Core-shell nanocrystals for targeted alpha therapy

Abstract

The present invention relates to a core-shell nanocrystal having a discrete core-shell structure comprising a core and a single crystalline shell, wherein the core comprises CeO2 and an alpha-emitter or a mother radionuclide of an alpha-emitter; and the single crystalline shell comprises a shell compound having a cubic space group with a lattice constant of 4.6 Å to 6.3 Å. The present invention further relates to the use of the core-shell nanocrystals in targeted alpha-therapy and to a method for synthesizing the core-shell nanocrystals.

Description

[0001] Our ref.: K07679WO - sbe / fha Muller-Bore

[0002] Description

[0003] Core-shell nanocrystals for targeted alpha therapy

[0004] The present invention relates to core-shell nanocrystals having a discrete core-shell structure comprising a core and a single crystalline shell, wherein the core comprises Ce02 and an alpha-emitter or a mother radionuclide of an alpha-emitter, and wherein the shell comprises a shell compound having a cubic space group with a lattice constant of 4.6 A to 6.3 A. The present invention further relates to the use of the coreshell nanocrystals in targeted alpha-therapy and to a method for synthesizing the coreshell nanocrystals. In addition, the present invention relates to a core-shell nanocrystal having a discrete core-shell structure comprising a core and a single crystalline shell, wherein the core comprises Ce02, and the single crystalline shell comprises CaF2.

[0005] Targeted alpha therapy (TAT) recently attracted interest from the scientific community as a new class of cancer therapy. Contrary to classical external radiotherapy, TAT can deliver high-energy alpha radiation directly to the tumor site. Over the past few years, preclinical and clinical research demonstrated that TAT is a powerful treatment modality that gives hope to patients suffering from life-threatening or difficult to treat cancers. Despite the high potential of TAT, the utilization of therapeutic alpha-emitters faces significant challenges. For instance, radium-223, which is the only therapeutic alpha-particle emitting radioisotope approved for clinical use, is not able to form stable complexes with biomolecules, thus strongly limiting its therapeutic utilization. Additionally, decay products (radioactive or stable) can accumulate in critical organs leading to high toxicity.

[0006] It has been attempted to address the above drawbacks by using nanocrystals. For example, the first article reported in the literature was published by Woodward and coworkers (Bioconjugate Chemistry 2011, 22, 766). Actinium-225 was doped in LaPCM core only nanocrystals. Soon after, McLaughlin and co-workers reported on the utilization of core-shell nanocrystals (double shell) for TAT (PLOS ONE 2013, 8, e54531 ). The core (Lao.5Gdo.5PO4) doped with actinium-225 was protected by pure GdPO4 as the inner shell while metal gold was used as the outer shell. It is worth noting that no experimental evidence proves the formation of hetero-structured core-shell nanocrystals in McLaughlin’s article. Since then, several research groups worldwide investigated the use of inorganic core-shell nanocrystals for TAT. Nevertheless, despite the claims, articles reporting on the utilization of core or core-shell nanocrystals for TAT often produce poor quality materials whose characteristics (e.g. very large size and shape distributions, highly agglomerated nanocrystals) are not compatible with human applications. Some articles also investigated the utilization of core or core-shell nanocrystals for TAT but by implementing surface doping with alpha-emitters ( / .e. surface adsorption) instead of intrinsic doping ( / .e. inside the crystal structure) thus losing all benefits of using inorganic nanocrystals compared to classical organic molecules and macro-molecules.

[0007] It is therefore an object of the present invention to provide core-shell nanocrystals that can incorporate an alpha-emitter or a mother radionuclide of an alpha-emitter while being stable under self-radiation and retaining recoiled daughters of the alpha-emitter or of the mother radionuclide of an alpha-emitter.

[0008] In particular, the present invention provides core-shell nanocrystals having a discrete core-shell structure comprising a core and a single crystalline shell, wherein the core comprises CeO2 and an alpha-emitter or a mother radionuclide of an alpha-emitter, and wherein the single crystalline shell comprises a shell compound having a cubic space group with a lattice constant of 4.6 A to 6.3 A.

[0009] According to the present invention, the core of the core-shell nanocrystals comprises CeO2. CeO2 is known to be stable under self-irradiation and thus can serve as a host matrix for various alpha-emitters of pharmaceutical interest. It crystallizes in a cubic crystal structure having space group Fm3m. According to the present invention, the lattice constant of CeO2 is taken as 5.418 A.

[0010] According to the present invention, the core of the core-shell nanocrystals further comprises an alpha-emitter or a mother radionuclide of an alpha-emitter. An alphaemitter is a radionuclide that undergoes alpha-decay in which an atomic nucleus emits an alpha particle (helium nucleus) and thereby decays into a different atomic nucleus with a mass number that is reduced by four and an atomic number that is reduced by two compared to the initial alpha-emitter.

[0011] According to the present invention, a mother radionuclide of an alpha-emitter is a nuclide that undergoes a radioactive decay and whose daughter nuclide is an alphaemitter. The kind of radioactive decay that the mother radionuclide undergoes is not particularly limited. However, in view of an intended pharmaceutical application, the mother radionuclide preferably undergoes an alpha- or a beta-decay to form the alphaemitter.

