WO2018127912A1

WO2018127912A1 - Mri tracers comprising inorganic nanofluorides

Info

Publication number: WO2018127912A1
Application number: PCT/IL2018/050009
Authority: WO
Inventors: Amnon Bar-Shir; Idan ASHUR; Dana Cohen; Reut MASHIACH
Original assignee: Yeda Research And Development Co. Ltd.
Priority date: 2017-01-03
Filing date: 2018-01-03
Publication date: 2018-07-12

Abstract

The present invention provides MR imaging tracers comprising inorganic fluoride nanoparticles and uses thereof in a liquid for ¹⁹F-NMR and ¹⁹F-MRI.

Description

MRI TRACERS COMPRISING INORGANIC NANOFLUORIDES

FIELD OF THE INVENTION

[001] The present invention provides MR imaging tracers comprising inorganic fluoride nanoparticles and uses thereof in a liquid for ¹⁹F-NMR and ¹⁹F-MRI.

BACKGROUND OF THE INVENTION

[002] Magnetic resonance imaging (MRI) is a bio-medical imaging technique used to obtain anatomical, physiological and biochemical information mostly based on relaxation of proton (1H) nuclear spin in a magnetic field. It is one of the imaging techniques capable of visualizing the body organs of a human or an animal in real time in a non-invasive manner.

[003] In MRI, information is most commonly obtained by enhancing the contrast between the target and its surrounding tissue. This contrast can be enhanced by either manipulating the imaging acquisition parameters or by introducing chemical entities also known as contrast agents. MRI contrast agents are used to manipulate the observed contrast in a region of interest (ROI). On MRI image, the contrast between tissues occurs due to differences in relaxation properties of the proton (1H) nuclear spin of water molecules. MR image contrast can also be obtained due to differences in water diffusion properties, perfusion or proton exchange of solutes with that of water. Most of the MRI contrast agents affect the relaxation properties of the surrounding water, thereby altering the relaxation times in different tissues, and induces the change in MRI signals, thereby enhancing contrast between tissues. The contrast enhanced using the contrast agent allows clearer imaging by intensifying or weakening image signals from tissues of a particular organ.

[004] The properties required for the MRI contrast agent include thermodynamic stability and water solubility. In addition, the MRI contrast agent should be chemically inert, have low cytotoxicity in vivo and be completely excreted after diagnostic examination.

[005] For many years, gadolinium (Gd³⁺)-based paramagnetic probes and superparamagnetic iron oxide (SPIO)-based particles have been used as MRI contrast agents, which affect water relaxation properties (Ti, T₂ or T₂*) and, consequently, the localized MR image contrast. Although Gd³⁺, (and other paramagnetic based materials) and SPIO-based NPs (and other metal oxide-based NPs) proved to be extremely sensitive for MRI applications, their lack of specificity (relaxation- based MRI signal alternation) and high background signals lead to data misinterpretations and false results mandate background-free and quantifiable alternatives. [006] Contrary to conventional contrast agents that affect water relaxation properties and thus the MR image contrast, ¹⁹F-based sensors may be used as imaging tracers where the MR signal is directly proportional to the number of the observed ¹⁹F-spins, with no dependency on the surrounding water content. Fluorine (¹⁹F)-based sensors for molecular and cellular MRI show several advantages over commonly used metal-based contrast agents. First, due to the negligible amounts of ¹⁹F contents in soft biological tissues, ¹⁹F MRI picks up no background signal from the host tissues, and therefore, ¹⁹F-probes can be used as imaging tracers and be displayed as "hot- spots" on an image in the region of interest. Thus, false positive detection is unlikely, which overcomes one of the major limitations of MRI contrast agents. Second, the ¹⁹F-MR signal can be absolutely and accurately quantified because it is directly correlated with the number of ¹⁹F atoms in the monitored ROI. Third, the optimal (for MRI applications) magnetic properties of ¹⁹F nucleus, which has a high gyromagnetic ratio (γ = 40.05 MHz/T compared to 42.8 MHz/T of 1H, which allows the use of the same hardware), spin 1/2, and 100% natural isotopic abundance. Based on the above, it was demonstrated by others (summarized in a recent review: Chem. Rev. 2015; 115: 1106-1129) that perfluorocarbons (PFCs) could be used as ¹⁹F-MRI tracers for molecular and cellular imaging applications.

[007] In such imaging setups, different formulations of PFCs as oil-in-water nanoemulsions were used as imaging agents and are mostly used for labeling therapeutic cells or inflammatory processes. In one example, such platform was already used in clinical setup in patients (Magnetic Resonance in Medicine, 2014; 72:1696-1701).

[008] However, although their great advantages PFCs cannot obey some very important features of inorganic nanoparticles. Among these, PFC do not have a well defined structure, they cannot be prepared as very small (<10 nm) sized nanoparticles, their stability is undefined, they have non- flexible design (i.e., cores sizes and shapes, or shell functionalization and charge modifications) and their ¹⁹F content density is lower compared to the density of ¹⁹F in inorganic fluoride NPs tracer of this invention.

[009] Inorganic fluoride-containing nanocrystals (nanofluorides) are being used in many fields due to their unique chemical and physical properties. Several applications have been proposed for the use of inorganic fluorides in the industry, from isotope separation of uranium, through the production of fluorinated polymers, to the synthesis and use of optical materials. Among other advantages, nanofluorides were found to be useful due to the ability to modify their content, composition, size, and shape for an application of need [Fedorov, P.P., et al., Nanofluorides. Journal of Fluorine Chemistry, 2011. 132(12): p. 1012-1039]. Recently, lanthanide-doped nanofluorides were investigated and used, particularly the alkaline-earth lanthanide-doped nanofluorides (MF₂, M= Mg, Ca, Sr, Ba), as materials with unique optical properties, such as up- conversion fluorescence [Xiaoming, Z., et al., Solvothermal synthesis of well-dispersed MF 2 (M = Ca,Sr,Ba) nanocrystals and their optical properties. Nanotechnology, 2008. 19(7): p. 075603]_. In addition to their optical properties and to the simple and versatile available synthetic routes, their ¹⁹F NMR properties, as revealed from solid-state NMR (ssNMR) studies, feature their morphological properties. Indeed, both fluoride atoms of the MF₂ crystals (having fluorite-like, face-centered cubic crystals) are magnetically equivalent, resulting in a single and intense ¹⁹F NMR singlet peak [Sadoc, A., et al., NMR parameters in alkali, alkaline earth and rare earth fluorides from first principle calculations. Physical Chemistry Chemical Physics, 2011. 13: p. 18539-18550].

[0010] Inorganic fluoride-containing nanocrystals (nanofluorides) are often capped by organic ligands that assist with their synthesis, allowing the particles colloidal stability, can be exchanged by other ligands, and can be used for surface chemistry [Dong, A., et al., A Generalized Ligand- Exchange Strategy Enabling Sequential Surface Functionalization of Colloidal Nanocrystals. Journal of the American Chemical Society, 2011. 133(4): p. 998-1006; Xie, T., et al., Monodisperse BaF2 Nanocrystals: Phases, Size Transitions, and Self-Assembly. Angewandte Chemie International Edition, 2009. 48(1): p. 196-200. Thus, such capping ligands play an important role, not just in the nanocrystal formations, but also are crucial for various applications.

[0011] Therefore, the ability to use imaging tracers for MRI applications that are not based on contrast agents (manipulate the water relaxation properties), but rather, generate their own MR signals (e.g., ¹³C-, or ¹⁹F-based agents), opens new opportunities for the design and use of novel imaging platforms for molecular and cellular MRI.

SUMMARY OF THE INVENTION

[0012] In one embodiment, this invention is directed to a MR imaging tracer for molecular and cellular imaging comprising inorganic fluoride nanoparticles. In another embodiment, the inorganic fluoride nanoparticles have a particle size of less than 20 nm and are water dispersed.

[0013] In another embodiment, the inorganic fluoride NPs comprise a first metal cation and a fluoride anion forming a metal fluoride composition. In another embodiment, the metal fluoride composition further comprises a second metal. In another embodiment, the metal fluoride composition is further coated by, encapsulated by, embedded by or coordinated to an organic material or an inorganic material. [0014] In one embodiment, this invention provides a method of molecular or cellular imaging comprising: administering to a subject or a cell an imaging tracer comprising inorganic fluoride nanoparticles (NPs) of this invention and scanning the subject or cell using diagnostic imaging.

[0015] In another embodiment, the diagnostic imaging is ¹⁹F-MRI or ¹⁹F-NMR.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which

[0017] Figure 1 shows dynamic light scattering (DLS) measurements of citrate coated CeF₃ nanoparticles in water.

[0018] Figure 2 shows dynamic light scattering (DLS) measurements of citrate coated ScF₃ nanoparticles in water.

[0019] Figure 3A shows dynamic light scattering (DLS) measurements of citrate coated LaF₃ nanoparticles in water, providing hydrodynamic diameter of 5.2 + 1.3 nm .

[0020] Figure 3B shows ¹⁹F-NMR spectrum of the water soluble LaF₃ nanoparticles, with a ¹⁹F chemical shifts of 25.9 ppm and -26.5 ppm.

[0021] Figure 3C shows XRD pattern of citrate coated LaF3 (features tysonite-type structure).

[0022] Figure 4A shows dynamic light scattering (DLS) measurements of citrate coated SrF₂ nanoparticles in water, providing hydrodynamic diameter of 12.4 +5.0 nm Inset shows the TEM images of the particles.

[0023] Figure 4B shows ¹⁹F-NMR spectrum of the water soluble SrF₂ nanoparticles, with a ¹⁹F chemical shift of -89.3 ppm.

Figure 4C shows XRD pattern of citrate coated SrF₂ (features fluorite-type structure).

[0024] Figures 5Ashows TEM micrographs of citrate-coated CaF₂ nanoparticles (left) with high- resolution image of a single particle (right).

[0025] Figure 5B shows dynamic light scattering (DLS) measurements of citrate-coated CaF₂ NPs in aqueous solution.

[0026] Figure 5C is a XRD pattern of the citrate-coated CaF₂ NPs. Schematic of the Ca²⁺ first coordination spere is shown. On the right panel a fast Fourier transform FFT processing of the single citrate-coated CaF₂ particles image.

[0027] Figure 5D presents high resolution ¹⁹F NMR (376 MHz) of citrate coated CaF₂ NPs in aqueous solution, showing a peak at -109.6 ppm referenced to CFC1₃ at 0 ppm. [0028] Figure 5E presents normalized F-NMR signal of citrate-coated CaF₂ NPs as a function of inversion time (TI) from inversion recovery for the ¹⁹F-NMR experiments and the calculated Ti value (left); and normalized ¹⁹F-NMR signal of citrate-coated CaF₂ NPs as a function of echo time (TE) from CPMG ¹⁹F-NMR experiments and the calculated T₂ value.

[0029] Figure 6 shows an Energy-dispersive analysis spectrum (EDS) of citrate coated CaF₂ NPs.

[0030] Figure 7 shows MRI of phantom (upper left panel) containing 2 tubes of (i) water and (ii) citrate-coated CaF₂ nanoparticles in water. ¹H-MRI (upper right panel), ¹⁹F-MRI (bottom left panel), and "hot-spot" representation of ¹⁹F-MRI overlaid over ¹H-MRI (bottom right panel). All experiments were performed on NMR/MRI scanners operating at 9.4 T.

[0031] Figure 8 shows ¹⁹F-NMR of cells following incubation with citrate coated CaF₂ nanoparticles.

[0032] Figures 9A-9B show characteristic of water-soluble PEGylated CaF₂ nanoparticles. Figure 9A presents schematics of the PEGylated CaF₂ nanoparticles. Figure 9B shows ¹⁹F-NMR response of PEGylated CaF₂ particles in aqueous solution; top right- ¹⁹F-NMR signal of PEGylated CaF₂ NPs as a function of inversion time (TI) from inversion recovery for the ¹⁹F- NMR experiments and the calculated Ti value; and bottom right- normalized ¹⁹F-NMR signal of PEGylated CaF₂ as a function of echo time (TE) from CPMG ¹⁹F-NMR experiments and the calculated T₂ value

[0033] Figure 9C, show DLS histograms of purified PEGylated CaF₂ NPs in aqueous solution after 0 and 40 days

[0034] Figure 9D presents XRD pattern of PEGylated CaF₂ NPs (cubic-phase, fluorite-type structural features, PDF card no. 00-035-0816).

