Cerium nitrate

Synthesis and Characterizations of CONPs: CONPs were synthesized using the hydrothermal process.34 In brief, cerium nitrate was dissolved in distilled water at 1 × 10⁻³m, with pH adjusted at 8.0 using ammonium hydroxide. Separately, CTAB was dissolved in distilled water at 0.1 × 10⁻³m. Two solutions were mixed dropwise in a Teflon hydrothermal vessel with a total volume of 60 mL. The Teflon vessel was transferred to a stainless steel autoclave and thermal‐treated at 140 °C for 24 h under autogenous pressure conditions to obtain hydrothermal‐processed CONPs. The nanoparticle morphology was examined by high‐resolution TEM (JEOL 7100, Jeol Ltd., Tokyo, Japan) after dispersing in anhydrous ethanol and then dropped on a copper grid. The size of nanoparticles was measured from TEM images (n = 30). The selected area electron diffraction pattern of the crystals was also analyzed. The crystal structure of nanoparticles was determined by X‐ray powder diffraction (Ragaku Co. Ltd., Tokyo, Japan). XPS (ESCA 2000, Thermo Fisher Scientific, Waltham, MA, USA) was carried out to analyze the chemical ionic status of Ce. The surface electrical properties of the nanoparticles were observed by means of ζ‐potential measurement (Zetasizer Nano, Malvern Instruments Ltd., Malvern, UK) at pH 7.0 and 25 °C. The hydrodynamic size of the nanoparticles was also characterized using the Zetasizer Nano by a DLS method. Nanoparticles of 100 µg dispersed in DW or in neurobasal medium were measured at 25 °C (n = 4). The enzyme mimetic activity of CONPs was evaluated by monitoring the redox reaction between TMB and H₂O₂ in the presence of CONPs.35 The reaction was first monitored time‐dependently (every 5 min up to 20 cycles) using UV–vis spectroscopy (Varian Cary 100, Varian Analytical Instruments, Walnut Creek, CA, USA) at a broad wavelength scan. A typical solution was made of 1 mL acetate buffer solution (50 × 10⁻³m, pH = 4.0), 25 µg nanoparticles, 0.5 × 10⁻⁶m TMB, and 1 mol H₂O₂,36 which was used when filtered through a syringe filter (pore size 0.45 µm; Hyundai Micro Co., Ltd., Seoul, Korea). Based on this, the reaction was again monitored at a wavelength of 652 nm with varying H₂O₂ concentrations (10 × 10⁻⁶, 20 × 10⁻⁶, 40 × 10⁻⁶, 60 × 10⁻⁶, 80 × 10⁻⁶, 100 × 10⁻⁶, 200 × 10⁻⁶, 400 × 10⁻⁶, 800 × 10⁻⁶, and 1000 × 10⁻⁶m) using UV–vis spectroscopy (Biochrom, Libra S22, Cambridge, UK).
Primary Cultures of Rat Cerebral Cortical Neurons: Cortical neurons were isolated and cultured using a modified method from the previous study.37 Cortex was removed from the Sprague‐Dawley rat embryos (embryonic day 16) and placed into Hank's balanced salt solution (HBSS) (GIBCO, Grand Island, NY, USA), and meninges were manually removed. Cortex was rinsed twice in HBSS medium and then transferred to a 15 mL conical tube containing 2 mL of 2.5 mg mL⁻¹ (in HBSS) papain solution (Sigma‐Aldrich, St. Louis, MO, USA). After incubation for 15 min at 37 °C, the papain solution was discarded and the remaining samples were rinsed twice in 2 mL HBSS, and then centrifuged at 1500 rpm for 3 min to discard HBSS. The samples were placed in 1 mL cortical neuron culture media containing Neurobasal medium (Gibco, Waltham, MA, USA) supplemented with B27 (Invitrogen Life Technologies, Carlsbad, CA, USA), Gluta‐MAX (Invitrogen Life Technologies), and 1% penicillin/streptomycin. The pellet was resuspended by triturating about 20 times through 1 mL pipette tips. The single cells were then plated onto 18 mm circular cover slips for immune staining and onto 96‐well plates for cell viability assay. The cover slips and plates were prepared by coating with 20 mg mL⁻¹ poly‐d‐lysine (Sigma‐Aldrich) overnight and 10 mg mL⁻¹ laminin (Sigma‐Aldrich) for 2 h at 4 °C.
