Inﬂuence of Stabilizing Ion Content on the Structure, Photoluminescence and Biological Properties of Zr 1–x Eu x O 2–0.5x Nanoparticles

: Quasi-spherical nanoparticles of ZrO 2 containing EuO 1.5 from 2 to 15 mol.% were synthesized from the chlorides of the corresponding metals under hydrothermal conditions. The structural changes of Zr 1–x Eu x O 2–0.5x (x = 0.02 ÷ 0.15) nanoparticles depending on the content of europium (III) ions were studied using the complementary methods (X-ray di ﬀ raction, electron microdi ﬀ raction, Raman and photoluminescence spectroscopy). It was shown that increasing the Eu 3 + concentration in the Zr 1–x Eu x O 2–0.5x nanoparticles leads to a transition from the equilibrium monoclinic zirconia phase to metastable tetragonal and cubic polymorphic modiﬁcations. In this case, the size of the nanoparticles decreases from 11.5 nm to 9 nm; the speciﬁc surface area grows from 80.2 to 111.3 m 2 / g, and the electrokinetic potential increases monotonously from − 8.7 to 16.3 mV. The evolution of the phase composition of Zr 1–x Eu x O 2-0.5x nanoparticles from monoclinic to tetragonal / cubic allomorphs with an increase in the molar fraction of stabilizer ions was correlated with changes in the sublevel structure of 5 D 0 → 7 F 2 and 5 D 0 → 7 F 4 optical transitions for Eu 3 + in the luminescence spectra. Besides, for the nanoparticles obtained by hydrothermal synthesis from chlorides, the quantum e ﬃ ciency does not exceed 3%. According to the M.T.T. assay, as a result of three-day human ﬁbroblast cultivation in the aqueous dispersion of Zr 1–x Eu x O 2–0.5x (x = 0.02 ÷ 0.15) nanoparticles, the proliferation activity of the cells is maintained, indicating that they do not have cytotoxic properties. Such nanoparticles can be used in organic–inorganic composites for medical applications in order to strengthen the polymer sca ﬀ olds and visualize changes in their structure within time.


Introduction
Recently, more and more attention has been paid to the development of highly sensitive and safe nanophosphors for probing and visualizing biological objects [1][2][3]. Inorganic nanoparticles doped with lanthanide ions (Ln 3+ ) for use in biomedical applications should be biocompatible, exhibit intense luminescence and low toxicity, and have good colloidal stability in water and biological solutions [4,5].
In addition, small diameter nanoparticles with a narrow size distribution are required, which can be easily functionalized using surface modifiers for subsequent conjugation with biomolecules [6,7]. Compared with conventional organic molecular phosphors, Ln 3+ -doped nanocrystalline oxides have long fluorescence lifetimes (in the µs and ms range), quasilinear emission bands, broad Stokes shift, high photobleaching resistance, and comparable photoluminescence (P.L.) efficiency [8,9].
When lanthanide ions are introduced into the oxide matrix, the 4f-4f radiative electronic transitions forbidden by the Laporte Rule become partially allowed due to the admixture of opposite parity configuration 4f states. [10]. As a result, there is the possibility of the indirect P.L. excitation of the Ln 3+ , with narrow f-f emission lines and long lifetime, through oxide nanoparticles, acting as nanoantennas [11,12]. If Ln 3+ ions are embedded into a suitable crystal lattice, the sequential absorption of several photons through the lanthanide energy levels can occur in oxide nanoparticles, which provides higher energy to the anti-Stokes luminescence [13].
The electronic excitation energy transferred to lanthanide ions is defined by the oscillator strength, with the latter depending on the bandgap width and the refractive index of the oxide matrix [14,15]. Crystalline ZrO 2 has a large bandgap (4-6 eV), a high refractive index (2.13-2.20), and a small absorption capacity in the visible and near-infrared spectral regions in combination with low phonon energy (470 cm −1 ), which reduces the probability of the nonradiative multiphonon relaxation of excited Ln 3+ activator ions [16][17][18]. These properties, combined with the bioinertness of crystalline zirconia, make it the optimal matrix for the introduction of Ln 3+ [19]. ZrO 2 at an ambient temperature and atmospheric pressure is present in the monoclinic phase. However, the tetragonal and cubic polymorphs can be formed by heat treatment, the inclusion of dopant metals with a lower valence into the crystal lattice, and also by increasing the surface energy of nanoparticles [20][21][22][23]. The heat treatment of zirconium dioxide at phase transition temperatures leads to an increase in crystallite size, while the phase stabilization of thermodynamically unstable tetragonal (t) and cubic (c) ZrO 2 using lanthanide ions helps to reduce the size of nanoparticles, which is preferable.
The distribution of lanthanide ions between the monoclinic and more symmetrical t and c phases of ZrO 2 can be seen in the example of europium (III) ions, used as local structural probes [24,25]. The latter fact is due to the luminescent properties of Eu 3+ , which are very sensitive to changes in the symmetry of the crystal field [26,27]. This is manifested in the Stark splitting of the electric dipole transition 5 D 0 -7 F 2 [28,29]. With an increase in the content of europium ions in the ZrO 2 crystal structure, the phase composition of nanoparticles, the possible concentration quenching, and the P.L. efficiency depend strongly on the synthesis method and the nature of the precursors [30][31][32][33][34][35][36][37][38].