[0035] Figure 9E presents TEM images of the fabricated water-soluble PEGylated CaF₂ NPs. Figure 9F presents particle size distribution of PEGylated CaF₂ NPs obtained by analysis of the TEM image (Figure 9E). Figure 9G presents EDS spectrum of PEGylated CaF₂ NPs, displaying prominent Ka peaks of Ca and F. Peak area analysis of three different sampling spots that produced an atomic Ca : F ratio of 1 : 2.01.

[0036] Figure 10 depicts MRI of phantom containing aqueous solutions with or without PEGylated CaF₂ NPs with two different concentrations, as labeled in the left panel ^H-MRI). Middle panel: ¹⁹F-MRI acquired with a 3D-UTE sequence (TR/TE= 150/0.02 ms). Right panel: ¹⁹F-data displayed as a "hot spot" map overlaid on a 1H-MR image.

[0037] Figure 11A-11C depict functionalized water soluble PEGylated CaF₂ NPs (=CFP). Figure 11A nanofluorides with FITC coupled to the COOH groups of PEGylated CaF₂ NPs(=CFP) through an ethylene diamine linker resulted in CFP-FITC NPs. Figure 11B depicts nanofluorides with FITC coupled directly to the -OH groups of PEGylated CaF₂ NPs(=CFP) resulted in CFP-OFITC NPs. Figure 11C depicts nanofluorides with SCY3 conjugated directly to the -COOH groups of PEGylated CaF₂ NPs(=CFP) resulted in CFP-SCY3 NPs. The Left panels show the fluorescent spectra (excitation as a solid line, emission as a dashed line) of functionalized CFP. Middle panels display the DLS histograms of purified functionalized CFP NPs in aqueous solution. Right panels show the corresponding high-resolution ¹⁹F-NMR of the NPs in water.

[0038] Figures 11D-11F depict Zeta-potential plots of Figure 11D: CFP-FITC; Figure 12E CFP- OFITC; and Figure 11F: CFP-SCY3 NPs in water showing surface charges of -6.1 mv + 5.5, - 11.2 mv + 3.8, and -36 + 9 mv respectively.

[0039] Figure 12A-12C depict DLS histograms. Figure 12A depicts a DLS histogram of citrate- coated CaF₂ nanoparticles (=CF-Cit). Figure 12B depicts a DLS histogram of CFP-FITC NPs and Figure 13C depicts a DLS histogram of CFP-OFITC NPs in aqueous solution. Data are normalized.

[0040] Figures 13A-13B present in vivo imaging of PEGylated CaF₂ NPs (specifically, CFP- SCY3) in a model of inflammation. Figure 13A presents a schematic depiction of the experimental sequence for the in vivo experiment. Figure 13B presents, anatomical ¹H-MR images of representative mouse (left panel) and matched ¹⁹F-MR images shown as pseudo-color maps overlaid on the anatomical ^-MR images (right panel). MRI data were acquired on a 9.4 T MRI scanner equipped with a ^lH/^l9F.

[0041] Figure 14A-14B present FACS analysis. Flow cytometry analysis of cells excised from lymph nodes (one hour post-CFP-SCY3 or PBS injections). Figure 14A presents dot plot data from PBS (left) or CFP-SCY3 (right) injections. Figure 14B presents analysis of specific cell populations with red and black histograms representing cells from mice subjected to injection of CFP-SCY3 or PBS, respectively. Staining for dendritic cells (left, stained for CDl lb CDl lc) and macrophages (right, stained for CDl lb CD45).

[0042] Figure 14C presents analysis of specific cell populations with red and gray histograms representing cells from mice subjected to injection with CFP-SCY3 and PBS, respectively. Staining for CD4⁺ T cells (stained for CD4, upper panel) and CD8⁺ T cells (stained for CD8, lower panel).

[0043] Figure 15 presents FTIR spectra of purified CFP NPs supported on KBr pellets. Data were acquired at a resolution of 4 cm^-1. The spectrum exhibited typical peaks for H-O-H bending at 3393 cm^"1 and a strong -CH stretching at 2886 cm^"1. The carboxyl bands at 1743 cm^"1 and 1595 cm^"1 were especially important for verifying the presence of carboxylated PEGs on the surface of the particles. The strong stretch around 1105 cm^"1 was consistent with the appearance of a sharp intense band at 1103 cm^"1 in the spectra of free PEG600 molecules and the PEG600-coated iron- oxide NPs.

[0044] Figures 16A-16B provide representative mass loss profiles of Citrate-coated CaF₂ (CF- Cit) in Figure 16A and of PEGylated CaF₂ (CFP) NPs in Figure 16B. The samples were dried under vacuum prior to the measurements and the measurements were carried out under a nitrogen atmosphere. The profile of CF-Cit already showed the onset of degradation below 100 °C, corresponding to 13.7% of the total mass. Degradation of the citrate coating is expected at much higher temperatures. The first degradation phase in Figure 16A was attributed to decomposition of tightly bound solvent molecules, as reported in previous studies. Using Equation 3 and Equation 4 the average mass losses of 11.5% in Figure 16A and 45.3% in Figure 16B were used to estimate the ligand density of citrate and the PEG molecules on the surface of CF-Cit and CFP NPs, respectively.

[0045] Figure 17 depicts percentage of lymphatic cells showing increased fluorescence (red filter) following injections of PBS (N=3 mice) or CFP-SCY3 (N=3 mice), as obtained from FACS experiments.

[0046] Figure 18 depicts the effect of CFP-SCY3 NPs on the viability of HeLa cells that were incubated with and without CFP-SCY3 NPs for 2 h at 37 °C. Viability was tested by the CellTiter method. Treatment with 1% Triton was used as a positive control to assess the normal functioning of the cells. The data represent survival rates relative to untreated cells.

[0047] Figure 19A and 19B present oleate-coated CaF₂ NPs. Figure 19A: TEM image, Figure 19B particle size distribution histogram with a mean diameter of D=8.1 + 2.5nm.

[0048] Figures 20A and 20B present NMR spectra of oleate-coated CaF₂ dispersed in cycloheaxane-dl2. Figure 20A: ¹⁹F NMR. Figure 20B: 1H NMR and the chemical structure of oleic acid.

[0049] Figures 20C and 20D: present 1H NMR of bound (to CaF₂) and free oleate coating in cyclohexane-dl2 solvent. Oleate-coated CaF₂ NPs. (Figure 20C). Free oleic acid (Figure 20D). Upper and lower insets in the tables are displayed for bound and free ligands, respectively. These tables demonstrate the relaxation and diffusion coefficient constant for each resonance peak.

[0050] Figure 21A and 21B present oleate-coated SrF₂ nanofluorides dispersed in cyclohexane. Figure 21A: TEM image; Figure 21B: ¹⁹F NMR spectra.

[0051] Figure 22A and 22B present oleate-coated MgF₂ nanofluorides dispersed in cyclohexane. Figure 22A: TEM image; Figure 22B: ¹⁹F NMR spectra.

[0052] Figure 23A and 23B present Oleate-coated NaYF₄ nanofluorides dispersed in cyclohexane. Figure 23A: TEM image; Figure 23B: ¹⁹F NMR spectra. [0053] Figure 24 presents schematic illustration of the rationale of using different nanofluorides (i.e., MgF₂, CaF₂, SrF₂, and BaF₂) for artificial multicolor MRI.

[0054] Figures 25A-25C: Axial plane MRI images of oleate-coated CaF₂ dispersed in cycloheaxane-dl2 in a 5mm closed tube submerged in 5% gelatin in a 15mm tube. ¹⁹F 3D UTE MRI scan, 64x64 matrix, 4 averages, TE=0.02msec, and TR=300msec, with a flip angle of 10°. The ¹⁹F concentration was 1.7mM (Figure 25A). 3D UTE 1H MRI scan, 128x128 matrix, 1 scan, TE=0.02msec, and TR=8msec (Figure 25B). Overlay of ¹⁹F (in cyan) and ^lH images (in white)(Figure 25C).

[0055] Figure 26A and 26B present oleate-coated BaF₂ NPs. TEM (Figure 26A) and HRTEM (Figure 26B).

[0056] Figure 27: XRD pattern of oleate-coated BaF₂ NPs; numbers represent the crystal structure of frankdiksonite (BaF₂).

[0057] Figure 28: Representative mass loss profile of oleate-coated BaF₂ NPs, as obtained from TGA experiments, with pronounced weight loss at ~400°C that corresponds to the decomposition of organic molecules.

[0058] Figure 29: High-resolution ¹⁹F-NMR of cyclohexane solutions of BaF₂ NPs doped with increasing levels of La³⁺.

[0059] Figure 30A-30C: TEM images of different %mol La³⁺ vs. Ba²⁺ 1.25La:BaF₂ (Figure 30A) 2.5La:BaF₂ (Figure 30B) and 5La:BaF_r (Figure 30C).

[0060] Figure 31: X-ray diffraction of 5La:BaF₂ (black XRD pattern) vs. pure BaF₂ (red XRD pattern).

[0061] Figure 32: DLS histogram (with the TEM image in the inset) of BaF₂ NPs prepared using polyol synthesis.

[0062] Figures 33A and 33B depict ¹⁹F NMR (Figure 33A) and DLS results (Figure 33B) of

AEP-coated BaF₂ in water as obtained by the co-precipitation synthetic approach, as well as the chemical structure of AEP.

[0063] Figures 34A-34B: DLS of AEP-La: BaF₂ NPs (Figure 34A). TEM and HRTEM images of AEP-coated BaF₂ NPs with La³⁺ as a stabilizing doping agent (Figure 34B).

[0064] Figures 35A-35C: MRI of nanofluorides. Axial MR images of two 5mm tubes containing AEP-coated La:BaF₂ NPs in D₂0 and two tubes containing only water. All the four tubes were submerged in 2% gelatin and placed in a 25 mm tube. 1H 3D UTE MRI scan,128xl28 matrix, TE=0.02 msec and TR=8 msec (Figure 35A) ¹⁹ F 3D UTE MRI, 4 averages, TE=0.02 msec, and TR=150 msec, with a flip angle of 21.9°(Figure 35B). Overlay of ¹⁹F (in pink) over ¹H-MRI data (from panel a) (Figure 35C).

[0065] Figure 36: Artificial multicolor MRI using water soluble nanofluorides. Left: ¹H-MRI of a phantom containing four tubes containing (BaF₂, SrF₂, CaF₂ and water as labeled). Right: ¹⁹F- MRI (pseudo-color) overlaid on ¹H-MRI with ¹⁹F-MR signals display as artificial colors of BaF₂ (center frequency at -13 ppm) SrF₂ (center frequency at -89 ppm) CaF₂ (center frequency at -109 ppm).

[0066] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0067] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

[0068] In one embodiment, this invention provides inorganic fluoride nanoparticles (nanofluorides) as soluble colloids for molecular or cellular imaging application. The nanofluorides of this invention are water soluble and experience longstanding colloidal stability in aqueous solutions.

[0069] In one embodiment, this invention provides an imaging tracer comprising an inorganic fluoride nanoparticles (nanofluorides) as soluble colloids. The nanofluorides of this invention are water soluble and experience longstanding colloidal stability in aqueous solutions.

[0070] In one embodiment, this invention provides an MR imaging tracer for molecular and cellular imaging comprising inorganic fluoride nanoparticles, wherein the inorganic fluoride nanoparticles have a particle size of less than 20 nm and are water dispersed.

[0071] In another embodiment, the molecular or cellular imaging refers to ¹⁹F NMR or ¹⁹F MRI.

Each represents a separate embodiment of this invention.

[0072] In one embodiment, the inorganic fluoride NPs of this invention are nanocrystallines. [0073] In one embodiment, the inorganic fluoride nanoparticles of this invention are used for imaging diagnosing. These applications include monitoring of: therapeutic cells migration and/or fate, inflammatory diseases, inflammation processes, immune-activation, changes in physiological conditions (pH, redox, temperature), enzymatic activity, specific receptor expression, circulation and body clearance, and/or multiplexed (i.e., artificial multicolor) imaging.

