Synthesis and Characterization of Photoluminescent Ce(III) and Ce(IV) Substituted Hydroxyapatite Nanomaterials by Co-Precipitation Method: Cytotoxicity and Biocompatibility Evaluation

Improved compounds of Ce(III) and Ce(IV)-doped hydroxyapatite (Ca10-xCex(PO4)6(OH)2) with different concentrations such as x = 0.5, 1, 2.5, 5, and 10%, obtained by the simple co-precipitation method were synthesized. The cerium (3+) and cerium (4+)-doped hydroxyapatite were evaluated for biocompatibility and fluorescence properties. It was found that the cerium-HAp powders were non-toxic, even at higher level of concentration. The synthesized powders were further characterized by FTIR spectrometry, UV-Vis spectroscopy, XRD diffraction, SEM and TEM analysis. Therefore, the present study proves that the developed cerium (3+) and cerium (4+)-doped hydroxyapatite, respectively can be widely used as luminescent labeling materials, with improved biological properties.


Introduction
In the world of bioceramics, chemical compounds such as calcium apatites and other calcium orthophosphates are widely used in many fields of science, including geology, chemistry, biology, and medicine due to their abundance and presence in living organisms [1]. Bioceramics can be defined as a biocompatible ceramic material, manufactured in a porous, dense, amorphous or crystalline form, which can be used for the repair and reconstruction of different parts of tissue. The main group of bioceramics is derived from calcium phosphate (CaP), the others are zirconium (ZrO 2 ), alumina (Al 2 O 3 ), carbon, silicates, and phosphate ceramics [2].
Photoluminescence is an important and useful mechanism for in situ investigation of tissues, fluorescent elements being used in clinical trials for years. Recently, various studies used nanoparticles or different components for this purpose [3].
Hydroxapatite (HAp) is the main inorganic compound of human hard tissue [4]. The composition, crystal size, morphology, and stoichiometry of biological apatite are different from synthetic HAp. The Ca/P molar ratio for HAp is 1.67 [5,6]. An important application of HAp is in the treatment with chemotherapeutic drugs and antibiotics, mainly due to its porous surface and highly biodegradable properties [7]. Various studies suggest that calcium phosphate nanoparticles can be successfully used as a fluorescent probe

Sample Characterization
The morphological, structural, and optical properties of the obtained samples were investigated by means of Fourier Transform Infrared spectrometry (FT-IR), scanning electron microscopy (SEM), bright field transmission electron microscopy (TEM), X-ray diffraction (XRD), UV-Vis and photoluminescence (PL) spectroscopy. The FTIR spectra of all the samples were recorded using a Nicolet iS50R spectrophotometer (Thermo Fisher, Waltham, MA, USA), at room temperature, with ATR mode at 4 cm −1 resolution, in the measurement range 4000-400 cm −1 . The degree of crystallinity and crystallites size of the samples were estimated by X-ray diffraction (XRD) (Thermo Fisher, Waltham, MA, USA) analysis using a PANalytical Empyrean diffractometer at room temperature, with a Cu X-ray tube (λ Cu Kα1 = 1.541874Ǻ) operating in-line focusing, with programmable divergent slit on the incident side and a programmable anti-scatter slit mounted on the PIXcel3D detector on the diffracted side. The XRD diffraction patterns, recorded in the 2θ range of 20 • -80 • , were collected in a Bragg-Brentano geometry, with a scan step of 0.02 • and a 255 s counting time per step. The lattice parameters were calculated using the HighScore Plus 3.0 e software and refined by the Rietveld method. The morphology of the samples was studied by scanning electron microscopy (SEM) using a Quanta Inspect F50 FEG (field emission gun) with 1.2 nm resolution (Thermo Fisher, Waltham, MA, USA). The bright field TEM images were obtained using a high resolution Titan Themis transmission electron microscope from Thermo Fisher Scientific (Waltham, MA, USA). The microscope operated in transmission mode at a voltage of 200 kV. UV-Vis diffused reflectance spectra were obtained using an Able Jasco V-560 spectrophotometer (PW de Meern, The Netherlands), with a scan speed of 200 nm/s, between 200 and 850 nm. The fluorescence spectra were measured using a Perkin Elmer LS 55 fluorescence spectrophotometer (Arkon, OH, USA). All the spectra were recorded with a scan speed of 200 nm/s between 350 and 800 nm, and with excitation and emission slit widths of 4, 7, and 10 nm, respectively. The used excitation wavelength was 320 nm.