Z.W. Quan and others showed that for nanoparticles obtained by spray pyrolysis, the cubic polymorphic ZrO 2 modification stabilizes, regardless of the content of europium ions, and concentration quenching is observed in crystal phosphors containing more than 10 mol.% Eu 3+ [32]. In the case of using the microwave hydrothermal synthesis method for ZrO 2 nanoparticles with a content of 0.1 mol.% (monoclinic phase), 5 mol.% (tetragonal phase) and 10 mol.% (cubic phase) Eu 3+ , the concentration quenching of P.L. in the first two compositions was not detected [33]. The solid-phase synthesis of nanoparticles promotes the growth of emission intensity with an increase in the concentration of Eu 3+ in the ZrO 2 matrix from 0.5 to 5 mol.%, and in the entire range of selected concentrations, the authors fixed only cubic phase zirconia [34]. ZrO 2 :Eu 3+ nanophosphors were obtained by complex polymerization in [35], and the authors noted that the tetragonal phase of zirconia was formed at 4 mol.% trivalent impurity, and a cubic one at 8 mol.%. The emission intensity of Eu 3+ reached a maximum when 4 mol.% stabilizing ions were introduced into the ZrO 2 structure. N. Tiwari and his colleagues synthesized nanophosphors with an m-ZrO 2 structure independently of the Eu 3+ embedded amount (0.5-2.5 mol.%) using the solution combustion technique [36], wherein a P.L. intensity maximum is observed for the sample with 2 mol.% of the lanthanide. Thus, the synthesis conditions play a vital role in the processes of the phase formation and stabilization of zirconia high-temperature modifications by introducing trivalent europium ions. For the same elemental compositions of Eu 3+ -doped ZrO 2 nanoparticles, the use of various approaches for the synthesis leads to a different ratio of polymorphic modifications. At the same time, the structure, size and morphology of the forming ZrO 2 nanocrystallites, as well as the quantity, distribution uniformity, and localization of the Eu 3+ active centers in their crystal lattice, determine the P.L. efficiency of the phosphors.
The work addresses the physicochemical problems of the synthesis and separation of various polymorphic modifications for nanoparticles of Zr 1-x Eu x O 2-0.5x solid solutions, which were formed under relatively soft hydrothermal conditions at a temperature of 250 • C without subsequent annealing. The use of complementary methods provides information on the predominance of one phase or another among the polymorphic modifications of the studied zirconia nanoparticles, including cubic, based on the profile of Raman spectra, X-ray diffraction patterns, and fine splitting in the P.L. spectra. Hydrothermal treatment also predetermined the presence of hydroxyl groups on the surface of the Eu 3+ -doped ZrO 2 nanoparticles, which can then be used for conjugation with biomolecules [6]. Therefore, in connection with the biomedical aspects of the use of synthesized phosphors in composite materials, one of the study objectives was to analyze how phase stabilization with europium (III) ions affects the size, surface area and charge. Since in our work, we consider Zr 1-x Eu x O 2-0.5x nanoparticles as a possible component in the creation of a bone tissue precursor, understanding the toxicity of nanomaterials is a crucial problem [39]. To know whether nanoparticles affect fibroblasts' ability to divide, differentiate, and produce collagen and elastin precursors, we used the colorimetric assay with the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (M.T.T. assay).

Synthesis of the Zr 1-x Eu x O 2-0.5x Nanoparticles
The reagents for the synthesis of Zr 1-x Eu x O 2-0.5x nanoparticles were used without additional purification. The content of EuO 1.5 during the synthesis of zirconia-based nanophosphors was 2, 5, 7, 10, and 15 mol.%. At the first stage, the co-precipitation of ZrO(OH) 2 -Eu(OH) 3 mixtures in the predetermined molar ratio from ZrOCl 2 × 8H 2 O and EuCl 3 × 6H 2 O solutions using ammonium hydroxide was carried out [37]. Then the zirconium and europium hydroxide compositions were dehydrated under hydrothermal conditions for 4 h at 250 • C and 15 MPa. All nanopowders after hydrothermal treatment were dried at 100 • C to a constant weight.

X-ray Diffraction of Zr
The X-ray diffraction (X.R.D.) method was used for the structural analysis of resulting nanophosphors. The powder diffractograms were recorded using the SmartLab X-ray diffractometer (Rigaku, Japan) at CuKα in the 5-150 • range with a scan rate of 0.5 • /min. LaB 6 was used as a reference to take into account the apparatus width. The MAUD software package (L. Lutterotti, Italy) was used to determine the phase composition and calculate the parameters of a unit cell of nanoparticles [40].

Energy-Dispersive X-ray Microanalysis of Zr
The elemental composition of synthesized nanoparticles was measured using a SUPRA 55VP (Carl Zeiss AG, Germany) scanning electron microscope (S.E.M.) with an INCA microanalysis system (Oxford Instruments, UK). Quantitative energy-dispersive X-ray (E.D.X.) microanalysis was carried out using the method of fundamental parameters. The standard accumulation time of the spectrum Crystals 2020, 10, 1038 4 of 19 was 60 s, while for the determination of the elements with small content, the time was increased to 300 s. The composition was determined from the maximum possible area of the test sample.