[0074] In one embodiment, the inorganic fluoride nanoparticles, or the imaging tracer of this invention comprise a first metal cation and a fluoride anion forming a metal fluoride composition. In another embodiment the first metal cation is paramagnetic. In another embodiment, the metal fluoride composition comprises a first metal cation and a fluoride anion (i.e. only one metal cation, not combination of metal cations). In another embodiment, the metal fluoride composition comprises a first metal cation, a second metal cation and a fluoride anion (i.e. combination of two different metal cations). In another embodiment, the metal fluoride composition comprises more than one metal cation. In another embodiment, the first and the second metal cations are different. In another embodiment, the first or the second metal cation is an alkaline earth metal cation. In another embodiment, the first or the second metal cation is an alkali metal cation. In another embodiment, the first or the second metal cation is a transition metal cation. In another embodiment, the first or the second metal cation is a lanthanide cation. In another embodiment, the metal fluoride composition comprises an alkaline earth metal, an alkali metal, a transition metal, a lanthanide or any combination thereof. In another embodiment, the metal fluoride composition comprises Ca, Ba, Ce, La, Sc, Mg, Y, Sr ions, or combination thereof. Each represents a separate embodiment of this invention.

[0075] In one embodiment, the second metal cation is a paramagnetic metal cation. In another embodiment, the second metal cation is a non-paramagnetic metal cation. In another embodiment, the second metal cation (dopant) is a lanthanum cation (La³⁺). In another embodiment the second metal cation (dopant) is any atom that is not the metal cation paired with fluoride in the metal composition, wherein the second metal cation (dopant) is selected from an alkaline earth metal cation, an alkali metal cation, a transition metal cation, a lanthanide cation or combination thereof.

[0076] In one embodiment, the second metal cation is a dopant. In another embodiment, the concentration of the second metal cation is between 0.1 to 50 mol% of the first metal cation. In another embodiment, the concentration of the second metal cation is between 0.1 to 1 mol%. In another embodiment, the concentration of the second metal cation is between 0.1 to 5 mol%. In another embodiment, the concentration of the second metal cation is between 1 to 10 mol%. In another embodiment, the concentration of the second metal cation is between 1 to 20 mol%. In another embodiment, the concentration of the second metal cation is between 1.25 to 50 mol%. In another embodiment, the concentration of the second metal cation is between 5 to 50 mol%.

[0077] In another embodiment, the inorganic fluoride nanoparticles, or the imaging tracer of this invention further comprise phosphorous, oxygen, boron, sulfur, silicon or combination thereof. In another embodiment, the inorganic fluoride nanoparticles, or the imaging tracer of this invention comprises a mineral comprising fluorine atom(s).

[0078] In one embodiment, the inorganic fluoride nanoparticles, or the imaging tracer of this invention comprise a metal cation and a fluoride anion, forming a metal fluoride composition, wherein the metal fluoride composition is further coated by, encapsulated by, embedded by or coordinated to an organic material or an inorganic material. In another embodiment, the metal fluoride composition is coated by an organic or by inorganic material. In another embodiment, the metal fluoride composition is encapsulated by an organic material or by inorganic material. In another embodiment, the metal fluoride composition is embedded by an organic material or by inorganic material. In another embodiment, the metal fluoride composition is coordinated to an organic material.

[0079] In another embodiment, the organic material comprises citric acid, oleic acid, fatty acid, polyethylene glycol (PEG), polyethylene imine, polysaccharide, polymers of amine, peptide, protein, phospholipid, lipid, amino ethyl phosphate (AEP), phosphate, polylysine, PLGA (poly(lactic-co-glycolic acid), cellulose, any sugar, amino acid, nucleoside, nucleotide, nucleic acids, gelatin a polar polymer, small molecule, drug or any combination thereof. Each represents a separate embodiment of this invention.

[0080] In another embodiment, the inorganic material comprises gold, silver, titanium, ceramic, silica (dense, porous, hollow or meso-porous silica), inorganic fluoride, any metal, any metal oxide.

[0081] In another embodiment, the inorganic material is composite as a shell layer.

[0082] To enable the dispersion of the prepared nanofluorides in water, a coating of hydrophobic organic material/ligand (oleate ligand, for example) needs to be replaced by a hydrophilic, water-soluble coating. In other embodiments, one hydrophobic organic material is replaced by another water-soluble organic material and may facilitate the preparation of water- dispersed colloids (ligand exchange approach). In other embodiments, amphiphilic ligands (a ligand with a hydrophilic head and a hydrophobic tail) are incorporated between the a coating of hydrophobic organic material of the inorganic fluoride nanoparticles (Ligand incorporation approach). [0083] In one embodiment, the inorganic fluoride nanoparticles of this invention are further functionalized by one or more functionalizing group comprising a fluorescent moiety or a targeting moiety. In another embodiment, the fluorescent moiety or the targeting moiety are chemically linked to the organic material (covalently or ionic). In another embodiment, the fluorescent moiety or the targeting moiety is the coating organic material.

[0084] In another embodiment, the functional group comprises a fluorescent moiety or a targeting moiety. In another embodiment, non-limiting examples of a fluorescent moiety include sulfo- Cyanine5, Cyanine5.5, sulfo-Cyanine3, Rhodamine, Cyanine3, Cyanine3.5, Cyanine5, Cyanine7, Cyanine7.5, Fluorescein, sulfo-Cyanine7, borondipyrromethene dyes, Coumarin 343, Pyrene, sulfo-Cyanine5.5 or sulfo-Cyanine7.5. Each represents a separate embodiment of this invention.

[0085] In another embodiment, the targeting moiety comprises a target ligand. Non limiting examples of a targeting moiety includes RGD peptide, a cyclic RGD peptide, an antibody, folic acid or a TAT peptide, cell penetrating peptides (CPPs, e.g., GRKKRRQRRRPPQ (SEQ ID NO: 1), RKKRRQRRR (SEQ ID NO: 2), RQIKIWFQNRRMKWKK (SEQ ID NO: 3), poly-L- arginine, VKRGLKLRHVRPRVTRMDV (SEQ ID NO: 4),

G ALFLGFLG AAGS TMGA WS QPKKKRKV (SEQ ID NO: 5),

KETWWETWWTEWS QPKKKRKV (SEQ ID NO: 6), LLIILRRRIRKQAHAHS K (SEQ ID NO: 7), MVRRFLVTLRIRRACGPPRVRV (SEQ ID NO: 8), MVKSKIGSWILVLFVAMWSDVGLCKKRP (SEQ ID NO: 9), KLALKLALKALKAALKLA (SEQ ID NO: 10), GWTLNS AG YLLGKINLK ALA ALAKKIL (SEQ ID NO: 11), LS T AADMQGV VTDGM AS GLDKD YLKPDD (SEQ ID NO: 12),

DPKGDPKGVTVTVTVTVTGKGDPKPD (SEQ ID NO: 13),

RRIRPRPPRLPRPRPRPLPFPRPG (SEQ ID NO: 14), CSIPPEVKFNKPFVYLI (SEQ ID NO: 15), PFVYLI (SEQ ID NO: 16), SDLWEMMMVSLACQY (SEQ ID NO: 17)), tumor receptors binding moieties (e.g., LHDH, SP5-2 (TDSILRSYDWTY(SEQ ID NO: 18)), NGR peptide, iRGD, RNA aptamer, DNA aptamer, A10 RNA aptamer, Sgc8c DNA aptamer, Monoclonal antibody A7, Transferrin antibody, DI17E6, 2C5 antibody, 5D4 antibody, Anti-HER2 scFv, Anti- VCAM-1, Anti-CD22 scFv, Affibody, Avimer, Nanobody).

[0086] In one embodiment, the inorganic fluoride nanoparticles or the imaging tracer have an average diameter size of less than 100 nm. In one embodiment, the inorganic fluoride nanoparticles or the imaging tracer have an average diameter size of less than 50 nm. . In one embodiment, the inorganic fluoride nanoparticles or the imaging tracer have an average diameter size of less than 20 nm. In one embodiment, the inorganic fluoride nanoparticles or the imaging tracer have an average diameter size of less than 15 nm. In another embodiment, the nanoparticles have an average diameter size of between 1-100 nm. In another embodiment, the nanoparticles have an average diameter size of between 1-80 nm. In another embodiment, the nanoparticles have an average diameter size of between 1-70 nm. In another embodiment, the nanoparticles have an average diameter size of between 1-60 nm. In another embodiment, the nanoparticles have an average diameter size of between 1-50 nm. In another embodiment, the nanoparticles have an average diameter size of between 1-40 nm. In another embodiment, the nanoparticles have an average diameter size of between 1-30 nm. In another embodiment, the nanoparticles have an average diameter size of between 1-20 nm. In another embodiment, the nanoparticles have an average diameter size of between 1-10 nm. In another embodiment, the nanoparticles have an average diameter size of between 5-20 nm. In another embodiment, the nanoparticles have an average diameter size of between 3-20 nm. In another embodiment, the nanoparticles have an average diameter size of between 1-15 nm. In another embodiment, the nanoparticles have an average diameter size of between 5-15 nm. In another embodiment, the nanoparticles have an average diameter size of between 4-5 nm. In another embodiment, the nanoparticles have an average diameter size of between 9-11 nm.

[0087] In one embodiment, the inorganic fluoride nanoparticles or the imaging tracer of this invention are soluble in water or an aqueous solution. In another embodiment, the inorganic fluoride nanoparticles or the imaging tracer of this invention form an emulsion in water or an aqueous solution. In another embodiment, the inorganic fluoride nanoparticles or the imaging tracer of this invention are form colloids in water or in aqueous solution. In another embodiment, the inorganic fluoride nanoparticles or the imaging tracer of this invention form a dispersion in water or in aqueous solution. In another embodiment, the inorganic fluoride nanoparticles or the imaging tracer of this invention are water soluble and experience longstanding colloidal stability in aqueous solutions.

[0088] In one embodiment, the inorganic fluoride nanoparticles or the imaging tracer comprise polyethyleneglycol (PEG), oleic acid, citric acid or amino ethyl phosphate (AEP) as a coating ligand.

[0089] In one embodiment, the inorganic fluoride nanoparticles or the imaging tracer comprise a metal fluoride composition. Non limiting examples include CaF₂, BaF₂, MgF₂, CeF₃, ScF₃, LaF₃, SrF₂, YF₃, NaYF₄, or combination thereof. In another embodiment the metal fluoride composition is doped by other lanthanide cation or non-lanthanide.

[0090] In one embodiment, inorganic fluoride nanoparticles and the imaging tracer of this invention have enhanced thermodynamic and kinetic stability. Methods of use

[0091] Fluorine- 19 is the second most NMR-sensitive nuclei (after 1H) and therefore it is favorable for MR-based studies (NMR and MRI) covers a wide range of fields from chemistry to structural biology, material sciences and even medicine. In addition, the lack of ¹⁹F-nuclei in soft biological tissues makes ¹⁹F-based materials potential quantitative tracers for molecular and cellular MR imaging applications. Although fluorine-containing nanoemulsions (mostly based on perfluorocarbons) were proposed for ¹⁹F-MRI applications and have been translated to clinical use, fluoride-based nanocrystals (M_xF_y; M is a metal cation, F is a fluoride anion) have not been studied in solutions with high-resolution ¹⁹F-NMR and were not used in ¹⁹F-MRI. This is because in nanocrystal-based formulations the restricted mobility of the elements within the crystal frequently results in NMR line-broadening. Therefore, high-resolution NMR signals from the nanoparticle core's nuclei that experience limited mobility and high dipole-dipole interactions cannot be obtained using solution NMR experiments. This invention shows that high-resolution ¹⁹F-NMR spectra can be achieved by sufficient averaging of homonuclear dipolar interactions of ¹⁹F-nuclear spins within freely tumbling fluoride-containing nanocrystals. Overcoming such limitation and successful performances of high-resolution ¹⁹F-NMR experiments in solutions, which are achieved by sufficient averaging of homonuclear dipolar interactions found in inorganic nanofluorides, this invention propose a novel type of ¹⁹F-nanotracers for ¹⁹F-MR imaging. The synthesized, purified and fully characterized nanofluorides aim to combine the advantages of inorganic nanocrystals (e.g., small and controllable sizes, dense fluoride content, monodispersity, colloidal stability, surface modifiability, etc.) with the merit of ¹⁹F-MRI. The ability to monitor MRI signals of pools of nuclear spins that have extremely short T₂ values (as those of ¹⁹F-nuclei in nanofluorides) using advanced MRI acquisition schemes such as ultrashort echo time (UTE) or zero echo time (ZTE) based schemes allow the use of inorganic nanofluorides as imaging tracers. The ability to study nanocrystals in solutions with high-resolution NMR could mark the dawn of a new scientific era in material sciences, biology and medicine.

