Cerium Oxide Nanoparticles: Synthesis and Characterization for Biosafe Applications

: Due to its excellent physicochemical properties, cerium oxide (CeO 2 ) has attracted much attention in recent years. CeO 2 nanomaterials (nanoceria) are widely being used, which has resulted in them getting released to the environment, and exposure to humans (mostly via inhalation) is a major concern. In the present study, CeO 2 nanoparticles were synthesized by hydroxide-mediated method and were further characterized by Scanning Electron Microscopy (SEM), Field Emission Scanning Electron Microscopy (FESEM), Energy Dispersive X-ray Spectroscopy (EDX), Fourier Transform Infrared Spectroscopy (FTIR), and X-ray Diffraction Spectroscopy (XRD). Human lung epithelial (Beas-2B) cells were used to assess the cytotoxicity and biocompatibility activity of CeO 2 nanoparticles. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Live/Dead assays were performed to determine the cytotoxicity and biocompatibility of CeO 2 nanoparticles. Generation of reactive oxygen species (ROS) by cerium oxide nanoparticles was assessed by ROS assay. MTT assay and Live/Dead assays showed no signiﬁcant induction of cell death even at higher concentrations (100 µ g per 100 µ L) upon exposure to Beas-2B cells. ROS assay revealed that CeO 2 nanoparticles did not induce ROS that contribute to the oxidative stress and inﬂammation leading to various disease conditions. Thus, CeO 2 nanoparticles could be used in various applications including biosensors,


Introduction
In nanotechnology, nanomaterials have been explored in various fields of science, such as physics [1], chemistry [2], biology [3], medicine [4], pharmacy [5], and materials sciences [6] because of their novel optical and electronic properties that differ from their bulk counterparts [7,8]. Nanomaterials are described as the elements with the diameter ranging from 1 nm to 100 nm. Reduction in particle size enhances the properties of those particles [9]. The physical and chemical characteristics determine the main strength of the nanomaterials to be utilized in various applications [10]. Nanomaterials can be used for the development of new cutting-edge applications in communications, energy storage, sensing, data storage, optics, transmission, environmental protection, cosmetics, biology, and medicine [8]. Furthermore, significant efforts have been made to produce many nanoparticles/nanocrystals with controlled morphologies, shapes, and sizes in the past few years [8]. Examples of some nanoparticles that are currently being explored are cerium, cellulose, silver, titanium, iron, aluminum, manganese, tantalum, gold, or a mixture of at least two thereof.
Of all the above nanoparticles, cerium is the first element in the lanthanide series which belongs to the rare earth family [11]. Cerium integrates with oxygen during the formation of cerium oxide nanoparticles; they acquire a fluorite structure [12]. Cerium oxide exists in 3+ and 4+ states, and redox cycling between cerium in these two states is the reason for the antioxidant properties [13]. Due to the significance of oxygen vacancies in cerium oxide, they can act as solid electrolyte in fuel cells [8]. The cerium oxide nanoparticles have got a significant consideration from the researchers in the field of nanotechnology because of their applications as catalyst converters, fuel additives and self-regenerating antioxidants [14].
Nanoceria has been found to possess great potential for a broad range of applications, particularly in the biomedical field. Nanoceria has been found to exert a profound antibacterial effect against different strains of bacteria. The usefulness of nanoceria depends upon its inherent property of showing variable oxidation states, due to which it can act as an excellent antioxidant agent and protect the healthy cells from oxidative stress [15]. Further, cerium oxide nanoparticles have been reported to act as synthetic antioxidant catalysts with measurable CNS penetrance, prolonged retention in the CNS, and high, regenerative catalytic activity that hold great promise for use in MS patients and, potentially, other ROS mediated disorders of the CNS [16].
Various methods used for the synthesis of cerium oxide nanoparticles include hydrothermal method, solvo-thermal method, and micro emulsification method [12]. Properties of nanomaterials determine the characterization techniques to be used to make sure that these techniques are sufficient to provide necessary information regarding those properties [17,18]. Parameters like size and morphology are analyzed by the means of microscope-based characterization methods. Parameters like crystal structure and elemental composition are determined by scattering and spectroscopy-based characterization techniques [17]. These methods help to determine the use of cerium oxide nanoparticles in various applications like low-temperature water-gas shift (WGS) reaction [19]; autoexhaust catalyst [20]; fuel cells [21]; oxygen sensors [22]; glass-polishing materials [23]; oxygen permeation membrane systems [24]; electrochromic thin-film application [25]; as well as environmental chemistry, biotechnology, and medicine [26,27].
CeO 2 nanomaterials are widely being used which has resulted in them getting released into the environment and exposure to humans (mostly via inhalation) is a major concern. Currently many publications are focused on mechanistic studies and potential therapeutic uses of CeO 2 nanoparticles. Insufficient toxicity and biocompatibility data is available on CeO 2 nanoparticles to support its biosafety [28]. Interactions between nanomaterials and living systems, including humans, may potentially have harmful effects [10]. For this reason, the nanomaterials must be assessed for toxicity and biocompatibility aspects. Results found in the publications reporting the toxicity of CeO 2 nanomaterials are contradictory. Low toxicity and inflammation effects of CeO 2 nanomaterials were reported in some publications [29][30][31]. On the contrary, toxic effects of CeO 2 nanomaterials triggering cell death was also reported [32]. The cause of toxic effect was found to be reactive oxygen species (ROS) generation which can damage the cell and ultimately lead to activation of cell death [32]. Some papers reported that CeO 2 nanomaterials induce oxidative stress both in vitro and in vivo whereas they also act as direct antioxidants and behave as free radical scavengers [33]. Findings have also shown CeO 2 nanomaterials to exhibit either antioxidant properties or pro-oxidative properties [34][35][36].