[0012] Suitable alpha-emitters that can be used in the nanocrystals of the present invention are thorium-227, radium-223, radium-224, actinium-225, terbium-149, lead-212, astatine-211 , bismuth-212, bismuth-213, and uranium-230, for instance. The alphaemitter comprised by the core of the core-shell nanocrystals of the present invention is preferably thorium-227 and / or radium-223 since thorium-227 is the mother radionuclide of radium-223, which has already been approved for clinical use. Since ThO2 and CeO2 form isomorph phases, ThO2 can be evenly distributed within the core of the core-shell nanocrystal. Due to the decay of thorium-227, radium-223 is generated evenly distributed within the core.

[0013] Since radionuclides may have more than one decay pathway, in the present invention the common notation is adopted where a radionuclide is denoted as alpha- or betaemitter according to which decay pathway is dominant.

[0014] According to the present invention, the alpha-emitter, the mother radionuclide of an alpha-emitter and the alpha-emitting daughter radionuclide of the mother radionuclide preferably have a half-life of 45 min to 30 days, more preferably 1 day to 20 days. A half-life in this range ensures that there is sufficient time for synthesizing the core-shell nanocrystals of the present invention with still sufficient alpha-emission taking place during TAT.

[0015] The core of the nanocrystals may contain one type of alpha-emitter or mother radionuclide of an alpha-emitter, or it may contain a mixture of two or more alphaemitters and mother radionuclides of an alpha-emitter. By combining a mixture of radionuclides having shorter and longer half-life, an initial high dose of alpha radiation can be delivered followed by a lower dose over an extended period of time. Similarly, by providing a mixture of an alpha-emitter and a mother radionuclide of an alphaemitter, alpha-radiation can be delivered over an extended period of time.

[0016] The concentration of the alpha-emitter and / or the mother radionuclide of an alphaemitter in the core is not particularly limited. Preferably, the ratio of the alpha-emitter and the mother radionuclide of an alpha-emitter to the Ce atoms is 0.0001 at.% to 5 at.%. The upper limit is preferably 0.5 at.% more preferably 0.01 at.%. If the ratio of the alpha-emitter and the mother radionuclide of an alpha-emitter to the Ce atoms is higher than 5 at.% the stability of the core structure may be compromised.

[0017] According to the present invention, the core of the core-shell nanocrystals comprises CeO2 and one or more alpha-emitters and / or mother radionuclides of an alpha-emitter.

[0018] The size of the core of the core-shell nanocrystals of the present invention is not particularly limited. In view of its intended pharmaceutical application and sizelimitations of the core-shell nanocrystals associated therewith, the core preferably has a diameter of 2 nm to 20 nm.

[0019] The core-shell nanocrystals of the present invention further comprise a single crystalline shell surrounding the CeO2 core. The shell comprises a shell compound having a cubic space group with a lattice constant of 4.6 A to 6.3 A. This range for the lattice constant of the shell compound corresponds to a lattice mismatch of 15% or less relative to the cell parameter of CeO2. Since the compound forming the shell has a small lattice mismatch relative to the cell parameter of CeO2, the core crystals can serve as nucleation sites for the shell compound and a shell connected to and surrounding the core can readily be formed. That is, according to the present invention, the single crystalline shell preferably consists of the shell compound (different than CeO2) having a cubic crystal system with a lattice constant of 4.6 A to 6.3 A. Preferably, the shell compound has the space group Fm3m, which is the same crystal structure as the core of the core-shell nanocrystals.

[0020] According to the present invention, the term “lattice mismatch” relates to the ratio of the difference between the lattice constant of the core compound and of the shell compound relative to the lattice constant of the core compound. Since both the core compound, i.e. CeO2, and the shell compound, which is different from CeO2, have a cubic crystal structure, they only have one lattice constant a. Thus, the lattice mismatch can be calculated according to the following formula:

[0021] The lattice constant a can be determined by X-Ray diffraction measurements followed by Rietveld-refinement of the obtained diffractograms. The lattice constant of the shell compound is preferably in the range of 4.9 A to 6.0 A, more preferably in the range of 5.0 A to 5.9 A. Accordingly, the lattice mismatch is preferably 10% or less, more preferably 8% or less relative to the cell parameter of Ce02.

[0022] The exact composition of the shell compound is not particularly limited as long as it is able to form a shell around the core and is stable to the alpha-emission of the core. Suitable examples of the shell compound include alkaline-earth fluorides as well as other binary and ternary compounds. According to the present invention, the shell compound may also include CeO2. However, according to a preferred embodiment of the present invention, the shell compound is different from CeO2.

[0023] Preferably, the shell compound is selected from the group consisting of CaF2, SrF2, BaF2, CeO2, AgBiS2, a-NaREF4with RE = Sc, Y, Ho, Er, Tm, Yb, Lu, NaBiS2, preferably from the group consisting of CaF2, SrF2, BaF2, CeO2, AgBiS2 and more preferably from the group consisting of CaF2, SrF2, and BaF2. In view of its known stability to alpharadiation and biocompatibility, the shell compound is most preferably CaF2.

