From Nanothermometry to Bioimaging: Lanthanide-Activated KY3F10 Nanostructures as Biocompatible Multifunctional Tools for Nanomedicine

Lanthanide-activated fluoride-based nanostructures are extremely interesting multifunctional tools for many modern applications in nanomedicine, e.g., bioimaging, sensing, drug delivery, and photodynamic therapy. Importantly, environmental-friendly preparations using a green chemistry approach, as hydrothermal synthesis route, are nowadays highly desirable to obtain colloidal nanoparticles, directly dispersible in hydrophilic media, as physiological solution. The nanomaterials under investigation are new KY3F10-based citrate-capped core@shell nanostructures activated with several lanthanide ions, namely, Er3+, Yb3+, Nd3+, and Gd3+, prepared as colloidal water dispersions. A new facile microwave-assisted synthesis has been exploited for their preparation, with significant reduction of the reaction times and a fine control of the nanoparticle size. These core@shell multifunctional architectures have been investigated for use as biocompatible and efficient contrast agents for optical, magnetic resonance imaging (MRI) and computerized tomography (CT) techniques. These multifunctional nanostructures are also efficient noninvasive optical nanothermometers. In fact, the lanthanide emission intensities have shown a relevant relative variation as a function of the temperature, in the visible and near-infrared optical ranges, efficiently exploiting ratiometric intensity methods for optical thermometry. Importantly, in contrast with other fluoride hosts, chemical dissolution of KY3F10 citrate-capped nanocrystals in aqueous environment is very limited, of paramount importance for applications in biological fluids. Furthermore, due to the strong paramagnetic properties of lanthanides (e.g., Gd3+), and X-ray absorption of both yttrium and lanthanides, the nanostructures under investigation are extremely useful for MRI and CT imaging. Biocompatibility studies of the nanomaterials have revealed very low cytotoxicity in dfferent human cell lines. All these features point to a successful use of these fluoride-based core@shell nanoarchitectures for simultaneous diagnostics and temperature sensing, ensuring an excellent biocompatibility.

the dispersion of KYF:Er,Yb core NPs (mass concentration of 100 mg ml −1 ) was added to the shell precursor solution. Subsequently, 1.7 mmol of NH4F (0.5 ml, 3.5M) were added to this dispersion and heat treated in the microwave reactor at 195°C for 10 minutes. The obtained core@shell NPs were precipitated and washed with acetone and redispersed in deionized water (with a typical concentration of 20 mg ml −1 ) for further characterization. The KY3F10:Er,Yb@KY3F10:Nd,Yb NPs are denoted in the paper as KYF:Er,Yb@KYF:Nd,Yb.
5. Preparation of -NaYF4:Er 3+ ,Yb 3+ @NaYF4 nanoparticles The NaYF4 core nanoparticles doped with Yb 3+ and Er 3+ ions were synthesized via a microwave assisted hydrothermal treatment, using a microwave reactor (Monowave 400, Anton Paar). Briefly, proper volumes of aqueous solutions (1 M) of yttrium and lanthanide chlorides (YCl3·6H2O, Strem Chemicals 99.9%, ErCl3·6H2O, Strem Chemicals 99.9%, YbCl3·6H2O, Acros organics 99.99+%) in order to obtain a metal molar ratio Y 3+ /Yb 3+ /Er 3+ = 0.78/0.2/0.02, were mixed with 13 ml of deionized water (total metal amount of 2.6 mmol). Then, 13.3 mmol of solid Na3C6H5O7·H2O (sodium citrate dihydrate, Alfa Aesar 99+%), were added as powder to the previous metal precursor solution. Then, an ammonium fluoride (NH4F, Sigma Aldrich > 98%) aqueous solution (3.56 ml, 3.5 M) was added to the previous mixture. The obtained transparent solution was then placed in a 30 ml glass vial (G30, Anton Paar) and heat treated in the above-mentioned microwave reactor at 170°C for 10 seconds (heating in 2 minutes). After washing with acetone, the synthesis product was stored under acetone. The synthesis of the shell was performed in the same way but using the core nanoparticles as seeds. In brief, a solution of 13.3 mmol of Na3C6H5O7·H2O (sodium citrate dihydrate, Alfa Aesar 99+%), 2.6 mmol of YCl3·6H2O (Strem Chemicals 99.9%) and 150 mg of Yb,Er doped NaYF4 NPs were mixed with ammonium fluoride (NH4F, Sigma Aldrich > 98%) aqueous solution (3.56 ml, 3.5 M) and subjected to a temperature treated in the above-mentioned microwave reactor at 170°C for 10 seconds (heating in 2 minutes). The nanoparticles were then washed with acetone and redispersed in water for further characterization.