[0092] In one embodiment, this invention provides a method of molecular or cellular imaging comprising: administering to a subject or a cell an imaging tracer or an inorganic fluoride nanoparticles (NPs) of this invention and scanning the subject or cell using diagnostic imaging.

[0093] In one embodiment, this invention provides a method of measuring ¹⁹F-NMR of inorganic fluoride in a liquid sample comprising coated inorganic fluoride nanoparticles of this invention in a liquid and measuring said sample by ¹⁹F NMR. [0094] In another embodiment, the liquid can be any solvent or liquid appropriate for the NMR. In another embodiment, the liquid is water or an aqueous solution. In another embodiment, the inorganic fluoride nanoparticles are soluble in the liquid or form an emulsion, dispersion or colloids in the liquid.

[0095] In one embodiment, this invention is directed to a method of molecular or cellular imaging comprising: administering to a subject or a cell an imaging tracer of this invention; and scanning said subject or cell using diagnostic imaging.

[0096] In one embodiment, this invention is directed to a method of molecular or cellular imaging comprising: administering to a subject or a cell inorganic fluoride nanoparticles of this invention; and scanning said subject or cell using diagnostic imaging. In another embodiment, the diagnostic imaging is ¹⁹F-MRI or ¹⁹F-NMR.

[0097] In another embodiment, a subject in this invention refers to a mammal, a human or an animal.

[0098] In one embodiment, the methods of this invention make use of inorganic fluoride nanoparticles. In another embodiment, the inorganic fluoride nanoparticles are in aqueous solution. In another embodiment, the inorganic fluoride nanoparticles form emulsion, dispersion or colloids in water.

[0099] In one embodiment, the imaging tracer of the present invention is used in a method of imaging, including methods of imaging in a subject comprising administering the imaging tracer to the subject by injection, infusion, or any other known method, and imaging the area of the subject wherein the event of interest is located.

[00100] The useful dosage to be administered and the particular mode of administration will vary depending upon such factors as age, weight, and particular region to be treated, as well as the particular contrast agent used, the diagnostic use contemplated, as will be readily apparent to those skilled in the art

[00101] In one embodiment, inorganic fluoride nanoparticles of this invention are used for diagnostic imaging. Non limiting examples of diagnostic imaging include ¹⁹F-MRI ¹⁹F-NMR or combination thereof. In another embodiment, the diagnostic imaging is used to monitor non limiting examples such as: therapeutic cells migration, inflammatory diseases, immune-activation, changes in physiological conditions (pH, redox, temperature), enzymatic activity, specific receptor expression, circulation and body clearance, multiplexed imaging, etc.

[00102] In one embodiment, this invention provides a diagnostic kit comprising the imaging tracer of this invention. [00103] The F MR imaging capacity of the imaging tracer of this invention allows a physician, radiologist, technician or scientist to monitor the chemotherapeutic/ therapeutic agent/drug directly in real time. Such real time feedback makes it possible to adjust treatment plans immediately. By determining the amount of a particular dose of therapeutic agent that has reached the target tissue or organ, further dosages for the patient can be determined.

[00104] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES Example 1

Synthesis and Characterization of Citrate Coated Inorganic Fluorides Nanoparticles

[00105] Citrate-coated water-soluble nanoparticles were synthesized using the co-precipitation approach. By introducing aqueous solution of CaCl₂ (or any other metal chloride) into a stirring solution of NaF and citric acid, CaF₂ (or any other metal fluoride) particles were formed. Following washing/ centrifugation procedures, pure monodispersed 5 nm CaF₂ (or any other metal fluoride) nanoparticles were obtained.

citrate-coated CaF₂ (CF-Cit) nanoparticles (NPs)

[00106] Sodium fluoride (43 mg) and citric acid (410 mg) were mixed in 25 ml of water (milli-Q grade). The pH of the mixture was adjusted to 7.0 by NH₄OH, which was followed by heating to 75 °C under vigorous stirring. A solution of CaCl₂ (55 mg in 1.5 ml of water) was rapidly injected into the reaction mixture, followed by its immediate removal from the heating element and was left to cool down. Fast injection of the precursor, together with the shortest reaction time possible, was found to yield the smallest particle core and hydrodynamic diameters. A similar procedure was applied in order to synthesize citrate-coated LaF₃ and SrF₂ NPs. For the latter, the reagents were left to react for 45 min after injecting the precursor.

[00107] Using the same procedure (with a different metallic salt as the cationic precursor) CeF₃, ScF₃ LaF₃, and SrF₂ nanofluorides were obtained (Figures 1, 2 3 and 4A-4C respectively) with hydrodynamic diameter of smaller than 10 nm. [00108] Purification: The colloid was centrifuged in a mixture of water and absolute ethanol and re-dispersed in water. Additional cycle of centrifugation was employed and the particles were dried under a stream of nitrogen to remove traces of ethanol. The dried colloid was re-dispersed in water and was purified by dialysis. The dispersion was stored at 4 °C for further use.

[00109] Small, water-soluble, citrate-coated CaF₂ (CF-Cit) NPs (Fig. 5A-5D) were synthesized to examine the hypothesis that high-resolution ¹⁹F-NMR spectra can be achieved by sufficient averaging of homonuclear dipolar interactions of ¹⁹F-nuclear spins within freely tumbling fluoride- containing nanocrystals. The obtained very small CF-Cit NPs were highly crystalline (Figure 5 A) and monodispersed (core size of 4.5 + 2 nm). Energy-dispersive X-ray spectroscopy (EDS) analysis revealed a -1:2 ratio between the Ca²⁺ and F^" ions (Figure 6). The NPs possessed preserved monodispersity and lack of aggregation in water, as determined by dynamic light scattering (DLS, Figure 5B), while their colloidal stability could be conserved for at least 25 days in water solution (Figure 12A). The XRD pattern of the synthetic small CF-Cit NPs (Figure 5C) features a typical cubic -phase, fluorite-type structure (JCPDS Card no. 87-0971) with peaks corresponding to <h k 1> values of (1 1 1), (2 2 0), (3 1 1), (4 0 0), (3 3 1), and (4 2 2). This observation was supported by fast Fourier transform (FFT, Figure 5C) analysis of the TEM image of a single particle shown on the right panel of Figure 5A. Following these validations that small, stable, and water-soluble CF-Cit NPs could be obtained, high-resolution ¹⁹F-NMR experiments of nanofluorides in water were performed and the obtained ¹⁹F-NMR spectrum is depicted in Figure 5D. In a face-centered cubic (fee) fluorite-type structure of CaF₂ (Figure 5C), all fluorides were expected to exhibit high magnetic equivalence, as reflected by the coordination scheme of the Ca and F atoms in the crystal (inset, Figure 5C). Indeed as shown in Figure 5D a singlet peak was observed at high-resolution ¹⁹F-NMR spectrum at a resonance of -109.6 ppm, similar to the frequency obtained previously by solid-state NMR for CaF₂ powder. This is an advantage when proposing novel ¹⁹F-based tracers for ¹⁹F-MRI applications where high magnetic equivalence of the ¹⁹F-source is desired for maximizing the ¹⁹F-MR signal intensity.

[00110] The mass loss profile of the citrate coated CaF₂ particles shows two major regions (Figure 16B). The region between 25 °C and 150 °C is typical to desorption of adsorbed moisture from the outer layer of the particles. The region between 300 °C and 600 °C is attributed to the loss of the citrate coating. Taking together the results from TGA and the evaluating the core size from TEM images, the estimated molecular weight of the citrate coated CaF₂ nanoparticle to be -114 KDa. citrate-coated SrF₂ and l il \ nanoparticles (NPs)

[00111] Citrate-coated SrF₂ and LaF₃ NPs were prepared as described above.

[00112] Citrate-coated water soluble and monodispersed SrF₂ NPs (Figure 4A), which shares similar fee crystal structure as CaF₂ (compare Figure 5C with Figure 4C) also features a clear, single high-resolution ¹⁹F-NMR peak in aqueous solution (Figure 4B), as expected from the high magnetic equivalence of the fluoride content in fluorite-like crystals (Figure 4C). Conversely, an aqueous solution of small monodispersed citrate-coated LaF₃ NPs (Figure 3A) with tysonite-type crystal structure (Figure 3C), exhibited a more complex high-resolution ¹⁹F-NMR spectrum in water. This spectrum (Figure 3B) depicts more than one ¹⁹F-resonance having the expected chemical shifts obtained with solid-state ¹⁹F-NMR studies of LaF₃ crystals (the two up-field peaks are merged due to line broadening), thus reflecting that the fluorides in the crystal are located at nonequivalent crystallographic sites (schematically depicted in the inset of Figure 3C). These findings (Figures 3A-3C, 4A-4C and 5A-5D) confirm that small nanofluorides in aqueous solution can be visualized and studied by high-resolution ¹⁹F-NMR approaches and may be proposed for ¹⁹F-MR applications.

Example 2

Citrate coated CaF₂ Nanoparticles as ¹⁹F-MRI tracers

[00113] To examine the feasibility of using the small water-soluble CaF₂ as ¹⁹F-MRI tracers, ¹⁹F- NMR experiments were performed on 9.4 T NMR spectrometer using citrate coated CaF₂. As shown in Figure 5D, a single ¹⁹F-NMR peak was obtained. The frequency of the CaF₂ was determined to be -109 ppm, which is in a good agreement with the frequencies obtained for CaF₂ powder using solid-state NMR. The Ti and T₂ of the ¹⁹F signal obtained from the particles were found to be 7.02+0.02 sec and 3.0+0.1 msec, respectively.

[00114] Figure 7 demonstrates that CaF₂ nanoparticles in water can be used as ¹⁹F-MRI tracers. The H-MRI image shows no difference between the examined tubes (contained either water or water with CaF₂ nanoparticles). As expected, in ¹⁹F-MRI only the CaF₂ containing tube could be imaged. The right bottom panel demonstrates the "hot-spot" display of the ¹⁹F-MRI signal overlaid on ^-MRI signal. Example 3

Citrate coated CaF₂ (CF-Cit) Nanoparticles in Cells

[00115] In order to examine the use the citrate coated CaF₂ platform in a biological setup we incubated live cells in the presence of the freshly synthesized citrate coated CaF₂ nanoparticles. Figure 8 demonstrates that following 2 hours incubation, typical CaF₂ ¹⁹F-NMR signal can be observed from the pelleted treated cells.

Example 4

Synthesis of PEGylated CaF₂ (CFP) Nanoparticles

[00116] In order to facilitate the use of the fluoride nanocrystals as tracers for ¹⁹F-MRI applications, CaF₂ NPs were coated with the biocompatible polyethylene-glycol (PEG) moieties (Figure 9A), which should provide NPs with the needed water solubility, stability, monodispersity, and surface modifiability. The pegylated CaF₂ NPs were synthesized using a solvothermal approach by mixing F^" and Ca²⁺ precursors in a mixture of PEG-hydroxyl and PEG- carboxylate (on average Mw 600).

Synthesis of PEGylated CaF2 NPs

[00117] Polyethylene glycol (PEG)-coated CaF₂ nanoparticles were synthesized using the solvothermal method. Ammonium fluoride (114 mg) and Ca(N(¾)₂x4H₂0 (236 mg) were mixed in a solution of PEG (24 g, average Mw 600) that contained poly(ethylene glycol)bis carboxymethylether (4.8 g, average Mw 600). The solution was heated to 80°C, under vacuum, and remained at that temperature for one hour. The mixture was then heated to 220°C (low pressure conditions were maintained until the temperature reached 160°C) and left to react at 220°C for 15 min under an inert atmosphere along with magnetic stirring. Unreacted reagents were removed by washing with a mixture of 1:5 ethanol diethyl-ether, followed by centrifugation for 10 min at 9000 g. This process was repeated three times, after which the product was dried under vacuum and kept for further use.