[0024] Since one function of the shell is to retain recoiled daughters of the alpha-emitter or of the mother radionuclide of an alpha-emitter stemming from the core of the core-shell nanocrystals, the shell is preferably substantially free of any alpha-emitters or mother radionuclides of an alpha-emitter. In this context, the term substantially free means that while some atoms may diffuse into the shell at the crystal boundary between the core and the shell (interface formation), the outermost region of the shell is free of any alphaemitters or mother radionuclides of an alpha-emitter.

[0025] The core-shell nanocrystals may comprise an agent that allows localization of the coreshell nanocrystals in a body. For example, the nanocrystals may comprise Ce-134, so as to monitor the position of the particles in the body during treatment via Positron Emission Tomography Scan. In this case, Ce-134 may be part of the CeO2 forming the core of the core-shell nanocrystals or it may be contained in the shell. Preferably, the agent that allows localization of the core-shell nanocrystals is comprised in the shell of the nanocrystal. In this way, the signal used for localizing the core-shell nanocrystals is less shielded and, thus, the core-shell nanocrystals can be localized with higher accuracy.

[0026] According to the present invention, the core-shell nanocrystals have a discrete coreshell structure. A discrete core-shell structure means that the single crystalline shell domain surrounds the core with no grain boundaries (neither intradomain nor interdomains). As will be discussed in more detail hereinbelow, the specific core-shell structure can be obtained by the controlled synthesis of the core-shell nanocrystals.

[0027] The shell of the core-shell nanocrystals is preferably dense. According to the present invention, dense means that no voids are present in the single crystalline shell. In this way, the stability and the retention of recoiled daughters of the alpha-emitter or of the mother radionuclide of an alpha-emitter in the core by the shell can be improved. Further, the shell is preferably in contact with the core of the core-shell nanocrystals.

[0028] The thickness of the shell is not particularly limited as long as it is thick enough to provide the required stability and retention capability of the shell material and thin enough to allow for sufficient alpha-radiation to pass through it. The thickness of the shell is preferably in the range of 2 nm to 80 nm. The lower limit of the thickness of the shell is preferably 5 nm, more preferably 10 nm, i.e., the thickness of the shell is more preferably in the range of 5 nm to 80 nm, even more preferably 10 nm to 80 nm. The upper limit of the thickness of the shell is preferably 50 nm, more preferably 20 nm, i.e., the thickness of the shell may be in the range of 2 nm to 50 nm, 5 nm to 50 nm, 10 nm to 50 nm, 2 nm to 20 nm, 5 nm to 20 nm, or 10 nm to 20 nm.

[0029] In view of the above, the size of the core-shell nanocrystals of the present invention is preferably in the range of 6 nm to 180 nm, more preferably in the range of 12 nm to 180 nm, even more preferably 22 nm to 180 nm. The upper limit of the size of the coreshell nanocrystals is preferably 100 nm, more preferably 50 nm, even more preferably 30 nm. The size of the core and core-shell nanocrystals is determined by powder x-ray diffraction (PXRD) and / or scanning transmission electron microscopy (STEM). The thickness of the shell is determined by performing energy dispersive x-ray spectroscopy chemical maps and line scan analyses.

[0030] The core-shell nanocrystal of the present invention may consist of the core and the shell as discussed above, or further shells and layers may be provided outside the shell. The further shells may provide additional functionality, stability and / or protection by further confining the radioactive decay products.

[0031] For example, core-shell nanocrystal of the present invention may further comprise a second shell surrounding the core and the single crystalline shell. The second shell may be crystalline or amorphous. In addition, the second shell may be dense or porous. The material for the second shell is not particularly limited and may be selected from metal oxides, such as silica, titania and alumina. In a specific embodiment, the second shell may be a mesoporous silica shell.

[0032] Moreover, the core-shell nanocrystal, with or without the second shell discussed above, may be surrounded by a lipid layer, preferably a lipid bilayer, a PEG (polyethylene glycol) layer, or a polyacrylic acid layer. In this way, the biocompatibility of the core-shell nanocrystals can be improved. In addition, or as an alternative, the core-shell nanocrystals may further comprise a targeting ligand on the surface thereof, the targeting ligand being adapted for targeting cancer cells. The targeting ligand can interact with specific receptors overexpressed in malignant cancer cells, such that the core-shell nanocrystals can accumulate in the cancer tissue and healthy tissue is less affected by the alpha-therapy. In general, targeting ligands that have been studied for and applied in targeted chemotherapy may also be used for the core-shell nanocrystals of the present invention. Suitable targeting ligands may include folic acid, hyaluronic acid, transferrin, carbohydrates, peptides, aptamers, antibodies and antibody fragments.

[0033] The present invention is further directed to the use of the core-shell nanocrystals as described above in targeted alpha therapy. That is, the core-shell nanocrystals according to the present invention are suitable for treating cancer in a human body.