(Scanning) Trasmission Electron Microscopy
The nanoparticles were spread on a holey carbon grid after being dispersed into water. (S)TEM images were obtained using a Talos F200X and a FEI Titan Cubed Themis Electron Microscopes, both operating at 200 KV.

Colloidal characterization
Hydrodynamic size and -potential measurements on water dispersions (mass concentration of 2 mg ml -1 ) were performed with a Malvern Zetasizer Nano ZS operating with a HeNe 633 nm laser.

X-Ray diffraction setup
XRD measurements were carried out using a Thermo ARL X-TRA diffractometer equipped with a Copperanode X-ray tube (Kα, λ=1.5406 Å) and a Peltier solid-stage that cools down the Si(Li) detector close to -100°C. For the measurements, the samples were dried in an oven at 80°C and homogenized in an agata mortar before deposition on a low background Si sample holder. The lattice constant for the KY3F10 cubic structure was determined from the XRPD patterns and the Bragg law (see Section S3 below).

Elemental analysis
An ICP-MS instrument was used for the measurement (Agilent 7700x, Agilent Technologies International Japan, Ltd., Tokyo, Japan). The instrument is equipped with an octupole collision cell operating in kinetic energy discrimination mode, which was used for the removal of polyatomic and Ar-based interferences. The ICP-MS was tuned daily using a tuning solution containing 1 µg/L 140 Ce, 7 Li,205 Tl, and 89 Y (Agilent Technologies, UK). The internal standard (IS) mixture (Agilent, 5183-4681) containing 6 Li,45 Sc,72 Ge,103 Rh,115 In,159 Tb,175 Lu and 209 Bi at 10 µg/mL each in 3.5% HNO3 was used through addition to the sample solution via a T-junction. IS is required for both matrix and instrumental drift corrections. Samples are weighed and digested in 15 mL S-5 Falcon (PP) tubes. Two digestion protocols were used, one with a mixture of 2.5 g of HNO3 (69%) + 2.5 g of HCl (37%) and the other with only 2.5 g of HNO3 (69%) both for 3h at 100°C. The digested solutions are suitably diluted to ensure that the concentrations of the elements enter the calibration line.