MTT Assay
The biocompatibility of cerium-doped hydroxyapatite samples was evaluated by means of MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) assay (Vybrant ® MTT Cell Proliferation Assay Kit, Thermo Fischer, Waltham, MA, USA), performed on human mesenchymal amniotic fluid stem cells (AFSC). The cells were grown in DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 1% antibiotics (penicillin and streptomycin) (Sigma-Aldrich, St. Louis, MO, MI, USA), which was changed twice a week. The method is based on the growing of AFSC cells in 96-well plates, with a seeding density of 3000 cells/well in the presence of the obtained powders for 72 h. Afterwards, 15 mL (12 mm) of MTT was added to the cells, and next were incubated at 37 • C for 4 h. A solution of 1 mg sodium dodecyl sulphate in 10 mL HCl, 0.01 M was added to the first one and pipetted vigorously to solubilize the formed formazan crystals. After 1 h, the optical density (OD) of solubilized formazan is evaluated spectrophotometrically using a TECAN Infinite M200 spectrophotometer (Männedorf, Switzerland) at 570 nm.

GSH-Glo Glutathione Assay
The oxidative stress assessment was evaluated using the GSH-Glo Glutathione Assay kit (Promega, WI, USA). For the GSH-Glo Assay analysis, AFSC were seeded at a density of 3000 cells in 300 µL of Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin, streptomycin/neomycin) in 96-well plates. After 24 h of seeding, cells are treated with the studied samples and then incubated for 72 h. To the obtained solution, 100 µL 1 × GSH-Glo Reagent was added, followed by incubation at 37 • C for 30 min and then 100 µL Luciferin Detection Reagent was added and incubated at 37 • C for an additional time of 15 min. The medium from the wells was well homogenized and then the plate was read on the luminometer (Microplate Luminometer Centro LB 960, Berthold, Germany) [37,38].

Cell Morphology and Viability Evaluation-Fluorescence Microscopy
The biocompatibility of the obtained cerium-doped HAp powders with AFSC was evaluated using fluorescence microscopy with a RED CMTPX fluorophore (Thermo Fischer, Waltham, MA, USA), a cell tracker for long-term tracing of living cells. The CMTPX tracker was added in cell culture, priory treated with the synthesized nanoparticles. After 5 days, the viability and morphology of the AFSC was evaluated. The CMTPX fluorophore, at a concentration of 5 µM and incubated for 30 min, was added in the culture medium, to allow the dye penetration into the cells. Finally, the AFSC were washed with PBS and visualized by fluorescent microscopy. The photomicrographs were taken using an Olympus CKX 41 digital camera driven by CellSense Entry software (Olympus, Tokyo, Japan) [38,39].

FTIR Analysis
The FTIR spectroscopy was used to identify the characteristic functional groups present in pure Hap, Ce(III)-HAp and Ce(IV)-HAp. FTIR spectra of pure HAp, Ce(III) x HAp and Ce(IV) x HAp samples are shown in Figure 1a,b. The FTIR spectrum of pure HAp (black line from Figure 1a,b) shows all the bands reported in literature [40]. The bands in the region 3000-3400 cm −1 are due to the existence of bending and stretching modes of the OH-group. The bands around 1093, 1022, and 956 cm −1 , respectively are due to the asymmetrical and symmetrical stretching modes of P-O bonds in the PO 3− 4 group [41]. The bands around 606, 562, and 473 cm −1 , respectively are attributed to the bending mode of O-P-O phosphate bands [42]. The bands around 872 and 1432 cm −1 which appear in FTIR spectra of HAp indicate the presence of carbonate moieties in the prepared HAp powder [43].
found to be decreased with the increase in the inclusion of Ce 3+ or Ce 4+ into the HAp lattice until the molar fraction of cerium (III) and cerium (IV) is 2.5%. Above this concentration the banding bands of PO4 3− increases. The replacement of calcium ions with cerium ions causes a change of bonding forces between the ions and leads to the weakening of the banding bands of O-P-O in the HAp structure [42]. The increasing concentration of Ce 3+ and Ce 4+ ions, which resulted in lowering the intensity of the bands, is associated with a decrease in HAp crystallinity. These results were further evidenced by the XRD analysis.

