Highly colloidally stable trimodal 125I-radiolabeled PEG-neridronate-coated upconversion/magnetic bioimaging nanoprobes

“All-in-one” multifunctional nanomaterials, which can be visualized simultaneously by several imaging techniques, are required for the efficient diagnosis and treatment of many serious diseases. This report addresses the design and synthesis of upconversion magnetic NaGdF4:Yb3+/Er3+(Tm3+) nanoparticles by an oleic acid-stabilized high-temperature coprecipitation of lanthanide precursors in octadec-1-ene. The nanoparticles, which emit visible or UV light under near-infrared (NIR) irradiation, were modified by in-house synthesized PEG-neridronate to facilitate their dispersibility and colloidal stability in water and bioanalytically relevant phosphate buffered saline (PBS). The cytotoxicity of the nanoparticles was determined using HeLa cells and human fibroblasts (HF). Subsequently, the particles were modified by Bolton-Hunter-neridronate and radiolabeled by 125I to monitor their biodistribution in mice using single-photon emission computed tomography (SPECT). The upconversion and the paramagnetic properties of the NaGdF4:Yb3+/Er3+(Tm3+)@PEG nanoparticles were evaluated by photoluminescence, magnetic resonance (MR) relaxometry, and magnetic resonance imaging (MRI) with 1 T and 4.7 T preclinical scanners. MRI data were obtained on phantoms with different particle concentrations and during pilot long-time in vivo observations of a mouse model. The biological and physicochemical properties of the NaGdF4:Yb3+/Er3+(Tm3+)@PEG nanoparticles make them promising as a trimodal optical/MRI/SPECT bioimaging and theranostic nanoprobe for experimental medicine.

does not allow for quantitative analysis. To optimize imaging sensitivity, contrast probes should preferably emit light in the red or near-infrared (NIR) region (~ 600-1000 nm) 6,8 . CT provides a superb hard tissue contrast; however, it has a low soft tissue contrast and produces harmful ionizing radiation 9 . As a result, no single imaging modality is able to provide thorough preclinical information, making it difficult to obtain precise disease diagnoses. Thus, merging several imaging modalities into an "all-in-one" system can tremendously improve sensitivity and provide the better resolution needed for the detection of diseases, while overcoming limitations of a single modality alone 10 .
Lanthanide-based upconversion nanoparticles (UCNPs) have recently attracted extensive scientific interest due to their unique photoluminescence properties [11][12][13][14] . UCNPs are able to upconvert low-energy NIR irradiation into high-energy visible or ultraviolet (UV) light via anti-Stokes emission. The ability to absorb NIR radiation allows for deep tissue penetration due to reduced light absorption in the tissue, lower autofluorescence and photon scattering 15 . Compared to conventional fluorescence nanomaterials (e.g., organic dyes or metal complexes), UCNPs have several advantages, such as high chemical stability, large anti-Stokes shift, narrow emission lines, no blinking, and no bleaching 16 . UCNPs have great potential in a variety of applications, from photovoltaics, photocatalysis, security, and display technology to in vitro and in vivo tissue imaging, background-free biosensors, light-triggered drug and gene delivery, optogenetics, photodynamics and photothermal therapy [17][18][19][20][21][22] . The nanoparticles are also beneficial for multimodal in vivo bioimaging because simple variations of dopant ions in the crystal lattice can induce the formation of particles suitable for down-and upconversion luminescence, MRI, and CT imaging 23,24 . As an example, UCNPs containing Gd 3+ ions in the host lattice exhibit a short longitudinal relaxation time T 1 , which makes their prospective use for luminescent/MR imaging very attractive 25 .