Luminescence measurements
The emission spectra were measured on the colloidal dispersion of the NPs using a laser diodes at 980 nm (MDLIII980, CNI), with variable laser power, and at 532 nm as the excitation sources. The emission signal was collected in the visible-NIR region using 4X and 40X microscope objectives and a short-pass filter with cutoff at 900 nm in the case of 980 nm laser. An half-meter monochromator (Sr-500i, ANDOR) equipped with a -80°C cooled CCD camera (iDus DU420A-BVF, ANDOR) was used as the recording setup. The optical resolution of the upconversion spectra is 5 cm −1 . The power density was calculated by measuring the irradiated area in the objective focal points for the two different microscope objectives (4X and 40X). For the thermometric measurements, emission spectra at different temperatures were acquired under 980 or 532 nm laser excitation, by heating the colloidal dispersions, while constantly stirring, with an aluminum heated stage, measuring the temperature with a K-type thermocouple (with a sensitivity of 0.2°C).
The decays of the UC emission were excited with the same diode laser at 980 nm, operating in TTL mode (square wave) using a waveform generator (FeelTech FY6900). The signal was collected with a 40X microscope objective and measured with the above mentioned monochromator, equipped also with GaAs photomultiplier cooled to -30°C (Hamamatsu) and a 500 MHz oscilloscope (LeCroy LT342). The emission decays were fit using the multiexponential formula: (S1) where τi, Ai represent respectively the i-th decay time and the weight for the i-th component of the multiexponential function. The average lifetimes τav were calculated using the formula (Li et al., 2020) (S2)
After 24 h, the medium was replaced with fresh medium containing increasing concentration (from 50 to 1000 µg/ml) of NPs or a fixed concentration (500 or 300 µg/ml) The MTT (3-(4,5-dimethylthiazol-2-yl) -2,5diphenyltetrazolium bromide cytotoxicity assay was performed after 2, 24, and 48 h of incubation: 100 µl of MTT (at 5 mg/ml concentration, purchased from Sigma, Italy) were added to each well and incubated for additional 4 h (37 •C, 5% CO2). Formazan crystals were dissolved in 100 µl of DMSO, and the absorbance was read at a wavelength of 570 nm using a microplate reader (HTX Microplate Reader BioTek Instruments,

Confocal Microscopy
For confocal microscopy HEK cells were seeded on glass coverslips (200.000 cells/well), incubated for 24h with KYF:Er,Yb @ KYF, KYF:Er,Yb @ KYF:Gd and KYF:Er,Yb @ KYF:Nd,Yb NPs at the concentrations found to be non toxic according to MTT tests (150 µg/mL) . After 24 h cells were fixed with 4% (v/v) paraformaldehyde, in PBS (pH 7.4) for 30 minutes at room temperature. To visualize the intracellular distribution of nanoparticles, the cells were washed in PBS, stained for DNA with Hoechst 33342 (1µg/mL in PBS), rinsed in PBS and finally mounted in 1:1 mixture of glycerol:PBS.
For confocal microscopy, a Leica TCS SP5 AOBS system (Leica Microsystems Italia) was used: for fluorescence excitation, a diode laser at 405 nm for Hoechst 33342 and a 514 nm laser diode for NPs were employed, by collecting images in blue region (410-460 nm), in the green (520-590 nm) and in the red (610-670 nm) and by merging the bright field images for cell morphology visualization.

Optical Imaging
Optical images were acquired by using the IVIS Spectrum Optical Imager (Perkin Elmer, Massachusetts, USA). This instrument is equipped with a cooled (-90°C) back-thinned, back-illuminated CCD camera (active array of 1920x1920 pixels with pixel size of 13x13 µm). The optical images were acquired and analyzed with Living Image 4.5 (Perkin Elmer) software and corrected for dark measurements. A Xe-lamp and a bandpass filter centered at 570 nm (optical width of 10 nm) was used as the excitation radiation. A longpass filter with a wavelength cutoff at 850 nm (Thorlabs, New Jersey, USA) was used to collect the NIR emission, using an exposure time of 20 seconds, with a Field of View (FoV) of 13.0 cm. An NMR tube filled with NPs colloidal dispersion was covered with a chicken breast slab (thickness of 4 mm) and directly imaged with the IVIS instrument.

CT scanner
CT images were acquired using a small animal CT scanner (SmART, Precision X-Ray). For the measurements, two acquisition setups were used: a) 40 kVp, current of 8 mA; b) 80 kVp, current of 3 mA. Images were acquired using a 3D phantom, made by a plastic based cylindrical container, with a diameter of 70 mm and 90 mm in length, housing six 63-mm diameter polycarbonate tubes. A dataset of 360 images was acquired as a function of the measurement depth. The Hounsfield values were determined as mean values over the image dataset, by calibrating the measured ROI on the Hounsfield values for water (0 HU) and that for air (-1000 HU).

