Upconverting SrF₂ nanoparticles doped with Yb³⁺/Ho³⁺, Yb³⁺/Er³⁺ and Yb³⁺/Tm³⁺ ions – optimisation of synthesis method, structural, spectroscopic and cytotoxicity studies

Abstract

For a number of years nanomaterials have been continuously devised and comprehensively investigated because of the growing demand for them and their multifarious applications, especially in medicine. This paper reports on the properties of SrF₂ nanoparticles (NPs) for applications in biomedicine, showing effective ways of their synthesis and luminescence under near infrared radiation - upconversion. NPs doped with lanthanide, Ln³⁺ ions (where Ln = Yb, Ho, Er, Tm) were prepared by the hydrothermal method and subjected to comprehensive studies, from determination of their structure and morphology, revealing small, 15 nm structures, through spectroscopic properties, to cytotoxicity in vitro. The effects of such factors as the reaction time, type and amount of precipitating compounds and complexing agents on the properties of products were characterized. The cytotoxicity of the synthesized and functionalized NPs was investigated, using human fibroblast cell line (MSU-1.1). The synthesized structures may decrease cells’ proliferation in a dose-dependent manner in the measured concentration range (up to 100 µg/mL). However, the cells remain alive according to the fluorescent assay. Moreover, the treated cells were imaged using confocal laser scanning microscopy. Cellular uptake was confirmed by the presence of upconversion luminescence in the cells.

Introduction

Lanthanides are known as excellent for application in luminescent materials, have unique physicochemical and optical properties, being a result of 4 f electronic configuration. Nowadays the growing interest in research is focused on upconverting nanoparticles (UCNPs) based on host materials doped with lanthanide ions (Ln³⁺). These materials can convert low energy photons from the near-infrared range (NIR) to higher energy ones, through the multiphoton absorption process¹. Upconverting materials can be used in various applications such as displays, solar cells, sensors, lasers, biosensors, drug delivery and many others^2,3,4,5,6. Especially interesting is the application of UCNPs as biomarkers what is possible due to their excitation within biological transparency window (in the range 700–1000 nm)⁴. Thanks to this property and by using of NIR radiation, harmful effects to healthy cells are reduced in comparison to those taking place under UV excitation.

As potential biomarkers, UCNPs should exhibit high emission intensity, small size (below 50 nm) and low cytotoxicity. Their surface should also allow conjugation with biomolecules. The most prominent phosphors, which exhibit efficient emission of light under NIR radiation are based on fluoride matrices, such as, MF₂ host materials doped with Ln³⁺ ions (where M = Ca, Sr, Ba; for more details see Table S1)^{3,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22}. These materials are characterized by low phonons energy and high chemical stability, which have direct influence on their potential applications^23,24.

The most important step, determining NPs utilization is their effective synthesis. For this purpose, the most promising for preparing MF₂ materials is the solvo(hydro)thermal method allowing carrying out the process under high pressure and temperature²⁴. The advantages of the method are: obtaining single-phased products with small sizes of NPs, possibility of carrying out the synthesis in water, easy control of the synthesis conditions and, what is the most important, good crystallinity of products which improves their luminescence efficiency²⁴.

Hydrothermal method is very common for synthesis of upconverting SrF₂ what is reflected by many of published articles^{12,14,15,23,25,26}. One of the first papers was published by J. Sun et al.²⁶, where particles were obtained in one-step hydrothermal method with different surfactants (citric acid, EDTA, PVP, OA) to change their size and morphology. The same group of researchers obtained SrF₂:Yb³⁺, Er³⁺ in oleate complex system in a mixture of water, ethanol and oleic acid, what allowed to obtain much smaller particles, around 5–25 nm¹¹. The most known and cited method for synthesis of SrF₂ NPs, was published by M. Pedroni et al.¹². This procedure allows obtaining very small particles with a size around 8 nm and intense emission in water colloids. Based on this synthesis route, a lot of research has been done, e.g. I. Villa et al. obtained SrF₂:Nd³⁺ particles for deep tissue, autofluorescence free, high resolution in vivo imaging using emission band at 1.340 μm¹³; M. Quintanilla et al. synthesized 9 nm SrF₂:Yb³⁺,Tm³⁺ NPs in water/D₂O colloids, with intense emission in ultraviolet range; S. Balabhada et al.¹⁷ based on SrF₂:Yb³⁺,Er³⁺ reported a straightforward method to predict the temperature calibration curve of any upconverting thermometer based on two thermally coupled electronic levels independently of the medium. Besides, many research groups modified Pedroni’s method of SrF₂ NPs synthesis, especially to obtain core@shell structures, e.g. S. Zanzoni et al.¹⁶ synthesized SrF₂:Yb³⁺,Tm³⁺@SrF₂:Yb³⁺,Er³⁺ in two-step hydrothermal synthesis for investigation of interactions between Ln-doped fluoride NPs and biomolecules whereas P. Cortelletti et al. published synthesis of multishell NPs¹⁹ used for optical thermometry. A lot of research was focused on different applications and properties of upconverting SrF₂, (Table S1). The aqueous environment of NPs synthesis may be responsible for quenching of luminescence by –OH modes, due to non-radiative relaxation process. Therefore, design of accurate nanosized bioimaging probe requires optimization of the standard synthesis methods. Apart from the appropriate composition of the starting mixture, time and temperature of reaction, also adequate co-regents present in the synthesis medium, e.g. sodium citrate, ethylenediaminetetraacetic acid (EDTA) or cetyl trimethyl ammonium bromide (CTAB) may influence luminescence intensity of the final product and have effect on the size, shape, and degree of agglomeration of particles^3,8,12,27. In MF₂ doped with Ln³⁺ ions, the composition of the starting mixture may also be crucial for charge compensation within the crystal structure^12,28.