[0034] The present invention further provides a method for synthesizing the core-shell nanocrystal described above. The method comprises: a) a step of synthesizing core crystals comprising CeO2 and an alphaemitter or a mother radionuclide of an alpha-emitter; followed by b) a step of synthesizing the single crystalline shell surrounding the core crystals and comprising a shell compound having a cubic space group with a lattice constant of 4.6 A to 6.3 A.

[0035] The synthesis of the core crystals may be achieved by modifying a known synthesis route. Specifically, in a first synthesis method, the step a) may comprise the following: a1 ) a step of dissolving cerium(lll) nitrate and a nitrate salt of the alphaemitter or of the mother radionuclide of an alpha-emitter in water; a2) a step of preparing a solution of oleic acid or a carboxylic acid having a hydrocarbon chain of at least 8 carbon atoms, tert-butylamine and a non-polar solvent; a3) a step of combining the solutions obtained in steps a1 ) and a2) in an autoclave in such a way that a layered two-phase system is formed; a4) a step of performing solvothermal treatment at a temperature in the range of 150°C to 220°C for a duration of about 12 h to about 96 h; and a5) a step of recovering the core particles.

[0036] The non-polar solvent used in step a2) of the first synthesis method is not particularly limited as long as it can dissolve the oleic acid and tert-butylamine. Preferably, the nonpolar solvent is selected from the group consisting of toluene, octadecene and n- hexadecane. The nitrate salt of the alpha-emitter is preferably thorium(IV) nitrate and / or radium(ll) nitrate and / or actinium(lll) nitrate.

[0037] In a second synthesis method, the step a) may comprise the following: a1 ) a step of dissolving a cerium salt and a salt of the alpha-emitter or of the mother radionuclide of an alpha-emitter in an organic mixture comprising an organic solvent and an organic capping agent under an inert atmosphere; a2) a step of heating the solution obtained in step a1 ) to a temperature in the range of 180 °C to 320 °C and maintaining the temperature for a time of 15 minutes to 60 minutes under an inert atmosphere, followed by natural cooling of the solution; a3) a step of recovering the core crystals.

[0038] The cerium salt used in step a1 ) of the second synthesis method is not particularly limited and may be a nitrate salt, an acetate salt and / or ammonium nitrate salt of Ce.. The salts may, for example, include Cerium(lll) nitrate (Ce(NO3)3), Cerium(lll) acetate (Ce(CH3COO)3), and Cerium(IV) ammonium nitrate ((NH4)2Ce(NO3)e).

[0039] The salt of the alpha-emitter or of the mother radionuclide of an alpha-emitter used in step a1 ) of the second synthesis method is not particularly limited and may be a nitrate salt, an acetate salt, an ammonium nitrate salt and / or a halide salt. The halide salt of the alpha-emitter or of the mother radionuclide of an alpha-emitter is preferably a chloride salt.

[0040] The organic solvent used in step a1 ) of the second synthesis method is not particularly limited as long as it can dissolve the cerium salt, the salt of the alpha-emitter or of the mother radionuclide of an alpha-emitter, and the organic capping agent. Suitable organic solvents include 1 -octadecene and benzyl ether. The capping agent may be selected from the group consisting of oleic acid, oleylamine, and trioctylphosphine oxide.

[0041] The dissolution of the salts in step a1 ) of the second synthesis method in the organic mixture is performed under an inert atmosphere, such as an atmosphere mainly consisting of Ar, He, or N2. The conditions used for dissolution of the salts in the organic mixture can be freely adjusted by a person skilled in the art depending on the cerium salt, the salt of the alpha-emitter or of the mother radionuclide of an alpha-emitter and the organic mixture used. Typically, dissolution is performed at a temperature from 100 °C to 130 °C for about 30 minutes.

[0042] Between the step a1 ) and the step a2) of the second synthesis method, the solution obtained in step a1 ) may be purified under vacuum / inert gas cycles. For example, the solution may be evacuated to a pressure of 5.1 O’2mbar at a temperature of 100 °C for 10 minutes, followed by repressurizing with an inert gas. This procedure may be repeated several times, for example five times.

[0043] In step a2) of the second synthesis method, natural cooling of the solution means that the heating source is removed and no active cooling is performed.

[0044] The step a3) of the second synthesis method may include the following steps a3-1 ) and a3-2): a3-1 ) a step of precipitating the core crystals by adding a non-solvent to the solution; and a3-2) a step of centrifuging the obtained colloidal solution, discarding the supernatant and redispersing the precipitate in a non-polar solvent.

[0045] The non-solvent in step a3-1 ) of the second synthesis method may be selected from acetone, ethanol, or a mixture of acetone and ethanol. The non-polar solvent in step a3-2) of the second synthesis method may be selected from toluene, octadecene and n-hexadecane.

[0046] In a third synthesis method, the core crystals may be prepared in supercritical water at 400 °C and pressure of about 250 bar for about 15 min to about 60 min followed by recovery of the core crystals. Details of the synthesis were published earlier (Zhang, J. et al., Advanced Materials 2007, 19, 203-206).