Despite huge progress in the synthesis and understanding of the properties of UCNPs during recent years 26,27 , a number of limitations still have to be solved before these unique particles are translated into clinical praxis. One of the main challenges is their poor colloidal stability in water, and more importantly, in physiological buffers (e.g., PBS). High-quality monodispersed UCNPs with size < 50 nm are typically prepared in high-boiling point organic solvents in the presence of stabilizers; however, the resulting particles are hydrophobic 28,29 . To make them water-dispersible, different surface modification strategies were utilized, including ligand oxidation, silica encapsulation, coating with polyelectrolytes via a layer-by-layer technique, and ligand exchange with hydrophilic and amphiphilic polymers, e.g., poly(acrylic acid), poly(ethylene glycol) (PEG), polyethyleneimine, poly(vinylpyrrolidone), dextran, chitosan, poly(maleic anhydride-alt-1-octadecene), etc. [30][31][32] . Polymers containing anchoring end-groups, e.g., carboxylic or sulfonic acid, amine, hydroxyl, phosphate or phosphonate groups have also been utilized to modify the UCNP surface 33 . While all of these coating strategies result in the UCNPs being dispersible in water, only coatings based on PEG terminated by bisphosphonate or tetraphosphonate groups proved to produce particles stable in PBS, avoiding aggregation 34,35 . The effectiveness of these coatings originates from a strong binding affinity of the phosphonate groups to the lanthanide ions 36 . Therefore in the present work, highly colloidally stable upconversion and magnetic NaGdF 4 :Yb 3+ /Er 3+ and NaGdF 4 :Yb 3+ /Tm 3+ nanoparticles coated by PEG-neridronate were designed as a trimodal contrast agent for photoluminescence, SPECT, and MRI, which was verified on a mouse model.
Synthesis of PEG-neridronate (PEG-Ner). PEG-neridronate (PEG-Ner) was prepared according to previously published report 35 . In a typical procedure, sodium neridronate (0.32 g; 1 mmol) was dissolved in 0.1 M PBS (10 ml; pH 7.4), the solution was cooled to 5 °C, PEG-NHS (1 g; 0.1 mmol of NHS groups) was added, Characterization methods. The size, composition, and crystal structure of the nanoparticles was analyzed using a Tecnai Spirit G2 transmission electron microscope (TEM; FEI; Brno, Czech Republic) 39 . The particles were deposited on a standard carbon-coated copper grid and visualized by means of bright field imaging at 120 kV. The microscope was equipped with an energy dispersive spectrometer (EDX; Mahwah, NJ, USA) to analyze elements in the nanoparticles and selected area electron diffraction (SAED) mode to verify the crystal structure 39 . The electron diffraction patterns were processed with ProcessDiffraction software 41 and compared with the diffraction patterns calculated with PowderCell software 42 ; the crystal structures for the diffraction pattern calculation were obtained from ICSD database 43 . Particle size and distribution was determined by measuring at least 300 nanoparticles from four randomly selected TEM micrographs using Atlas software (Tescan; Brno, Czech Republic) 44 . Number-(D n ), weight-average diameter (D w ), and the uniformity (dispersity Ð ) were calculated as follows 39 : where N i and D i are number and diameter of the nanoparticle, respectively. Hydrodynamic particle diameter (D h ), size distribution (polydispersity PD), and ζ-potential were analyzed by a dynamic light scattering (DLS) on a ZEN 3600 Zetasizer Nano Instrument (Malvern Instruments; Malvern, UK) 39 . The nanoparticle dispersion was measured at 25 °C, and the D h and PD were calculated from the intensity-weighted distribution function obtained by CONTIN analysis of the correlation function embedded in Malvern software.
Thermogravimetric analysis (TGA) was performed from 30 to 600 °C using a Perkin Elmer TGA 7 analyzer (Norwalk, CT, USA) at a heating rate of 10 °C/min under air atmosphere 45 .
ATR FTIR spectra were recorded on a Nexus Nicolet 870 FTIR spectrometer (Madison, WI, USA) equipped with a liquid nitrogen cooled mercury cadmium telluride detector. The spectra were measured using a Golden Gate single reflection ATR cell (Specac; Orpington, UK) equipped with a diamond internal reflection ATR crystal 39 .
Inductively coupled plasma mass spectrometry (ICP-MS) was performed on a mass spectrometer with an inductively coupled plasma Elan DRC-e (Perkin Elmer; Concord, Canada); 158 Gd isotope was measured with 103 Rh as an inner standard.