Synthesis of Cerium Oxide Nanoparticles
The synthesis process was carried out using hydroxide-mediated approach which is dominant over other methods in such a way that uniform distribution of particle size can be achieved [37,38]. At the end of the process, the solution was obtained with precipitate which was then centrifuged for 15 min at 8000 rpm to get the precipitate to settle down at the bottom. The supernatant was discarded, and the precipitate was further washed thrice with water and once with ethanol and isopropanol.
After this process, the precipitate was collected in a glass plate and dried in a hot air vacuum oven at the temperature of 200 • C. At the end of this process, mortar and pestle was used to crush the dried precipitate into fine particles.

Scanning Electron Microscopy
Phenom G2 Pro Tabletop SEM (Phenom, FEI Company, Hillsboro, OR, USA) was used to perform the scanning electron microscopy analysis. SEM is a multipurpose instrument which is able to provide qualitative information of the material including its morphology, topography, composition, and crystallographic information [9]. To prepare the sample for SEM, a double sided carbon adhesive tape was cut for the required shape and the side with exposed adhesive was adhered to the specimen holder and the other side has a thin layer of liner on the top. A small amount of cerium oxide nanoparticles was dissolved with ethanol and sonicated for uniform dispersion of nanoparticles in ethanol. Immediately after sonication, the top layer of liner in the tape was removed and 10 µL of the sonicated solution was pipetted on the adhesive part of the specimen holder. The sample was dried and was loaded in the equipment to start the SEM analysis.

Field Emission Scanning Electron Microscopy
Characterization of nanoparticles using Field Emission Scanning Electron Microscopy is carried out using Hitachi S-4700 Field Emission Scanning Electron Microscope (Hitachi, Chiyoda, Tokyo, Japan) with Energy Dispersive X-ray Spectroscopy. Sample was prepared [39] and loaded and a vacuum environment was created to confirm the absence of any air molecules in the microscope. The primary electrons released from field emission source were focused on the sample, and as a result secondary electrons were discharged from the sample which generates signals and those signals were converted into images to perform the further analysis. FESEM is advantageous over SEM in such a way that the source of electrons being a sharp field emission gun generates elevated resolution images with low acceleration voltages [40].
The elemental composition of cerium oxide nanoparticles was determined by Energy Dispersive X-ray Spectroscopy (Phenom, FEI Company, Hillsboro, OR, USA). The energy dispersive spectrometer records the characteristic X-ray which determines the composition of elements in cerium oxide nanoparticles along with the concentration of those elements [40].