X-ray Analysis
The phase formation by the co-precipitation method of pure HAp, Ce(III)-doped hydroxyapatite, and Ce(IV)-doped HAp, respectively were shown in Figure 2a,b. The XRD spectra for pure and Ce-doped hydroxyapatite were compared and revealed only a pure and crystalline phase with well-defined diffraction peaks that match with the standard pattern of a hexagonal structure (according to ICDD PDF4+ card no. 00-068-0738) [45] with space group P63/m, in agreement with literature data [46][47][48]. The XRD patterns from  Comparing the FTIR spectra from Figure 1a,b, it was found that the FTIR spectra of Ce(III)-doped HAp and Ce(IV)-doped HAp powders, respectively with various cerium concentrations are similar to that of pure HAp. In all FTIR spectra of Ca 10-x Ce(III)x(PO 4 ) 6 (OH) 2 and Ca 10-x Ce(IV)x(PO 4 ) 6 (OH) 2 samples, the absorption bands which appear around 872 and 1432 cm −1 are assigned to CO 3 2− . This can be attributed to the carbonate CO 3 2− groups that replaced the PO 4 3− groups, indicating a reaction between HAp and carbon dioxide in the air [44]. The band intensity of PO 4 3− bands at 1093-956 cm −1 were found to be decreased with the increase in the inclusion of Ce 3+ or Ce 4+ into the HAp lattice until the molar fraction of cerium (III) and cerium (IV) is 2.5%. Above this concentration the banding bands of PO 4 3− increases. The replacement of calcium ions with cerium ions causes a change of bonding forces between the ions and leads to the weakening of the banding bands of O-P-O in the HAp structure [42]. The increasing concentration of Ce 3+ and Ce 4+ ions, which resulted in lowering the intensity of the bands, is associated with a decrease in HAp crystallinity. These results were further evidenced by the XRD analysis.
The lattice parameters a = b and c obtained from the XRD pattern of pure Hap shows a value of a = b = 9.42 Å and c = 6.88 Å, in agreement with literature data [49,50]. The changes in the lattice parameters are due to the ionic radius of ions: Ce 4+ (0.97Å); Ce 3+ (1.19 Å); compared with Ca 2+ is 1Å. When Ca 2+ ions are replaced by larger ions, such as Ce 3+ , the most significant change in lattice parameters is an increase in the a and c lattice parameters [47]. The differences observed for smaller ions than Ca 2+ ion, such as Ce 4+ are caused by their small differences in the ionic radius. When an ionic radius is greater than others, a slow lattice expansion is observed. For the same rare earth ion, the differences at a low concentration are not observed, but a higher substitution degree induces significant changes in the unit cell parameters. The metallic ion exchanges can lead to minimal distortion of bonds in the Hap structure [51,52]. The lattice of the Hap contains two Ca atoms with different crystal configurations: Ca(1)-nine coordinated with an ionic radius of 0.118 and Ca(2)-octahedral coordinated with an ionic radius of 0.106 nm, respectively [53]. From the literature, it is known that ions with larger ionic radius and smaller charge preferentially occupy the Ca(1) site, while ions with a smaller ionic radius and greater charge occupy the Ca(2) site [54,55]. In the present research, the differences in the ionic radius of the substitution ions suggest that the Ce 3+ ion favors the substitution in the Ca(1) site, and the Ca(2) site would be occupied by Ce 4+ ions, since it has a smaller ionic radius than Ce 3+ .
The variation of lattice microstrain values, crystallite size and crystallinity degree with cerium ions content are shown in Figure 4.  The crystallinity degree decreases with the increase in cerium concentration due to the slower kinetics of solid solution formation with multiple cations on the Ca 2+ site [56].