Upconversion luminescence and single nanoparticle imaging. For the steady state photoluminescence, a 980-nm laser (Shanghai Dream Lasers Technology SDL-980-LM-1000 T) was used as an excitation source. An HR4000 spectrometer (Ocean Optics; Ostfildern, Germany) was used as a detection system for measurements in a visible spectral range. The emission spectra were not corrected spectrally. Photoluminescence decays were measured using an Opolette pulsed laser (Opotek; Carlsbad, CA, USA) at 978 nm, 7 ns, 20 Hz, and average optical power P av = 3 mW. The laser was coupled to a gated detection system with a time resolution of 1 μs. Single UCNPs were imaged using a custom-build wide-field fluorescence microscope equipped with Magnetic resonance. Magnetic resonance imaging (MRI) was performed on phantoms containing particles at 25 °C on a 4.7 T Bruker Biospec spectrometer equipped with a commercially available resonator coil. Standard two-dimensional rapid acquisition with a relaxation enhancement multispin echo (RARE) sequence was used with the following parameters: repetition time 280 ms, echo time 12 ms, turbo factor 2, spatial resolution 137 × 137 mm 2 , slice thickness 0.7 mm, number of acquisitions 16, and acquisition time 9 min 36 s. The signal-to-noise ratio (SNR) was 0.655•S/σ, where S is signal intensity in a region of interest, σ is the standard deviation of the noise in background, and constant 0.655 reflects Rician distribution of the background noise in a magnitude MR image.
Magnetic resonance relaxometry of NaGdF 4 :Yb 3+ /Er 3+ (Tm 3+ ) particles with and without PEG-neridronate modification was performed on a Minispec mq60 (Bruker; Germany) at 37 °C and magnetic field B 0 = 1.41 T. The experimentally determined solvent relaxation rate R (concentration 0 mM) was subtracted as a starting value from the nanoparticle relaxation rates prior to the linear regression analysis. Summary of relaxation results are shown in Tables S1 and S2.
Cytotoxicity of the particles. Cytotoxicity of the NaGdF 4 :Yb 3+ /Er 3+ and NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoparticles was evaluated on a cell line derived from human cervical carcinoma (HeLa) and human dermal fibroblast (HF) 46-48 kindly provided by Dr. Mělková and Dr. Dvořánková, respectively, First Faculty of Medicine, Charles University, Prague. 5•10 3 of HeLa or 8•10 3 of HF cells were seeded in 100 µl of media into 96-well flat-bottom TPP plates (Merck; Darmstadt, Germany) for 24 h. Subsequently, the nanoparticles (0.4-400 µg/ml) were added, the cells were cultivated at 37 °C for 72 h under 5% CO 2 atmosphere, AlamarBlue cell viability reagent (10 µl) was added to each well, and the incubation continued at 37 °C for 4 h. The active component of the AlamarBlue reagent (resazurin) was reduced to the highly fluorescent resorufin only in viable cells. Fluorescence was detected using a Synergy Neo plate reader (Bio-Tek; Winooski, VT, USA) at 570 nm (excitation) and 600 nm (emission). Cells cultivated in medium without the nanoparticles served as a control. The assay was repeated two to three times in tetraplicates.

Experimental animals. Four 12 weeks old female adult BALB/C mice (Charles River Laboratories;
Erkrath, Germany) were maintained in individually ventilated cages (12:12 h light-dark cycle, 22 ± 1 °C, 60 ± 5% humidity) with standard food and access to water ad libitum. Animals were regularly observed during the whole experiment for changes in their behavior and health status. First two mice were measured on an Albira SPECT/ PET/CT imaging system (Bruker BioSpin; Ettlingen, Germany), while the next two mice were imagined on MRI preclinical scanner (ICON; Bruker BioSpin).
All Biodistribution of the NaGdF 4 :Yb 3+ /Er 3+ @PEG-125 I nanoparticles in mice on SPECT/CT. Dispersion of the NaGdF 4 :Yb 3+ /Er 3+ @PEG-125 I nanoparticles in PBS buffer (200 µl; 30 MBq/mouse) was intravenously injected into two mice via the tail vein. 125 I with a long half-life (59.49 days) was selected as a radioisotope because it is cheap, easily available on the market, readily chemically modifiable, and very suitable especially for small-animal SPECT with an Albira imaging system used in our work. The low-energy emission (35 keV) of 125 I is also a great advantage for the longitudinal studies. Biodistribution of the particles in mice was evaluated under anesthesia (2% isoflurane in air); both mice were scanned at 30, 50, 70, and 260 min and 1, 2, 6, and 8 days after the injection and acquisitions were obtained using multi-pinhole collimators. Acquisition time was 45 min (90 s/projection) and followed by a single CT scan (15 min). Image analysis and co-registration was done using PMOD analysis software (PMOD Technologies; Zürich, Switzerland).