Fourier Transform Infrared Spectroscopy
FTIR spectroscopy is a significant method for determining the presence of compounds. The nanoparticles sample was subjected to irradiation from which the presence of functional groups and the presence of respective compound in the fingerprint region are identified along with the intensity of their stretching bands [41]. These obtained intensities were plotted to their respective wavenumbers in the FTIR spectrum. Fourier Transform Infrared Spectroscopy was carried out using the equipment called Nicolet Nexus 670 (Nicolet instruments, Madison, WI, USA) which was set in absorbance mode in the range of 650 cm −1 to 4000 cm −1 to analyze the nanoparticles with multiple number of scans.

X-ray Diffraction Spectroscopy
XRD (Panalytical Empyrean, Malvern Panalytical, Malvern, United Kingdom) was used for determining and analyzing the crystal structure of the cerium oxide nanoparticles. Preparation of sample for XRD is simple compared to other methods. The sample holder had a well in which the synthesized nanoparticles were filled. Extra powders were removed by using a glass slide which also makes the surface even. The sample was then analyzed using the X-rays with the source of Cu Kα having the wavelength of 1.5418 Å to determine the crystal structure and phase composition of those particles which provides a diffraction pattern from 20 • to 80 • in the series of 2θ [42].

In Vitro Cell Culture and Experimental Conditions
Beas-2B is a non-tumorigenic human lung epithelial cell line which was immortalized via transfection with an adenovirus 12-SV40 hybrid virus. However, recent investigations revealed that those cells exhibit characteristics of mesenchymal stem cells [43].
Beas-2B was cultured in DMEM media containing 4500 mg/L D-glucose, L-glutamine, and sodium pyruvate supplemented with 10% fetal bovine serum (FBS), 1% 100 IU/mL of penicillin, and 100 g/mL of streptomycin and incubated at 37 • C in a CO 2 incubator with 5% CO 2 and 95% humidified atmosphere for at least 24 h. before the appropriate treatments. In this study, cerium oxide nanoparticles and SWCNTs stock solutions were prepared in the cell culture medium (DMEM) and required concentrations was added to the wells to perform all experiments. In the control experiments, cells without nanoparticles were considered as negative control and cells treated with SWCNTs were considered as positive control.

Measurement of Cell Viability by MTT Assay
The effect of cerium oxide nanoparticles on cell viability was assessed by using a yellow tetrazolium salt, MTT, as described earlier with slight modification [44]. Cerium oxide nanoparticles were prepared at selected concentrations (10,25,50, and 100 µg) in 100 µL supplemented DMEM and were homogenized. Beas-2B cells suspension was seeded in a 96-well microplate (at 5000 cells/well), in 100 µL of supplemented DMEM and incubated for 48 h at 37 • C under a 5% CO 2 atmosphere. Then, medium was discarded, and washed with DPBS. 100 µL of varying concentrations of cerium oxide nanoparticles test solutions were added to the cell culture and incubated at 37 • C, 5% CO 2 for 48 h 100 µL of a negative control (cells without nanoparticles) and a positive control (25 µg of SWCNTs in supplemented DMEM) were also tested. Each treatment was performed 6 times independently. Thereafter, the supernatant was removed and 100 µL of MTT solution (125 µg/well in DMEM supplemented) was added to each well and placed in a 5% CO 2 incubator at 37 • C for 3 h, to allow the formation of purple formazan crystal. Afterward, the medium was discarded, and purple formazan crystals formed inside the cells were solubilized using 100 µL of DMSO. The culture plates were placed on a rocker for 30 min to completely solubilize purple formazan crystals. The enzymatic reduction of yellow tetrazolium dye, MTT to a purple formazan is catalyzed by mitochondrial succinate dehydrogenase [45] which was measured by a Multi-mode Microplate Reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany) at 570 nm.