[0047] Once the core crystals have been recovered, they can be used as nucleation sites for forming the shell around the core, thus forming the core-shell nanocrystals according to the present invention. Specifically, the step b) comprises the following: b1 ) a step of combining a precursor compound for the shell with oleic acid, a dispersion of the core particles, and toluene or a non-polar solvent having a hydrocarbon chain of at least 8 carbon atoms; b2) a step of heating the mixture obtained in step b1 ) at a temperature in the range of 100 °C to 160 °C until a clear solution is formed; b3) a step of removing the dispersion solvent of the core crystals under vacuum; b4) a step of heating the reaction solution obtained in step b3) at a temperature in the range of 250 °C to 350 °C for about 45 min to about 90 min; and b5) a step of recovering the core-shell nanocrystals.

[0048] The non-polar solvent having a hydrocarbon chain of at least 8 carbon atoms used in step b1 ) is not particularly limited as long as it can dissolve the oleic acid and the precursor compound for the shell. The non-polar solvent having a hydrocarbon chain of at least 8 carbon atoms is further not particularly limited as long as it is liquid at room temperature and normal pressure and has a boiling point of 200°C or more. Preferably, the non-polar solvent having a hydrocarbon chain of at least 8 carbon atoms is selected from the group consisting of octadecene, n-hexadecane and squalene.

[0049] The precursor compound for the shell can be freely selected by a person skilled in the art depending on the type of shell to be formed. In case of forming a CaF2 shell, fluorinated organic carboxylates of Ca(ll) can be used as precursor such as, for example, Ca(ll) trifluoroacetate. For recovery and purification of the core-only and core-shell nanocrystals in step a5) of the first synthesis method and step b5), known protocols can be applied. For example, at the end of the reaction, the nanocrystals are dispersed in an organic solvent, such as toluene, chloroform, n-hexane or cyclohexane. They are then precipitated by addition of a polar solvent, such as methanol, ethanol or acetone. The precipitate is isolated and purified by re-dispersion and re-precipitation. The core-only and core-shell nanocrystals can be stored as colloidal solution.

[0050] The present invention further provides a core-shell nanocrystal having a discrete coreshell structure comprising a core and a single crystalline shell, wherein the core comprises CeC ; and the single crystalline shell comprises CaF2. The size and layer thickness limitations outlined above also apply to these core-shell nanocrystals.

[0051] Brief description of the figures

[0052] Figure 1 HAADF-STEM image of CeO2-CaF2 core-shell nanocrystals (core crystals obtained according to the first synthesis method). The bright spots in the center of the particles indicate a CeO2 domain while the less-contrasted outer shell indicates the CaF2 domain.

[0053] Figure 2: HAADF-STEM image of ThO2-CaF2 core-shell nanocrystals (core crystals obtained according to the first synthesis method as a proof of concept). The bright spots in the center of the particles indicate a ThO2 domain while the less-contrasted outer shell indicates the CaF2 domain.

[0054] Figure 3: HAADF-STEM image of CeO2 core crystals synthesized by the second synthesis method.

[0055] Figure 4: HAADF-STEM image of ThO2 core crystals synthesized by the second synthesis method (as a proof of concept).

[0056] Figure 5: HAADF-STEM image of UO2 core crystals synthesized by the second synthesis method (as a proof of concept).

[0057] Figure 6: HAADF-STEM image of ThO2-CaF2 core-shell nanocrystals (core crystals obtained according to the second synthesis method as a proof of concept). The bright spots in the center of the particles indicate a CeO2 domain while the less-contrasted outer shell indicates the CaF2 domain.

[0058] Figure ?: HAADF-STEM image of UO2-CaF2 core-shell nanocrystals (core crystals obtained according to the second synthesis method as a proof of concept). The bright spots in the center of the particles indicate a Ce02 domain while the less-contrasted outer shell indicates the CaF2 domain.

[0059] In summary, the present invention provides the following advantages:

[0060] 1 ) The core-shell nanocrystals according to the present invention constitute a unique carrier suitable for radium-223 and its mother radionuclide thorium-227, for instance. The former is, to date, the only alpha-emitter approved for clinical utilization for cancer treatment. Radium-223 is commercialized by the German company Bayer under the name Xofigo. Nevertheless, Xofigo is a simple salt of223RaCl2, which considerably limits its utilization for very specific cancer types involving bone metastases due to the chemical similarity with Ca. There is no obvious solution for radium-223 because it does not form stable complexes with organic molecules and macromolecules. Therefore, the core-shell nanocrystals, which can trap radium in their crystal structure, constitute the first realistic alternative to Xofigo. This can considerably extend the cancer types that could be treated by radium-223, which is already approved. Note that thorium-227 is a promising alpha emitter with a long half-life (18.7 days) that is important for centralized production and direct shipping to hospitals worldwide. Nevertheless, as thorium-227 generates radium-223, it is poorly investigated due to the stability problems generated by the latter.