X-ray Fluorescence Analysis of Zr 1-x Eu x O 2-0.5x Nanoparticles
A qualitative/quantitative elemental analysis of nanoparticle powders was also carried out using a software-analytical complex based on the Spectroscan Max-GF2E portable X-ray fluorescence crystal diffractive spectrometer (Spectron, Russia Q5). Raman measurements were carried out using LabRAM HR800 with a 1800 gr/mm diffraction grade in a backscattering geometry. Raman spectra (R.S.) were recorded using an Ar + laser with an excitation wavelength of 488 nm in the range of 110-700 cm −1 . The spectrometer was calibrated using a 520.7 cm −1 line of silicon standard before measurement. At the sample surface, the laser power was 13 mW under the 10x lens. Each spectrum was recorded 10 times for 10 s. Biological material from a skin biopsy of a young, conditionally healthy donor obtained with the observance of the septic and antiseptic rules was used to isolate dermal fibroblasts. A standard enzymatic technique was used to isolate the primary culture of dermal skin fibroblasts [42]. The cell suspension obtained during the enzymatic treatment was cultured in a complete DMEM nutrient medium (Paneco, Russia) supplemented with L-glutamine, 10% bovine fetal serum, and antibiotics in a CO 2 incubator (Thermo Fisher Scientific, 3423) at a temperature of 37 • C, a CO 2 concentration of 5%, and high humidity. Cells of 3-12 passages were used for research. The dispersion of Zr 1-x Eu x O 2-0.5x nanoparticles obtained by ultrasonic treatment was disinfected and placed in the wells of a culture plate. A cell suspension was added to the wells in a complete culture medium containing 25,000 cells, and the dermal fibroblasts were incubated for 3 days. The growth medium was replaced with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide solution and incubated for 2 h. Subsequently, the solution was removed, and the formazan crystals were extracted by pouring dimethylsulfoxide into the wells. After thoroughly mixing, the resulting solution was spread over The P.L. quantum yield of nanophosphors was measured by the modified de Mello method using a Fluorolog-3 fluorescence spectrometer equipped with a Quanta-j integrating sphere [44]. Tablets with a 10 mm diameter and thickness of 3 mm were compressed from a mixture of dry KBr, with spectroscopic-grade purity, and Zr 1-x Eu x O 2-0.5x nanoparticles in the mass ratio of 100:1. Potassium bromide without nanophosphors was used as a reference. The P.L. quantum yield measurements were carried out at an ambient temperature. The P.L. emission spectra for Zr 1-x Eu x O 2-0.5x nanoparticle powders were studied using a luminescence spectrophotometer LS-100 (PTI ® , Canada) and a Horiba LabRAM HR Evolution system. The P.L. intensity (I lum ) was recorded from the side of the incidence light in the 100-2000 µs range of an integration window for LS-100. Here, the grazing angle of the exciting light to the sample was ca. 30 • . To excite photoluminescence in LS-100, we used an EG&G FX 280V 91 xenon lamp. The geometric slit width for excitation and luminescence monochromators was 1 mm, with a reciprocal linear dispersion of 4 nm/mm. A Horiba LabRAM HR Evolution system was equipped with a He-Cd excitation laser emitting at 325 nm with a maximum power of 1 mW at the sample, and it possessed a 100 grooves/mm grating and a Peltier-cooled charge-coupled device detector. The light was focused onto the sample surface thanks to a 15× (0.32 numerical aperture) objective. The emission spectra of the Zr 1-x Eu x O 2-0.5x nanoparticles were registered from 560 to 770 nm. The P.L. excitation spectra of the systems under investigation were recorded using LS-100 in the range of 205-400 nm. The P.L. intensities for the correct comparison led to an internal laboratory standard.

Transmission Electron
The luminescence lifetime of the Zr 1-x Eu x O 2-0.5x nanoparticles was measured in the pulsed mode of a xenon lamp based on the P.L. intensity's decay. According to the excitation spectra, we excited the P.L. at 247 and 255 nm, and its observation was carried out at 614 and 608 nm, starting from the emission spectra. The values of lifetimes (τ pl ) were calculated with an iterative fitting procedure using QtiPlot software [45]. Model approximation functions for the P.L. intensity decay were selected based on the χ 2 criterion.

Structural Study
The carried-out elemental analysis revealed that the ZrO 2 /EuO 1.5 ratios specified in the synthesis pertained for the selected nanoparticle compositions. The deviation in the content of elements was about 0.5 mol.%, according to X-ray fluorescent analysis ( Table 1). The S.E.M.-E.D.Х. microanalysis gave a higher difference in the EuO 1.5 concentration for nanophosphors of Zr 1-x Eu x O 2-0.5x , which may have been caused by the applied powders screening due to the surface relief formed as a result of sample preparation, or the dissimilarity in the approaches to processing the spectra via software. Figure 1 illustrates the X-ray diffraction patterns for the Zr 1-x Eu x O 2-0.5x (x = 0.02-0.15) nanoparticles with different phase compositions. It shows a tetragonal/cubic crystal lattice, except for the lowest europium (III) ion content in the Zr 1.98 Eu 0.02 O 1.99 nanoparticles obtained from chlorides ( Figure 1, pattern 1; Figure 2). The distinction between tetragonal and cubic phases when the system consists of the three polymorphic modifications is not reliable. One can talk about the predominating tetragonal phase, which is more plausible, especially if we take into account the (102) diffraction peak of t-ZrO 2 . (Along with the tetragonal phase, at high concentrations of europium (III) ions in ZrO 2 , the cubic one is observed using Rietveld refinement. See Table S1 in the Electronic materials' support (E.M.S.) file.) Crystals 2020, 10, x 6 of 19 ( Figure 1, pattern 1; Figure 2). The distinction between tetragonal and cubic phases when the system consists of the three polymorphic modifications is not reliable. One can talk about the predominating tetragonal phase, which is more plausible, especially if we take into account the (102) diffraction peak of t-ZrO₂. (Along with the tetragonal phase, at high concentrations of europium (III) ions in ZrO₂, the cubic one is observed using Rietveld refinement. See Table S1 in the Electronic materials' support (E.M.S.) file.) As the content of Eu³⁺ in the Zr₁₋ₓEuₓO₂₋₀.