figure 4b, for PEG-CNPs (phosphate ion concentration 5−20 mM), is due to complete saturation of the available Ce3+ on the surface of the CNPs. Analysis of Surface Chemical Modification of Nanoparticles Post Incubation. The analysis of the oxidation state of the cerium element in both cerium phosphate and the CNPs was obtained using XPS. This experiment was completed in 18999

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Figure 12. Catalase mimetic activity of treated and untreated CNPs. (a) CNPs’ versus the control. (b) PEG-CNPs versus the control. (c) DEXCNPs versus the control. Throughout the results obtained, it can be noted that DEX-CNPs exhibit the greatest catalase mimetic response of all samples.

The depth profile study was compiled in order to detect the presence of cerium and phosphorus on the surface of ceria in DEX-CNPs. Figure 10a contains the spectrum of DEX-CNPs prior to sputtering. Figure 10b contains the Ce’s 3d spectrum after one cycle of sputtering. After the second sputtering cycle, a prominent peak structure of the Ce 3d was observed in the treated DEX-CNPs (Figure 10c). Due to the polymer coating in DEX-CNPs, the peaks of the 3d emission are more difficult to interpret. In Figure 10c, after the second sputtering cycle, the presence of Ce is enhanced in DEX-CNPs’ spectrum. Sputtering of sample may modify the binding energy and peak structure of the emission envelope due to ion bombardment; therefore, the determination chemical states is very difficult under these circumstances. After a series of sputtering cycles, a weak peak off phosphorus can be observed. In Figure 10d, there exists no apparent evidence that phosphorus is on the surface of the polymer matrix. Figure 10e and 10f shows a weak P 2p3/2 spectral line, an indication that there may exist a small concentration of phosphorus beneath the surface of the polymer matrix. Deduced Transformation of XPS Peak Envelopes. Ce 3d has one of the most complex structures due to both the Ce3+ and Ce4+ emission. In Figure 5a, the prominent peaks of Ce3+ oxidation state can be observed. CNPs have Ce3+ and Ce4+ oxidation states; therefore, primary peaks and satellite can be seen in the envelopes of Figure 6c and 6d. The transition of cerium oxide to cerium phosphate is also supported by the O 1s spectrum of Figure 8a and 8b. The spectra in Figure 8a and 8b show the change in asymmetry of

in treated WB2, and this indicates that WB2 has minimal interaction with phosphate ions. The effects of phosphate ion on the surface of WB1 sample are also shown in Figure 8. Figure 8a shows the generated O 1s emission envelope of WB1 after incubation of the nanoparticle with phosphate ions. Figure 8b shows the O 1s emission envelope of the untreated WB1 particles. A prominent asymmetry structure was observed in treated WB1 sample, which is due to the chemical modification of WB1’s surface. Figure 8c and 8d is the emission envelopes of the Ce 3d emissions of treated and untreated WB1 sample, respectively. Figure 8c shows the high concentration of Ce3+ oxidation states post treating. Figure 8d shows the intense peak at 880.2, 885.0, 899.2, and 903.4 eV corresponding to ceria Ce3+ oxidation in ceria. In Figure 8c the emission envelope is very similar to that of cerium phosphate in Figure 5a. Notice that there exists a shift (to lower biding energy) in the Ce3+ peaks of the treated sample in Figure 8c as compared to 8d due to the change in Ce3+ chemical configuration. A significant amount of phosphate can be observed on the surface of the WB1 particle after incubating the sample with phosphate buffer. Figure 9 shows the XPS P 2p envelope before and after treating WB1 particles. Figure 9a shows the prominent peak envelope of phosphorus after treating the particles with ions. Figure 9b shows that prior to treating the particle there is no existing phosphorus on the surface of the CNPs. Hence, post treating the WB1 sample cerium phosphate is generated on the surface of the nanoparticles. 19000

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Figure 13. SOD mimetic activity of bare CNPs and polymer-coated CNPs before and after treatment. (a) WB1 versus the control sample. (b) PEGCNPs’ response versus the control sample. (c) DEX-CNPs versus the control sample. In all analyses of CNPs, the SOD mimetic activity exceeds that of the control sample, but interestingly, treated DEX-CNPs exhibit a similar SOD response to that of DEX-CNPs. (The shaded region is the standard deviation or error band of the respective plots).

