Radioactive medical isotope Ra-223 stably-labeled rare earth nano diagnosis and treatment preparation as well as preparation method and application thereof

Abstract

The invention discloses a radioactive medical isotope Ra-223 stably-labeled rare earth nano diagnosis and treatment preparation as well as a preparation method and application thereof. According to the invention, by constructing a dual protection mechanism of the shell layer and the functional biomolecular layer, the Ra-223 is effectively packaged, the recoil nuclei of the Ra-223 are reserved at the tumor part and are prevented from escaping to the surrounding environment of the tumor, the safety of the Ra-223 treatment process can be enhanced, the radioactive energy is ensured to accurately act on the focus, and the treatment effect of the Ra-223 is improved. Meanwhile, the damage risk to healthy tissues is minimized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomedicine technology, and particularly relates to a rare earth nanotherapeutic agent stably labeled with the radioactive medical isotope Ra-223, its preparation method and application. Background Technology

[0002] With the continuous updates in modern medical treatment concepts, cancer treatment has evolved towards targeted therapy. Besides targeted chemotherapy drugs, radionuclide-based targeted therapy is receiving increasing attention. Suitable radionuclides for targeted cancer therapy mainly include beta-radionuclides and alpha-radionuclides. Alpha particles possess a very high linear energy transfer density (LET, 80–100 keV / μm) and high energy, with a range of 40–100 μm in human soft tissue, equivalent to 5–10 cell diameters. Because alpha particles deposit high energy over a very short distance when passing through cells, they can simultaneously break the double strand of DNA molecules, causing irreparable damage. Beta particles, on the other hand, can only break single strands of DNA molecules; therefore, the cytotoxicity of alpha particles is far greater than that of beta particles. Furthermore, unlike beta particles, the radiation damage to cells caused by alpha particles is almost unaffected by the cell's absorbed dose rate and intracellular oxygen levels. These advantages make alpha-radionuclides particularly attractive for treating non-solid tumors and micrometastases. In recent years, alpha-radionuclides have become a hot topic in radiopharmaceutical research. In 2013, the first alpha radionuclide drug, Xofigo (… 223 RaCl injection has been marketed in the United States and the European Union and has achieved good clinical efficacy.

[0003] Ra-223 belongs to the same family as calcium and can mimic calcium ions, possessing natural bone-targeting properties in areas of increased bone regeneration. It easily binds to pathological bone formation sites, precisely targeting bone lesions and concentrating the emission of high-energy alpha particles without harming surrounding tissues. It cleaves the double-stranded DNA of tumor cells, inducing tumor cell apoptosis, while simultaneously destroying osteoblasts and osteoclasts, thus inhibiting the vicious cycle of bone metastasis. However, the off-target effect of daughter nuclei recoil must be considered when using Ra-223. During alpha decay, daughter nuclei recoil, producing high kinetic energy (tens to hundreds of keV). These recoiled nuclei easily break free from chemical bonds (energy of several eV) and detach from the target site. As they redistribute throughout the body via the circulatory system, they cause radiation damage to normal tissues and organs. Low-level Ra-223 daughter nuclei redistribution has been reported in mice and has also been confirmed in humans. Therefore, improving the therapeutic efficacy of Ra-223 while limiting its daughter nuclei's fate has a profound impact on the development of Ra-223 radionuclide radiotherapy. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0005] A rare earth nano-therapeutic agent, comprising a radionuclide rare earth luminescent core, a shell, and a functional biomolecule layer; the shell is located on the surface of the radionuclide rare earth luminescent core, and the functional biomolecule layer is located on the surface of the shell.

[0006] The rare-earth nuclide luminescent core comprises an alkaline earth metal fluoride doped with the radioactive medical isotope Ra-223 and rare-earth luminescent ions; the general structural formula of the material of the rare-earth nuclide luminescent core is M. a F2:Ln b , 223 Ra; where,

[0007] M a It is an alkaline earth metal element;

[0008] Ln b Rare earth luminescent ions;

[0009] The structural formula of the shell material is M. c F2, where M c It is an alkaline earth metal element.

[0010] In this invention, Ra-223 can also be used 223 Ra said.

[0011] According to an embodiment of the present invention, the M a It is selected from at least one or more of Mg, Ca, Sr and Ba, for example Ca.

[0012] According to an embodiment of the present invention, the Ln b It is selected from at least one or more of Y, Sc, Yb, Er, Tm, Ho, Gd, Eu, Tb, Sm, Dy, Ce, Nd, La, Pr and Lu, preferably at least one or more of Y, Yb, Er, Gd, Eu and Lu, for example Yb, Er and Ce.

[0013] According to an embodiment of the present invention, the M c It is selected from at least one or more of Mg, Ca, Sr and Ba, for example Ca.

[0014] According to an embodiment of the present invention, the rare earth nuclide luminescent core has a crystal structure, for example, the rare earth nuclide luminescent core has a trigonal phase, cubic phase, tetragonal phase or monoclinic crystal phase structure, preferably a cubic phase crystal phase structure.

[0015] According to an embodiment of the present invention, the amount of doped rare earth luminescent ions in the rare earth nuclide luminescent core can be selected from the content known in the art, for example, the molar content of doped rare earth luminescent ions is 0.1-30 mol%, for example, 1 mol%, 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%.

[0016] According to an embodiment of the present invention, in the rare earth nuclide luminescent core, 223 The abundance of Ra is at least 0.1 μCi / g, preferably at least 0.15 μCi / g, for example 0.2 μCi / g, 0.3 μCi / g, 0.4 μCi / g, or 0.5 μCi / g. In this invention, the abundance refers to the weight ratio of the radiation dose of Ra-223 to the luminescent nucleus of the rare-earth nuclide.