₅ₓ nanoparticles increases, the (101/111) diffraction peak corresponding to the metastable zirconia phases gradually shifts from 30.17 to 29.96°, which indicates the incorporation of europium (III) ions into the t-/c-ZrO2 crystal lattices, with the formation of solid solutions ( Figure 2). Phase composition changes of nanoparticles, following the increase in the Eu 3+ content in the ZrO2 structure, were also analyzed using normalized R.S. (Figure 4). Nanoparticles with 2 mol.% EuO1.5 are characterized by Raman shifts at 178, 335, 474, 533, 556, 613, and 636 cm⁻¹, corresponding to the m-ZrO2 phase. Strong peaks appear at 178, 335, and 474 cm⁻¹ ( Figure 4, curve 1). The set of peaks at 149, 224, 266, 324, 407, 459 and 639 cm⁻¹ makes it easy to identify the tetragonal phase of ZrO2, which is present in all the nanoparticle compositions under study [46]. The Raman spectra of the high-temperature metastable cubic phase of ZrO2 have a narrow peak at 149 cm⁻¹, and wide, poorly resolved ones in the ranges 230-290 cm⁻¹, 400-430 cm⁻¹ and 530-670 cm⁻¹, which appear when the EuO1.5 content is 10 or 15 mol.% (Figure 4, curves 3-5) [47]. The observed Raman peak of 149 cm⁻¹ is common to both the tetragonal and cubic phases [48]. It should be noted that the cubic ZrO₂ demonstrates an amorphous spectrum of Raman scattering with one wide band at 530-670 cm⁻¹ [49]. We observed the above band with a maximum at 611 cm⁻¹ in the Raman spectrum for ZrO₂ nanoparticles containing 15 mol.% EuO1.5 ( Figure 4, curve 5), whereas the predominantly tetragonal ZrO₂ shows clearly visible bands at 460 and 640 cm⁻¹ due to its lower symmetry [50] (Figure 4, curves 1-3). Phase composition changes of nanoparticles, following the increase in the Eu 3+ content in the ZrO 2 structure, were also analyzed using normalized R.S. (Figure 4). Nanoparticles with 2 mol.% EuO 1.5 are characterized by Raman shifts at 178, 335, 474, 533, 556, 613, and 636 cm −1 , corresponding to the m-ZrO 2 phase. Strong peaks appear at 178, 335, and 474 cm −1 (Figure 4, curve 1). The set of peaks at 149, 224, 266, 324, 407, 459 and 639 cm −1 makes it easy to identify the tetragonal phase of ZrO 2 , which is present in all the nanoparticle compositions under study [46]. The Raman spectra of the high-temperature metastable cubic phase of ZrO 2 have a narrow peak at 149 cm −1 , and wide, poorly resolved ones in the ranges 230-290 cm −1 , 400-430 cm −1 and 530-670 cm −1 , which appear when the EuO 1.5 content is 10 or 15 mol.% (Figure 4, curves 3-5) [47]. The observed Raman peak of 149 cm −1 is common to both the tetragonal and cubic phases [48]. It should be noted that the cubic ZrO 2 demonstrates an amorphous spectrum of Raman scattering with one wide band at 530-670 cm −1 [49]. We observed the above band with a maximum at 611 cm −1 in the Raman spectrum for ZrO 2 nanoparticles containing 15 mol.% EuO 1.5 (Figure 4, curve 5), whereas the predominantly tetragonal ZrO 2 shows clearly visible bands at 460 and 640 cm −1 due to its lower symmetry [50] (Figure 4, curves 1-3). Along with the X.R.D. data obtained by the Rietveld refinement (Figure 2), the Raman spectra indicate the c-phase increases as the content of Eu 3+ grows, which is seen in Figure 4 at 585 cm⁻¹. Unfortunately, the cubic (single) F2g mode around 630 cm⁻¹ is rather weak and hidden in the much stronger phonon bands of the tetragonal phase. Here, the tetragonal ZrO₂ behaves anti-symbaticly with the cubic phase as the Eu 3+ content increases. In R.S., this corresponds to a decrease in t-ZrO₂ contribution at 266 cm⁻¹ ( Figure 4).
Besides this, the scattering band in the range 530-670 cm⁻¹ shifts to the smaller wave numbers, from the peak at 640 cm -1 for zirconia nanoparticles containing 2 mol% EuO1.5 (Figure 4, curve 1) to the peak with a maximum at 611 cm⁻¹ for the Zr0.85Eu0.15O1.925 composition (Figure 4, curve 5). This result is also confirmed by a decrease in the intensity of scattering at 460 cm⁻¹ with a Eu 3+ content increase in the ZrO2 crystal lattice (Figure 4).

Photoluminesence Properties
The P.L. spectra for Zr₁₋xEuxO₂₋₀.₅x (x = 0.02÷0.15) nanoparticles are presented in Figure 5. The peaks observed at 591, 606, 614, 624, 633, 651, 657, 701 and 748 nm, as well as the shoulder at 580 nm, can be compared with the spectral terms of the Stark splitting 5 D0 → 7 FJ (J=0-6) for the radiative transitions in the Dieke diagram of the Eu 3+ 4f n configuration [14,51,52].  Along with the X.R.D. data obtained by the Rietveld refinement (Figure 2), the Raman spectra indicate the c-phase increases as the content of Eu 3+ grows, which is seen in Figure 4 at 585 cm −1 . Unfortunately, the cubic (single) F 2g mode around 630 cm −1 is rather weak and hidden in the much stronger phonon bands of the tetragonal phase. Here, the tetragonal ZrO 2 behaves anti-symbaticly with the cubic phase as the Eu 3+ content increases. In R.S., this corresponds to a decrease in t-ZrO 2 contribution at 266 cm −1 (Figure 4).