A pivotal aspect that can be deduced from the UV−vis and PL analysis is that the surface of WB1 is more inclined to form cerium phosphate than that of WB2. The XPS analysis of the bare particles (WB1 and WB2) shows a significant difference in surface Ce3+:Ce4+ ratios. In Figure 11, the deconvoluted spectrum of WB1 and WB2 Ce 3d emission can be observed. Surface Ce3+ percentage of WB1 was calculated to be 59% whereas 21% in case of WB2, using the method of calculations published elsewhere.14 In conjunction with prior data such as UV−vis, Figure 11 shows that samples with higher concentration of Ce3+ are more likely to react with phosphate ions. The synthesis of PEGCNPs and DEX-CNPs, as described earlier, is directly related to that of WB1 and WB2, respectively. The exception is the fact that the polymers were added to the synthesis process in order to coat CNPs. WB1 and PEG-CNPs were synthesized through a similar process, which tends to obtain higher concentration of Ce3+. Figure 11a shows the deconvolution of WB1 sample where the Ce3+ surface concentration is 59%. Therefore, a relatively high surface concentration Ce3+ is expected on PEGCNPs. The DEX-CNPs and WB2 samples have a greater surface concentration of Ce4+. The WB2 sample in Figure 11b has 21% of its surface cerium states assigned to Ce3+ prior to ion incubation. Through other experiments conducted such as UV−vis, conductivity measurements, and PL, it is apparent that these particles have a reduced interaction with phosphate ions. In addition, after treating bare particles with phosphate ions, the surface concentration of Ce3+ in WB1 increased from 59% to 80% and WB2’s surface Ce3+ concentration increased from 21% to 25%. The notion is that the polymeric coatings of the nanoparticle are aiding biocompatibility, but the oxidation state

the O 1s structure of WB1 after incubation. Deconvoluting the O 1s emission envelopes in Figure 8a and 8b would have shown the presence of other oxygen species. Species such as oxygen from ambient H2O/hydroxide adhered to the surface of ceria (533.1 eV)32 and oxygen from SiO2 (532.8 eV)33 substrate overlapping the region. The spectra of cerium phosphate in Figure 5c show the O 1s region and its intense peak at 531.4 eV which is associated with the chemical bonding of oxygen in CePO4. Figure 8a also shows an increase in the intensity of the 531.4 eV position after treating WB1 nanoparticles with phosphate ions. In comparison to WB2 (Figure 6c and 6d), Figure 8c and 8d shows that post treating the nanoparticle with phosphate ions, there is a slight increase in the concentration of Ce3+ states. Treating the WB1 sample with phosphate ions creates an intense peak in the P 2p region because of the surface chemical modification of the nanoparticle. The depth profile analysis in Figure 10 is very complex due to the sputtering process. In addition, the polymer coating and nanoparticle are not in a monolayer arrangement, but rather a matrix, which extensively attenuates the electron emission. The XPS results obtained with use of ion bombardment confirm the successful coating of DEX-CNPs. Analysis of the chemical makeup of CNPs, after the sputtering process, is difficult due to the low concentration and possible induced chemical shifts from bombardment. However, presence of very weak peak of phosphate indicates minimal presence or chemical modification of DEX-CNPs in the treated sample. This slight modification may be due to presence of very less numbers of Ce3+ sites on the surface of DEX-CNPs. In addition, there is a possibility of entrapping ions in the polymer network, post dialysis. 19001

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Figure 14. Comparative semiquantification of H2O2 scavenged by WB1, PEG-CNPs, and DEX-CNPs. (a) The quantity of H2O2 consumed by treated and untreated WB1 particles. A 72% reduction in initial catalase inhibition rate was observed for WB1 sample. (b) The quantity of H2O2 consumed by treated and untreated PEG-CNPs. The inhibition rate of H2O2 by PEG-CNP was a reduced by 87% reduction once particles were treated with phosphate ions. (c) The quantity of H2O2 consumed by treated and untreated DEX-CNPs. Treated DEX-CNPs consume less peroxide due to ion interaction and express 22% reduction in initial catalase inhibition rate.