Biodistribution of the NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoparticles in mice on MRI. NaGdF 4 :Yb 3+ / Er 3+ @PEG nanoparticles in PBS buffer (200 µl; 4 mg/ml) were intravenously injected into two mice and ICON imager with magnetic field 1 T was used for MRI imaging. Biodistribution of the nanoparticles was determined under anesthesia (2% isoflurane in air) with monitoring of vital functions (breathing and cardiac rate). The imaging parameters were following: T 1 RARE sequence, repetition time/echo time 350/12 ms, thickness 1 mm, coronal orientation, matrix 256 × 256 pixels, field of view 90 × 35 mm, 8 averages, duration 4.5 min. Image and data analysis was performed using ParaVision 6.0 software (Bruker BioSpin).

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| (2020) 10:20016 | https://doi.org/10.1038/s41598-020-77112-z www.nature.com/scientificreports/ The crystal structure and elemental composition of the nanoparticles was verified by TEM microscopy using selected area electron diffraction (SAED) and energy-dispersive analysis of X-rays (EDX). The SAED diffractogram corresponded to the hexagonal phase of NaGdF 4 , as confirmed by the transformation of the experimental 2D diffraction pattern (Electronic supplementary information, Fig. S1a) to a 1D diffractogram that was compared to the theoretically calculated 1D X-ray diffractogram (XRD) of hexagonal NaGdF 4 (Fig. S1b) 43 . The agreement between the experimental and theoretically calculated diffractograms was very good. The positions of the four most intense diffraction rings corresponded exactly to the strongest diffractions of NaGdF 4 (Fig. S1a), and the same was observed for the less intense diffraction peaks (Fig. S1b). The small differences between the XRD and SAED diffraction intensities could be attributed to the following three facts: (i) the first SAED ring was partially masked by the strong primary beam, (ii) the second SAED maximum (corresponding to a pair of XRD diffractions) was used for normalization, and (iii) the remaining SAED intensities were somewhat lower than those of the corresponding XRD diffractions probably due to the intrinsic differences of the two methods and/or possible preferred orientation effects 45 .
An elemental analysis of the NaGdF 4 :Yb 3+ /Er 3+ nanoparticles with TEM/EDX confirmed the electron diffraction results (Fig. S1c). Except for the peaks corresponding to the carbon-coated copper grid (C, Cu), the www.nature.com/scientificreports/ strongest peaks (Na, F, Gd, and Yb) in the EDX spectrum corresponded to the expected composition of the prepared NaGdF 4 :Yb 3+ /Er 3+ nanocrystals. Erbium peaks (expected at positions Mα = 1.4 keV and Lα = 6.9 keV) could not be detected due to a negligible amount of Er and possible overlaps with the stronger Gd and Yb peaks at given energies.
Surface modification of NaGdF 4 :Yb 3+ /Er 3+ (Tm 3+ ) nanoparticles with poly(ethylene glycol) (PEG). The OA-stabilized upconversion nanoparticles were hydrophobic and therefore not dispersible in aqueous media. However, thorough washing of the particles with hexane, ethanol, an ethanol/water mixture, and treatment with 0.01 M HCl made them dispersible and colloidally stable in water (Fig. S2a). The hydrodynamic diameter of the NaGdF 4 :Yb 3+ /Er 3+ nanoparticles in water was D h = 151 nm and PD = 0.22 with highly positive charge (ζ-potential = 41 mV) due to the presence of lanthanide ions on the particle surface; however, the particles aggregated in PBS. After modification with PEG-Ner, the number-average diameter did not change (Fig. 1b,d).