In this paper, we present structural and spectroscopic properties of SrF₂:Yb³⁺,Ln³⁺ NPs. Most of the published articles present spectroscopic properties of SrF₂:Yb³⁺,Er³⁺ or just single Ln³⁺-doped NPs^{9,11,13,15,26,29}. Herein we report comparison of three upconverting systems with Ho³⁺, Er³⁺ or Tm³⁺ ions as luminescence activators. NPs were prepared by hydrothermal synthesis, with two surfactants - trisodium citrate (NaCit) or ammonium citrate tribasic (NH₄Cit) as chelating agents. Alternative synthesis procedure, for the most common, NaCit-based was developed. Also, the effects of such factors as time and amount of precipitation agent on size, the shape and agglomeration of particles, and especially spectroscopic properties were investigated. Because of potential biological applications of the synthesized NPs also their cytotoxicity was assessed. The prepared NPs were also examined as potential biomarkers. NPs may interfere with the functioning of cells, e.g. by plasma membrane integrity disruption, disturbance with organelle function or damage of the cytoskeleton³⁰. The cytotoxicity depends on a lot of traits, such as size, shape, functionalization, coating, cell line used during the experiment, etc.³¹. Therefore, it is essential to assess the toxicity of newly produced NPs, which was one of the aims of the presented study. Toxicity analysis distinguishes our article from many other. Moreover, the cellular uptake of the nanostructures was imaged using confocal microscopy. Our article presents complex and wide report covering optimization of synthesis procedure, comparison of luminescence properties between products prepared in various conditions as well as with three different emitting lanthanide ions. Additionally cytotoxicity of prepared products was analysed for bare and functionalised NPs.

Results

Structure and morphology

Strontium fluoride doped with Ln³⁺ crystallized as cubic crystals with ${Fm}\bar{3}m$ space group (Fig. 1). Samples showed single-phased structure, however, they had different crystal sizes, which was reflected in the width of XRD peaks. NPs’ sizes determined from Scherrer equation and DLS measurements are collected in Tables S2 and S6 (for experimental results see Figs S1 and S2). Synthesis of the samples with 1.5× excess of NH₄F within 6 and 12 h resulted in similar NPs with 11–14 nm diameter, no matter which co-regent was used. By increasing the excess of F⁻ ions to 3×, more than twice larger NPs with sizes between 28 and 39 nm were obtained. These differences in the size and morphology were caused by changes in the kinetics of the reaction and rapid growth of crystallites in the presence of higher concentration of F⁻ ions. Determined hydrodynamic diameters of the obtained NPs also confirmed the growth of their sizes from around 20–40 nm to above 100 nm when a higher amount of NH₄F was used. The results indicate also, the tendency to agglomeration of NPs prepared with threefold excess of NH₄F.

Figure 1

XRD patterns of the SrF₂ samples synthesized by hydrothermal method. The patterns are described according to the scheme: source of citric ions, time of reaction, excess of NH₄F precipitating compound (the used concentrations were: 20% Yb³⁺, 1% Ho³⁺, 1%Er³⁺ and 0.25% Tm³⁺).

TEM images show small spherical NPs with narrow size distribution when 1.5× excess of NH₄F was used (Fig. 2a,b). The determined sizes were: 14.7 ± 2.5 nm for products obtained in the presence of NaCit as a co-reagent and 13.3 ± 2.5 nm when NH₄Cit was used. With a 3× excess of NH₄F much larger NPs precipitated: 42.4 ± 9.7 nm for NaCit used as a co-reagent and 38.4 ± 10.6 nm when NH₄Cit was used. NPs prepared with higher excess of NH₄F were of irregular shape with a wider distribution of their sizes and with visible agglomeration.

Figure 2

TEM images and nanocrystals size distribution of hydrothermally synthesized samples: (a) SrF₂:Yb³⁺,Er³⁺, NaCit, 12 h, 1.5 × NH₄F (b) SrF₂:Yb³⁺,Er³⁺, NH₄Cit, 12 h, 1.5 × NH₄F (c) SrF₂:Yb³⁺,Er³⁺, NaCit, 12 h, 3 × NH₄F (d) SrF₂:Yb³⁺,Er³⁺, NH₄Cit, 12 h, 3 × NH₄F.