Live/Dead Cell Imaging Assay
To assess and reconfirm the cell viability and cytotoxicity of cerium oxide nanoparticles with MTT assay results, a two-color assay, Live/Dead cell imaging kit was used [39]. This assay helps to distinguish between live and dead cells with the help of non-fluorescent cell-permeable dye (calcein AM) for staining of live cells and cell-impermeable dye (ethidium homodimer-1) for staining of dead and dying cells. Calcein-AM gets converted to green-fluorescent dye to indicate intracellular esterase activity in a live cell and ethidium homodimer-1 binds to nucleic acids to produce enhanced red fluorescence to indicate loss of plasma membrane integrity in a dead cell [46].
Beas-2B cells suspension was seeded in a 6-well microplate (at 10,000 cells/well) in 2 mL of supplemented DMEM and incubated for 48 h at 37 • C under a 5% CO 2 atmosphere. Then, medium was discarded, and washed with DPBS. Cerium oxide nanoparticles (10, 25, 50, and 100 µg) test solutions prepared in 2 mL supplemented DMEM were added to the cell culture and incubated at 37 • C, 5% CO 2 for 48 h 2 mL of a negative control (cells without nanoparticles) and a positive control (25 µg of SWCNTs in supplemented DMEM) were also tested. Each treatment was performed 6 times independently. Then, the supernatant was removed and a dye mixture containing 4 µM ethidium homodimer and 2 µM calcein-AM was prepared by adding 20 µL of the 2 mM EthD-1 stock solution and 5 µL of the supplied 4 mM calcein AM stock solution to 10 mL of sterile DPBS. 2 mL of the dye mixture was added to the each well and incubated for 30 min at room temperature. After incubation, the green and red color-stained cells were observed under a fluorescence microscope (Nis Element, Nikon Instruments Inc, Melville, NY, USA) at an excitation (ex)/emission (em) 495 nm/515 nm and at an ex/em 495 nm/635 nm, respectively and the image of each well was captured.

Detection of Reactive Oxygen Species by ROS Assay
The levels of intracellular reactive oxygen species (ROS) activity within the Beas-2B cells in the presence of cerium oxide nanoparticles were measured using the ROS assay as described earlier [39]. The assay uses a cell permeable compound, DCF-DA which after diffusion into the cells is deacetylated by cellular esterases to a non-fluorescent compound, and later oxidized by ROS into a highly fluorescent molecule, 2 , 7 -dichlorofluorescin (DCF) [47].
Beas-2B cells suspension was seeded in a black 96-well microplate (at 10,000 cells/well), in 100 µL of supplemented DMEM and incubated for 48 h at 37 • C under a 5% CO 2 atmosphere. Ten microliters of 10 µM DCF-DA was added to the cultured cells and incubated in the chamber for 3 h. The cells were then washed with DPBS and treated with 100 µL of different concentration of cerium oxide nanoparticles (5, 10, 25, 50, and 100 µg) dispersed in supplemented DMEM. After 48 h, the intensity of fluorescence was measured at excitation/emission wavelength of 485/527 nm, respectively, and the values represent the fluorescence unit.

Synthesis
The cerium oxide nanoparticles were successfully synthesized using the hydroxide mediated approach and yellowish white nanoparticles were obtained. This is one of the simple and economic method to synthesize the cerium oxide nanoparticles. Cerium nitrate hexahydrate is one of the significant precursors used in several synthesis methods. In hydroxide-mediated approach, cerium nitrate hexahydrate is dissolved in deionized water to get a homogeneous solution through which the conversion of Ce 3+ state to Ce 4+ state takes place which then gets reacted with sodium hydroxide to synthesize the cerium oxide nanoparticles [48].