The final physicochemical characterization of the OA-NaGdF 4 :Yb 3+ /Er 3+ , NaGdF 4 :Yb 3+ /Er 3+ , and NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoparticles involved ATR FTIR spectroscopy (Fig. S3c). The spectrum for the freshly prepared OA-NaGdF 4 :Yb 3+ /Er 3+ nanoparticles contained the characteristic OA bands, i.e., asymmetric and symmetric stretching vibrations of CH 2 groups at 2,925 and 2,853 cm −1 , stretching vibrational mode of C=O of COOH groups in OA at 1,712 cm −1 , bending vibrations of CH 2 groups at 1,462 cm −1 , and out-of-plane bending of C-OH at 1,411 cm −149, 50 . The spectrum for the NaGdF 4 :Yb 3+ /Er 3+ nanoparticles confirmed the efficacy of washing, as the intensity of the bands for OA significantly decreased. As expected, the particle surface modification with PEG-Ner changed the spectrum, and the characteristic bands of PEG at 2,885, 1,342, 1,280, 1,241, 1,106, 963, and 844 cm −1 appeared. The band at 2,885 cm −1 was assigned to symmetric stretching of CH 2 groups, the band at 1,342 cm −1 to wagging vibrations of CH 2 groups, the bands at 1,280 and 1,241 cm −1 to twisting vibrations of CH 2 groups of PEG chains that adopted helical and trans planar structures, the band at 1,106 cm −1 to coupled C-O and C-C stretching vibrations of PEG backbone, and the bands at 963 and 844 cm −1 to rocking vibrations of CH 2 groups of PEG when these chains adopted trans planar and helical structures 51,52 . The bands were strong, with intensities comparable to those in the spectrum of neat PEG 51 . ATR FTIR spectroscopy thus confirmed the successful modification of the NaGdF 4 :Yb 3+ /Er 3+ nanoparticles with PEG, which was in agreement with results from the other experimental methods used in this work.
Cytototoxicity of NaGdF 4 :Yb 3+ /Er 3+ and NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoparticles. The cytotoxicity of the particles was determined on HeLa cells and primary fibroblasts (HF) using an AlamarBlue viability assay. The nanoparticles did not cause toxicity in HeLa cells, the viability of which did not go below 90% (Fig. S5a). At the two highest particle concentrations (200 and 400 μg/ml), a slight decrease of HF cell viability was observed; however, it did not decline to < 80% (Fig. S5b). At the highest concentration, the NaGdF 4 :Yb 3+ / Er 3+ @PEG nanoparticles showed significantly lower toxicity than those without PEG, reflecting their high biocompatibility.
Upconversion photoluminescence of NaGdF 4 :Yb 3+ /Er 3+ (Tm 3+ ) nanoparticles. The photoluminescence spectra of colloidal NaGdF 4 :Yb 3+ /Tm 3+ nanoparticles at 980 nm diode laser excitation (nominal laser power on the sample P = 150 mW; power density 7.5 kW/cm 2 ) showed the main bands at 450, 480, and 800 nm (Fig. 2a), corresponding to the 1 D 2 → 3 F 4 , 1 G 4 → 3 H 6 , and 3 H 4 → 3 H 6 transitions of the Tm 3+ ions, respectively. Much lower intensity bands were observed at 330, 366, and 650 nm, corresponding to the 1 I 6 → 3 F 4 , 1 D 2 → 3 H 6 , and 1 G 4 → 3 F 4 transitions, respectively. The emission spectra of the NaGdF 4 :Yb 3+ /Er 3+ particles recorded under the same experimental conditions showed typical Er 3+ emission lines at 375 nm ( 4 G 11/2 → 4 I 15/2 ), 520 nm ( 2 H 11/2 → 4 I 15/2 ), 540 nm ( 4 S 3/2 → 4 I 15/2 ), 650 nm ( 4 F 9/2 → 4 I 15/2 ), and 840 nm ( 4 I 9/2 → 4 I 15/2 ); moreover, there was a strong blue band at 410 nm (Fig. 2b). This emission could be related to 2 H 9/2 → 4 I 15/2 transition in the Er 3+ ions. Only a small difference was observed in the NaGdF 4 :Yb 3+ /Tm 3+ and NaGdF 4 :Yb 3+ /Er 3+ luminescence spectra on the low energy side of the spectrum after the nanocrystal surface modification. Since the particles were relatively small (18 nm), the surface Tm 3+ ions could have a visible contribution to the total emission intensity 53,54 . Thus, the surface modifications changed the surface ion properties by varying excited carrier relaxation processes. These alterations were visible in the relative change of emission intensities. In the luminescence spectrum of the NaGdF 4 :Yb 3+ /Tm 3+ @PEG particles on the high energy level, only a small difference was found compared to the spectrum of the unmodified particles (Fig. 2a,b). In contrast, this change was much more significant in the NaGdF 4 :Yb 3+ /Er 3+ -based nanoparticles. This could be explained by different positions of energy levels of the ions www.nature.com/scientificreports/ relative to those of organic modifying agents on the particle surface. Other effect that should be considered is associated with spectrally dependent absorption of water and/or hexane, resulting in different quenching of the emitted light depending on the spectral range.