The composition of obtained products analysed by the ICP-OES technique (results in Table S3) revealed higher than expected amount of Yb³⁺ in all samples: between 21–24% when a 1.5× excess of NH₄F was used and between 23–28% when the excess was 3×. The explanation of the extra amount of Yb³⁺ ions is complex. The smallest ionic radius of Yb³⁺ ions from all the elements used in the synthesis allowed efficient incorporation of these ions into the SrF₂ crystal lattice (rSr²⁺ = 1.26 Å, rTm³⁺ = 0.9994 Å, rEr³⁺ = 1.004 Å, rHo³⁺ = 1.0015 Å, rYb³⁺ = 0.985 Å for coordination number CN = 8)³². High amount of charge compensating F⁻ ions presented when 3× excess of NH₄F was added, additionally allowed embedding Yb³⁺ into the SrF₂ structure to a higher extent. Another factor increasing the concentration of dopant ions, is the difference in solubility between SrF₂ and LnF₃. The latter one is less soluble in water, which favours incorporation of Ln³⁺ into the forming fluoride structure. The presence of higher amount of F⁻ ions also leads to this process (according to the solubility constant).

Elemental analysis revealed higher amount of nitrogen atoms in the samples synthesised in the presence of NH₄Cit as co-reagent (see Table S4) in comparison with those obtained in presence of NaCit. The increased amount of nitrogen is related to NH₄⁺ ions presented on the surface of NPs as well as ions embedded into structure of particles.

Higher concentration of Yb³⁺ in the materials resulted in reduced crystal cell volumes in comparison to those doped to a lower degree (for more see Table S5). The smallest cell volumes were calculated for the samples with 3× excess of NH₄F and prepared with NH₄Cit as co-reagent (for Yb³⁺/Ho³⁺, V = 188.09(4) ų; Yb³⁺/Er³⁺ V = 188.10(5) ų; Yb³⁺/Tm³⁺ V = 188.16(5) ų). The reference, taken from the ICSD database, shows the cell volume for the pure SrF₂ V = 194.50(7) ų.

In the fluorite-type structure of MF₂, the cations are located at the centre of a cubic unit cell, surrounded by eight F⁻ anions. The doping by Ln³⁺ replaces M²⁺ ions introducing a local charge. Charge balance may be achieved by the addition of interstitial F⁻ ions, occurring simultaneously or by introduction of M⁺ ions^12,23,28. Along with charge compensation process, structural and spectroscopic properties of NPs may be affected. Monovalent cations (e.g. Na⁺), lead to a reduction in interatomic distance which can significantly improve upconversion properties of these materials^23,33. In the prepared SrF₂ NPs, there are two possibilities of charge compensation. The first, with creation of interstitial fluorine ions: Sr²⁺ → Ln³⁺ + F⁻, and the second with incorporation of monovalent cations, which were Na⁺ or NH₄⁺ depending on the type of reagent: 2Sr²⁺ → Na⁺/NH₄⁺ + Ln^3+ 28. However, NH₄⁺ ions are larger than Na⁺ and Sr²⁺, thus their ability to compensate charge imbalance is rather low (rNH₄⁺  = 1.54 Å, rNa⁺  = 1.18 Å)³².

Zeta potentials were measured to determine the surface charge of the prepared NPs. All samples exhibited negative surface charge from −14.9 ± 5.4 mV to −33.3 ± 4.4 mV (Table S7) at physiological pH, which indicates their good stability in water as colloids and confirm the presence of negative COO⁻ groups on the surface. Furthermore, the FT-IR spectra also revealed citrate anions on the surface of the obtained particles via the presence of stretching vibrations assigned to the –CO, -CH, -COO⁻ and–OH bonds (Supplementary Materials, Fig. S3). As the result of higher amount of NH₄⁺ ions in the materials prepared in presence of NH₄Cit, absorption band located at around 1610 cm⁻¹ revealed shift towards -NH bending range in comparison to samples prepared in the presence of NaCit.

Spectroscopic properties

Under excitation with the NIR radiation (at 976 nm) all samples exhibited emission that resulted from the used Ho³⁺, Er³⁺ or Tm³⁺ dopant ions (Fig. 3). The brightest emissions were observed for the samples, obtained in 12 h synthesis with 3× excess of NH₄F. The weakest emissions were recorded for the samples obtained in shorter reaction time (6 h) and with 1.5× excess of F⁻ ions.

Figure 3

(a) Luminescence (300–900 nm) and excitation (>900 nm) spectra of the NPs obtained under pulsed excitation source (at 15 mJ·cm⁻²). (b) Emission of SrF₂:20%Yb³⁺,1%Er³⁺ samples, where: (i) NaCit, 12 h, 3 × NH₄F, (ii) NaCit, 12 h, 1.5 × NH₄F, (iii) NH₄Cit, 12 h, 3 × NH₄F, (iv) NH₄Cit, 12 h, 1.5 × NH₄F, (v) NaCit, 12 h, 1.5 × NH₄F, excited by laser (λ_ex = 976 nm) in water (i-iv) or phosphate-buffered saline (v) with concentration 0.1 mg/mL. (c) Simplified scheme of upconversion mechanism for SrF₂: Yb³⁺, Ln³⁺ systems.