Magnetic Resonance Imaging
MRI was performed using a Bruker Biospin 7T Scanner (Bruker Biospin, Ettlingen, Germany). To evaluate in vitro the longitudinal and transversal relaxivities (r1 and r2) of KYF:Er,Yb @ KYF:Gd NPs, longitudinal and transversal relaxation times of water solutions containing different amounts of Gd were measured using a T1map-RARE sequence with the following parameters: TR = 800.0 ms, TE = 13.3 ms, FOV = 4.20 cm, matrix size = 256 x 256, slice thickness = 1.5 mm, number of echoes = 6. Transversal relaxation rates (1/T1) were plotted as a function of the Gd concentration and r1 relaxivity was obtained by the slope of the fitting straight line. To measure the transverse relaxation time (T2), a multislice multiecho (MSME) imaging sequence was used with the following parameters: TR = 5000.0 ms, TE = from 7.47 to 186.7 ms (number of echoes = 25), FOV = 6.00 cm, matrix size = 256 x 256, slice thickness = 1.000 mm. To evaluate the presence of KYF:Er,Yb@KYF:Gd NPs, MRI images of chicken breast were acquired using T1w FLASH sequences with the following parameters: TR = 200.0 ms, TE = 2.8 ms, FOV = 3.00 cm, FA = 30.0 deg, matrix size = 256 x 256, slice thickness = 1.000 mm. S-7

In vivo biodistribution
N=3 Athymic Nude mice (Envigo, Udine, Italy) were maintained under standard environmental conditions (temperature, humidity, and 12h/12h light/dark cycle, with water and food ad libitum) and veterinarian control in the animal facility of the University of Verona. Animal experiments were conducted following Italian law (D.L. 4 March 2014 n°26) and the European Union normative (2010/63/EU). NPs (100 µl, 20 mg ml -1 ) were intravenously injected in healthy mice and monitored up to 24 hours, using Bruker Biospin Scanner operating at 7T (Bruker, Ettlingen, Germany). T2 weighted images were acquired before, 10 minutes, 30 minutes, 1 hour and 24 hours after NPs injection.

Hystological analysis
Mice were euthanized after 72 post-injection and liver, kidneys and spleen were dissected out, washed with PBS 0.1M and fixed in 10% formalin for 4 hours. Tissues were embedded in paraffin, cut into 5µm thick sections with a microtome and dried at 37°C for 24 hours. In order to evaluate tissue morphology, sections were stained with Hematoxylin and Eosin and examined under a light microscope (Olympus BXS1) equipped with a charge-coupled device camera.

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Section S1 -Morphology and size distribution analysis by TEM and high resolution HAADF-STEM Figure S1. TEM (a-e) and HAADF-STEM images (f-g) of core and core@shell and relative size distribution plot with the average size (nm).   Figure S5. Hydrodynamic diameter and -potential for aqueous colloidal dispersions of core and core@shell nanoparticles.

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Section S3 -XRD analysis and calculation of the lattice constant for a cubic system from XRD patterns Using the Bragg law (Hammond, 2015) = 2 (S3) Where  is the wavelength of the X-ray beam (for Cu, 1.54 Å). The interplanar spacing dhkl can be calculated and used to determine the lattice constant a. In case of a cubic system: where (hkl) are the Miller indices.
For the core nanoparticles four peaks were chosen for the calculation of lattice constant, after fitting each peak with a gaussian function for the determination of 2θ values in the experimental pattern.   Figure S7. Experimental XRD pattern for the α-NaYF4:Er,Yb core nanoparticles (light green line) and core@shell NPs (dark green line), compared to the corresponding databased pattern from ICSD.