Hydrogen Peroxide (H₂O₂)‐Induced Neuronal Injury and CONP Treatments: Cortical neurons were cultured in 96‐well plates at a density of 5 × 104 cells per well in the cortical culture medium for 2 h. Then the cortical neuron medium was replaced with H₂O₂ that was prepared at different concentrations in cortical neuron culture medium (100 × 10⁻⁶, 250 × 10⁻⁶, and 500 × 10⁻⁶m) for 30 min or 1 h. After this, the cortical neuron culture medium was replaced with various concentrations of CONPs (1, 10, 25, 50, 100, 250, 500, 1000, 2000, and 4000 µg mL⁻¹ for MTT assay, and 10, 250, and 1000 µg mL⁻¹ for live/dead cell assay) and then incubated in 5% CO₂ at 37 °C for 24 h. To perform MTT assay, MTT was dissolved in cortical neuron culture medium and added at final concentration of 0.5 mg mL⁻¹ at 37 °C for 4 h. Afterward, the MTT contained cortical neuron culture medium was replaced by 100 µL dimethyl sulfoxide (Sigma‐Aldrich). Optical density was measured at 570 nm by a Universal Microplate Reader. For live and dead cell assay, a fluorescent live/dead cell assay kit (L3224, Invitrogen Life Technologies) was used. Treated cells were incubated with 2 × 10⁻⁶m Calcein‐AM and 4 × 10⁻⁶m EthD‐1 in Dulbecco's phosphate‐buffered saline (DPBS) for 20 min at room temperature, and the prepared samples were visualized under a confocal microscope (Carl Zeiss Inc., Oberkochen, Germany) at 488 nm excitation (green) and 555 nm (red) wavelengths. Three images were taken from each well (three wells for each condition), and the number of cells labeled with green (live) or red (dead) color was counted using an ImageJ software (1.37 v, National Institutes of Health, Bethesda, MD, USA).
iNOS Immunostaining and Analysis: After the treatment of 500 × 10⁻⁶m H₂O₂ solution for 30 min, the CONPs were administered at varying concentrations (1, 10, 25, 50, 100, 250, and 500 µg mL⁻¹) and then incubated in 5% CO₂ at 37 °C. After 12 h, the cortical neuron samples were fixed with 4% paraformaldehyde for 30 min, and then rinsed three times for 5 min each with phosphate‐buffered saline (PBS)g. The cells were permeabilized in 0.2% Triton X‐100 (dissolved in 2% normal goat serum/PBS solution) for 5 min, washed three times in PBS for 5 min, and blocked in 2% normal goat serum/PBS solution for 1 h. Primary antibodies (mouse anti‐SMI312, 1:1000, Covance, Princeton, NJ, USA; rabbit anti‐iNOS, 1:100, Abcam, Cambridge, MA, USA) diluted in 2% normal goat serum/PBS solution were incubated overnight at 4 °C and washed three times in PBS. A secondary antibody (FITC‐conjugated goat anti‐mouse IgG, 1:200; Rhodamine Red‐X‐conjugated affinipure goat anti‐rabbit IgG, 1:200, Jackson Immuno‐Research Labs, Inc., West Grove, PA, USA) diluted in 2% normal goat serum/PBS solution was incubated at room temperature for 2 h, then washed three times with PBS. The coverslips were treated with 4′‐6‐diamidino‐2‐phenylindole containing PBS at room temperature for 10 min, washed three times with PBS, covered with fluorescent mounting medium (Dako Cytomation, Carpinteria, CA, USA), and then observed under a confocal microscopy (Carl Zeiss Inc.). For the quantification of iNOS fluorescence intensity, four randomized images at each group were captured with 400× magnification and the average intensity of iNOS fluorescence was measured using ImageJ software (National Institutes of Health).
In Vivo Models of Spinal Cord Contusion and Local Delivery of CONPs: Adult female Sprague‐Dawley rats (12‐week old, 230–250 g) were used in all experiment. All procedures complied with Dankook University's Institutional Animal Care and Use Committee (Approval No. DKU‐14‐035). Animals were housed individually in a temperature‐controlled environment (23–25 °C) and humidity (45%–50%) under 12 h light/dark cycle with ad libitum water and food access.