For all samples, spectroscopic measurements revealed the emission typical of Ln³⁺ ions and energetic processes taking place in the products, such as energy transfer from Yb³⁺ to Ln³⁺ ions, excitation into higher energetic levels or emission quenching. The excitation spectra (Fig. 3a 900–1050 nm range) measured for the samples show broad and intense bands with the maximum at 976 nm, which are characteristic of the ²F_7/2 → ²F_5/2 transition of Yb³⁺ ions. These ions play a role of sensitisers in the studied systems, absorbing laser light. Yb³⁺ ion is perfect for this function because of its simple energy structure: one excitation energy level (²F_7/2), produced by the absorption of radiation with a wavelength at around 980 nm. The intensity of this band was the highest for the samples obtained in 12 h synthesis, with 3× excess of NH₄F, where NaCit was used as a co-reagent. The excitation band is broad in each sample, which is the effect of crystal field on the local environment of the ion resulting in Stark-splitting of the ground ²F_7/2 multiplet.

The strongest emissions, similarly to the excitation spectra, were recorded for the products obtained in the 12 h synthesis with NaCit and 3× excess of NH₄F, (see also Fig. S4). High luminescence intensity was also observed for the samples doped with Yb³⁺/Ho³⁺ and Yb³⁺/Er³⁺ obtained in 12 h synthesis with NaCit and 1.5 × NH₄F or NH₄Cit and 3× NH₄F. The above results can be explained by a relatively large size of NPs, obtained during longer reaction time and with excess of fluorine ions. Small NPs are known to be prone for surface- and defects-related quenching to a greater extent than large ones³⁴. Also, an increased amount of sensitiser ions (Yb³⁺), found in the samples prepared during longer synthesis (Table S5) is a factor which improved luminescence of the described structures.

The used co-reagent had a significant effect on the emission of NPs prepared. When NH₄Cit was used, luminescence quenching was observed. This effect was caused by a larger number of NH₄⁺ ions present on the surface of NPs in comparison to that on the products prepared in the presence of NaCit. The N-H vibrations are more efficient quenchers than O-H and our results confirm this fact.

Analysis of the ratio of intensities of the two most intense bands present in the emission spectra brings additional information about the studied systems (see Fig. 3a for the spectra or Fig. S5 for comparison of integrated emission intensities). The samples doped with a Yb³⁺/Ho³⁺ pair of ions showed the ⁵S₂,⁵F₄ → ⁵I₈ band as the most intense, which resulted in green colour of emission (for CIE chromaticity diagram see Fig. S5). In the products whose emission was of lower intensity, the green (⁵S₂,⁵F₄ → ⁵I₈) and red (⁵F₅ → ⁵I₈) emission bands had similar intensities, which was the result of decreased efficiency of excitation to the ⁵S₂,⁵F₄ higher excited state. Excitation into higher levels of Ln³⁺ ions usually require good crystallinity of the material and lack of quenching factors, such as NH₄⁺ ions.

NPs doped with Yb³⁺/Er³⁺ pair ions, showed yellowish-green emission colour, as an effect of mixing of the transitions ⁴F_9/2 → ⁴I_15/2 (in the red range) and ⁴S_3/2 → ⁴I_15/2 (green range). The first one was the most intense from all observed transitions. NPs doped with Yb³⁺/Er³⁺ pair ions were characterized by the highest emission intensity from the whole studied group of compounds. The high upconversion intensity of Yb³⁺ and Er³⁺ doped samples is connected with the best match of the energies of the excited levels of the used activator ions (Yb^{3+ 4}F_5/2 → Er^{3+ 4}I_11/2), thanks to which the energy transfer between these ions is more efficient. Depending on the synthesis conditions, the ratio between red and green transition bands was slightly different, revealing the influence of NPs’ sizes and charge compensation issues on the UC mechanism and quenching processes efficiency (see Figs S5 and S6). Increased red emission band is usually connected with non-radiative relaxation form the ⁴S_3/2 excited state what in the presented results is an effect of NH₄⁺ ions presence and lower crystallinity of the products obtained at shorter time and with smaller NPs’ sizes.

The samples doped with Yb³⁺/Tm³⁺ pairs of ions showed blue or pink-blue UC with the domination of ³H₄ → ³H₆ transition (NIR band) in the spectra (Figs 3a, S5 and S6). The ratio between ¹G₄ → ³H₆ and ³H₄ → ³H₆ transitions was similar for each type of co-reagent used. However, for the systems in which quenching through to the presence of –NH oscillators was noticeable in the emission intensity, the colour of emission was slightly shifted to the red (Fig. S6). The ¹D₂ and ¹G₄ excited states of Tm³⁺ are more sensitive to the quenching factors as evidenced by a change in the emission colour. The observed spectroscopic properties of SrF₂:Yb³⁺,Tm³⁺ NPs shows that colour of luminescence may be controlled during the synthesis, by co-reagents affecting the quenching processes.

Figure 3b shows the luminescence of SrF₂:20%Yb³⁺,1%Er³⁺ NPs in the form of water colloids under excitation with 976 nm laser light. The intense emission of water colloids allow a description of prepared NPs as potentially useful for biomedical applications especially for bioimaging as we show below. A scheme of the upconversion mechanism responsible for the observed emissions is presented in Fig. 3c.