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Section S4 -Spectroscopic characterization for core and core@shell nanostructures  Figure S9. Absorption spectra of the KYF:Er,Yb (core) and the KYF:Er,Yb@KYF:Gd (core@shell) colloidal dispersions in H2O and D2O. The integrated areas of the Yb 3+ absorption bands were used to normalize the UC spectra of the colloids, for the same solvent (H2O or D2O) (see Figure 3 and Figure S10).  In order to further characterize the luminescence properties of the Ln 3+ -doped KY3F10, two excitation power ranges have been considered for power-dependence study: in particular, a lower power density is obtained using a 4X microscope objective with wider spot-size, while a higher power density is achieved using a 40X microscope objective with smaller spot-size. The green and red UC emissions are here considered in a separate way for better explanation.
The green emission band arises from the radiative emission of three different Er 3+ energy levels, as represented in Figure 3b in the main paper. 2 H11/2 and 4 S3/2 levels are populated through a two-photons process. On the other hand, the blue and green emissions originating from 2 H9/2 require a three photons mechanism (see Figure 3b). At low P (i.e. using the 4X microscope objective), no three photons up-conversion mechanism are detected, as shown in Figure S12. In fact, for power density around 1.2 W cm −2 there is no observable blue emission UC band, and the green one lacks the low energy component, around 555 nm. On the other hand, in the high power density regime, from a power density of 20 W cm −2 , both the blue emission band (around 400 nm) and the lower energy component of the green band are evident, pointing out that three photons absorption mechanism is active. The power dependence (log-log plot) of the green UC bands at low and high P results in slope values of 1.32 and 1.49, respectively ( Figure S13, a and b). The steeper slope observed for high power densities suggests that also three photons up-conversion processes are involved in the green emission.
For what concerns the red band, it arises from the radiative emission of the 4 F9/2 energy level of the Er 3+ ion. This energy level is populated through at least two different two-photon excitation pathways (Würth et al., 2017). The competition between the upconversion process and non-radiative relaxation is at the base of the different power dependence at low and high P for the red UC emission of Er 3+ : at low P, depopulation of the thermally coupled green emitting levels down to 4 F9/2 level is predominant, leading to an increase of red emission. Differently, at high P, energy transfer upconversion (ETU) is the most favorite process, leading to a preferential population of the 4 F9/2 energy level and depopulation of the red emitting energy level (Pollnau et al., 2000), as clearly evidenced from the comparison of the UC spectra shown in Figure S10. This behavior not only affects the P dependence of the red band, but also explains the higher intensity of the red band in comparison to the green one at lower P (when using a 4X microscope objective). This is also confirmed by the calculated value of slopes for the power study of the red emission, resulting to be 1.32 at low P and 1.14 at high P ( Figure S13, a and b). Finally, as a general consideration, the slope value n smaller than 2 can be related to different concurrent absorption and emission mechanisms, especially non-radiative de-excitation processes, cross relaxations, energy migration between active ions, all concurring in significant lowering of the experimental value of the slope (Pollnau et al., 2000).
S-17 low P high P x5 * Figure S12. Upconversion spectra of the KYF:Er,Yb@KYF colloidal dispersion under 980 nm laser excitation, at low power density P (1.2 W cm -2 , blue line) and high P (20 W cm -2 , red line). The band on which the normalization is carried out is indicated with *. Figure S13. Power plots of the upconversion emissions (green and red emissions) for KYF:Er,Yb@KYF sample under 980 nm laser excitation and collected in backscattering mode by using a 4X microscope objective (minimum power density 1.2 W cm -2 ) (a) and a 40X microscope objective (minimum power density 20 W cm -2 ) (b), with the relative pictures of KYF:Er,Yb@KYF:Gd sample in acqueous colloidal dipersion under 980 nm laser irradiation, using the two different microscope objectives.
The emission spectra in the 20-50 °C temperature range have been acquired for water colloids of core@shell nanoparticles with a concentration of 20 mg ml −1 ( Figure S15a). The relevant thermometric parameter LIR (Luminescence Intensity Ratio, defined in Equation S6) is calculated as the ratio of the integrated intensities I517 and I540 of the two above mentioned bands, and it is shown in Figure S15b. The ln(LIR) vs 1/T curve for the Er 3+ green emissions was fitted according to the Boltzmann distribution formula (see Figure S16): where A, ∆E, kB and T are respectively the pre-exponential term, the energy gap between the two thermalized energy levels, the Boltzmann constant and the absolute temperature, respectively (Vetrone et al., 2010). From the Boltzmann fit, shown in Figure S16, the energy gap ∆E between the 2 H11/2 and 4 S3/2 levels results of 858 cm −1 K −1 , while Solanki et al. found a value around 800 cm −1 for the same host (Solanki et al., 2021). This small difference between the energy gaps could be related to a slightly different local environments around the lanthanide ions, on the average slightly more distorted, especially at the particle surface, in the present case, due to the smaller size and to the presence of coordinating water molecules.
The thermometric performance of the nanoparticles is evaluated by calculating the relative thermal sensitivity, defined as (S7) and the S rel (Er) values as a function of the temperature are shown in Figure S15c. The S rel (Er) values range from 0.9%K −1 @343 K to 1.47%K −1 @ 307. The maximum value is slightly higher than the value reported in Solanki et al. (Solanki et al., 2021) for the same core@shell structure (1.245%K −1 @300K), although the sample is analyzed in powder form of much larger nanoparticle size. The average ΔTmin is evaluated to be 1.0°C, according to eq. 4, main text. = −