Characterization

[00118] The purified CaF₂ coated with PEG (CFP, schematically shown in Figure 9A) NPs were fully characterized and found to have a core diameter that is slightly larger (7.2 ± 1.3 nm, Figure 9E and Figure 9F) compared with CF-Cit NPs. The XRD pattern exhibited cubic -phase, fluorite- type structural features similar to those of the citrate coated CaF₂ (CF-Cit) NPs (compare Figure 9D with Figure 5C). A ratio of -1:2 between the Ca²⁺ and F ions was confirmed by EDS analysis (Figure 9G). The DLS histograms of CFP NPs in aqueous solution indicate that the particles are small and monodispersed (13.5+4.4 nm) and with retained colloidal stability for at least 40 days (Figure 9C). High-resolution ¹⁹F-NMR experiments of CFP NPs in aqueous solution revealed a clear singlet peak at the expected chemical shift of CaF₂ (-109.6 ppm, Figure 9B), similarly to the spectrum obtained for CF-Cit (Figure 5D). In a purpose to optimize the acquisition parameters in future ¹⁹F-MRI experiments, the longitudinal (TO and transverse (T₂) relaxation times of the ¹⁹F- content of the water-soluble CFP NPs were determined (Figure 9B) and the relaxation rates were found to be Ri (I/TO = 0.1027 + 0.0003 s^"1 and R₂ (1/T₂) = 345 + 35 s .

Example 5

PEGylated CaF₂ (CFP) Nanoparticles as ¹⁹F-MRI tracers ¹⁹F-MRI Experiments

[00119] Both in vitro and in vivo MRI experiments were performed on a vertical 9.4 T wide-bore MR scanner (Bruker Avance system). A double-resonant (^lH/^l9F), 25-mm, birdcage radiofrequency (RF) coil was used to acquire both ^lH and ¹⁹F-MR images.

[00120] Phantom studies: Four 5 mm NMR tubes containing (1) water, (2) water, (3) 2.5 μΜ CFP in aqueous solution, and (4) 25 μΜ CFP in aqueous solution were placed in a 25 mm NMR tube containing a 4% gelatin (w/w) solution in water. ¹H-MRI: A FLASH sequence was used to acquire the 1H-MR images with the following parameters: TR/TE=360/4 ms, flip angle=30°, 32 slices of 1 mm thickness, FOV=3.2x3.2 cm², matrix size=128xl28, and 2 averages (NA=2). ¹⁹F-MRI: (i) A three-dimensional gradient echo (3D-GRE) protocol with a flip angle of 10° was used with the following parameters: TR/TE=150/3.1 ms, FOV=3.2x3.2x3.2 cm³, matrix size=32x32x32, and 32 averages (NA=32). (ii) A three-dimensional ultrashort TE (3D-UTE) protocol with a flip angle of 10° was used with the following parameters: TR/TE= 150/0.02 ms, FOV=3.2x3.2x3.2 cm³, matrix size=32x32x32, and 8 averages (NA=8).

[00121] A phantom composed of reference samples (no ¹⁹F-content) and samples containing two different concentrations of CFP (i.e., -25 μΜ and -2.5 μΜ of CFP) was prepared and imaged on a 9.4 T MRI scanner (Figure 10). As expected, no significant difference could be observed between the four tubes in 1H-MRI (Figure 10 left panel). By using the ultrashort TE (UTE) sequence that enables MRI of nuclear spin pools having an extremely short T₂, a clear F-MR signal were observed from the CFP-containing tubes (Figure 10, middle panel). These data were acquired with a TE of 20 μ8 (TR = 150 ms with a flip angle of 10° to achieve Ernst angle conditions) and allow a "hot-spot" representation of the distributions of nanofluorides, thus demonstrating their potential to be used as imaging tracers for MRI applications (Figure 10 right panel). This ability to monitor extremely short T₂ values using conventional MRI setups, in addition to our small, water-soluble and monodispersed CaF₂-NPs, encouraged us to move forward and perform in vivo imaging experiments. Note that the use of a zero TE (ZTE) pulse sequence could be considered as well since such protocols were used to monitor metallated-PFC nanoemulsions in vivo with ¹⁹F-MRI.

Example 6

Functionalization of PEGylated CaF₂ (CFP) Nanoparticles

[00122] In order to ease the validation of future experiments in biological setups, CFP NPs were further functionalized with fluorescent moieties by capitalizing on both the -OH and -COOH groups of their PEG coating.

[00123] Fluorescein isothiocyanate isomer I (FrTC), and sulfo-cyanine3 amine (SCY3) were used to demonstrate the feasibility of CFP functionalization (Figures 11A-11C). One option is to functionalize CFP's surface -COOH group. Ethylene diamine was used as a linker between CFP (formation of amide after coupling to -COOH) and FrTC (amine binding to isothiocyanate group resulting in thiourea) to form FrTC-coupled CFP (CFP-FrfC, Figure 11A). The isothiocyanate- FrrC could be also couple to the hydroxyl end of the PEG coating resulting in CFP-OFrTC NPs (Figure 11B). Finally, the primary amine group of SCY3 allows direct coupling of the CFP carboxylic end resulting in the formation of the CFP-SCY3 conjugate (Figure 11C).

Coupling of fluorescein isothiocyanate (FITC) to amine-functionalized CFPs

[00124] FrTC fluorophore was coupled to CFP, based on published procedure for the preparation of PEGylated iron oxide [K. M. Yang, H-I Cho, H. J. Choi, Y. Piao. Synthesis of water well- dispersed PEGylated iron oxide nanoparticles for MR/optical lymph node imaging. Mater Chem B 2, 3355-3364 (2014)]., with the following minor modifications. Briefly, ca. 100 mg of dried CFP precipitate were re-dispersed in 15 mL of DMF, followed by the addition of 500 mL of Ν,Ν'-Dicyclohexylcarbodiimide (DCC, 10 mg in 1 ml DMF) and N-Hydroxysuccinimide (NHS,

5.5 mg in 1 mL). The solution was stirred for 10 min at RT, after which 50 mL of 1,2- ethylenediamine (EDA) was added and left to react overnight under stirring. The crude solution of amine-functionalized CFPs was purified by the ethanol-ether washing process and was re- dispersed in 15 ml of 0.01 M sodium bicarbonate buffer solution at pH 9.5 for the coupling reaction with FITC. The FITC dye (5 mg in 1 mL DMSO) was added to the solutions, which were left to react overnight at RT under stirring. The FITC-coupled CFPs (CFP-FITC) were washed in ethanol-ether as in previous steps. The obtained precipitant was re-dispersed in pH 7.4 PBS (Gibco) that contained 50 mM tri-sodium citrate. Three dialysis cycles of at least eight hours each were performed in order to remove traces of unreacted reagents and precursors. The first dialysis cycle was performed vs. water and the subsequent cycles were vs. the citrate-containing PBS buffer.

Coupling of sulfo-cyanine3 amine (SCY3) to the free carboxylate groups of CFP (CFP- SCY3)

[00125] An amine group of SCY3 dye was coupled directly to the free-surface carboxylate groups of the CFPs using DCC/NHS. Approximately 100 mg of CFPs were re-dispersed in 15 ml of DMF and were mixed with DCC (10 mg in 1 ml of DMF) and NHS (5 mg in 1 ml of DMF), 0.5 ml of each. After 10 min, a solution of 2 mg of SCY3 in 1 ml of DMSO was added and the mixture was left to react overnight under stirring. The product was washed in ethanol and precipitated by centrifugation. This process was repeated until the coloration of the supernatant by traces of dye was cleared. Further purification was achieved by applying three dialysis cycles of at least eight hours each. The first dialysis cycle was performed vs. water and the subsequent cycles were vs. the 50 mM pH 7.2 HEPES buffer.

Coupling of FITC to the hydroxyl groups of CFP (CFP-OFITC)

[00126] Approximately 100 mg of dried CFPs were mixed with 5 mg of FITC in 30 ml of THF. The mixture was refluxed overnight at 67°C under an inert atmosphere. The product was purified by washing/centrifugation cycles with a diethylethenethanol mixture as described above.

[00127] Estimation of ligand density and molecular composition

Characterization

[00128] High-resolution ¹⁹F-NMR properties of the functionalized NPs were maintained (Figures 11A-11C, right panels). The DLS histograms of all functionalized CFP NPs in aqueous solution show colloids monodispersity with hydrodynamic sizes ranging between -10 and 13 nm that were stable for several weeks (Figures 11A-11C, middle panels). This is an important result for future applications, demonstrating that high-resolution ¹⁹F-NMR characteristics are preserved even after further chemical modifications. Interestingly, while all functionalized NPs exhibited high fluorescence (Figures 11A-11C, left panels), their zeta-potentials were found to be different and were altered from almost neutral for CFP-FITC, to negative for CFP-SCY3 (Figures 11D and 11F, respectively). Note that surface charges may contribute considerably to NPs biodistribution and also play an important role in colloids stabilization in aqueous solutions.

Example 7

In vivo ¹⁹F-MRI Experiments using functionalized CFP nanocrystals

[00129] The potential of using the proposed ¹⁹F-nanocrystals (specifically the negatively charged CFP-CY3, Figure 11C) as imaging tracers for in vivo ¹⁹F-MRI was evaluated in a mouse model of inflammation. Figure 13A schematically shows the in vivo study flow that included inflammation induction, nanofluoride injection followed by MRI acquisition and post-MRI validation using fluorescent activated cell sorting (FACS) analysis.

In vivo MRI studies

[00130] Ten days post immunization, immunized animals were anesthetized by intraperitoneal injection of lmg/kg medetomidine (Dormitor) and 75 mg/kg ketamine (Vetmarket, Israel). Anesthetized mice were restrained in a custom-built mouse-holder, and centered at both the center of the RF coil and the center of the magnet. Using a localizer protocol (tripilot GRE available for the Bruker MRI scanners), the region of the lymph nodes of interest was identified. ^-MRI and ¹⁹F-MRI data sets were collected before and after CY3-CFP injection (5 mg NPs in 80 μί), subcutaneously into the footpad. Four mice were subjected to NP injection and two more mice were injected with PBS as controls. After 1H-MRI, a 3DUTE sequence was used to acquire the 1H- MR images with the following parameters: TR/TE=8/0.02 ms, flip angle=5°, FOV=3.2x3.2x3.2 cm³, matrix size=128xl28xl28, and 2 averages (NA=2). For ¹⁹F-MRI, a three-dimensional ultrashort TE (3D-UTE) protocol with a flip angle of 10° was used with the following parameters: TR/TE=150/0.02 ms, FOV=3.2x3.2x3.2 cm³, matrix size=32x32x32, and 8 averages (NA=8).

Lymph node cell suspension and flow cytometric analysis

[00131] Ten days post-immunization, CFP-FITC or CFP-SCY3 nanoparticles were subcutaneously injected into the footpads. One hour post-injection, mice were euthanized and popliteal lymph node cells were immediately harvested and suspended in phosphate buffered saline (PBS) for FACS analysis. Suspended cells were stained with fluorochrome-labeled monoclonal antibodies for surface antigen detection. APC -conjugated anti-mouse CD8, FITC-conjugated anti- mouse CD4, FITC-conjugated anti-mouse CD1 lb, pacific blue-conjugated anti-mouse CD1 lc, and APC-conjugated anti-mouse CD45 were purchased from BioLegend and used according to the manufacturer's protocols. Cells were analyzed on a CytoFLEX S flow cytometer (Beckman Coulter). Quantitative analysis of fluorescent cells was performed using Flowjo software (version 10, TreeS tar, Oregon, USA).

Results

[00132] Ten days post-immunization, when extensive inflammatory activity is expected, the immunized mice were subjected to two 1H- and ¹⁹F-MRI sessions that acquired pre- and post- CFP-SCY3 NPs injection (5 mg in 80 μL· into mouse footpads). For obtaining a ¹⁹F-MR signal from the injected nanofluorides, a UTE-based sequence was used. A clear ¹⁹F-signal was observed at the region of the popliteal lymph node (LN) of NP-injected mice (N=4) within the same leg of the injection site (Figure 13B) 1 hour post NP injection with no ¹⁹F-MR signal observed from contralateral regions.