coated nanoparticles’ catalytic mimetic reactivity after ion incubation. The intent of this study is to engineer nanoparticles with better biological compatibility, in addition to, retaining CNPs’ catalytic response after being exposed to ions. Therefore, it is imperative to check the catalytic responses (SOD and catalase mimetic activities) of the nanoparticles to track the effects of ion interaction on CNPs. Comparative Catalase Mimetic Reactivity. The catalase mimetic activity of the nanoparticles determines how efficient CNPs can convert hydrogen peroxide to H2O and oxygen molecules. In Figure 12a, the catalase mimetic activity of WB1 and treated WB1 versus that of the control are displayed. The results in Figure 12a show that CNPs and treated CNPs exhibit better catalase mimetic activity than the control sample. In Figure 12a, note that the treated CNPs show a slight decrease in catalase mimetic activity in comparison to that CNPs but still retain greater mimetic capability than the control sample. In Figure 12b, the comparative catalase mimetic activity of PEGCNPs and treated PEG-CNPs can be observed. Results indicate that the treated PEG-CNPs were less catalase active than PEGCNP. The reactivity of treated PEG-CNPs was comparable to that of the control sample, which means that essentially no catalase mimetic activity. Catalase mimetic performances of DEX-CNPs with and without phosphate treatment are displayed in Figure 12c. DEX-CNPs and treated DEX-CNPs both exhibit significantly greater catalase mimetic activity than that of the control sample. In Figure 12c, the catalase mimetic

Figure 15. Reduction in the comparatively quantified amount of H2O2 scavenged. Treated DEX-CNPs have outperformed treated WB1 and treated PEG-CNP samples by retaining most of it catalytic mimetic response. The treated PEG-CNPs were almost entirely neutralized due to ion interaction.

is determining whether ceria surface is going to react with phosphate ions. Catalytic Reactivity of Particles. This section proceeds with the intent of understanding the response of the polymeric 19002

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Figure 16. Comparatively quantified superoxide radicals scavenged by WB1, PEG-CNPs, and DEX-CNPs. (a) Quantified scavenging of treated and untreated WB1 particles. Treated WB1 shows an initial rate of inhibition was reduced by 55%. (b) Quantified scavenging of treated and untreated PEG-CNPs samples. Treated PEG-CNPs samples exhibited less reduction in inhibition rate (12%). (c) Quantified scavenging of treated and untreated DEX-CNP samples.

control sample as described elsewhere.25 The SOD mimetic activity of treated WB1 is less in comparison to that of untreated WB1 particles’ performance. Figure 13b shows the SOD mimetic activity of PEG-CNPs and ion treated PEGCNPs. Figure 13b shows the scavenging capability of both preand post-treated coated particles, though untreated PEG-CNPs exhibit greater SOD mimetic activity in comparison. Figure 13c shows the SOD mimetic reactivity of DEX-CNPs and treated DEX-CNPs. Interestingly, in Figure 13c, the minimum or no change in SOD mimetic activity was observed in treated DEXCNPs as compared to untreated DEX-CNPs. Catalase Mimetics of CNPs and Their Correlation to Oxidation State. In prior studies, the higher surface concentration of Ce3+ oxidation states has proven to be less effective in the catalase mimetic activity, whereas higher concentration of Ce4+ oxidation states have proven to be more catalase mimetic.20 WB1 and PEG-CNPs’ preparations are tailored to have higher concentration of Ce3+ surface oxidation states. In the case of DEX-CNPs, these nanoparticles are tailored to have higher concentrations of Ce4+ oxidation states. In Figure 12, comparatively, DEX-CNP (Figure 12c) exhibits more catalase mimetic activity than WB1 CNPs (Figure 12a) and PEG-CNP (Figure 12b). This correlation is primarily due to the higher concentration of Ce4+ on its surface.34 Interestingly, the most prominent aspect is that DEXCNPs, once incubated with phosphate ions, retains catalase mimetic activity. The surface chemistry of DEX-CNPs plays an

Figure 17. Percent reduction in SOD mimetic activity due to ion incubation of nanoparticles. Interestingly, DEX-CNPs SOD mimetic activity had 0% reduction in dismutation. WB1 expressed the highest reduction in SOD mimetic activity with a 35.1% decrease in dismutation post treating with phosphate ions.