Characterization
The cerium oxide nanoparticles synthesized by hydroxide mediated approach were characterized by various methods such as SEM, FESEM, EDX, FTIR, XRD, and the results of these methods are discussed here.

SEM Analysis
The cerium oxide nanoparticles were analyzed through Scanning Electron Microscope and as a result, the image with cluster of uniformly distributed particles was obtained as shown in the Figure 1. On measuring the nanoparticles using the image J software, the average size of the particles obtained was in the range of 40 nm to 50 nm thereby confirming the existence of nanoparticles.

FESEM-EDX Analysis
The cerium oxide nanoparticles were characterized by Field Emission Scanning Electron Microscopy to obtain a more elevated image to determine if there are particles with decreased size as compared to the one obtained in SEM. The image as shown in the Figure 2 obtained was analyzed and as a result, the average size of the nanoparticles was found to be between 10 nm and 30 nm. After obtaining the image from FESEM analysis, the sample was subjected to Energy Dispersive X-ray Spectroscopy where the elemental composition of the cerium oxide nanoparticles along with their weight concentration was determined as shown in the Figure 3. It is evident from the EDX spectrum that cerium and oxygen are present making sure that the obtained cerium oxide nanoparticles are almost pure which is assured by the weight concentration of cerium and oxygen which accounts to a total of 99.8%.

FTIR Analysis
The Fourier Transform Infrared spectra generated in the range of 4000-650 cm −1 are shown in the Figure 4. In the functional group region, two intense peaks are found, in which one is the broad peak at 3361.24 cm −1 corresponding to the OH group, and the other one is the peak at 1556.36 cm −1 representing the N-O band because of the presence of nitrate [50]. The peak corresponding to 1556.36 cm −1 represents the C-H stretching and the peaks corresponding to 1051.13 cm −1 and 841 cm −1 represent the C-O band. In the fingerprint region, the peak corresponding to 658 cm −1 represents the O-Ce-O band thereby confirming the presence of cerium oxide nanoparticles [51].

Effect of Cerium Oxide Nanoparticles on Cell Viability and Cytotoxicity
Cerium oxide nanoparticles have various applications including biomedical, cosmetics, sensors, bio-supercapacitors, etc., further study is necessary to obtain additional information about their toxicological attributes and their probable impact on human health [32].
Beas-2B cells were exposed to different concentrations of cerium oxide nanoparticles for 48 h and were analyzed for the cell damage using MTT assay. Figure 6 displays the concentration-dependent effects of the cerium oxide nanoparticles produced by hydroxidemediated method on Beas-2B cell viability. The cells were treated with the concentrations of the cerium oxide nanoparticles ranging from 10-100 µg per 100 µL for 48 h. Cells with 25 µg of SWCNTs were considered as positive control. The cells were treated with SWCNTs showed significant decrease in cell viability compared to the cells with no nanoparticles (negative control). Whereas decrease in the cell viability was not observed in case of the cells treated with CeO 2 nanoparticles indicating that the nanoparticles did not exhibit any cytotoxic effect. In fact, the cell viability is better than the negative control in the cells treated with CeO 2 nanoparticles. According to several research reports [54,55], the amount of formazan crystals generated is not primarily determined by cell number. The MTT assay is based on mitochondrial succinate dehydrogenases converting the tetrazolium dye to formazan. However, cytosolic enzymes like nicotinamide adenine dinucleotide (NADH) reductase and flavin oxidase may potentially be involved in this process [56]. Furthermore, the rate of formazan conversion has been connected to cellular metabolic activity as well as the number of mitochondria present in the cell. As a result, larger cells with more mitochondria convert tetrazolium at a faster pace [57]. Thus, the apparent rise in the percentage of viable cells seen in Beas-2B cells could be explained by an influence on mitochondrial activity, which is not always associated with increases in cell viability and proliferation. Statistical evaluation of differences was made using the Student T test at significance levels of p 0.05 with respect to control (*) and SWCNT (¥). Beas-2B cells treated with 10 µg, 25 µg, and 100 µg of CeO 2 nanoparticles showed significant difference with respect to negative control (cells with no nanoparticles) and the cells treated with 10 µg, 25 µg, 50 µg, and 100 µg of CeO 2 nanoparticles showed significant difference with respect to the cells treated with SWCNTs.
We observed more than 100% of cells being viable at concentration of 100 µg per 100 µL CeO 2 nanoparticles, which was reconfirmed by Live/Dead cell imaging assay (Figure 7). Images of Live/Dead assay showed no cell death where the Beas-2B cells treated with concentrations of the cerium oxide nanoparticles ranging from 10-100 µg. Considerable cell death was observed with the cells treated with SWCNTs for 48 h as shown in Figure 7. The obtained outcomes were consistent with the MTT assay. Previous studies demonstrated selective toxicity of nanoceria towards the cancer and the normal cell lines. Prostate cancer cell lines (PC-3) treated with nanoceria induced toxic effects, whereas non-toxic towards normal mouse fibroblast cell line (L929) which was confirmed by MTT assay [58]. Significantly low levels of cytotoxicity were observed in L929 at the concentrations from 250-500 µg/mL in MTT assay [59].