Surgical procedures have been previously described in detail.38 Briefly, rats were deeply anesthetized by isoflurane (Forane; Choongwae Pharma, Seoul, Korea) inhalation and laminectomy was performed at T9–T10 level. All animals received a moderate contusion injury (200 kdyn) to expose T9 spinal cord using the Infinite Horizon impactor (IH‐400, Precision Systems and Instrumentation, LLC, KY, USA). CONPs with different concentrations (50, 100, 250, 500, 1000, 2000, and 4000 µg mL⁻¹) were prepared in distilled water immediately before use. At 30 min following contusion, a total volume of 10 µL CONPs solution directly injected into the lesion cavity at T9 spinal cord (subdural, and exactly intralesional) via Hamilton syringe at a rate of 1 µL min⁻¹ (Hamilton Company, Reno, NV, USA). Control animals received the same amount of distilled water without CONPs. After delivering the solution, the cord was then covered with a piece of hemostatic agent (Surgicel, Johnson and Johnson, Arlington, TX, USA), and the muscle and subcutaneous layers, skin were closed by layer. Intramuscular injection of 40 mg kg⁻¹ cefotiam hydrochloride (Fontiam, Hanmi Pharma, Seoul, Korea) was performed to all operated rats for 3 d and intraperitoneal injection of normal saline (3 mL) was made just after surgery. Animals also received oral administration of 10 mg kg⁻¹ acetaminophen syrup (Tylenol, Janssen Pharmaceutica, Titusville, NJ, USA) for 3 d, and bladder expression was performed twice a day and continued until the amount of expressed urine was less than 0.5 mL per day. These groups of rats were sacrificed at one week (n = 9 per group). Based on the results on one week, additional SCI models were made for one day (n = 8 per group) and for eight weeks (n = 9 per group), with the optimal concentrations of CONPs.
Histology and Immunofluorescence: Frozen sections were used for hematoxylin and eosin staining (H&E) and immunohistochemistry. Five rats in each group were perfused with 0.9% saline followed by 4% paraformaldehyde (Hushi Inc., Shanghai, China) in 0.1 m PBS (pH 7.4). The spinal cord was then dissected, post‐fixed overnight in 4% paraformaldehyde at 4 °C, and transferred to 30% sucrose in 0.1 m PB for 3 d. The cord was embedded M1 compound (Thermo Fisher Scientific Inc.) and cryosectioned into 16 µm in the sagittal plane. H&E stain was performed to examine the lesion site of the injured spinal cord at one week and eight weeks postinjury. The sections were stained with hematoxylin for 5 min, rinsed in running tap water for 3 min, and then stained with eosin for 1 min. The stained sections were dehydrated through a graded series of ethanol, cleared with xylene, and then imaged under a microscope (Nikon, Tokyo, Japan). The lesion cavity in H&E‐stained sections (n = 3 per group) was outlined manually under a light microscope at X100 magnification, and the area was calculated using the National Institutes of Health ImageJ software (National Institutes of Health), as described elsewhere.39Immunohistochemistry was used to analyze the inflammatory response in contused spinal cord. The primary antibodies, rabbit anti‐iNOS (1:100, Abcam), rabbit anti‐GFAP (1:1000, Dako), mouse anti‐ED1 (1:400, Merck Millipore, Temecula, CA, USA), were incubated overnight at 4 °C. After the sections were washed three times, goat anti‐rabbit (Alexa Fluor 546) and goat anti‐mouse (Alexa Fluor 488) secondary antibodies were used at a dilution of 1:200 in 2% normal gout serum in PBS. Following 2 h incubation, the sections were washed three times with PBS. Stained tissue sections were imaged using a confocal microscopy (Carl Zeiss Inc.). For quantitation of ED1+ monocytes and macrophages in the sagittal section, the images were captured at the lesion site using 100× magnification on a confocal microscope, and then counting the expressed cell numbers (per 1 mm²) manually. For quantification of iNOS fluorescence intensity in the sagittal section, three representative images from the lesion site per animal (n = 3 per group) were captured with 400× magnification and fixed acquisition settings using confocal microscope and iNOS fluorescence intensity was analyzed using ImageJ software (National Institutes of Health). The background subtraction was performed using a rolling ball algorithm of ImageJ tools and the average intensity was measured with ImageJ measurement.