activities are comparable in treated and untreated samples. In addition, the initial rate of scavenging of H2O2 by DEX-CNPs is greater than that of the treated DEX-CNPs. Comparative SOD Mimetic Activity of CNPs. In Figure 13a, the scavenging of superoxide radicals by WB1 and ion incubated WB1 proved to be more efficient than that of the 19003

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point after subtracting the CNPs’ response from that of the control sample. The total consumed H2O2 was calculated as the integral of the absorption post subtraction from the control sample using eq 2:

Table 1. Summary of experimentally observed results along with quantifications of CNPs characteristics sample

TEM

sample

251, 300 252, 300 296 283 259, 277

251, 271, 300 252, 271, 300 296 283 null

emission peak, untreated sample (nm)

sample

PL

3−6 5−8 3−5 8−10 absorbance peaks, treated sample (nm)

WB1 PEG-CNPs WB2 DEX-CNPs cerium phosphate

UV− vis

WB1 PEG-CNPs WB2 DEX-CNPs cerium phosphate sample percentage of

XPS

WB1 WB2 sample

catalase

Ce3+

0.187 0.041)

1.07

0.935

initial reduced rate of consumption

reduction in consumed H2O2

72% 87%

50.4% 92.3%

22%

12.6%

WB1 PEGCNPs DEXCNPs sample

inhibited superoxide, untreated sample (au)

inhibited superoxide, treated sample (au)

6.26 6.37

4.06 4.73

6.79

6.80

WB1 PEGCNPs DEXCNPs sample

reduction in inhibiting radicals

percentage of Ce3+ of treated sample

0.377 0.543

WB1 PEGCNPs DEXCNPs

initial reduced rate

reduction consumed H2O2

55% 12%

35.1% 25.7%

0%

0.0%

imperative role in circumventing the neutralization of the surface reactive sites of the particles. Treated PEG-CNPs expressed significantly less catalase mimetic activity, similar to the control sample. Semi-Quantified Catalase Mimetics Activity. The rates of the reaction are quantified based on eq 1: inhibition ratecatalase = Δabs/Δtime

(2)

due to the discrete sampling frequency. In eq 2, f(a)i and Δi are the value of absorption at distinct interval (i) and the value/size of the interval, respectively. There was a 50.4% reduction in the quantity of H2O2 consumed by WB1 due to phosphate ion interaction, as seen in Figure 14a. In Figure 14b, the treated PEG-CNPs show a 87% reduction in initial activity. Figure 14b shows an 87% of reduction in treated PEG-CNPs as compared to the control. Thereby, the PEG-CNPs have expressed the highest reduction in catalase mimetic activity due to ion interaction. However, in Figure 14c, DEX-CNPs also showed a reduction in the quantity of consumed H2O2 (12.6%, in Figure 15). The initial inhibition rate of the treated DEX-CNPs has only reduced by 22%, the least of all treated samples. These results again indicate that due to the presence of less Ce3+ site on the surface of DEX-CNPs, the particles minutely altered the catalase response due to ion interaction. Semi-Quantified SOD Mimetic Activity. In previously noted literature, CNPs have proven to exhibit the capability to mimic superoxide dismutase.19 CNPs containing higher concentrations of Ce3+ surface oxidation states are more efficient at superoxide dismutation.19,35 Interestingly, DEXCNPs (Figure 13c) show significant SOD mimetic activity, regardless of the fact that it has the higher concentration of Ce4+ on its surface. PEG-CNPs (Figure 13b) and WB1 (Figure 13a) exhibit the same tendency as they are exposed to phosphate ions; the SOD mimetic activity is diminished due to formation of cerium phosphate on the surface by passivation of the active sites. The comparatively quantified amount of free radicals that are consumed by the CNPs is detailed in Figure 16. Comparative Free Radical Consumption. The method of calculation of the rate of reaction is similar to that previously done in the catalase experiments. The nanoparticles absorption was subtracted from the control sample. The derivatives and integrals were then calculated using eqs 1 and 2. Figure 16a shows the overall reaction rate and naturalized superoxide free radicals. The initial reaction rate of the WB1 sample was decreased by 55% after the introduction of phosphate ions. In addition, the reduction in the quantity of the converted free radicals was calculated to be 35.1% (Figure 17) in comparison to the untreated sample. The performance of PEG-CNPs (Figure 16b) was relatively better than that of WB1. However, PEG-CNPs exhibited only a 12% reduction in initial inhibition rate after ion incubation, and the overall free radical scavenged was reduced by only 25.7% percent of the untreated PEG-CNPs sample. In Figure 16c, DEX-CNPs show no change in inhibition rate or the quantity of consumed free radicals. A complete summary of the experimental data can be found in Table 1 with information in regards to all characterization, catalytic responses, and quantification done in the experiments.