On the basis of luminescence decays measured for the obtained product (Supplementary Materials, Fig. S7), the luminescence lifetimes were calculated and are collected in Table 1. The values of lifetimes for the Yb³⁺/Ho³⁺ doped samples are strongly related to the luminescence intensity. The longest lifetimes (147 μs for the ⁵F₅ → ⁵I₈ and 118 μs for ⁵S₂, ⁵F₄ → ⁵I₈ transition) were determined for the best emitting sample prepared in 12 h synthesis, with NaCit as co-reagent and 3× excess of NH₄F. An analogous sample, but prepared in the presence of NH₄Cit, had a similar value of lifetime. Luminescence lifetimes determined for the ⁵F₅ → ⁵I₈ transition, which occurred in the red spectral range, were usually the longest ones, which is related to the necessity of non-radiative transition between the ⁵S₂, ⁵F₄ and ⁵F₅, excited states. The shortest decay time was measured for the sample obtained in 6 h synthesis and with NaCit as co-reagent. The reason is very weak luminescence of this highly quenched sample.

Table 1 Emission lifetimes calculated on the basis of the measured luminescence decays of SrF₂:Yb³⁺,Ln³⁺ NPs under 976 nm laser excitation (for decays see Fig. S8, err < 0.1 μs).

Analysis of luminescence decays measured for the samples doped with Yb³⁺/Er³⁺ ions, revealed that the longest lifetimes were typical of products obtained in 12 h synthesis in the presence of NaCit and 3× excess of NH₄F, similarly to the Yb³⁺/Ho³⁺ doped samples. The lifetime assigned to the ⁴F_9/2 → ⁴I_15/2 transition was 236 μs for the sample prepared in the presence of NaCit and 174 μs when NH₄Cit was used as co-reagent. Lifetimes were slightly shorter for the compounds prepared with NH₄Cit as a chelating agent, because of the earlier mentioned quenching effect of -NH modes. The longest lifetimes calculated for the red band can be related to the Er³⁺ excitation mechanism to the ⁴F_9/2 level. There are two possibilities of the excitation: one connected with non-radiative transition from ²H_11/2 level and the other with non-radiative transition from ⁴I_11/2 to ⁴I_13/2 excited state followed by absorption of a photon to ⁴F_9/2 level (⁴I_13/2 (Er³⁺) + ²F_5/2 (Yb³⁺) → ⁴F_9/2 (Er³⁺) + ²F_7/2 (Yb³⁺)). The first excitation pathway occurs when the emission lifetime of ⁴F_9/2 → ⁴I_15/2 transition is similar to that of ⁴S_3/2 → ⁴I_15/2, which is observed for the compound synthesized in a 6 h process with 1.5× excess of NH₄F and in the presence of NaCit. The second mechanism can explain the observed spectroscopic properties if the decay time of the transition ⁴F_9/2 → ⁴I_15/2 is twice as long (or even more) as the ⁴S_3/2 → ⁴I_15/2 transition lifetime. That is true for all samples except the one mentioned above. If the first pathway of the ⁴F_9/2 population dominates, the decay time of ⁴F_9/2 level should be similar to that of ⁴S_3/2. But if the energy transfer prevails, the decay time of the ⁴F_9/2 level depends on the decay times of ⁴I_13/2 (Er³⁺) and ²F_5/2 (Yb³⁺).

The SrF₂:Yb³⁺,Tm³⁺ NPs showed the longest decays from all obtained samples. Furthermore, similarly as for the samples doped with Yb³⁺/Ho³⁺ or Yb³⁺/Er³⁺ ions described above, the sample prepared in 12 h synthesis, with 3 × NH₄F and with the use of NaCit, was characterized by the highest value of emission lifetime (596.6 μs for ¹G₄ → ³H₆ and 592.3 μs ¹G₄ → ³F₄). Similarly to the samples with Yb³⁺/Ho³⁺ and Yb³⁺/Er³⁺, those prepared with NH₄Cit as a complexing agent showed shorter lifetimes in comparison to that of the analogous product prepared with NaCit. Decay times strongly depended on the population mechanism of the excited states. Also, quenching factors present in the system affected the determined emission lifetimes.

For better understanding of the upconversion mechanism, dependencies of the luminescence intensity on laser powers were measured. The relation between the UC intensity I and the pumping excitation power density, P is given by the equation³⁵:

$$I\propto {P}^{n}$$

where n is the number of photons required to populate the excited state. The results of the calculations are collected in Table 2 (see Fig. S8 for the experimental data).

Table 2 Number of photons involved in the upconversion mechanism, determined from the dependencies of luminescence intensity on laser power for SrF₂:Yb³⁺,Ln³⁺ NPs (for experimental results see Fig. S9, err < 0.15).