Assessments of Locomotor Functions: For the evaluation of locomotor functions of paralyzed hindlimb after spinal cord injury, two scales were used: Basso, Beattie, and Bresnahan (BBB) scale and horizontal ladder test. The BBB scale of no hindlimb movement is 0, and that of normal hindlimb movement is 21.40 Rats were analyzed by two observers who were blinded to the treatment received by each rat and positioned across from each other to observe both sides of the rats during 4 min walking in the open field (cylindrical‐shaped acrylic box; 90 cm diameter, 15 cm high) with a smooth floor. Horizontal ladder test was performed on a runway made of acryl walls (10 cm tall, 127 cm long, 8 cm wide between walls, 1 cm between rungs).41 All rats were trained to walk from left to right on a runway several times for adaptation before testing and then captured with a digital camcorder. The ladder score was calculated as below
$\mathrm{Ladder}\mathrm{ }\hspace{0.17em}\mathrm{score}=\mathrm{Erroneous}\mathrm{ }\hspace{0.17em}\mathrm{steps}\hspace{0.17em}\mathrm{ }\mathrm{of}\mathrm{ }\hspace{0.17em}\mathrm{hind}\hspace{0.17em}\mathrm{ }\mathrm{limb}\mathrm{ }\mathrm{/}\mathrm{ }\mathrm{total}\hspace{0.17em}\mathrm{ }\mathrm{steps}\hspace{0.17em}\mathrm{ }\mathrm{of}\hspace{0.17em}\mathrm{ }\mathrm{hind}\hspace{0.17em}\mathrm{ }\mathrm{limb}\times \mathrm{100}\mathrm{ }\left(\%\right)$
The locomotor function of each group was examined every 7 d until sacrifice. All locomotor tests were recorded for at least 4 min with a digital camcorder for coupling score and ladder score and were interpreted by two observers who were blinded to the identity of the rats.
RNA Isolation and Real‐Time PCR: To examine the effects of CONPs on the reactive oxygen species (ROS), apoptosis and inflammation in SCI rat models, the expression level of nine genes; iNOS, Cox2, Nr‐f2, p53, Casp3, IL‐1β, IL‐6, IL‐10, and TNF‐α were evaluated in spinal cord tissues using real‐time PCR (Table1). Briefly, total RNA was extracted from spinal cord by using an RNeasy mini kit (Qiagen, Hilden, Germany). cDNA was synthesized using random hexamer primers and SuperScript III (Invitrogen, Thermo Fisher Scientific). All primers pairs were designed using the UCSC Genome Bioinformatics and the NCBI database. Real‐time PCR was performed using Fast SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific) on a StepOne Real‐Time PCR system (Thermo Fisher Scientific). Each real‐time PCR was performed on at least triplicate assay (n = 3 for each group). The expression of each target gene was normalized to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and expressed as the fold change relative to the control groups.
Statistical Analyses: All numeric data were reported as means ± SDs, and IBM SPSS Statistics 21 (International Business Machines Corp., Armonk, NY, USA) was used for the analysis. The Shapiro‐Wilk test was performed to check normal distribution of all quantified histological and functional data from each group, and according to the result, parametric or nonparametric tests were chosen. For histological, immunohistochemical and quantitative PCR data, Mann–Whitney U test was used to detect the differences between control and CONPs‐treated experimental groups. To compare the anti‐iNOS intensity and the relative cell viability of CONPs‐treated groups with untreated and H₂O₂‐treated controls, the Kruskal–Wallis test with Bonferroni correction method was used. The repeated measures one‐way analysis of variance were used to compare locomotor functions including the BBB and the ladder score tests among the control and experimental groups, and then the Kruskal–Wallis test with Bonferroni correction method was used at each time point. Significance was determined at p < 0.05.