[0061] 2) The core-shell nanocrystals according to the present invention can address one of the critical limitations associated with TAT regarding the uncontrolled release of toxic decay products. This is due to the devastating recoil effect after alpha disintegration that destroys the integrity of molecular and macromolecular carriers. This can lead to significant poisonous effects due to the uncontrolled release and body dissemination of “free” radionuclides. Consequently, the benefit-to-risk ratio can be significantly altered. The core-shell nanocrystals according to the present invention are stable under self-irradiation and can accommodate all decay products in their crystal structure. This constitutes a unique benefit compared to organic molecules and macromolecules.

[0062] 3) Contrary to organic molecules and macromolecules, the core-shell nanocrystals according to the present invention can accommodate more than one alpha-emitter within a single carrier ( / .e. single nanocrystal). Such a feature is extremely important when the target tissue has just a few receptors per cell. The utilization of the core-shell nanocrystals according to the present invention can easily adjust the dose that will be delivered and thus be more efficient compared to organic molecules and macromolecules.

[0063] Synthesis of CeO2:Th-CaF2 core-shell nanocrystals

[0064] The synthesis of CeO2-CaF2 core-shell nanocrystals doped with alpha-emitters of pharmaceutical interest involves firstly the synthesis of the starting core nanocrystals ( / .e. CeO2) and secondly the shell ( / .e. CaF2) deposition or growth on the core nanocrystals.

[0065] Step C1 : Dissolve cerium(lll) nitrate (Ce(NO3)3 xH2O - 0.120 mmol) and thorium(IV) nitrate (Th(NO3)4 xH2O - 0.05 mmol) in deionized water. The dissolution is performed under air and is complete in less than 3 minutes. The obtained solution is perfectly clear.

[0066] Step C2: The aqueous solution obtained in step C1 is transferred into a 23 mL Teflon (PTFE) liner.

[0067] Step C3: Oleic acid (300 pL), tert-butylamine (75 pL), and toluene (7000 pL) are added in a 10 mL glass vial under air to obtain a perfectly clear solution.

[0068] Step C4: The organic solution obtained in step C3 is carefully transferred into the 23 mL PTFE liner that contains the aqueous solution obtained in step C1 . The transfer is performed carefully to obtain a two-phase system (aqueous phase at the bottom and organic phase on the top) and avoid emulsion.

[0069] Step C5: The 23 mL PTFE liner is closed with a PTFE cap and introduced into a stainless-steel autoclave. The latter is sealed according to manufacturer’s recommendations.

[0070] Step C6: The sealed autoclave is placed in an oven and heated up to 180°C. The temperature is maintained at 180°C for 48h. Step C7: After 48h at 180°C, the autoclave is removed from the oven and cooled down to room temperature with compressed air.

[0071] Step C8: Once the autoclave is at room temperature, the PTFE liner is removed from the autoclave.

[0072] Step C9: The organic solution that contains the CeO2 core nanocrystals doped with thorium is carefully removed from the PTFE liner and introduced in a 50 mL centrifuge tube.

[0073] Step C10-1 : The core nanocrystals are extracted and purified by adding a non-solvent such as acetone, ethanol, or a mixture of acetone and ethanol.

[0074] Step C10-2: The colloidal solution obtained in step C10-1 is centrifuged.

[0075] Step C10-3: After centrifuging (step C10-2), the supernatant is discarded (waste) while the precipitate is re-dispersed in a non-polar solvent (e.g. toluene).

[0076] Step C11 : The purification step (steps C10-1 to C10-3) is repeated until the product is clean.

[0077] Step C12: After the last purification step and re-dispersion of the core nanocrystals in toluene, the dispersion is transferred into a glass vial with a screw cap and stored under air until needed.

[0078] Synthesis of MO2 core nanocrystals with M= Ce, Th, or U

[0079] The synthesis of pure Ce, Th and U core nanocrystals is described as proof of concept. The synthesis of pure compounds demonstrates the possibility of synthesizing mixed phases and doped compounds, such as Cei-xThxO2 or Cei-xllxO2, core crystals.

[0080] Step C1 : Dissolve the metal precursor (0.5 mmol of a nitrate salt, acetate salt or, ammonium nitrate salt of Ce, Th or U) in a reactor containing an organic mixture composed of an organic solvent (1 -octadecene and / or benzyl ether) and organic capping agents (oleic acid, oleylamine, and / or trioctylphosphine oxide) between 100°C and 130°C until a clear solution is obtained (typically 30 min). The dissolution is performed under inert gas.

[0081] Step C2: The solution obtained in step C1 is purified under vacuum / inert gas cycles (typically 5.1 O’2mbar) at 100°C for 10 minutes (typical for five cycles). Step C3: The solution obtained in step C2 is heated up to the target temperature (typical range 180°C - 320 °C) under inert atmosphere.

[0082] Step C4: After reaching the target temperature, the solution is maintained at the same temperature for a given aging time (from 15 minutes up to 60 minutes) under inert atmosphere.

[0083] Step C5: After aging, the reactor is cooled down naturally by removing the heating source.

[0084] Step C6: The organic solution that contains the MO2 (M= Ce, Th, U) core nanocrystals doped is carefully removed in a 50 mL centrifuge tube.

[0085] Step C7-1 : The core nanocrystals are extracted and purified by adding a non-solvent such as acetone, ethanol, or a mixture of acetone and ethanol.