To achieve ⁵S₂/⁵F₄ or ⁵F₅ excited state of Ho³⁺ ions two photons are required. The result of experiments, shown in Table 2, are in some cases different from the theoretical ones. For the samples prepared in 6 h synthesis and with NaCit as well as with NH₄Cit as co-reagents, the number of photons was lower than expected, which was caused by non-radiative relaxation to the ⁵F₅ lower excited state. The highest slope value was obtained for the sample prepared with 3× excess of NH₄F, which is also connected with intense emission and long lifetimes determined for this sample. The slope coefficients determined for NPs doped with a Yb³⁺/Er³⁺ pair of ions are higher than those for almost all samples (except the samples prepared in the presence of NH₄Cit), which confirms the two-photon excitation process. For the samples doped with Yb³⁺/Tm³⁺ ions, the dependencies of the emission intensity on laser energies were measured for four transitions. According to the scheme in Fig. 3c, the ¹D₂ energy level requires four photons in the excitation process, ¹G₄ three photons, and ³H₄ two photons. From the experimental data only for the ³H₄ → ³H₆ transition (two-photon process), the number of photons was close to the theoretical value.

Summarising, for almost all samples the slope coefficient values are lower than expected, especially for Yb³⁺/Tm³⁺ dopants. There are many factors which can affect the UC process and lower slope values, e.g. saturation effect, heating of samples or cross-relaxation process between dopants. Nanomaterials are particularly prone to quenching processes, and their crystal structure is often defected which yields luminescence deactivation centres.

On the basis of the collected and literature data, the UC mechanism proposed for SrF₂:Yb³⁺,Ln³⁺ NPs is presented in Fig. 3c. In these systems, Yb³⁺ ions absorb photons, which results in excitation of the Yb³⁺ ion from ²F_7/2 to ²F_5/2 excited state. In the next step, the energy may be dissipated, leading to Yb³⁺ ion in its ground state or, as a result of energy transfer upconversion (ETU), to the Ln³⁺ ion (⁵I₆ energy level for Ho³⁺, ⁴I_11/2 for Er³⁺ and ³H₅ for Tm³⁺). To achieve an appropriate energy level for Ho³⁺ and Er³⁺ in the samples, the two-photon process is required. For Tm³⁺ from two to four photons must be transferred.

Biological properties

Cell health and growth can be determined by quantifying different parameters. In the presented study, the viability of cells treated with the studied NPs was investigated using the WST-1 and Live/Dead cell viability assays (see Supplementary Materials for more details). The WST-1 assay determines the cell metabolic activity and proliferation, whereas the Live/Dead assay determines the ratio of alive to dead cells in the population. Fluorescent Live/Dead cell viability assay showed that the proportions of alive and dead cells incubated with NPs were similar to the control cells (Fig. 4a). Representative images of the cells exposed to the analysed NPs are displayed in Fig. 5 and Figs S9–11. However, the WST-1 test showed contrary results. Namely, NPs caused a significant decrease in the proliferation rate in a dose-dependent manner in the measured concentration range (up to 100 µg/mL) (Fig. 4b). Moreover, the cytotoxic effect differs between the NPs doped with various ions. From among bare NPs (not coated with PEG–(COOH)₂ or PAA) the structures doped with Tm³⁺ were more cytotoxic than those doped with Ho³⁺ and Er³⁺. Namely, the cells treated with SrF₂:20%Yb³⁺,0.25%Tm³⁺ at concentration 25 µg/mL showed significantly lower proliferation rate than the control cells, whereas the other two bare nanostructures showed a negative influence on the cells at 50 µg/mL. The obtained results are in contrast to some of the earlier reports claiming that the synthesized fluorides doped with Ln³⁺ ions had no or low impact on mammalian cells^36,37. However, other authors reported that some of the NPs may decrease cell viability even at relatively low concentrations³⁸.

Figure 4

(a) Influence of SrF₂:Yb³⁺,Ln³⁺, SrF₂:Yb³⁺,Ln³⁺@PEG(COOH)₂, SrF₂:Yb³⁺,Ln³⁺@PAA on human fibroblasts’ viability, determined by the Live/Dead cell viability assay. The data are shown as mean values with the standard deviation (means ± SD). (b) Influence of SrF₂:Yb³⁺,Ln³⁺, SrF₂:Yb³⁺,Ln³⁺@PEG(COOH)₂, SrF₂:Yb³⁺,Ln³⁺@PAA on human fibroblasts’ proliferation, determined by WST assay. Asterisks indicate results significantly different from those obtained for the control (Kruskal-Wallis test, significance at p < 0.05). The data are shown as mean values with the standard deviation (means ± SD).

Figure 5

Representative high-content images of MSU1.1 cells exposed to SrF₂:20%Yb³⁺,0.25%Tm³⁺ nanoparticles (6.25–100 µg/mL). Images were obtained using different filters to detect nuclei (DAPI), live cells (FITC), and dead cells (TexasRed). The scale bars denote 100 µm.