349.8, 503.8 344.4, 503.8 504.8 null null

80% 41% consumed H2O2, treated sample (au)

sample

free radicals consumed

349.8 349.2 504.8 null 503.4

∑ f (a)iΔi

emission peak, treated sample (nm)

59% 21% consumed H2O2, untreated sample (au)

WB1 PEGCNPs DEXCNPs

reduction in catalase

size distribution (nm)

WB1 PEG-CNPs DEX-CNPs WB2-CNPs absorbance peaks, untreated sample (nm)

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CONCLUSION CNPs were successfully engineered with polymeric coating to reduce ion interaction in addition to increasing the biocompatibility of CNPs. XPS, UV−vis, ζ-potential, con-

(1)

Where Δabs is change in absorption and Δtime is change in time of catalase response (the derivative) for each individual 19004

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(8) Heckman, K. L.; Decoteau, W.; Estevez, A.; Reed, K. J.; Costanzo, W.; Sanford, D.; Leiter, J. C.; Clauss, J.; Knapp, K.; Gomez, C.; et al. Custom Cerium Oxide Nanoparticles Protect against a Free Radical Mediated Autoimmune Degenerative Disease in the Brain. ACS Nano 2013, 7, 10582−10596. (9) Cimini, A.; D’Angelo, B.; Das, S.; Gentile, R.; Benedetti, E.; Singh, V.; Monaco, A. M.; Santucci, S.; Seal, S. Antibody-Conjugated PEGylated Cerium Oxide Nanoparticles for Specific Targeting of Aβ Aggregates Modulate Neuronal Survival Pathways. Acta Biomater. 2012, 8, 2056−2067. (10) Zholobak, N. M.; Ivanov, V. K.; Shcherbakov, A. B.; Shaporev, A. S.; Polezhaeva, O. S.; Baranchikov, A. Y.; Spivak, N. Y.; Tretyakov, Y. D. UV-Shielding Property, Photocatalytic Activity and Photocytotoxicity of Ceria Colloid Solutions. J. Photochem. Photobiol., B 2011, 102, 32−38. (11) Colon, J.; Herrera, L.; Smith, J.; Patil, S.; Komanski, C.; Kupelian, P.; Seal, S.; Jenkins, D. W.; Baker, C. H. Protection from Radiation-Induced Pneumonitis Using Cerium Oxide Nanoparticles. Nanomedicine 2009, 5, 225−231. (12) Briggs, A.; Corde, S.; Oktaria, S.; Brown, R.; Rosenfeld, A.; Lerch, M.; Konstantinov, K.; Tehei, M. Cerium Oxide Nanoparticles: Influence of the High-Z Component Revealed on Radioresistant 9L Cell Survival under X-Ray Irradiation. Nanomedicine 2013, 9, 1098− 1105. (13) Gojova, A.; Lee, J.-T.; Jung, H. S.; Guo, B.; Barakat, A. I.; Kennedy, I. M. Effect of Cerium Oxide Nanoparticles on Inflammation in Vascular Endothelial Cells. Inhal. Toxicol. 2009, 21 (Suppl 1), 123− 130. (14) Deshpande, S.; Patil, S.; Kuchibhatla, S. V.; Seal, S. Size Dependency Variation in Lattice Parameter and Valency States in Nanocrystalline Cerium Oxide. Appl. Phys. Lett. 2005, 87, 133113. (15) Dutta, P.; Pal, S.; Seehra, M. S.; Shi, Y.; Eyring, E. M.; Ernst, R. D. Concentration of Ce 3+ and Oxygen Vacancies in Cerium Oxide Nanoparticles. Chem. Mater. 2006, 18, 5144−5146. (16) Sayle, T. X. T.; Parker, S. C.; Sayle, D. C. Oxidising CO to CO2 Using Ceria Nanoparticles. Phys. Chem. Chem. Phys. 2005, 7, 2936− 2941. (17) Pagliari, F.; Mandoli, C.; Forte, G.; Magnani, E.; Pagliari, S.; Nardone, G.; Licoccia, S.; Minieri, M.; Di Nardo, P.; Traversa, E. Cerium Oxide Nanoparticles Protect Cardiac Progenitor Cells from Oxidative Stress. ACS Nano 2012, 6, 3767−3775. (18) Dowding, J. M.; Dosani, T.; Kumar, A.; Seal, S.; Self, W. T. Cerium Oxide Nanoparticles Scavenge Nitric Oxide Radical (·NO). Chem. Commun. (Cambridge, U. K.) 2012, 48, 4896−4898. (19) Korsvik, C.; Patil, S.; Seal, S.; Self, W. T. Superoxide Dismutase Mimetic Properties Exhibited by Vacancy Engineered Ceria Nanoparticles. Chem. Commun. (Cambridge, U. K.) 2007, 1056−1058. (20) Pirmohamed, T.; Dowding, J. M.; Singh, S.; Wasserman, B.; Heckert, E.; Karakoti, A. S.; King, J. E. S.; Seal, S.; Self, W. T. Nanoceria Exhibit Redox State-Dependent Catalase Mimetic Activity. Chem. Commun. (Cambridge, U. K.) 2010, 46, 2736−2738. (21) Dowding, J. M.; Das, S.; Kumar, A.; Dosani, T.; McCormack, R.; Gupta, A.; Sayle, T. X. T.; Sayle, D. C.; von Kalm, L.; Seal, S.; et al. Cellular Interaction and Toxicity Depend on Physicochemical Properties and Surface Modification of Redox-Active Nanomaterials. ACS Nano 2013, 7, 4855−4868. (22) Singh, S.; Dosani, T.; Karakoti, A. S.; Kumar, A.; Seal, S.; Self, W. T. A Phosphate-Dependent Shift in Redox State of Cerium Oxide Nanoparticles and Its Effects on Catalytic Properties. Biomaterials 2011, 32, 6745−6753. (23) Karakoti, A.; Monteiro-Riviere, N. Nanoceria as Antioxidant: Synthesis and Biomedical Applications. Jom 2008, 60, 33−37. (24) Hirst, S. M.; Karakoti, A. S.; Tyler, R. D.; Sriranganathan, N.; Seal, S.; Reilly, C. M. Anti-Inflammatory Properties of Cerium Oxide Nanoparticles. Small 2009, 5, 2848−2856. (25) Wason, M. S.; Colon, J.; Das, S.; Seal, S.; Turkson, J.; Zhao, J.; Baker, C. H. Sensitization of Pancreatic Cancer Cells to Radiation by Cerium Oxide Nanoparticle-Induced ROS Production. Nanomedicine 2013, 9, 558−569.