To better understand the optical properties of the NaGdF 4 :Yb 3+ /Tm 3+ and NaGdF 4 :Yb 3+ /Er 3+ nanoparticles, emission decay traces of the surface-modified particles have also been recorded (Fig. 2c,d) and the average values of the decay times have been recorded. To analyze the results, the simplest approach was used and for all the decay curves an average decay time τ was calculated according to the Eq. (4): where I(t) represents the photoluminescence intensity at time t after cutoff of the excitation light, t 0 = 0 s is the initial time, when the signal starts to decay, and t 1 is the time, when the luminescence intensity reaches the background. The average decay time was longer for the NaGdF 4 :Yb 3+ /Tm 3+ nanoparticles (τ 800 = 340 μs) than for the NaGdF 4 :Yb 3+ /Er 3+ nanoparticles (τ 540 = 180 μs). In addition, the recombination process for the Er 3+ ions was much less exponential compared to the recombination for the Tm 3+ ions. At the same time, a small decrease in the emission decay time of the NaGdF 4 :Yb 3+ /Tm 3+ (Er 3+ ) and NaGdF 4 :Yb 3+ /Tm 3+ (Er 3+ )@PEG nanoparticles compared to the OA-NaGdF 4 :Yb 3+ /Tm 3+ (Er 3+ ) nanoparticles was observed (Fig. 2c,d). This could be because the NaGdF 4 :Yb 3+ /Tm 3+ (Er 3+ ) and NaGdF 4 :Yb 3+ /Tm 3+ (Er 3+ )@PEG particles were dispersed in water, while the OA-NaGdF 4 :Yb 3+ /Tm 3+ (Er 3+ ) particles were dispersed in hexane. Thus, this small decrease could be due to photonic effects (induced by the surface ions) related to the differences in refractive index for the two solvents (water or hexane). Nevertheless, a more probable explanation for the reduced emission decay time of the NaGdF 4 :Yb 3+ / Tm 3+ (Er 3+ ) and NaGdF 4 :Yb 3+ /Tm 3+ (Er 3+ )@PEG particles compared to the OA-NaGdF 4 :Yb 3+ /Tm 3+ (Er 3+ ) particles is the appearance of surface defects formed during OA removal or the formation of new ligands, which can introduce new nonradiative channels for the RE 3+ ions present on the surface.
To investigate the effect of surface engineering on the emission intensity, the luminescence intensity maps of single OA-NaGdF 4 :Yb 3+ /Er 3+ , NaGdF 4 :Yb 3+ /Er 3+ , and NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoparticles were measured. In highly diluted colloids, it was possible to spatially separate individual nanoparticles by a distance larger than the point spread function of the optical system. The maps looked very similar, the intensity distribution was similar www.nature.com/scientificreports/ as well, with only a small difference in average values. As an example, fluorescence intensity map of single OA-NaGdF 4 :Yb 3+ /Er 3+ nanoparticles was shown on Fig. 3a. The luminescence intensity of the OA-NaGdF 4 :Yb 3+ /Er 3+ particles in hexane and the NaGdF 4 :Yb 3+ /Er 3+ and NaGdF 4 :Yb 3+ /Er 3+ @PEG particles in water was determined for fifty single particles each. Based on the histogram of the particle luminescence intensity, its mean value was estimated (Fig. 3b) and the distribution of single-particle luminescence intensities determined (Fig. 3c).
The calculated relaxivity r 1 and r 2 and results obtained from imaging on phantoms suggest that tested MR probes could be successfully used for in vivo MR imaging. Relaxometry study revealed interesting effect of modification with PEG-neridronate. Instead of negligibly decreased r 1 relaxivity of the NaGdF 4 :Yb 3+ /Er 3+ @PEG particles, r 1 was ~ 70% higher compared to that for the unmodified particles. On the other side, relaxivity r 2 of NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoparticles decreased by ~ 10%.