To improve the bio-application and decrease the cytotoxicity NPs are usually coated and functionalized as described previously^31,39. These procedures were also generally used to improve other properties of nanostructures, such as dispersion ability and stability of NPs in aqueous solutions, luminescence properties, shape or protection from the surrounding environment. The studied NPs were coated with two different organic compounds, namely PEG–(COOH)₂ and PAA. In the case of NPs doped with Ho³⁺ their modification with the PEG–(COOH)₂ and PAA caused a slight increase of the cytotoxicity (Fig. 4b). This effect was not observed when the cells were incubated with SrF₂:20%Yb³⁺,1%Er³⁺ and SrF₂:20%Yb³⁺,0.25%Tm³⁺. The obtained results are in accordance with those Das and co-authors, who claim that PEG-oleate capped NaYF₄:Yb³⁺,Er³⁺ significantly decrease the viability of Human Aortic Endothelial Cells in comparison to bare NPs⁴⁰, although in most of the previous research, NPs coated with PEG–(COOH)₂ and PAA were considered as a less toxic than bare NPs^38,39.

The confocal microscopy study demonstrated that all of the synthetized NPs were easily internalized by the fibroblast cells (Fig. 6 and Figs S12, S13), despite their negative charge, as confirmed by the presence of upconversion luminescence. High cellular uptake of negatively charged NPs results from strong and nonspecific interactions with the plasma membrane⁴¹. The results are in accordance with previous research, showing that upconverting fluorides doped with Ln³⁺ ions are suitable for imaging^42,43.

Figure 6

Human fibroblasts after 24 h incubation with the: (A) SrF₂:20%Yb³⁺,0.25%Tm³⁺, (B) SrF₂:20%Yb³⁺0.25%Tm³⁺@PEG(COOH)₂, (C) SrF₂:20%Yb³⁺0.25%Tm³⁺@PAA, imaged using confocal laser scanning microscopy equipped with a tuneable infrared laser. Red colour - cell membrane (concanavalin 647, exc. 633 nm), blue colour – cell nuclei (DAPI, exc. 405 nm), green colour – NPs (NPs’ luminescence, infrared excitation).

Discussion

SrF₂:Yb³⁺,Ln³⁺ nanoparticles (where Ln = Ho, Er, Tm) can be synthesised by hydrothermal method using metal salts and NH₄F as reagents, NaCit or NH₄Cit used as co-reagents allowing for control reaction kinetics. The influence of complexation agents, as well as reaction time and amount of ammonium fluoride on morphology and spectroscopic properties, was studied in details. The size of obtained NPs was slightly depended on the reaction time, which was 6 or 12 h. Much more important was the concentration of NH₄F precipitating reagent. Two concentrations were used: 1.5× and 3× excess to the stoichiometric amount. Lower concertation of NH₄F resulted in NPs with sizes around 11–15 nm, however, higher excess caused precipitation of slightly agglomerated NPs with sizes between 28–39 nm. The used synthesis conditions also affected dopants concentrations what was especially visible in case of Yb³⁺ ions. Smaller particles, prepared in the presence of 1.5× excess of NH₄F, usually had between 20–23% mol. of Yb³⁺ ions, where those prepared with 3× excess of NH₄F 23–28% mol. The applied conditions also had the influence of hydrodynamic diameter of NPs in water colloids. 1.5× excess of NH₄F resulted in NPs with diameter around 18–47 nm, whereas 3× excess in the formation of larger clusters with diameter 100–250 nm, most probably consisted of several NPs. The prepared NPs were negatively charged at physiological pH, what was a result of the presence of citrate groups on their surface. Determined zeta potentials were in the range of −15 to −33 mV.

The applied synthesis conditions had a great influence on the spectroscopic properties of obtained NPs. The most intense emission under NIR wavelengths was obtained for products prepared in 12 h synthesis, with 3 × NH₄F excess and NaCit as a complexing agent. Furthermore, reaction time and added co-reagent had also effect on the upconversion colour, what was a result of changes in the intensity of emission bands. Measured luminescence decays as well as dependences of luminescence intensity on laser powers confirmed energy transfer between Yb³⁺ and Ho³⁺, Er³⁺ or Tm³⁺ as process responsible for observed upconversion.

Summarising, high upconversion intensity can be achieved only if a large excess of NH₄F reagent is used, the reaction time is long (12 h) and as co-reagent, NaCit is used. On the other hand, small NPs with narrow size distribution can be obtained when reaction time is short (6 h) and NH₄F precipitating agent is at low concentration.

The cytotoxicity of the synthesized and functionalized NPs was investigated, using WST-1 cell proliferation assay and Live/Dead cell viability assay. We demonstrated that the synthesized structures exhibited proliferative inhibition in fibroblast cells in a dose-dependent manner, whereas they appeared to be alive according to fluorescent assay. Further in vitro toxicity evaluation with the aim to discover the mechanisms of impact of NPs on the cells has to be performed. Cellular uptake of the NPs was confirmed by the presence of upconversion luminescence in the cells. The luminescent properties of the nanoparticles allow them to be used as fluorescent agents in bio-imaging applications.

Methods

Materials

Rare earth (RE) oxides: Er₂O₃ and Yb₂O₃ (99.99%, Stanford Materials, United States) were dissolved separately in hydrochloric acid, HCl (ultra-pure, Sigma-Aldrich, 37%, Poland) in order to obtain respective rare earth chloride solutions in a concentration of 1 or 0.25 M. Ammonium fluoride, NH₄F (98+%, Sigma-Aldrich, Poland) was used as the source of fluoride ions. Strontium chloride hexahydrate SrCl₂·6H₂O (Sigma-Aldrich, 99+%, Poland), citric acid trisodium salt dihydrate, (Sigma-Aldrich, 97%, Poland) and ammonium citrate tribasic, (Sigma-Aldrich, ≥97%, Poland), phosphate buffered saline (BioShop), poly(ethylene glycol) bis(carboxymethyl) ether (average M_n 250, Sigma-Aldrich, Poland), polyacrylic acid (average M_w 1800, Sigma – Aldrich, Poland) were used as received, without further purification. Deionized water was used for the synthesis.