The PAA-stabilized CeO₂ nanoparticles were synthesized using the low-temperature precipitation method. A mixed solution containing 30 mM cerium(III) nitrate hexahydrate salt and ammonium cerium(IV) nitrate salt with 10% by weight of PAA (MW 5100) was prepared, and 30% ammonium hydroxide solution was added to this in a dropwise manner. Eu-doped cerium nanoparticles were synthesized similarly. During the synthesis of Eu-doped CeO nanoparticles, the concentration of ammonium cerium(IV) nitrate salt ((NH₄)₂[Ce(NO₃)₆]) remained constant and amounted to 30 mM, whereas the concentration of cerium(III) nitrate hexahydrate salt (Ce(NO₃)₃·6H₂O) was changed depending on the degree of doping. The amount of europium(III) nitrate hydrate salt (Eu(NO₃)₃·5H₂O) was added so that the total concentration of cerium nitrate hexahydrate and europium nitrate hydrate salts was 30 mM. For example, for 20% Eu doping, 0.006 M Eu salt and 0.024M Ce salt were used and similarly for other levels of Eu doping. Several syntheses of Eu-CeONPs with 5%, 10%, 15% and 20% doping were conducted to compare properties and select the most optimal suspensions. After continuous stirring for 24 h, the obtained suspensions of doped Eu-CeO nanoparticles were dialyzed against 5L of distilled water at pH 7 for 2 days. The water was changed 3 times a day.
The hydrodynamic particle size and zeta potential of Eu-CeONPs were determined by Dynamic Light Scattering (DLS) (Zetasizer Nano Series, Malvern Instruments, Malvern, Worcestershire, UK). The sample was measured at 25 °C in triplicate with at least 20 measurements using water as dispersant with parameters set for cerium oxide (refractive index = 2.2 and absorption = 0.001). The zeta potential measurements were conducted using LDE (Laser Doppler Electrophoresis) and Zetasizer Nano Series (Malvern Instruments, Malvern, Worcestershire, UK). The X-ray Photoelectron Spectroscopy (XPS) measurements were carried out in a multi-chamber UHV system equipped with a hemispherical analyzer (SES R4000, Gammadata Scienta, Uppsala, Sweden), and the numerical analysis was performed with CasaXPS 2.3.23 software after subtracting the Shirley-type background. The experimental results were fitted using a profile with a variable ratio (70:30) of Gaussian and Lorentzian lines [35 (link)]. The absorption spectra were recorded with a UV-Vis spectrometer (Shimadzu Corporation, Duisburg, Germany) in the range of 200–800 nm. Photoluminescence spectra of Eu-CeONPs were recorded using a Fluorolog^®-3 spectrofluorometer (HORIBA Jobin Yvon, Longjumeau, France) at the excitation wavelength of 380 nm and emission spectra with the scan range from 400 to 660 nm with 5 nm slit widths and an integration time of 0.1 s. All samples were characterized as synthesized. To confirm the imaging abilities of synthesized nanoparticles by optical modalities, the nanoprobes were deposited on the surface of positively charged latex microparticles and visualized by Carl Zeiss LSM780 (Carl Zeiss, Jena, Germany) confocal microscopy.

Nanoceria samples were prepared by
three different procedures reported elsewhere^{28 (link)} with some modifications. The samples were denoted according to the
procedure used as CeAMM, CePER, and CeUREA. Briefly, the CeAMM sample
was prepared by precipitation of cerium(III) nitrate aqueous solution
with an ammonia solution followed by aging for 4 h at 60 °C in
CO₂-free ambient air without any calcination. The CePER
sample was prepared by precipitating an aqueous cerium(III) nitrate
solution with sodium hydroxide solution followed by treatment with
hydrogen peroxide and refluxing at 100 °C for 24 h without any
calcination. The CeUREA sample was synthesized by homogeneous precipitation
of aqueous cerium(III) nitrate solution with urea at 90 °C and
subsequent calcination at 500 °C/2 h. All prepared samples were
dried (or calcined) in atmospheric air and stored in vials under ambient
air. See details in the Supporting Information (SI).

All materials utilized in this study were of analytical grade. The materials used for synthesizing the Ce-MOF were cerium nitrate hexahydrate (Ce(NO₃)₃·6H₂O, Fluka 99%) as the cerium precursor, benzene-1,3,5-tricarboxylic acid (H₃BTC, Aldrich 98%) as ligand precursor and ethanol (Merck, CH₃CH₂OH), as solvent.