SEM Analysis
The surface morphologies of pure HAp, Ce(III)-doped hydroxyapatite and Ce(IV)doped hydroxyapatite with various concentrations were shown in Figures 5-7. The SEM image of pure HAp (Figure 5a) reveals that the particles are in the spherical shape with a dense nature, and the size was found to be in the range of 10-20 nm. The crystallinity degree decreases with the increase in cerium concentration due to the slower kinetics of solid solution formation with multiple cations on the Ca 2+ site [56].

SEM Analysis
The surface morphologies of pure Hap, Ce(III)-doped hydroxyapatite and Ce(IV)doped hydroxyapatite with various concentrations were shown in Figure 5. The SEM image of pure Hap (Figure 5a) reveals that the particles are in the spherical shape with a dense nature, and the size was found to be in the range of 10-20 nm.  The crystallinity degree decreases with the increase in cerium concentration due to the slower kinetics of solid solution formation with multiple cations on the Ca 2+ site [56].

SEM Analysis
The surface morphologies of pure HAp, Ce(III)-doped hydroxyapatite and Ce(IV)doped hydroxyapatite with various concentrations were shown in Figures 5-7. The SEM image of pure HAp (Figure 5a) reveals that the particles are in the spherical shape with a dense nature, and the size was found to be in the range of 10-20 nm.

TEM Analysis
In order to further evaluate nanodimensions of Hap, TEM investigations were made. Thus, Figure 6 shows the bright field TEM of pure HAp, Ca9.5Ce(III)0.5(PO4)6(OH)2 and Ca9.5Ce(IV)0.5(PO4)6(OH)2, and particle size distributions. (d) (e) (f) By doping with Ce(III) and Ce(IV) ions, respectively a little influence can be observed in the morphology of substituted Hap. Figure 5b,c show the SEM images of Ca 9.5 Ce(III) 0.5 (PO 4 ) 6 (OH) 2 and Ca 9.5 Ce(IV) 0.5 (PO 4 ) 6 (OH) 2 . Both samples exhibit the morphologies of particles of spherical shapes with a tendency of forming agglomeration. The observed particle sizes range from about 9 to 10 nm for Ce(III)-doped Hap, and 4 to 5 nm for Ce(IV)-doped Hap, respectively.

TEM Analysis
In order to further evaluate nanodimensions of Hap, TEM investigations were made. Thus, Figure 6 shows the bright field TEM of pure HAp, Ca9.5Ce(III)0.5(PO4)6(OH)2 and Ca9.5Ce(IV)0.5(PO4)6(OH)2, and particle size distributions. (d) (e) (f) From the bright field TEM images obtained on all three samples, we can observe the rod-like morphology of the nanoparticles, typical for HAP. The size distributions obtained on the width of the particles reveal the monomodal distribution of HAp. Ce doping has the effect of reducing the particle size in both cases. The particle size distribution varies from 8.59 to 7.70 nm. The length of the Hap particles are in the range of 10 to 25 nm.

UV-Vis and PL Spectra
The UV-Vis spectra of the two cerium(III)-doped hydroxyapatite and cerium(IV)doped hydroxyapatite, respectively are shown in Figure 7a,b. A general trend was observed for all the studied compounds: An increase in the intensity of absorption peaks and a broadening of them with increases in cerium ions concentrations in HAp.
From the bright field TEM images obtained on all three samples, we can obse rod-like morphology of the nanoparticles, typical for HAP. The size distributions o on the width of the particles reveal the monomodal distribution of HAp. Ce dop the effect of reducing the particle size in both cases. The particle size distribution from 8.59 to 7.70 nm. The length of the Hap particles are in the range of 10 to 25 n