Biodistribution of NaGdF 4 :Yb 3+ /Er 3+ @PEG and NaGdF 4 :Yb 3+ /Er 3+ @PEG-125 I nanoparticles. Biodistribution of the particles was investigated in mice; the particles were evidently nontoxic, as animal's feed habits were unchanged after the particle administration, the mice were active and interacted with each other during the study. The weight of animals did not significantly change, nor altered posture and/or fur appearance (typical signs of discomfort or toxicity of the injected compound) has been observed. The in vivo MRI results revealed an intense signal in the liver caused by the accumulation of the NaGdF 4 :Yb 3+ /Er 3+ @PEG particles (Fig. 5). Comparing the reference region of interest (ROI; shoulder muscle) versus three different ROI in the liver revealed a maximum signal increase at 72 h after particle application (Fig. 6). Additionally, SPECT/ CT in vivo measurements demonstrated the fast accumulation of NaGdF 4 :Yb 3+ /Er 3+ @PEG-125 I nanoparticles in the liver. The particles circulated for at least 4 h after the application. The activity remained in the liver and the signal peaked after 48 h. The signal then started to decline due to detoxication of the particles via the hepatobiliary route (Fig. 7) that is a typical excretion way from the liver. A weak signal was also observed in the thyroid gland, which was caused by free 125 I leaking from the iodinated shell of the particles. The SPECT measurement proved to be more sensitive for determining the particle biodistribution than MRI. The MRI results reported only the peak values at longer times. The initial accumulation that was observable on the SPECT image within 2 h was not seen on the MRI image. Nevertheless, MRI provides indispensable information about the exact position of the nanoparticles within the soft tissues.

Discussion
High-temperature coprecipitation of lanthanide chlorides produced NaGdF 4 :Yb 3+ /Er 3+ and NaGdF 4 :Yb 3+ /Tm 3+ UCNPs. Compared to other conventional synthetic procedures, this approach brings several benefits regarding the precise control of the crystal structure, morphology, and uniformity of the resulting nanoparticles. The particle size distribution was quite narrow and more uniform compared to that in other recent studies 56,57 . The particle uniformity is a key criterium for the subsequent application of the particles in biomedicine 18 . The thoroughly washed nanoparticles could not be directly transferred in a phosphate buffer, which is required for biomedical applications, because they immediately aggregated and formed a turbid colloidal solution (Fig. S2b). Therefore, the particles were modified with PEG-Ner to provide them with very good colloidal stability not only in water (Fig. S2c) but also in PBS (Fig. S2d). This stability was higher than that of cancer cell membrane-coated   59 . Upconversion photoluminescence measurements of the NaGdF 4 :Yb 3+ /Er 3+ and NaGdF 4 :Yb 3+ /Tm 3+ nanoparticles confirmed the characteristic strong green/red (520, 540, and 650 nm) and UV/blue/NIR emissions (447, 473, and 800 nm). The NaGdF 4 :Yb 3+ /Er 3+ @PEG single-particle investigation revealed that the high luminescence intensity was not compromised by the surface modification. The luminescence intensity map of tens of individual PEG-functionalized upconversion nanoparticles monitored by the widefield microscopy made statistical analysis of their optical properties possible. The single-particle studies indicated that the surface engineering did not affect the luminescence intensity of the NaGdF 4 :Yb 3+ /Er 3+ nanoparticle, which confirmed that their high homogeneity was due to the equal width of the luminescence intensity distribution. Single particle imaging is thus a powerful tool that has huge potential in bioimaging and tracing of specific proteins in theranostic applications. Considering in vivo upconversion imaging, it is much more challenging compared to in vitro studies especially due to relatively low emission wavelengths of UCNPs leading to significant absorption of emitted light in the tissues. The efficient optical imaging of deeper tissues needs excitation and emission wavelengths that fit into the "NIR imaging window" 60 . Our NaGdF 4 :Yb 3+ /Tm 3+ particles show strong emission at 800 nm, which is the optimal wavelength for imaging with low hemoglobin and water absorption. The particles are thus a potential candidate also for in vivo optical imaging. The NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoparticles were found to be biocompatible, not significantly affecting viability of the HeLa or HF cells at concentrations < 400 μg/ml. At 400 μg/ml, the HF cells were slightly more sensitive than the HeLa cells. The difference in the behavior of the HeLa and HF cells was attributed to their different metabolism. HF cells are primary fibroblasts that are more sensitive to incubation with potentially toxic agents than the tumor HeLa cells, which are more adaptable to various anticancer drugs, for instance. Similar results, where noncancer cell lines, especially HF cells, showed different metabolic activity compared to cancer cell lines 61 or higher sensitivity to treatment with various nanomaterials, were demonstrated also in other studies 35,62 .