Synthesis of SrF₂:Ln³⁺ nanoparticles

To obtain 3.5 mmol of SrF₂ doped with 20% of Yb³⁺ and 1% of Ho³⁺, 2.77 mL of 1 M SrCl₂ solution and YbCl₃ mixed with HoCl₃ (0.70 mL of 1 M YbCl₃ and 0.14 mL of 0.25 M HoCl₃) were added to 20 mL of 1 M sodium citrate, NaCit solution (anti-agglomeration and complexing agent) or 1 M solution of ammonium citrate, NH₄Cit. Then, 5 mL of 2.10 M or 4.20 M solution of NH₄F (depending on the NH₄F excess) were added to the solution containing SrCl₂ and LnCl₃ salts. The pH of the final mixture was equal to 7.5. The as-prepared transparent solution was transferred into the 50 mL Teflon-lined vessel and hydrothermally treated for 6 h or 12 h (180 °C, 15 bars), in an externally heated autoclave (Berghof DAB-2). When the reaction was completed, the obtained white precipitate was purified by centrifugation and rinsed several times with water and ethanol. The final product was dried under ambient conditions. NPs doped with Er³⁺ or Tm³⁺ were prepared analogously.

Surface functionalization of SrF₂:Yb³⁺,Ln³⁺ nanoparticles

Three samples: SrF₂:20%Yb³⁺,1%Ho³⁺, SrF₂:20%Yb³⁺,1%Er³⁺ and SrF₂:20%Yb³⁺,0.25%Tm³⁺ prepared by 12 h synthesis in the presence of NaCit and with 1.5× excess of NH₄F were selected for cytotoxicity studies. The samples showed necessary parameters for bioimaging such as good spectroscopic properties and small NPs’ sizes, around 15 nm.

To modify surface of NPs, 100 mg of each sample was dissolved in 30 mL of phosphate-buffered saline (PBS). After that, the solution was ultrasonicated for 1 h. Next, 4 mL of each NPs solution was mixed with 4 mL of 1% solution of poly(ethylene glycol) bis(carboxymethyl) ether (PEG–(COOH)₂) in PBS or 4 mL of 1% solution of polyacrylic acid (PAA) in PBS. After another 0.5 h of sonification, samples where centrifuged, washed with PBS solution three times, and dissolved in PBS. As prepared colloids were diluted by PBS solution to receive 100 µg/mL concertation of NPs.

Characterization

Powder diffractograms were recorded on a Bruker AXS D8 Advance diffractometer in the Bragg-Brentano geometry, with Cu K_α1 radiation λ = 1.5406 Å, in the 2θ range from 6 to 60°. The reference data was taken from the Inorganic Crystal Structure Database (ICSD). The composition of prepared materials was analysed by Inductively Coupled Plasma-Optical Emission Spectrometer Varian ICP-OES VISTA-MPX and EA Vario EL III. Transmission-electron-microscopy (TEM) images were recorded on an FEI Tecnai G2 20 X-TWIN transmission electron microscope, which used an accelerating voltage of 200 kV. Fourier transform infrared spectra (FT-IR) were recorded using a JASCO 4200 FT-IR spectrophotometer. DLS and zeta potential measurements were performed by using a Malvern Zetasizer Nano ZS instrument.

The luminescence characteristics (excitation, emission spectra, luminescence decays) of the prepared samples were measured on a QuantaMaster^TM 40 spectrophotometer equipped with an Opolette 355LD UVDM tunable laser, with a repetition rate of 20 Hz and a Hamamatsu R928 photomultiplier used as a detector for emission/excitation spectra and decay time measurements. A continuous Dragon Lasers DPSS 980 nm laser was used as the excitation source, coupled to a 200 µm optical fibre and collimator to determine dependencies between emission intensity and laser power.

Cytotoxicity

Cell culture

Human fibroblast (MSU-1.1) cell line from Prof. C. Kieda (CBM, CNRS, Orleans, France), was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin-streptomycin antibiotics (Sigma-Aldrich). The cells were maintained at 37 °C in a humidified atmosphere supplemented with 5% CO₂. The cell morphology was checked using an inverted microscope (Leica DMIL LED).

Cytotoxicity assays

For the cytotoxicity test, 5·10³ cells/well were seeded at 96-well plate and incubated for 24 h. Afterwards, 50 µl of several different concentrations of the NPs diluted in PBS were added to 150 µl of culture medium in each well resulting in a final concentration of 100, 50, 25, 12.5 and 6.25 µg/mL, and the cells were further incubated for 48 h. Phosphate buffered saline (PBS, Sigma-Aldrich) was used as a control. For more details see Supporting Information.