[00133] Following the MRI experiments, excised cells from the lymph node (LN) of the imaged mice were subjected to FACS analysis to classify the cell population that took up the injected nanofluorides (Figures 14A and 14B). As expected, the lymphatic cells of CFP-CY3 injected mice showed much higher fluorescence as compared to PBS injected mice with 22+6% of the LN's cells being labeled (Figure 14A). A double- staining analysis of the labeled cells reveled that injected nanofluorides were accumulated mostly in macrophages and dendritic cells (Figure 14B), with a minimum accumulation in the CD4⁺ and CD8⁺ T cells (Figure 14C). These findings can be explained by the fact that both macrophages and dendritic cells are phagocytic cells, whereas T cells lack phagocytic receptors and would not be likely to accumulate injected NPs.

Example 8

Calculation of the crystalline CaF₂ core

[00134] As indicated in the TEM tomograms (Figure 5A and Figure 9E), the fabricated CaF₂ NPs featured a well-defined spherical shape. This allowed us to evaluate the volume of the core of a single nanoparticle, V_core , by calculating the volume of a sphere (Eq. 1).

V ^Ycore = — TFT core

Eq. 1 [00135] In equation 1, r_core is the radii of the core part of the particles, i.e., the uncoated CaF₂

-7 -7

crystal. Using the r_core values of 2.3 x 10^" cm for CF-Cit, and 3.6 x 10^" cm for CFP, the obtained

-20 3 -19 3

corresponding volumes are 5 x 10^" cm and 2 x 10^" cm , respectively. The weight of a single particle, w_core, was calculated by using the density of a fluorite-type crystal, d_Cap₂ , multiplied by V_core (Eq 2).

Eq. 2

[00136] In equation 2, d_CaFz is the density of the core that is 3.18 g xcm^" for a fluorite-type crystal. The obtained weights of the uncoated cores of CF-Cit and CFP were 1.6 x 10^"19 g and 6.4 x 10^"19 g, respectively. Multiplying w_core in the Avogadro constant provides the molecular weight of the cores (MW_core), which are 96 KDa and 385 KDa for CF-Cit and CFP, respectively.

Example 9

Calculation of the weight of the coating layer of the ¹⁹F-nanocrystals

[00137] Figures 16A and 16B present representative mass loss profiles of CF-Cit and CFP NPs (respectively) measured by TGA. The ligand density on the surface of the NPs, p_t, was derived using Eq 3

w_core

A_core

Eq. 3.

[00138] In this equation, mi and m_core are the mass fractions of the coating ligand and the crystalline core of the NPs, respectively, NA is the Avogadro constant, w_core is the weight of the core, MWi is the molecular weight of the ligand, and A_core is the surface area of the core. The latter is approximated as a surface of a sphere that can be calculated by 4 r_Co_re . The molecular weight of the coating on the surface of the NPs {MW_coati_ng) is given by Eq. 4.

MW_coati = - - ^■ N_A ^■ w_core

Eq. 4 [00139] Table 1 summarizes the obtained calculated values and the estimated molecular composition of the NPs.

Example 10

Cell toxicity assay

[00140] Serial twofold dilutions of purified CFP-SCY3 NPs were prepared in triplicate with ΙΟΟμΙ/well in a 96-well plate and cultured for 2 hours at 37°C in DMEM medium containing 10% FBS and 1% Penicillin-Streptomycin. Untreated cells and cells in 1% Triton solution were used as negative and positive controls, respectively. After incubation, the cells were washed and then incubated with CellTiter-Blue reagent (20μ1 reagent in ΙΟΟμΙ fresh medium per well) for 4 h to allow viable cells to convert resazurin to resorufin. The viability was then tested by recording the fluorescence emission of resorufin at 590 nm using a Tecan Spark 10M microplate reader. As inferred by the presented data (Figure 18), no significant loss of cells could be observed, when compared to cells that were not treated with CFP-SCY3 NPs. As expected, the Trition-treated cells exhibited a substantial drop in viability, indicating the normal functioning of the cells that were tested in this experiment.

Example 11

Synthesis and Characterization of Oleate Coated Inorganic Fluorides Nanoparticles Oleate-coated CaF₂ NPs

[00141] Oleate-coated CaF₂ NPs were synthesized following the solvothermal method:

[00142] 4.2 ml of oleic acid, 12 ml of ethanol, and 0.1 gr of sodium hydroxide were mixed together until a homogeneous milky solution was formed. Then, 5 ml of an aqueous solution with 2mmol M(N(¾)₂ (M= Ca, Sr, Mg or Y) of the appropriate stoichiometric amount of NaF were added to the mixture under vigorous stirring in a 50 ml flask. After lh, it was transferred to the Teflon liner in the reactor. The reactor was sealed and heated to 160°C for 16h. Then, the system was cooled to room temperature and the solution was centrifuged at 8500 rpm for 10 min to obtain the powder products. This powder was washed in ethanol/ cyclohexane two times to remove impurities. The obtained NCs that are capped with a long-alkyl oleate chain were dispersed in an organic solvent, such as cyclohexane.

[00143] The oleate coated CaF₂ were dispersed in cyclohexane and TEM image shown in Figure 19A indicates that the CaF₂ NPs have a hexagonal shape with a mean diameter of D=8.1 + 2.5nm, with a relatively narrow size distribution, as presented in Figure 19B. High-resolution TEM was used to measure the crystal lattice constant, which was found to be S acing = 0.32+0.02nm.

[00144] The size of the oleate-coated CaF₂ NPs was measured with two other techniques, XRD and DLS, as summarized in Table 2.

Table 2: Size measurements of oleate coated CaF₂ NP based on various techniques

[00145] XRD verified the face-centered cubic (FCC) crystal structure of CaF₂ and also measured the grain size. Moreover, the diameter measured with both XRD and TEM was approximately 2 nm smaller than the DLS measurements. This is consistent with the fact that the DLS is a dynamic measurement performed in solution and the hydrodynamic diameter measured includes the thickness of the ligand layer.

[00146] Another characterization technique is TGA, which was utilized to measure the amount of organic coating absorbed on the surface of the NP. It was found that 20% of the dry weight was organic material. Accordingly, the number of ligands on each NP at about 450 .

[00147] NMR Characterization: Solution ¹⁹F- and ¹H-NMR measurements were performed on the oleate-coated CaF₂ dispersed in cycloheaxane-dl2 and are presented in Figures 20A and 20B. Figure 20A shows, for the first time, the ¹⁹F-NMR spectrum of oleate-coated nanofluorides in solution. Importantly, the main peak's resonance at the ¹⁹F-NMR spectrum in Figure 20A was found to be at a frequency of -109ppm, which is consistent with the ssNMR measurements of dry powder of CaF₂. Surprisingly, in addition to this peak, two more resonances were found at - 105ppm and at -120ppm. Figure 20B shows the ¹H-NMR of the protons of the oleate, along with its chemical structure (Figure 20B, inset).

[00148] Relaxation measurements were performed using two methods: an inversion recovery pulse sequence for Ti measurements; and Carr-Purcell-Meiboom-GiU sequences (CPMG) to measure the transverse relaxation constants, T₂. [00149] Figures 20C and 20D show 1H NMR of the bound (Figure 20C) and free (Figure 20D) oleate dissolved in cyclohexane-dl2. The peaks of the bound oleate are broadened (compare free to bound oleate, Figures 20C and 20D),

Oleate coated SrF2 NPs

[00150] Oleate coated SrF₂ NPs (Figures 21A and 21B) were synthesized using solvothermal process: . 4.2 ml of oleic acid, 12 ml of ethanol, and 0.1 gr of sodium hydroxide were mixed together until a homogeneous milky solution was formed. Then, 5 ml of an aqueous solution with 2mmol Sr(N0₃)₂ of the appropriate stoichiometric amount of NaF were added to the mixture under vigorous stirring in a 50 ml flask. After lh, it was transferred to the Teflon liner in the reactor. The reactor was sealed and heated to 160°C for 16h. Then, the system was cooled to room temperature and the solution was centrifuged at 8500rpm for 10 min to obtain the powder products. This powder was washed in ethanol/ cyclohexane two times to remove impurities. The obtained NCs were capped with a long-alkyl oleate chain outside and were dispersed in an organic solvent, such as cyclohexane.

[00151] The size of the SrF2 (d_H=7.2nm) was about the same size of CaF₂, the same surface chemistry, and the same crystallinity. The ¹⁹F-NMR spectrum of SrF₂ in Figure 21B revealed a broad main peak at -87 ppm, which consisted of the single ssNMR peak of powder SrF₂. Similar to the case of CaF₂, a "shoulder" peak was observed downfield of the main peak, at -84 ppm.

Oleate coated NaYF₄ NPs

[00152] Oleate-coated NaYF₄ NPs were synthesized (same process as described above for Oleate coated SrF₂ NPs ) using solvothermal conditions with a size of d=15nm (Figure 22A). These NPs have a different crystal structure compared to the fluorite crystal of CaF₂ and SrF₂. The bulk material crystallizes into two distinct phases: the cubic phase, a-NaYF_4; and the hexagonal phase, P"NaYF_4i leading to more than one magnetically distinct fluorine environment and resulting in a much broader ¹⁹F-NMR peak. The NaYF₄ NPs showed a different ¹⁹F-NMR profile compared to that obtained for CaF₂, with an exceptionally broad line of ~8KHz at -77ppm (Figure 22B). These results imply the correlation between the crystal structures and the ¹⁹F-NMR profile.

Oleate coated BaF₂ NPs

[00153] Oleate-coated BaF₂ NPs were synthesized using the solvothermal approach with optimized conditions. In a typical synthesis, 4.2 ml of oleic acid, 12 ml of ethanol, and 0.1 g of sodium hydroxide were mixed together under vigorous stirring in a round-bottom flask at room temperature for 16 hrs to obtain sodium oleate. The resultant homogenous milky mixture, 5 ml of aqueous solution of 2 mmol of Ba(N0₃)2, and 5 ml of aqueous solution of 4 mmol of NaF were combined at once. The obtained mixture was vigorously stirred for an additional one hour, and then heated to 110°C for four hours. After spontaneously cooling to room temperature (RT), the mixture was transferred to a centrifuge tube and centrifuged for 10 min at 8500 rpm. The obtained precipitate was washed twice with a 1:10 cyclohexane: ethanol solution, followed by 10 min centrifugation (6500 rpm) and washout removal. The white solid final product was dispersed in 2 ml cyclohexane and centrifuged for additional three minutes at 2000 rpm to remove remaining residues, aggregates, or large impurities, and the clear solution was then filtered through a 0.22 μιη PTFE membrane and kept in a glass vial for further characterization

Characterizations of oleate coated BaF2 NPs

[00154] Electron microscopy studies were performed. Both TEM and HRTEM images were acquired to characterize the synthetic BaF₂ nanocrystals. As shown in Figures 26A and 26B, the obtained fluoride nanocrystals tend to form rectangle- sheets and reflect both size and shape transformations that might explain the fact that their NMR properties and DLS characteristics change with time.

[00155] To determine the crystal structure of the obtained BaF₂ NPs, XRD measurements were performed on the dried powder of the purified synthetic product. The diffraction pattern from the XRD experiments is shown in Figure 27 and the extracted crystal structure was found to be in a good agreement with that reported for the mineral frankdicksonite (ICDD, 01-085-1342), which is a known form of BaF₂ in nature. The obtained crystal adopts the fluorite crystal structure with the cubic symmetry of fee (face-centered cubic).

[00156] TGA measurements were performed to verify the presence of organic molecules (oleic acid) on the surface of the nanoformulations and to evaluate the number of oleate coating ligands per NP. Generally, organic molecules have a decomposition temperature of ~400°C, while inorganic nanocrystals cannot be decomposed at such temperatures. Figure 28 depicts a representative TGA plot from which one can see a single drop that represents mass loss and implies on the presence of a monolayer of organic molecule coating (i.e., oleate). Example 12

Synthesis and Characterization of doped BaF₂ NPs coated by organic ligand PEGylated La:BaF2 NPs (BaF₂ doped by La³⁺)

[00157] In a three-neck round-bottom flask, 74 mg NH₄F, 260 mg Ba (Ν0₃)₂, and 43 mg La (N0₃)₃ were mixed into a mixture of 24 g PEG (average Mw 600) and 4.75 g PEG- bis(carboxymethyl)ether (average Mw 600) along with magnetic stirring. The solution was heated to 100°C under a vacuum and remained at that temperature for 30 min. The mixture was then heated to 220°C (the vacuum was turned off at 160°C) and left at 220°C under an inert atmosphere for 16 hr. After the mixture was spontaneously cooled to RT, the resultant mixture was washed with 30 ml of ethanol/diethyl ether, 1:5, followed by centrifugation at 8000 rpm for 10 min. The precipitate was washed twice with 20 ml of the same solvent mixture at the same ratio, 6500 rpm for 5 min. The resultant precipitate was dried under condensed air and the final product obtained as a brown powder was suspended in 1.5 ml of water and kept for further characterization. Relatively small particles (-16 nm) were obtained as shown by both DLS measurements (Figure 32) and from the TEM image (Figure 32, inset), with no indication of massive NPs aggregation.