UV-Vis and PL Spectra
The UV-Vis spectra of the two cerium(III)-doped hydroxyapatite and ceriu doped hydroxyapatite, respectively are shown in Figure 7a,b. A general trend w served for all the studied compounds: An increase in the intensity of absorption and a broadening of them with increases in cerium ions concentrations in HAp. The UV-Vis absorption spectra of Ca10-xCe(III)x(PO4)6(OH)2 and xCe(IV)x(PO4)6(OH)2, with different concentrations represented in Figure 7a,b are The UV-Vis spectra of Ca10-xCe(III)x(PO4)6(OH)2 has maxima which covers a length range from 364 to 378 nm, and for Ca10-xCe(IV)x(PO4)6(OH)2 from 343 to 3 respectively. The intensities of this maxima increase with the increasing cerium co Ce ions display strong UV absorbtion [57]. Pure hydroxyapatite presents no abs band, therefore the UV absorbtion band of the doped powders can be attributed presence of Ce ions. The UV-Vis spectra of Ca 10-x Ce(III)x(PO 4 ) 6 (OH) 2 has maxima which covers a wavelength range from 364 to 378 nm, and for Ca 10-x Ce(IV)x(PO 4 ) 6 (OH) 2 from 343 to 361 nm, respectively. The intensities of this maxima increase with the increasing cerium content as Ce ions display strong UV absorbtion [57]. Pure hydroxyapatite presents no absorbtion band, therefore the UV absorbtion band of the doped powders can be attributed to the presence of Ce ions. Figure 8a,b presents the photoluminescence emission spectra of Ca 10-x Ce(III) x (PO 4 ) 6 (OH) 2 and Ca 10-x Ce(IV) x (PO 4 ) 6 (OH) 2 samples excited with 320 nm wavelength. It was observed that the intensities of peaks decrease with the increasing cerium ions content. All emission peaks which appear at 399, 446, 457, 482, 513, and 527 nm are associated with various oxygen vacancies, interstitial or oxygen antisites. xCe(IV)x(PO4)6(OH)2, with different concentrations represented in Figure 7a,b are similar.
The UV-Vis spectra of Ca10-xCe(III)x(PO4)6(OH)2 has maxima which covers a wavelength range from 364 to 378 nm, and for Ca10-xCe(IV)x(PO4)6(OH)2 from 343 to 361 nm, respectively. The intensities of this maxima increase with the increasing cerium content as Ce ions display strong UV absorbtion [57]. Pure hydroxyapatite presents no absorbtion band, therefore the UV absorbtion band of the doped powders can be attributed to the presence of Ce ions.

MTT Assay
The MTT assay revealed that the synthesized Ce (III)-doped HAp and Ce(IV)-doped HAp nanoparticles reveal no cytotoxic effect, the absorption values being close, lower or higher compared to the control sample or any toxic effects even at higher concentrations ( Figure 9). The tested samples stimulated cellular metabolism, with a significant increase in proliferation compared to the control at 72 h.