Effect of Cerium Oxide Nanoparticles on Oxidative Stress
Reactive oxygen species (ROS) is a collection of oxygen radicals and certain nonradical oxidizing agents, that can be converted easily into radicals [60]. Major site for ROS production is mitochondria in mammalian cells. When cellular production of ROS overpowers its antioxidant capacity, cellular macromolecules for example lipids, protein, and DNA may get damaged. Such an imbalance is called "oxidative stress" which may cause pathogenesis of a several human diseases [61].
The synthesized cerium oxide nanoparticles were tested for its effect on oxidative stress by ROS assay. Results revealed that the nanoparticles did not affect viability of the Beas-2B cells shown by the constancy in nearly all dosages. Our data showed that there was no significant increase in the ROS level formed with 5-100 µg of CeO 2 nanoparticles and remained same as the negative control till 48 h. Measurement of the intensity of fluorescence was done at excitation and emission of wavelength at 485/527 nm, respectively, and expressed as fluorescence units (Figure 8). No significant difference was observed between the tested/treated cells and the control in a statistical evaluation of differences made using the Student T test at significance levels of p 0.05 with respect to control (*). Previous studies showed increase in DCF fluorescence intensity in lung adenocarcinoma (A549) cells to 171%, 200%, and 259% after 3 h and 240%, 266%, and 286% after 6 h exposure of 25 µg/mL, 50 µg/mL, and 100 µg/mL of CeO 2 nanoparticles, respectively, as compared to control. However, ROS production decreased (115%, 118%, and 109% at 25 µg/mL, 50 µg/mL, and 100 µg/mL, respectively) after 24 h exposure [62].

Conclusions
The current work discussed the synthesis and characterization of cerium oxide nanoparticles. The CeO 2 nanoparticles were successfully synthesized by hydroxide mediated approach and pale-yellow color nanoparticles were obtained. The synthesized nanoparticles were characterized by SEM, FESEM, EDX, FTIR, and XRD. The SEM analysis determined the size of the nanoparticles to be in the range of 10 to 30 nm and the EDX analysis indicated the elemental composition of the nanoparticles without any other impurities. The FTIR spectra determined the presence of CeO 2 nanoparticles. The XRD analysis determined the crystal structure of the nanoparticles. Biocompatibility studies performed using Beas-2B cells in MTT assay, Live/Dead viability assay, and ROS assay showed that the CeO 2 nanoparticles are compatible with the cells and there was no cell death even at higher concentrations (100 µg per 100 µL) of the CeO 2 nanoparticles. Thus, CeO 2 nanoparticles could be used in various biomedical applications including biosensors and cancer therapy. CeO 2 nanomaterials may also be used in filters and ultraviolet absorbers to produce products such as catalytic converters and sunscreen. Furthermore, as CeO 2 nanoparticles can cross cell membranes due to their small size, they could be effective for drug delivery.