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Section S9 -Calculation of the induced heat and estimate of the temperature variation in a water sample and measure of temperature changes in a biological tissue under laser excitation at 980 nm First, for evaluating the heating induced by the excitation radiation, we consider a laser beam at 980 nm wavelength hitting a water sample contained in a laboratory cuvette (4×1×1 cm), kept under constant stirring. The increase of the temperature of the water sample in these experimental conditions can be calculated assuming that the absorbed power is completely converted into heat, depending on the incident laser power and the absorption coefficient of water at 980 nm.
In particular, we consider our actual experimental conditions involved in our UC nanothermometry investigation on KYF:Er,Yb@KYF nanostructures, i.e. an incident laser power of 0.45 W and an irradiation time of 2 seconds.
We consider that the heat induced in the system Q is proportional to the absorbed power due the exciting laser radiation Ilas and to the irradiation time t according to:

= *
On the other hand, the induced variation of the temperature T depends on the heat capacity of the system Cs according to the:

= *
In our thermometry experiment (see Section S8), an incident laser power (I0) of 0.45 W was used, with an irradiation time of 2 seconds, for measuring the UC spectra. The heat capacity of our system (assuming pure water), can be calculated from: where cs is the specific heat capacity of water, 4.18 J g -1°C-1 , m is the mass of the sample (water).
If we consider the absorption coefficient of H2O at 980 nm, α(980), the actual power absorbed by the sample Ilas can be estimated according to: where l is the optical path or the radiation, in our case 1 cm considering a glass square cuvette and α(980)=0.482 cm -1 . Therefore, we obtain a value of Ilas = 0.3 W.
Assuming a mass of water of 4 g inside the cuvette, the estimated temperature increase for a 10 seconds irradiation time, which we will consider as the maximum ∆T (∆Tmax) for this irradiation experiment, is ΔT ≈ 0.04°C.
As a further proof of concept of the reliability of our thermometric analysis, we carried out an experiment using a biological tissue, i.e. a chicken breast slice. In particular, 20 l of the KYF:Er,Yb@KYF colloidal dispersion (concentration of 20 mg/ml) was injected into a chicken breast slice (about 6 cm 2 ) and the temperature variation of the biological tissue was monitored as a function of the incident laser powers and irradiation times. It is worth noting that the UC emission is clearly visible at the naked eye (see Figure S17a). Thermal images taken with a thermal camera of the chicken slice under different irradiation powers and times are shown in Figure S17b. From the thermal pictures, the temperature values of the biological system have been extracted and they are plotted vs time in Figure S18, where the behaviors as a function of the incident laser power have been also shown. It can be clearly noted from the Figure S18 that before 50 seconds under CW 980 nm laser irradiation the maximum induced temperature variation is close to 1°C. Section S10 -Thermometric parameters for the Visible-to-NIR thermometry of KYF:Er,Yb@KYF:Nd,Yb colloidal dispersion Table T5. Thermometric parameter for the three LIR involving emissions in the NIR region of the Nd 3+ and Yb 3+ ions.