Oleate-coated La*³ -doped BaF₂ (La.-BaFijnanoparticles

[00158] Oleate-coated lanthanum (La³⁺)-doped BaF₂ (La:BaF₂) NPs were synthesized using the solvothermal approach. In a typical synthesis, 4.2 ml of oleic acid, 12 ml of ethanol, and 0.1 g of sodium hydroxide were mixed together under vigorous stirring in a round-bottom flask at room temperature for six hours. The resultant homogenous milky mixture, 5 ml of aqueous solution of 2 mmol of Ba(N0₃)₂, an aqueous solution of a relevant % mol of La(N03)₃, and 5ml of aqueous solution of 4 mmol NaF were combined together at once. The obtained mixture was vigorously stirred for additional 30 min, and then heated to 110°C in a Teflon-lined sealed autoclave reactor for 16 hrs. The reaction mixture was left for spontaneous cooling to RT and then transferred to a centrifuge tube, followed by centrifugation at 8500 rpm for 10 min. The obtained precipitate was washed three times with cyclohexane/ethanol (1:10 ratio), followed by 10 min centrifugation at 6500 rpm. The final product was obtained as a white solid and dispersed in 2 ml of cyclohexane followed by additional three-minute / 2000 rpm centrifugation to remove any remaining impurities and aggregates. Finally, the resultant clear solution was filtered through a 0.22 μιη PTFE membrane and then kept in a glass vial for further characterization. Example 13

Doping the Metal Fluoride-Concentration Study

[00159] Different concentrations of La3+ were added to BaF₂ NPs. La was added to BaF₂ as a dopant to stabilize the above-mentioned BaF₂ nanoformulations with the aim to establish them as robust, stable, and well-characterized nanofluorides for ¹⁹F-MRI applications. Different amounts of La³⁺ dopant relative to Ba²⁺ were introduced into the BaF₂ NPs reaction mixture based on previously reported methodology [Chen, D., et al., Modifying the Size and Shape of Monodisperse Bifunctional Alkaline-Earth Fluoride Nanocrystals through Lanthanide Doping. Journal of the American Chemical Society, 2010. 132(29): p. 9976-9978.]. Using the solvothermal approach with oleate as the capping ligand, a series of La-doped BaF₂ (La:BaF₂) NPs were prepared with La dopant molar ratio ranging from 1.25% to 10%. The optimum reaction conditions for such preparations were found to be 18 hours at 110°C. Figure 29 summarizes the high-resolution ¹⁹F- NMR spectra of purified La:BaF₂ NPs dispersed in cyclohexane, with a variable %mol (1.25%- 10%) of dopant relative to the Ba²⁺ precursor.

[00160] Interestingly, for all La-doped formulations (1.25La:BaF₂, 2.5La:BaF₂, 5La:BaF₂, and 10La:BaF₂), the main ¹⁹F-NMR characteristics of BaF₂ (singlet high intense peak at -11.5 ppm) were preserved. In addition, as depicted from the NMR spectra in Figure 29, the highest SNR was obtained for NPs prepared with the highest dopant levels, either with 5% or 10% La dopant (i.e., 5La:BaF₂ or 10La:BaF₂).

[00161] In order to evaluate the contribution of the doping agent to the stability of the pure synthetic BaF₂ NPs, the hydrodynamic diameter of samples of the doped-NPs, with different %mol of the doping agent was evaluated using DLS measurements. Two weeks after their synthesis and purification, all La:BaF₂ NPs showed preserved and stable sizes. The colloidal stability of the doped NPs was preserved for at least 14 days after their preparation. This observation is unique to La:BaF₂ NPs, while, for BaF₂ NPs, such stable formulation could not be obtained and aggregation occurred immediately following their synthesis. These findings demonstrate the requirement of La- dopant as a key feature in any final BaF₂-based nanocrystals.

[00162] For further characterization of the La-doped BaF₂ nanocrystals, and to better understand their morphologies, TEM data were performed. As shown in Figure 30A-30C, for a low amount of doping agent, i.e., for 1.25% mol and 2.5% mol lanthanum, non-spherical nanoparticles that probably went structural transformation (similar to pure BaF₂ NPs, see Figure 26A, 26B), could be still observed. While for 1.25La:BaF₂ spherical NPs could not be detected (Figure 30A) some round-shaped depositions could be found for the 2.5La:BaF₂ NPs sample (Figure 30B). However, by increasing the amount of the doping agent to 5%mol and 10%mol, well-defined homogenous round-shaped NPs were obtained (Figure 30C).

[00163] To evaluate the number of coating oleate ligands per nanoparticle of oleate-coated 5La:BaF₂ NPs, TGA measurements were performed on a dry powder of freshly synthesized and pure NPs. The percentile of organic ligand (oleate coating) mass from the whole particle mass was found to be almost equivalent for both cases (48% for BaF₂ NPs and 44 % for 5La:BaF₂ NPs). Both parameters may rely on the fact that the ligand-coating characteristics are not changed upon the addition of lanthanide dopant to the nanofluorides. The number of coating ligands was found to be -270.

[00164] The crystal structure of the purified doped La:BaF₂ NPs, XRD measurements were performed. The XRD patterns of 5La:BaF₂ revealed no difference when compared to non-doped BaF₂ nanocrystals, as depicted in Figure 31 where a clear overlap of the two patterns is observed. Such observations may have two explanations: (i) The actual amount of the doping agent was too small (less than what had been added during the reaction), with a negligible effect on the XRD pattern; and (ii) Lanthanum cations (La³⁺) remain on the surface of the BaF₂ crystal and align in a core- shell mode without affecting the crystal structure of the BaF₂ core.

[00165] When increasing the concentration of the lanthanum cations (La³⁺) up to 40% mol, the 40La:BaF₂ showed a shift in the XRD pattern and high-resolution ¹⁹F-NMR revealed unique NMR characteristic of 40La:BaF2. When comparing between the ¹⁹F-NMR spectra of cyclohexane solutions of LaF₃, BaF₂, and 40La:BaF₂ it was found that both LaF₃ and BaF₂ solutions are well- defined and correspond to their crystal structures However, the¹⁹F-NMR spectrum obtained for the 40La:BaF₂ solution was centered at the chemical shift offset of BaF₂ (-11.5 ppm) but with a features of the ¹⁹F-NMR characteristics of LaF₃ (line broadening and a larger range of chemical shifts). This characteristics observation confirms that the studied nanoparticles contain both Ba and La elements.

Example 14

Synthesis and Characterization of Amino-Ethyl-Phosphate (AEP) coated BaF₂ NPs Co-precipitation approach (BaF₂ NPs)

[00166] The co-precipitation approach for preparing nanomaterials is based on concurrent precipitation of soluble ions from their solution, which results in non-soluble nano-crystals. Therefore, two soluble salts of Ba²⁺ (e.g., BaCl₂) and F^" (e.g., NaF) may result in BaF₂ nanocrystals in a water solution. The use of the proper ligand may assist in dispersing the obtained nanoparticle in aqueous solutions. Citric acid ligands are commonly used ligands in co -precipitation-mediated nanoparticle synthesis. Surprisingly, citrate-coated water-soluble BaF₂ NPs could not be prepared. Therefore, the ligand was replaced the same synthetic approach was used with amino ethyl phosphate (AEP) as the coating ligand. Amino ethyl phosphate (AEP), BaCl₂, and NaF were mixed together in water to obtain AEP-coated and water-soluble BaF₂. ¹⁹F-NMR of BaF₂ NPs in water was detected (Figure 33A). This first observation of ¹⁹F-NMR of water-soluble BaF₂ NPs was accompanied by DLS measurements that revealed small (-12 nm) and monodispersed colloids in aqueous solution (Figure 33B). The data obtained from TEM experiments confirmed the mono- dispersity of the AEP-coated BaF₂. The water-soluble AEP coated BaF₂ NPs were not stable after 2 days in aqueous solution, and, La³⁺ cations were added as a stabilizing dopant to the nanocrystals.

Co-precipitation for the preparation of (La: BaF₂ NPs) coated by AEP

[00167] In a round-bottom flask, 25 ml of water, 300 mg of AEP, and 42 mg of NaF were added and mixed until completely dissolved. The pH of the resultant solution was adjusted to 7 using N¾OH, followed by heating to 75°C using an oil bath. After the temperature was stabilized at 75°C, a solution of 121 mg BaCl₂-2H₂0 in 2 ml water was added at once. The temperature of the mixture was left at 75 °C for an additional 20 min until the mixture became gradually opaque and was then left to cool spontaneously to RT, washed twice with water/ ethanol solution, and spun down once for 10 min at 6000 rpm and then for 15 min at 7000 rpm. The resultant product was dissolved in 1 ml D₂0 (for NMR measurements), and then, a relevant amount of La (N0₃)₃ was added and the solution was left at RT for a minimum of 16 hrs. The clear solution was filtered through a 0.22 μιη PVDF membrane.

[00168] The high-resolution ¹⁹F-NMR spectra obtained of the resultant aqueous solutions after the doping procedure, performed with increasing concentrations of La³⁺. Importantly, in all La³⁺ amounts used (% mol of La³⁺ per BaF₂: 5%, 10%, 20% and 30%) a clear ¹⁹F-NMR spectrum was detected, which confirmed the existence of BaF₂ NPs.

[00169] The results from the DLS and TEM experiments are depicted in Figure 34A-34B and show NPs with an average size of 8+0.5nm and 6+0.5 nm, respectively. The addition of the La³⁺ ions stabilizes (up to one month) the AEP-coated BaF₂ NPs, which are the first water-soluble BaF₂ nanocrystals investigated with high-resolution ¹⁹F NMR and show the potentiality to be used as imaging tracers for ¹⁹F-MRI. Example 15 'F Relaxation Measurements

[00170] To obtain high-resolution F-NMR spectra for nanofluorides in solution is a requirement for their use in ¹⁹F-MRI studies. The relaxation properties (i.e., Ti and T₂) of the ¹⁹F-nuclear spins within the obtained nanoformulation are crucial parameters. By knowing both the longitudinal and transverse relaxation times of the nuclei of interest, one can optimize the acquisition parameters for maximal performance (minimum time and maximum SNR). For future biological applications, the ideal relaxation properties of NPs are a short Ti (to shorten the experiment time, especially for in vivo studies) and long T₂ (for increased SNR). Both ¹⁹F-NMR inversion recovery (IR) and Carr- Purcell-Meiboom-Gill (CPMG) experiments were performed to evaluate the Ti and T₂ values of the synthetic nanofluorides, respectively.

Relaxation measurements of oleate coated CaF₂ NPs

[00171] Relaxation measurements on oleate coated CaF₂ NPs dispersed in solution were performed. Both Ti and T₂ relaxation constants are presented in Table 3.

[00172] Table 3. Longitudinal, Ti, and transverse, T₂, relaxation times for an oleate-coated CaF₂ NP dispersed in cyclohexane-dl2. The resonance peaks correspond to the ¹⁹F spectrum in Figure.20A:

[00173] A 80%:20% ratio between the long and the short Ti, respectively were obtained, for each peak. This biexponential fitting may be attributed to multiple exchanging components having the same resonance. The transverse relaxation times, T_2, were measured with the Carr-Purcell- Meiboom-Gill sequences (CPMG) and are presented in Table 3. A single T₂ time value for each resonance peak in the ¹⁹F spectrum is shown, due to a monoexponential fitting. The calculated T₂ value is on the order of msec compared to the hundreds of msec of commonly used imaging probes and tracers. Relaxation measurements of oleate coated BaF ₂ and LaBaF '₂ NPs

[00174] The resultant Ti and T₂ for both oleate coated doped-BaF₂ and non-doped particles are summarized in Table 4

Table 4: Ti and T₂ values of oleate-coated non-doped and doped BaF₂.