[0086] Step C7-2: The colloidal solution obtained in step C7-1 is centrifuged.

[0087] Step C7-3: After centrifuging (step C7-2), the supernatant is discarded (waste) while the precipitate is re-dispersed in a non-polar solvent (e.g. toluene).

[0088] Step C8: The purification step (steps C7-1 to C7-3) is repeated until the product is clean.

[0089] Step C9: After the last purification step and re-dispersion of the core nanocrystals in toluene, the dispersion is transferred into a glass vial with a screw cap and stored under air until needed.

[0090] All core nanocrystals are characterized at least by powder x-ray diffraction (PXRD) and scanning transmission electron microscopy (STEM). If PXRD and STEM reveal the formation of the pure cubic phase (PXRD) with monodisperse size and shape distributions (STEM), the core nanocrystals are considered as suitable for the CaF2 shell deposition.

[0091] PXRD samples are prepared by drop casting 10-20 pL of the concentrated colloidal solution of the core nanocrystals onto a low background (911 ) oriented Si substrate. X- ray diffraction patterns are acquired using a Bruker D8 Discover diffractometer in Bragg-Brentano geometry, equipped with a copper anticathode, a quartz monochromator (Cu Ka1 1.540562 A), and a 1 -dimensional LynxEye XE-T detector. The patterns are collected in 29 from 10° to 135° with a total acquisition time of 9 h. Grids for STEM characterization are prepared at room temperature by drop casting 10 pL of a diluted suspension of core nanocrystals in toluene onto an ultrathin, amorphous 3 nm carbon film mounted on a 400 pm mesh Cu grid (Ted Pella Inc.). The shape, size, and chemical composition of the core nanocrystals are investigated by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) using a FEI Osiris ChemiSTEM microscope operated at 200 keV.

[0092] All syntheses performed with radionuclides are performed in a dedicated facility where radionuclides can be handled with all requested safety measures.

[0093] CaF2 shell deposition / qrowth on CeC>2 core nanocrystals doped with alpha-emitters

[0094] Step S1 : In a glovebox under inert atmosphere (N2), anhydrous calcium(ll) trifluoroacetate (Ca(OOCCF3)2 - 1 mmol) is introduced into a 50 mL three-neck round bottom flask together with oleic acid and octadecene. The three necks of the round bottom flask are sealed with rubber septa and the flask is removed from the glovebox.

[0095] Step S2: Once removed from the glovebox, the three-neck round bottom flask is maintained under argon (Ar) by connecting it to a Schlenk line (first side neck). The central neck is connected to a water-cooled condenser.

[0096] Step S3: Under Ar-flow, a PTFE coated magnetic bar is added into the three-neck round bottom flask. The colloidal solution of the core nanocrystals (typically 20-30 mg of core nanocrystals) obtained in step C13 is also added. The core nanocrystals act as seeds for the deposition / growth of CaF2.

[0097] Step S4: The second side neck is connected to a thermocouple that is inserted in the liquid without blocking the rotation of the magnetic bar.

[0098] Step S5: The mixture obtained in step S4 is heated up to 120°C under Ar-flow and kept until all Ca(OOCCF3)2 precursor is dissolved.

[0099] Step S6: Toluene is removed under vacuum.

[0100] Step S7: The three-neck round bottom flask is placed under Ar and heated up to 300°C. The temperature is maintained for 60 minutes.

[0101] Step S8: After 60 minutes at 300°C, the three-neck round bottom flask is cooled down to room temperature with compressed air. Step S9: The organic solution that contains the CeO2:Th-CaF2 core-shell nanocrystals doped with thorium is introduced in a 50 mL centrifuge tube.

[0102] Step S10-1 : The core-shell nanocrystals are extracted and purified by adding a nonsolvent (acetone, ethanol, or a mixture of acetone and ethanol, total volume 5-10 mL).

[0103] Step S10-2: The colloidal mixture obtained in step S10-1 is centrifuged.

[0104] Step S10-3: After centrifuging (step S10-2), the supernatant is discarded (waste) while the precipitate is re-dispersed in a non-polar solvent (e.g. toluene).

[0105] Step S11 : The purification step (steps S10-1 to S10-3) is repeated at least three times.

[0106] Step S12: After the last purification step and re-dispersion of the core-shell nanocrystals in toluene, the toluene solution is transferred into a glass vial with a screw cap and stored under air until needed.

[0107] All core-shell nanocrystals are characterized at least by powder X-ray diffraction (PXRD) and scanning transmission electron microscopy (STEM) under the same conditions as for the core nanocrystals.

[0108] The shell thickness can be determined, in a first approximation, by direct measurements on STEM images based on the contrast difference. The difference between the average size of the core and core-shell NCs can also be used to determine the shell thickness. More advanced measurements based on EDX line scan analysis as reported by Hudry et al. should be used for accurate shell thickness determination (Advanced Materials 2019, 31 , 1900623).

[0109] All syntheses performed with radionuclides are performed in a dedicated facility where radionuclides can be handled with all requested safety measures.