MTT Assay
The MTT assay revealed that the synthesized Ce (III)-doped HAp and Ce(IV)-doped HAp nanoparticles reveal no cytotoxic effect, the absorption values being close, lower or higher compared to the control sample or any toxic effects even at higher concentrations ( Figure 9). The tested samples stimulated cellular metabolism, with a significant increase in proliferation compared to the control at 72 h. In the presence of cerium-doped nanomaterials, the glutathione a marker of oxidative stress, is able to prevent cellular damage caused by reactive oxygen species, such as free radicals, peroxides, lipid peroxides, and heavy metals (Figure 10), as well as responds similarly to the control cells, indicating that the analyzed materials do not induce cellular stress. In the presence of cerium-doped nanomaterials, the glutathione a marker of oxidative stress, is able to prevent cellular damage caused by reactive oxygen species, such as free radicals, peroxides, lipid peroxides, and heavy metals (Figure 10), as well as responds similarly to the control cells, indicating that the analyzed materials do not induce cellular stress. In the presence of cerium-doped nanomaterials, the glutathione a marker of oxidative stress, is able to prevent cellular damage caused by reactive oxygen species, such as free radicals, peroxides, lipid peroxides, and heavy metals (Figure 10), as well as responds similarly to the control cells, indicating that the analyzed materials do not induce cellular stress.  Fluorescence microscopy ( Figure 11) shows that AFSC cells are viable and ceriumdoped HAp nanomaterial samples have no cytotoxic effect, thus confirming the biochemical results. Therefore, the fluorescent synthesized nanoparticles could be used as fluorescent probing samples for cellular imaging and also as an antioxidant agent against oxidative stress-induced cell damages. Fluorescence microscopy ( Figure 11) shows that AFSC cells are viable and ceriumdoped HAp nanomaterial samples have no cytotoxic effect, thus confirming the biochemical results. Therefore, the fluorescent synthesized nanoparticles could be used as fluorescent probing samples for cellular imaging and also as an antioxidant agent against oxidative stress-induced cell damages. After 5 days in the presence of cerium-doped HAp powders, the AFSC showed normal morphology with a fibroblast-like characteristic appearance. Fluorescence images showed that the AFSC were viable, no dead cells or cell fragments were observed. Moreover, the cells formed filopodia to move and establish contacts with neighboring cells, suggesting that AFSC exhibited an active phenotype. Furthermore, the picture of a nega- After 5 days in the presence of cerium-doped HAp powders, the AFSC showed normal morphology with a fibroblast-like characteristic appearance. Fluorescence images showed that the AFSC were viable, no dead cells or cell fragments were observed. Moreover, the cells formed filopodia to move and establish contacts with neighboring cells, suggesting that AFSC exhibited an active phenotype. Furthermore, the picture of a negative control (dead cells) for the fluorescent microscopy images was represented in Figure 12. The negative control is represented by the AFSC treated with H 2 O 2 , 10 mm.

Conclusions
Pure hydroxyapatite, Ce 3+ -doped HAp, and Ce 4+ -doped HAp were prepared using the co-precipitation method. Analysis of pure HAp and cerium-doped HAp using spectral and scanning electron microscopy were performed. The FTIR spectroscopy and XRD diffraction analysis confirmed the formation of HAp. The results of FTIR spectroscopy and XRD analysis demonstrate that the calcium ions were successfully replaced by cerium ions in the hydroxyapatite crystal lattice. The SEM micrographs showed typical spherical morphologies for all the studied samples. The Ce 3+ -doped HAp and Ce 4+ -doped HAp samples exhibit a high fluorescent emission peak. The MTT assay showed that with cerium doping no cytotoxic effect was observed when the dose cells were incubated with fluorescent nanoparticles. Therefore, the obtained samples in this present research could be used as a fluorescent probing for cellular imaging. Funding: The support received from the project "Academic excellence and entrepreneurial values -scholarship system to ensure opportunities for training and development of entrepreneurial skills of doctoral students and PhD-ANTREPRENORDOC", Project no. 36355/23.05.2019 POCU/380/6/13-ID SMIS: 123847" is highly acknowledged.

Conclusions
Pure hydroxyapatite, Ce 3+ -doped HAp, and Ce 4+ -doped HAp were prepared using the co-precipitation method. Analysis of pure HAp and cerium-doped HAp using spectral and scanning electron microscopy were performed. The FTIR spectroscopy and XRD diffraction analysis confirmed the formation of HAp. The results of FTIR spectroscopy and XRD analysis demonstrate that the calcium ions were successfully replaced by cerium ions in the hydroxyapatite crystal lattice. The SEM micrographs showed typical spherical morphologies for all the studied samples. The Ce 3+ -doped HAp and Ce 4+ -doped HAp samples exhibit a high fluorescent emission peak. The MTT assay showed that with cerium doping no cytotoxic effect was observed when the dose cells were incubated with fluorescent nanoparticles. Therefore, the obtained samples in this present research could be used as a fluorescent probing for cellular imaging.