Relaxation measurements ofAEP coated BaF ₂ and LaBaF ₂ NPs

[00175] The relaxation properties of the obtained AEP-coated BaF₂ nanocrystals were studied. The results of the relaxation times (Ti calculated from the IR experiments and T₂ values extracted from the CPMG experiments) were summarized in Table 5. While, for non-doped BaF₂ NPs, Ti values were comparable for oleate-coated and AEP-coated NPs (4.7 sec), different Ti values were obtained upon doping (1.1 sec for AEP-La:BaF2 vs. 5.8 sec for oleate-La:BaF2). This unique observation may be attributed to differences in the doping procedure. For AEP-coated NPs, the dopant was added after the synthesis and might have resulted in mobile fluoride anions on the NP surface between the core and the ligand. These anions may be more mobile than those in the crystal, and, upon their exchange, may affect the obtained Ti values. This explanation also aligns with the increased T₂ values obtained for the AEP-coated NPs upon La³⁺ doping. Importantly, regardless of the reason for the obtained shorter Ti values, this is an advantage for future ¹⁹F-MRI applications, especially for in vivo MRI studies where short experimental times are crucial.

[00176] Table 5: Ti and T₂ results of AEP-coated BaF₂, with and without La³⁺ as a dopant

Example 16

AEP-coated 5La:BaF₂ Nanoparticles as ¹⁹F-MRI tracers

[00177] Once the size, the composition, and the MR properties of the NPs, such as chemical shift and relaxation times were determined, the MRI measurements were carried out.

Due to the short transverse relaxation times, ¹⁹F MRI data were acquired using a 3D ultra-short TE (3D-UTE) pulse sequence for the sample that contained the NP. UTE-based sequences are generally used to monitor the MRI of tissues, such as tendons, ligaments and menisci, with a T₂ of about 1-lOmsec. Figure 35A depicts the ¹⁹F-MR images of this potential new generation of MR tracers, i.e., nanofluorides. Both ¹⁹F MRI (Figure 35B-C) and 1H (Figure 35A) were obtained with a 3D UTE sequence using different parameters.

[00178] The ability to map the distribution of the developed water-soluble 5La:BaF₂ nanocrystals with ¹⁹F-MRI was examined. A phantom composed of reference samples (water, no ¹⁹F-content) and two samples containing nanofluorides were prepared and imaged on a 9.4 T MRI scanner (Figure 35A-35C). The lower SNR obtained for the nanofluoride samples in the ¹H-MRI (Figure 35A) can be attributed to the fact that they were prepared in D₂0 for NMR studies, and thus, contain much less H₂0. The relatively short T₂ values found for La:BaF₂ NPs in water (3.7 msec, Table 5) necessitated the use of an ultrashort TE (UTE) sequence that enabled the acquisition of MRI signals of nuclear spin pools with an extremely short T₂. From the UTE ¹⁹F-MRI, indeed, a pronounced ¹⁹F-MR signal could be detected from the nanofluoride-containing tubes (Figure 35B). This ¹⁹F-MR image was acquired with a TE of 20 μβ. In order to shorten the experimental time, TR was set to 150 ms along with a flip angle of 21.9° to achieve Ernst angle conditions, which allow shorter experiments while maintaining the SNR. The ¹⁹F-MRI data obtained can be overlaid on high-resolution ^-MR images and can be presented as a "hot-spot" map of the fluoride tracer distributions (Figure 35C). This is the first demonstration in which BaF₂ based nanocrystals are being proposed as imaging tracers for ¹⁹F-MRI applications.

Example 17

Inorganic Fluorides as Imaging Tracers for Multiplexed Imaging

[00179] A phantom composed of reference samples (no ¹⁹F-content) and samples containing either BaF₂ (5% La doped and 2-aminoethylphosphate as the ligand), SrF₂ (coated with citric acid), and CaF₂ (coated with PEG, i.e. CFP) were prepared and imaged on a 9.4 T MRI scanner (Figure 36). By using the ultrashort TE (UTE) sequence that enables MRI of nuclear spin pools having an extremely short T₂, a clear F-MR signal could be observed from the nanofluorides -containing tubes but not from the reference tube. For ¹⁹F-MRI, the center frequency (Oi) was set at the frequency of the ¹⁹F atom at -13 ppm for monitoring BaF₂, at -89 ppm for monitoring SrF₂, and at - 109 ppm for monitoring CaF₂ These data were acquired with a TE of 20 and allow a "hot-spot" multicolor representation of the distributions of nanofluorides, thus demonstrating their potential to be used as imaging tracers that feature artificial multicolor characteristics for MRI applications

[00180] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMS What is claimed is:

1. A method of molecular or cellular imaging comprising: administering to a subject or a cell an imaging tracer comprising inorganic fluoride nanoparticles (NPs) and scanning said subject or cell using diagnostic imaging.

2. The method of claim 1, wherein the diagnostic imaging is ¹⁹F-MRI, ¹⁹F-NMR or a combination of them.

3. The method of claim 1 or claim 2, wherein the inorganic fluoride NPs comprise a first metal cation and a fluoride anion forming a metal fluoride composition.

4. The method of claim 3, wherein the inorganic fluoride NPs further comprise phosphorous, oxygen, boron, sulfur, silicon or combination thereof.

5. The method of claim 3 or claim 4, wherein the metal fluoride composition further comprising a second metal cation wherein the second metal cation is a paramagnetic metal ion.

6. The method of claim 3 or claim 4 wherein the metal fluoride composition further comprising a second metal cation wherein the second metal cation is a non- paramagnetic metal ion.

7. The method of claims 5 or claim 6, wherein the concentration of the second metal cation is between 0.1 to 50 mol% of the first metal cation.

8. The method of claims 5 or claim 6, wherein the concentration of the second metal cation is between 0.1 to 1 mol% of the first metal cation.

9. The method of any one of claims 1 to 8, wherein the inorganic fluoride NPs comprise alkaline earth metal ion, alkali metal ion, transition metal ion, lanthanide metal ion or combination thereof.

10. The method of any one of claims 1 to 9, wherein the inorganic fluoride NPs are nanocrystallines.

11. The method of any one of claims 1 to 10, wherein the inorganic fluoride NPs comprise a first metal cation and a fluoride anion, forming a metal fluoride composition, wherein the metal fluoride composition is further coated, encapsulated, embedded or coordinated to an organic material.

12. The method of claim 11, wherein the organic material comprises citric acid, fatty acid, polyethylene glycol, polyethylene imine, polysaccharide, polymers of amine, peptide, protein, phospholipid, lipid, AEP (amino ethyl phosphate), phosphate, polylysine, PLGA (poly(lactic-co-glycolic acid), cellulose, any sugar, amino acid, nucleoside, nucleotide, nucleic acids, gelatin, a drug, a polar polymer or any combination thereof.

13. The method of any one of claims 1 to 10, wherein the inorganic fluoride NPs comprise a first metal cation and a fluoride anion, forming a metal fluoride composition, wherein the metal fluoride composition is further coated, encapsulated, embedded or coordinated to an inorganic material.

14. The method of claim 13, wherein the inorganic material comprises gold, silica, titanium, silver, any metal, metal oxide, inorganic fluoride or any combination thereof.

15. The method of any one of claims 1 to 14, wherein the inorganic fluoride NPs are further functionalized by one or more functionalizing group comprising a fluorescent moiety or a targeting moiety.

16. The method of claim 15, wherein the fluorescent moiety comprises sulfo-Cyanine5, Cyanine5.5, sulfo-Cyanine3, Rhodamine, Cyanine3, Cyanine3.5, Cyanine5, Cyanine7, Cyanine7.5, Fluorescein, sulfo-Cyanine7, borondipyrromethene dyes, Coumarin 343, Pyrene, sulfo-Cyanine5.5 or sulfo-Cyanine7.5.

17. The method of claim 15, wherein the targeting moiety comprises a RGD peptide, a cyclic RGD peptide, an antibody, folic acid, TAT peptide, cell penetrating peptides or tumor receptors binding moieties.

18. The method of any one of claim 15 to 17, wherein the fluorescent moiety or the targeting moiety are chemically linked to the organic material.

19. The method of any one of claim 15 to 17, wherein the fluorescent moiety or the targeting moiety is the organic material.

20. The method of any one of claims 1 to 19, wherein the inorganic fluoride NPs have a diameter of less than 20 nm.

21. A MR imaging tracer for molecular and cellular imaging comprising inorganic fluoride nanoparticles, wherein the inorganic fluoride nanoparticles have a particle size of less than 20 nm and are water dispersed.

22. The MR imaging tracer of claim 21, wherein the particle size of the inorganic fluoride nanoparticle is between 3 to 20 nm.

23. The MR imaging tracer of claim 21, wherein the inorganic fluoride NPs comprise a first metal cation and a fluoride anion forming a metal fluoride composition.

24. The MR imaging tracer of claim 23, wherein the inorganic fluoride NPs further comprise phosphorous, oxygen, boron, sulfur, silicon or combination thereof.

25. The MR imaging tracer of claim 23 or claim 24, wherein the metal fluoride composition further comprises a second metal cation, wherein the second metal cation is a paramagnetic metal ion or a non-paramagnetic metal ion.

26. The MR imaging tracer of claim 25, wherein the concentration of the second metal cation is between 0.1 to 50 mol% of the metal cation.

27. The MR imaging tracer of claims 25 or claim 26, wherein the concentration of the second metal cation is below 0.1 to 1 mol% of the metal cation.

28. The MR imaging tracer of any one of claims 20 to 27, wherein the inorganic fluoride NPs comprise alkaline earth metal ion, alkali metal ion, transition metal ion, lanthanide metal ion or combination thereof.

29. The MR imaging tracer of any one of claims 20 to 28 wherein the inorganic fluoride NPs are nanocrystallines.

30. The MR imaging tracer of one of claims 20 to 29, wherein the inorganic fluoride NPs comprise a metal cation and a fluoride anion, forming a metal fluoride composition, wherein the metal fluoride composition is further coated by, encapsulated by, embedded by or coordinated to an organic material.

31. The MR imaging tracer of one of claims 20 to 29, wherein the inorganic fluoride NPs comprise a metal cation and a fluoride anion, forming a metal fluoride composition, wherein the metal fluoride composition is further coated by, encapsulated by, embedded by or coordinated to an inorganic material.

32. The MR imaging tracer of claim 31 wherein the organic material comprises citric acid, fatty acid, polyethylene glycol, polyethylene imine, polysaccharide, polymers of amine, peptide, protein, phospholipid, lipid, AEP (amino ethyl phosphate), phosphate, polylysine, PLGA (poly(lactic-co-glycolic acid), cellulose, any sugar, amino acid, nucleoside, nucleotide, nucleic acids, gelatin, a drug, a polar polymer or any combination thereof.

33. The MR imaging tracer of claim 31 wherein the inorganic material comprises gold, silica, titanium, silver, any metal, metal oxide, inorganic fluoride or any combination thereof.

34. The MR imaging tracer of any one of claims 30-33, wherein the inorganic fluoride NPs are further functionalized by one or more functionalizing group comprising a fluorescent moiety or a targeting moiety.

35. The MR imaging tracer of claim 34, wherein the fluorescent moiety comprises sulfo- Cyanine5, Cyanine5.5, sulfo-Cyanine3, Rhodamine, Cyanine3, Cyanine3.5, Cyanine5, Cyanine7, Cyanine7.5, Fluorescein, sulfo-Cyanine7, borondipyrromethene dyes, Coumarin 343, Pyrene, sulfo-Cyanine5.5 or sulfo-Cyanine7.5.

36. The MR imaging tracer of claim 34, wherein the targeting moiety comprises a RGD peptide, a cyclic RGD peptide, an antibody, folic acid, TAT peptide, cell penetrating peptides or tumor receptors binding moieties.

37. The MR imaging tracer of any one of claims 34 to 36, wherein the fluorescent moiety or the targeting moiety are chemically linked to the organic material.

38. The MR imaging tracer of any one of claims 34 to 36, wherein the fluorescent moiety or the targeting moiety is the organic material.

39. The MR imaging tracer of any one of claims 20 to 38, wherein the inorganic fluoride NPs have a diameter size of less than 20 nm.