ductivity measurements, and PL characterization indicate the formation of cerium phosphate or phosphate complex on the surface of CNPs due to ion interaction. This chemical modification of CNPs can significantly influence the catalytic response of the particles’ antioxidant properties. Catalase and SOD mimetic activities analyzed showed that WB1 and PEGCNPs are unable to retain its surface physiochemical properties in the presence of phosphate ion, hence showing significant reductions in catalase and SOD responses. Alternatively, DEXCNPs and WB2 particles exhibit a more sustained catalase and SOD mimetic response when incubated with phosphate ions. This is because WB2 and DEX-CNPs are more viable at inhibiting phosphate ion interaction due to its higher concentration of Ce4+ but still minimally react due to residual Ce3+ state on its surface. Therefore, DEX-CNPs and WB2 particles are preferred due to their reduced interaction with phosphate ions in the presence of buffer in biological system.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Tel: 1-407-823-5277. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding from NSF CBET-1261956 and EECS-0901503 are acknowledged. R.M. and P.M. were funded by the NSF REU Program.

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ABBREVIATIONS: CNP, cerium oxide nanoparticle; SOD, superoxide dismutases; ROS, reactive oxygen species; PBS, phosphate buffered saline; PEG, polyethylene glycol; PEG-CNP, polyethylene glycol coated CNPs; DEX-CNP, dextran coated-CNPs

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