Besides this, the scattering band in the range 530-670 cm −1 shifts to the smaller wave numbers, from the peak at 640 cm −1 for zirconia nanoparticles containing 2 mol% EuO 1.5 (Figure 4, curve 1) to the peak with a maximum at 611 cm −1 for the Zr 0.85 Eu 0.15 O 1.925 composition (Figure 4, curve 5). This result is also confirmed by a decrease in the intensity of scattering at 460 cm −1 with a Eu 3+ content increase in the ZrO 2 crystal lattice ( Figure 4).

Photoluminesence Properties
The P.L. spectra for Zr 1-x Eu x O 2-0.5x (x = 0.02 ÷ 0.15) nanoparticles are presented in Figure 5. The peaks observed at 591, 606, 614, 624, 633, 651, 657, 701 and 748 nm, as well as the shoulder at 580 nm, can be compared with the spectral terms of the Stark splitting 5 D 0 → 7 F J (J=0-6) for the radiative transitions in the Dieke diagram of the Eu 3+ 4f n configuration [14,51,52]. The excitation spectra ( Figure 6) for the Zr1-xEuxO2-0.5x nanoparticles cover the range of intraconfiguration 4f n →4f n transitions in the region of 270-490 nm. Besides this, the excitation band with the maximum peak at 240 nm and several additional local extremums at 227, 256 and 265 nm corresponds to the O 2 ⁻-Eu 3 ⁺ charge transfer originating from electronic transitions from the 2p orbital of O 2 ⁻ to the 4f orbital of the Eu 3 ⁺ [30, 53,54]. In our case, the excitation wavelengths (λexc) of 255, 270 and 397 nm were chosen based on the excitation spectra needed to obtain P.L. It is known that the contributions of intensity in the P.L. spectrum at 606 and 633 nm are associated with the tetragonal and cubic phases of zirconia, the maximum at 614 with the shoulder at 624 nm being assigned to the ZrO2 monoclinic allomorph [29,55]. The fact that the crystalline field of different allomorphs may split the luminescence band Eu³⁺ in different ways was previously shown in [28]. The presence of Eu 3+ multisites across a single phase and the co-presence of multiple allomorphs complicates any reliable analysis of its Stark structure. Besides, it should be remarked that the 5 D0→ 7 F2 peak does not split in the statistically non-symmetric environment formed by the Eu 3+ -polymeric complexes, either in bulk or solution [52].
The appearance of the high symmetry phases (tetragonal/cubic) of ZrO2 is evident in the P.L. spectra when the content of EuO1.5 in nanoparticles is 5 mol.% or more ( Figure 5, curves 2-5). Its fraction is shown to rise upon increasing the Eu 3+ content, according to the P.L. spectra at all used values of λexc. In this case, the Ilum contribution grows at 606 nm and 633 nm in the P.L. spectra (Figure  5a-c). The spectral term 7 F4 splitting into several components with clear peaks at 684, 692, 701, and  The excitation spectra ( Figure 6) for the Zr 1-x Eu x O 2-0.5x nanoparticles cover the range of intraconfiguration 4f n →4f n transitions in the region of 270-490 nm. Besides this, the excitation band with the maximum peak at 240 nm and several additional local extremums at 227, 256 and 265 nm corresponds to the O 2− -Eu 3+ charge transfer originating from electronic transitions from the 2p orbital of O 2− to the 4f orbital of the Eu 3+ [30,53,54]. In our case, the excitation wavelengths (λ exc ) of 255, 270 and 397 nm were chosen based on the excitation spectra needed to obtain P.L. It is known that the contributions of intensity in the P.L. spectrum at 606 and 633 nm are associated with the tetragonal and cubic phases of zirconia, the maximum at 614 with the shoulder at 624 nm being assigned to the ZrO 2 monoclinic allomorph [29,55]. The fact that the crystalline field of different allomorphs may split the luminescence band Eu 3+ in different ways was previously shown in [28]. The presence of Eu 3+ multisites across a single phase and the co-presence of multiple allomorphs complicates any reliable analysis of its Stark structure. Besides, it should be remarked that the 5 D 0 → 7 F 2 peak does not split in the statistically non-symmetric environment formed by the Eu 3+ -polymeric complexes, either in bulk or solution [52]. 11 monoclinic phases [28]. We found that in the luminescence spectra, the m-ZrO₂ phase manifests itself against a tetragonal/cubic contribution, which is associated with the possible uneven segregation of europium (III) ions. According to the phase diagram given in [56], we can talk about the uneven isolation (segregation) of europium (III) ions along the grain of the different phases. In the P.L. spectra at excitations of 270, 300 and 325 nm, the segregation of Eu³⁺ between phases is made particularly evident for Zr₀.₈₅Eu₀.₁₅O₁.₉₂₅ by comparing the peaks at 614 nm with 606 and 633 nm (cf. curves 4 and 5 in Figure 5b, as well as 5d, curve 2'; cf. curve 4 with 2 and 3 in Figure S2). Moreover, the profile of the P.L. spectra depends on the excitation wavelengths. The higher symmetry phases, such as t-and c-ZrO2, show themselves at 606 and 633 nm (Figure 5a and b) in the range of interconfiguration and charge-transfer state transitions upon excitation at 255 and 270 nm (Figure 6a). The significant contribution of the less symmetric m-ZrO2 phase appears at 397 nm as a result of the intrinsic absorption of Eu 3+ (Figure 5c). Changing the excitation wavelength proves this effect for Zr₀.