MRI and relaxometric studies suggested that the particles possessed suitable paramagnetic properties that will allow for the application of the NaGdF 4 :Yb 3+ /Er 3+ @PEG particles as a novel contrast probe for MRI; this is in agreement with an earlier published report on sodium gluconate-coated lanthanide-doped upconversion nanoparticles 63 . Moreover, we decided to add the third imaging modality to the NaGdF 4 :Yb 3+ /Er 3+ @PEG particles by their radiolabeling that is a rarely used modification of UCNPs 64,65 . This approach enabled us to use SPECT method, rather sparsely exploited technique for the determination of particle biodistribution, which allows higher spatial resolution and more reliable and accurate nanoparticle quantification in deeply located organs than fluorescence employed in most current studies 56,57,66 . Pilot in vivo MRI/SPECT multimodal imaging of the NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoparticles proved that they circulated in the blood stream of an experimental animal for at least 4 h, though rapid uptake to the liver was observed with a maximum signal 2-3 days after intravenous application. The blood circulation time was prolonged compared to that achieved with poly(ethylene Figure 6. (a) MRI relative signal intensity of targeted tissues in regions of interest (ROIs) in liver (ROI 1-ROI 3) and muscle (ROI 4) before and after the intravenous application of NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoparticles. The results show the mean value ± standard deviation, which were evaluated from T 1 -weighted images in selected liver ROIs and related to the reference signal (ROI 4 in muscle). (b) Liver position marked in mouse body on respective image. (c) Regions of interest (ROIs) were chosen in three different parts of the liver (ROI 1-3) and compared with a reference ROI 4 (muscle in the left or right limb). Both liver and ROI images were acquired from precontrast scans.

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| (2020) 10:20016 | https://doi.org/10.1038/s41598-020-77112-z www.nature.com/scientificreports/ glycol) diglycidyl ether-crosslinked poly(maleic anhydride-alt-1-octadecene)-coated NaYF 4 :Yb 3+ Tm 3+ @NaYF 4 nanoparticles that circulated for up to 1 h 67 . Our multimodal probes are constructed to allow SPECT imaging with 125 I. SPECT is more available and widely used and cheaper than PET in systems for NIR/MRI/PET applications reported earlier 68 . 125 I-labelled SPECT probes have also advantage of considerably lower energy than PET probes (35 keV vs. 511 keV) and relatively long half-time allowing to use them in longitudinal studies that cannot be performed with short-living high-energy PET isotopes. Moreover, PET intrinsic resolution cannot go under positron range 1-3 mm 69 , while ultra-SPECT resolution can be significantly better 70 . The multimodal character of the NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoparticles allows to use them in laboratories equipped with any of the imaging methods (optical imaging, SPECT, MRI). Applying two or more of these methods to the imaged object allows very precise characterization of the diseased tissue.

Conclusion
Ultimately, the NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoprobes were not only luminescent, but also enabling MR imaging. Depending on the selection of the dopant ions, the size of the particles was controlled in the range of 13-18 nm with a relatively narrow particle size distribution. The particles proved to be biocompatible, dispersible, and stable in phosphate buffer media. The colloidal stability of the NaGdF 4 :Yb 3+ /Er 3+ particles in PBS was achieved by a unique, rapid, and facile surface modification with PEG-neridronate, which amounted to 20 wt.% according to www.nature.com/scientificreports/ TGA; this is in agreement with our previous report on the NaYF 4 :Yb 3+ /Er 3+ @PEG nanoparticles 35 . The particles exhibited NIR-to-UV/Vis photoluminescence, sufficient relaxivity enabled contrast in MRI, 125 I labeling allowed SPECT imaging, and thus their biodistribution and fate could be monitored in vivo. Prolonged blood circulation time, targeting specific tissues or cell labeling applications would require additional functionalization of the particles. Multimodality of NIR luminescence/MRI/SPECT/CT successfully applied in one system can potentially reduce overall cost and combine advantages of the three individual imaging techniques. The recent availability of a new generation of optical imagers allowing imaging in NIR I and NIR II makes the newly developed NaGdF 4 :Yb 3+ /Er 3+ @PEG nanoparticles perspective for use in NIR bioimaging and theranostics. This makes the particles a prospective diagnostic and/or theranostic platform for the multimodal imaging of serious disorders.