Claims

Claims1 . A core-shell nanocrystal having a discrete core-shell structure comprising a core and a single crystalline shell, wherein the core comprises CeO2 and an alpha-emitter or a mother radionuclide of an alpha-emitter; and the single crystalline shell comprises a shell compound having a cubic space group with a lattice constant of 4.6 A to 6.3 A.

2. The core-shell nanocrystal according to claim 1 , wherein the shell compound has the space group Fm3m.

3. The core-shell nanocrystal according to claim 1 or claim 2, wherein the alphaemitter or the mother radionuclide of an alpha-emitter is at least one selected from the group consisting of thorium-227, radium-223, radium-224, actinium-225, terbium- 149, lead-212, astatine-211 , bismuth-212, bismuth-213, and uranium-230, preferably wherein the alpha-emitter is thorium-227 and / or radium-223.

4. The core-shell nanocrystal according to any one of claims 1 to 3, wherein the shell compound is selected from the group consisting of CaF2, SrF2, BaF2, CeO2, AgBiS2, a-NaREF4 with RE = Sc, Y, Ho, Er, Tm, Yb, and Lu, and NaBiS2, preferably from the group consisting of CaF2, SrF2, BaF2, CeO2, AgBiS2, more preferably from the group consisting of CaF2, SrF2, and BaF2.

5. The core-shell nanocrystal according to any one of claims 1 to 4, wherein the single crystalline shell is dense.

6. The core-shell nanocrystal according to any one of claims 1 to 5, wherein the core has a diameter of 2 nm to 20 nm.

7. The core-shell nanocrystal according to any one of claims 1 to 6, wherein the single crystalline shell has a thickness of 2 nm to 80 nm.

8. The core-shell nanocrystal according to any one of claims 1 to 7, further comprising a second shell surrounding the core and the single crystalline shell.

9. The core-shell nanocrystal according to any one of claims 1 to 8, wherein the core-shell nanocrystal further comprises a lipid layer, a polyethylene glycol layer or a polyacrylic acid layer surrounding the nanocrystal.

10. The core-shell nanocrystal according to any one of claims 1 to 9, wherein the core-shell nanocrystal further comprises a targeting ligand on the surface thereof, the targeting ligand being adapted for targeting cancer cells.11 . The core-shell nanocrystal according to any one of claims 1 to 10, wherein the single crystalline shell is substantially free of any alpha-emitter or mother radionuclide of an alpha-emitter.

12. The core-shell nanocrystal according to any one of claims 1 to 11 for use in targeted alpha therapy.

13. A method for synthesizing a core-shell nanocrystal according to any one of claims 1 to 11 , the method comprising: a) a step of synthesizing core crystals comprising CeO2 and an alphaemitter or a mother radionuclide of an alpha-emitter; followed by b) a step of synthesizing the single crystalline shell surrounding the core crystals and comprising a shell compound having a cubic space group with a lattice constant of 4.6 A to 6.3 A.

14. The method according to claim 13, wherein the step a) comprises: a1 ) a step of dissolving cerium(lll) nitrate and a nitrate salt of the alphaemitter or the mother radionuclide of an alpha-emitter in water; a2) a step of preparing a solution of oleic acid or a carboxylic acid having a hydrocarbon chain of at least 8 carbon atoms, tert-butylamine and a non-polar solvent; a3) a step of combining the solutions obtained in steps a1 ) and a2) in an autoclave in such a way that a layered two-phase system is formed;a4) a step of performing solvothermal treatment at a temperature in the range of 150°C to 200°C for a duration of about 12 h to about 96 h; and a5) a step of recovering the core crystals.

15. The method according to claim 13, wherein the step a) comprises: a1 ) a step of dissolving a cerium salt and a salt of the alpha-emitter or of the mother radionuclide of an alpha-emitter in an organic mixture comprising an organic solvent and an organic capping agent under an inert atmosphere; a2) a step of heating the solution obtained in step a1 ) to a temperature in the range of 180 °C to 320 °C and maintaining the temperature for a time of 15 minutes to 60 minutes under an inert atmosphere, followed by natural cooling of the solution; a3) a step of recovering the core crystals.

16. The method according to claim 13, wherein the step a) comprises a supercritical synthesis in water at 400 °C at a pressure of about 250 bar for about 15 min to about 60 min followed by recovery of the core crystals.

17. The method according to any one of claims 12 to 16, wherein the step b) comprises: b1 ) a step of combining a precursor compound for the shell with oleic acid, a dispersion of the core crystals, and toluene or a non-polar solvent having a hydrocarbon chain of at least 8 carbon atoms; b2) a step of heating the mixture obtained in step b1 ) at a temperature in the range of 100 °C to 160 °C until a clear solution is formed; b3) a step of removing the dispersion solvent of the core crystals under vacuum; b4) a step of heating the reaction solution obtained in step b3) at a temperature in the range of 250 °C to 350 °C for about 45 min to about 90 min; and b5) a step of recovering the core-shell nanocrystals.

18. A core-shell nanocrystal having a discrete core-shell structure comprising a core and a single crystalline shell, wherein the core comprises CeC ; and the single crystalline shell comprises CaF2.

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