₈₅Eu₀.₁₅O₁.₉₂₅ nanoparticles clearly (Figure 5d). It turned out that in the P.L. band, we could observe six components (606, 611, 614, 618, 624, and 633 nm) in the splitting of 5 D0→ 7 F2 for nanoparticles with 15 mol.% EuO1.5 at λexc = 300 nm. This result is accounted for by the europium (III) ions found in more than one local environment. They work as luminescent probes for investigating the complex media environment [57]. In the term 7 F2 of the P.L. band, the two lines of Stark splitting are responsible for the cubic symmetry, and the four ones are because of the occurrence of the t-phase environment [27]. This Stark splitting evidences the two regular ionic arrangements in the The appearance of the high symmetry phases (tetragonal/cubic) of ZrO 2 is evident in the P.L. spectra when the content of EuO 1.5 in nanoparticles is 5 mol.% or more ( Figure 5, curves 2-5). Its fraction is shown to rise upon increasing the Eu 3+ content, according to the P.L. spectra at all used values of λ exc . In this case, the I lum contribution grows at 606 nm and 633 nm in the P.L. spectra (Figure 5a-c). The spectral term 7 F 4 splitting into several components with clear peaks at 684, 692, 701, and 712 nm (Figure 5a,b) proves the high symmetry of the t-/c-ZrO 2 phase. However, we should note the splitting of nine lines for monoclinic symmetry, seven for tetragonal, and four for cubic indicates the single center distribution and the multisite one, which is the case for Eu 3+ in ZrO 2 [26,27]. As a result, it is difficult to conclude on this or that symmetry for the zirconia crystal lattice. However, what needs to be noted is that the index characterizing the environmental symmetry of Eu 3+ in the zirconia crystal lattice was estimated as the ratio of intensities of the magnetic (I 591 ) and electric (I max 606, 614 ) dipole transitions from the P.L. spectra (Figure 5a-c) [25]. This systematically increases from 0.65 to 0.87 at λ exc = 255 nm, from 0.41 to 0.71 at λ exc = 270 nm, and from 0.29 to 0.54 at λ exc = 397 nm for the t-/c-ZrO 2 phase.
The presence of monoclinic zirconia along with high symmetry phases of Zr 1-x Eu x O 2-0.5 x nanoparticles at x = 0.05-0.15 is confirmed in the P.L. spectra by a significant contribution at 614 and 624 nm (Figure 5b-c) [29,55]. The existence of m-ZrO 2 was also inferred from the peaks in the Raman spectra at 178, 336 and 474 cm −1 (Figure 4). The X.R.D. method has shown that its quantity at x ≥ 0.05 can be expected to be no more than 2%. Tiseanu C. et al. in their article discuss ordered and disordered monoclinic phases [28]. We found that in the luminescence spectra, the m-ZrO 2 phase manifests itself against a tetragonal/cubic contribution, which is associated with the possible uneven segregation of europium (III) ions. According to the phase diagram given in [56], we can talk about the uneven isolation (segregation) of europium (III) ions along the grain of the different phases. In the P.L. spectra at excitations of 270, 300 and 325 nm, the segregation of Eu 3+ between phases is made particularly evident for Zr 0.85 Eu 0.15 O 1.925 by comparing the peaks at 614 nm with 606 and 633 nm (cf. curves 4 and 5 in Figure 5b, as well as Figure 5d, curve 2'; cf. curve 4 with 2 and 3 in Figure S2).
Moreover, the profile of the P.L. spectra depends on the excitation wavelengths. The higher symmetry phases, such as t-and c-ZrO 2 , show themselves at 606 and 633 nm (Figure 5a,b) in the range of interconfiguration and charge-transfer state transitions upon excitation at 255 and 270 nm (Figure 6a). The significant contribution of the less symmetric m-ZrO 2 phase appears at 397 nm as a result of the intrinsic absorption of Eu 3+ (Figure 5c). Changing the excitation wavelength proves this effect for Zr 0.85 Eu 0.15 O 1.925 nanoparticles clearly (Figure 5d). It turned out that in the P.L. band, we could observe six components (606, 611, 614, 618, 624, and 633 nm) in the splitting of 5 D 0 → 7 F 2 for nanoparticles with 15 mol.% EuO 1.5 at λ exc = 300 nm. This result is accounted for by the europium (III) ions found in more than one local environment. They work as luminescent probes for investigating the complex media environment [57]. In the term 7 F 2 of the P.L. band, the two lines of Stark splitting are responsible for the cubic symmetry, and the four ones are because of the occurrence of the t-phase environment [27]. This Stark splitting evidences the two regular ionic arrangements in the nanoparticles' crystal lattice, at least. Stated differently, varying the excitation wavelength is sufficient to enable the m-ZrO 2 to be recognized against the prevalent metastable phases.
It should be noted that when the intraconfigurational transitions are excited in the region of 270-397 nm, the contribution of P.L. intensity at 614 nm grows in the excitation spectra (Figure 6a) upon increasing the Eu 3+ content. Moreover, in the excitation region of 395-405 nm for ZrO 2 nanoparticles with 5 mol.% EuO 1.5 , one can infer the increase in the contribution of the m-phase from the P.L. intensities observed at 614 nm, as compared with the t-/c-ZrO 2 which appeared at 606 nm (cf. curve 1 and 2, Figure 6b). Figure 7 shows the P.L. intensity decays I lum (t) of Eu 3+ in the ZrO 2 matrix. The systems under study are sensitive to the wavelength of excitation; in addition, they consist of particles in metastable states with microstrains and excess energy at the border. In this regard, it is possible to expect the wide spectrum of times characterizing the luminescence lifetime for Eu 3+ as a probe. Therefore, in this work, we used the "stretched" exponential function to describe luminescence decay. The parameters for the obtained P.L. lifetimes are given in Table 2. The stretched exponential function is used following A. Werner (1907) and T. Förster (1949) to approximate the P.L. decay for phosphors dwelling in multiple-type environments [58]: where t is decay time, I 0 is the luminescence intensity at t = 0, τ PL is the photoluminescence lifetime, t 0 is the time shift in the observation channels, and B is background of a signal under a "dark" condition. The value of βis the stretched exponential parameter (0 ≤ β ≤ 1), with which the width of the luminescence lifetime spectrum in the excited states can be characterized as D f = 2 − β.
Crystals 2020, 10, x 12 of 19 nanoparticles' crystal lattice, at least. Stated differently, varying the excitation wavelength is sufficient to enable the m-ZrO2 to be recognized against the prevalent metastable phases. It should be noted that when the intraconfigurational transitions are excited in the region of 270-397 nm, the contribution of P.L. intensity at 614 nm grows in the excitation spectra (Figure 6a) upon increasing the Eu 3+ content. Moreover, in the excitation region of 395-405 nm for ZrO2 nanoparticles with 5 mol.% EuO1.5, one can infer the increase in the contribution of the m-phase from the P.L. intensities observed at 614 nm, as compared with the t-/c-ZrO2 which appeared at 606 nm (cf. curve 1 and 2, Figure 6b). Figure 7 shows the P.L. intensity decays Ilum(t) of Eu 3+ in the ZrO2 matrix. The systems under study are sensitive to the wavelength of excitation; in addition, they consist of particles in metastable states with microstrains and excess energy at the border. In this regard, it is possible to expect the wide spectrum of times characterizing the luminescence lifetime for Eu³⁺ as a probe. Therefore, in this work, we used the "stretched" exponential function to describe luminescence decay. The parameters for the obtained P.L. lifetimes are given in Table 2. The stretched exponential function is used following A. Werner (1907) and T. Förster (1949) to approximate the P.L. decay for phosphors dwelling in multiple-type environments [58]: where t is decay time, I0 is the luminescence intensity at t = 0, τPL is the photoluminescence lifetime, t0 is the time shift in the observation channels, and B is background of a signal under a "dark" condition. The value of β is the stretched exponential parameter (0 ≤ ≤ 1Ошибка! Ожидалась цифра.), with which the width of the luminescence lifetime spectrum in the excited states can be characterized as Df = 2 -β.    1 The reduced Chi-square parameter, χ red 2 , which equals the residual sum of squares divided by the degree of freedom and is used in Pearson's chi-square test of fit goodness to establish whether an observed experimental curve differs from a theoretical one.
It was found that the values of τ PL occur in the range of 0.27-0.84 ms. This fact can be connected with the low P.L. quantum yield (ϕ). The widths of the lifetime spectra were almost equal, according to the value of D f ( Table 2).
It turned out that the quantum yield of Zr 1-x Eu x O 2-0.5x nanoparticles (x = 0.02-0.15), obtained from zirconyl and europium chlorides by ammonia co-precipitation and hydrothermal treatment, is no more than 3%.

Surface Properties
The values of specific surface area (S BET ) for the nanocrystalline samples of the ZrO 2 -x mol.% EuO 1.5 composition with x = 2, 5, 7, 10 and 15, obtained by hydrothermal synthesis, are presented in Table 3. The data indicate a slight growth of the S BET value from 80.2 to 87.4 m 2 /g with an increase in the EuO 1.5 concentration in the ZrO 2 crystal lattice from 2 to 10 mol.%. The introduction of 15 mol.% Eu O 1.5 leads to a drastical increase (ca. 20%) in the S BET to 111.3 m 2 /g. At the same time, the form of sorption isotherms for these powders is practically independent of the stabilizing ion concentration. All isotherms belong to the IV type with a strongly pronounced loop of capillary-condensation hysteresis of H2 type, characteristic for mesoporous materials with bottle-shaped pores (Figure 8). The pore size distribution of ZrO2 nanopowders with different Eu 3+ contents calculated for the desorption branch according to the B.J.H. algorithm is shown in Figure 8b. The lognormal distribution of mesopores in the range of 4 to 20 nm, with a maximum of 8 nm, is observed for all samples, except nanoparticles with a concentration of europium (III) ions equal to 15mol.%. For the latter, the pore size maximum is fixed as 6 nm.
On the surface of undoped ZrO2 nanoparticles, there are terminal hydroxyl groups associated with zirconium (IV) ions, as well as the tribridged Zr3OH moieties (Figure 9) [59,60]. The monocoordinated ZrOH bonds exhibit acidic properties, which lead to the negative charge of the nanoparticle's surface, i.e., to the formation of the ZrOinner coating of the electrical double layer   1 The specific volume of pores is determined by the limiting filling (P/P 0 = 0.99).
The pore size distribution of ZrO 2 nanopowders with different Eu 3+ contents calculated for the desorption branch according to the B.J.H. algorithm is shown in Figure 8b. The lognormal distribution of mesopores in the range of 4 to 20 nm, with a maximum of 8 nm, is observed for all samples, except nanoparticles with a concentration of europium (III) ions equal to 15mol.%. For the latter, the pore size maximum is fixed as 6 nm.
On the surface of undoped ZrO 2 nanoparticles, there are terminal hydroxyl groups associated with zirconium (IV) ions, as well as the tribridged Zr 3 OH moieties ( Figure 9) [59,60]. The mono-coordinated ZrOH bonds exhibit acidic properties, which lead to the negative charge of the nanoparticle's surface, i.e., to the formation of the ZrO − inner coating of the electrical double layer with negative ϕ-potential [61]. In the outer coating, the positive counterion-presented protons (hydroxonium ions) are adsorbed. Thus, a negative ζ-potential exists at the surface of the hydrodynamic shear, which separates the Stern layer of the positive ions strongly attached to the negatively charged nanoparticle from the counterions' diffuse layer. In the case of the isomorphic substitution of Zr 4+ ions by Eu 3+ , during the synthesis, the stabilization of the ZrO 2 high-temperature phases occurs. The formation of t-/c-ZrO 2 is accompanied by a decrease in the size of nanoparticles, and an increase in their surface energy, as well as their S BET values. Moreover, the isomorphic incorporation of Eu 3+ into the ZrO 2 matrix reduces the surface concentration of both negatively charged sites and terminal hydroxyls, which is manifested in a charge sign alteration for the electrokinetic potential ( Figure 9) [62]. This effect is caused by lower europium (III) electrovalence compared to zirconium (IV). Besides this, the Eu 3+ ions can also coordinate protons [61]. At a high local concentration on the surface, they can exhibit the properties of Lewis acid as well, attaching a water molecule [63]. This circumstance leads to the appearance of an excess of positive charge on the surface and the emergence of a positive ζ-potential (Table 3). Therefore, a further increase in the europium (III) content in the zirconia matrix contributes to a monotonous growth in the electrokinetic potential from 3.1 to 16.3 mV, with a fixed background electrolyte concentration (10 −3 M NaCl) and a pH of 6.4 for the nanoparticle dispersion.

Biological Properties
The changes in the phase composition, size, specific surface area and electrokinetic potential are reflected in the biological activity of the nanoparticles under study. The proliferation activity of human fibroblasts is preserved after three cultivation days of cells in the aqueous dispersion of Zr 1-x Eu x O 2-0.5x nanoparticles following the M.T.T. assay. This fact indicates the absence of cytotoxic properties in nanoparticles ( Figure 10). However, compared to the control sample, the proliferation activity of cells in aqueous dispersions of Zr 1-x Eu x O 2-0.5x nanoparticles is lower. An increase in the nanoparticle concentration from 5 to 500 µg/mL leads to a reduction in the survival of fibroblast cells by approximately one and half times ( Figure 10

Biological Properties
The changes in the phase composition, size, specific surface area and electrokinetic potential are reflected in the biological activity of the nanoparticles under study. The proliferation activity of human fibroblasts is preserved after three cultivation days of cells in the aqueous dispersion of Zr₁₋ₓEuₓO₂₋₀.₅ₓ nanoparticles following the M.T.T. assay. This fact indicates the absence of cytotoxic properties in nanoparticles ( Figure 10). However, compared to the control sample, the proliferation activity of cells in aqueous dispersions of Zr1-xEuxO2 0.5x nanoparticles is lower. An increase in the nanoparticle concentration from 5 to 500 μg/mL leads to a reduction in the survival of fibroblast cells by approximately one and half times ( Figure 10, column 1). A much higher survival rate is recorded for suspensions of zirconia nanopowders with a high content of europium ions, which is due to both the ζ-potential increasing and the nanoparticle size decreasing in the range of EuO₁.₅ content from 2 to 15 mol.% (Figure 10, Columns 3). The above two parameters can impact the absorption of nanoparticles by transfer through the cell membrane using

Biological Properties
The changes in the phase composition, size, specific surface area and electrokinetic potential are reflected in the biological activity of the nanoparticles under study. The proliferation activity of human fibroblasts is preserved after three cultivation days of cells in the aqueous dispersion of Zr₁₋ₓEuₓO₂₋₀.₅ₓ nanoparticles following the M.T.T. assay. This fact indicates the absence of cytotoxic properties in nanoparticles ( Figure 10). However, compared to the control sample, the proliferation activity of cells in aqueous dispersions of Zr1-xEuxO2 0.5x nanoparticles is lower. An increase in the nanoparticle concentration from 5 to 500 μg/mL leads to a reduction in the survival of fibroblast cells by approximately one and half times ( Figure 10, column 1). A much higher survival rate is recorded for suspensions of zirconia nanopowders with a high content of europium ions, which is due to both the ζ-potential increasing and the nanoparticle size decreasing in the range of EuO₁.₅ content from 2 to 15 mol.% (Figure 10, Columns 3). The above two parameters can impact the absorption of nanoparticles by transfer through the cell membrane using A much higher survival rate is recorded for suspensions of zirconia nanopowders with a high content of europium ions, which is due to both the ζ-potential increasing and the nanoparticle size decreasing in the range of EuO 1.5 content from 2 to 15 mol.% (Figure 10, Columns 3). The above two parameters can impact the absorption of nanoparticles by transfer through the cell membrane using the mechanism of pinocytosis, as one of the two types of endocytosis, which is the absorption of liquid and dissolved substances with small bubbles up to 150 nm in diameter. Thus, all five types of Zr 1-x Eu x O 2-0.5x nanoparticles under investigation can penetrate fibroblasts due to pinocytosis. The data obtained are consistent with those previously published by Patil et al. [64], as well as Chen et al. [61]. Patil et al. suggested that negatively charged nanoparticles may interact with the positive sites on the surface of the mainly negatively charged cellular membrane [64]. In the locus of interaction, the absorbed substance is gradually surrounded by a small area of plasma membrane, which is first