[0017] According to an embodiment of the present invention, the rare earth nuclide luminescent core is a nanocrystalline particle with a size of 1-50 nm, preferably 2-10 nm, for example 5 nm, 10 nm, 20 nm, 30 nm, or 40 nm.

[0018] According to an embodiment of the present invention, the thickness of the shell layer is 1-50 nm, preferably 1-5 nm, for example 1 nm, 2 nm, 3 nm, 4 nm.

[0019] According to an embodiment of the present invention, the thickness of the shell is preferably less than 50% of the size of the rare earth nuclide luminescent core, for example, 10%-50%, or even 20%, 30%, or 40%.

[0020] According to an embodiment of the present invention, the shell is obtained by growing an alkaline earth metal fluoride on its surface using the rare earth nuclide luminescent core as a seed crystal.

[0021] According to an embodiment of the present invention, the rare earth nuclide luminescent core and shell constitute nanoparticles with a core-shell structure.

[0022] According to an embodiment of the present invention, the size of the core-shell structured nanoparticles is 2-200 nm, for example 2-100 nm, preferably 2-30 nm, and for example 2 nm, 5 nm, 7 nm, 8 nm, 10 nm, 12 nm, 15 nm, 18 nm, 20 nm, 25 nm, 30 nm, or any range between the above values.

[0023] According to an embodiment of the present invention, the rare earth luminescent ions are distributed inside the rare earth nuclide luminescent core by being doped in alkaline earth metal fluorides.

[0024] According to an embodiment of the present invention, the radioactive medical isotope Ra-223 is distributed inside the rare earth nuclide luminescent core by being doped in an alkaline earth metal fluoride.

[0025] According to an embodiment of the present invention, the rare earth nanotherapeutic agent has, for example, a transmission electron microscope image as shown in FIG2.

[0026] According to embodiments of the present invention, in order to enhance the biocompatibility and targeting of the rare-earth nanotherapeutic agents while preventing leakage of the recoil nuclei of the radioactive medical isotope Ra-223 at the target site in the organism, those skilled in the art can select the required material for the functional biomolecular layer according to imaging needs. The present invention does not specifically limit the material of the functional biomolecular layer. Exemplarily, the material of the functional biomolecular layer may be selected from at least one of organic materials (e.g., water-soluble ligands and / or targeting ligands), inorganic materials (e.g., silica), or organic / inorganic hybrid materials (e.g., silicon-based hybrid micelles), for example, a functional biomolecular layer composed of water-soluble ligands and targeting ligands.

[0027] According to embodiments of the present invention, the water-soluble ligand may be selected from water-soluble functional ligands, oil-soluble functional ligands (such as oleic acid), and amphiphilic functional ligands (such as liposomes), with examples selected from amphiphilic functional ligands. For instance, the amphiphilic functional ligand is selected from one or more of distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), dilaurylphosphatidylethanolamine-polyethylene glycol (DLPE-PEG), dimyristoylphosphatidylethanolamine-polyethylene glycol (DMPE-PEG), and dioleoylphosphatidylethanolamine-polyethylene glycol (DOPE-PEG).

[0028] For example, the number average molecular weight of the polyethylene glycol in this invention is preferably 300-50000, more preferably 500-10000, such as 300, 500, 1000, 2000, 3000, 4000, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 20000, 25000, 30000, 40000, 50000, or any range between any two values.

[0029] According to embodiments of the present invention, the targeting ligand may be selected from peptides, proteins, DNA, RNA, antibodies, cytokines, growth factors, enzymes, carbohydrate molecules, other targeting small molecules, etc., with antibodies being an example. For instance, the antibody may be selected from at least one or more of tri-GalNAc biotin, Anti-CEA McAb, Anti-AFP McAb, Anti-Free-PSA McAb, Rabbit anti-NSEPcAb, Anti-NSE McAb, Anti-Cyfra21-1(CK19)McAb, etc.

[0030] According to a preferred embodiment of the invention, the functional biomolecular layer, for example, partially or completely covers the surface of the shell layer.

[0031] According to an embodiment of the present invention, the thickness of the functional biomolecular layer is 1-100 nm, preferably 5-50 nm. For example, 1 nm, 5 nm, 10 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, or any range between the above values.

[0032] According to an embodiment of the present invention, the hydrated particle size of the rare earth nanotherapeutic agent is 3-1000 nm, preferably 3-100 nm. For example, 3 nm, 10 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, or any range between the above values.

[0033] The present invention also provides a method for preparing the above-mentioned rare earth nano-therapeutic agents, wherein the preparation method may employ at least one of the following: hot injection method, high-temperature co-precipitation method, high-temperature thermal decomposition method, hydrothermal method, or sol-gel method.

[0034] According to an embodiment of the present invention, a high-temperature thermal decomposition method is preferred; specifically, the preparation method includes:

[0035] S1, Preparation containing 223 Ra solution;

[0036] S2, the contents of step S1 223 Ra solution and removal 223 The core materials other than Ra are obtained by high-temperature thermal decomposition to produce alkaline earth metal fluorides doped with radioactive medical isotope Ra-223 and rare earth luminescent ions, which are called radionuclide rare earth luminescent cores.

[0037] S3. Using the rare earth luminescent nucleus as a seed crystal, an alkaline earth metal fluoride is grown on the surface of the rare earth luminescent nucleus as a shell layer by a layer-by-layer epitaxial growth method. The rare earth luminescent nucleus and the shell layer form nanoparticles with a core-shell structure.

[0038] S4. Load a functional biomolecular layer onto the surface of the nanoparticles in step S3 (preferably on the surface of the shell) to obtain the rare earth nanotherapeutic agent.

[0039] According to an embodiment of the present invention, in step S1, the containing 223 Ra solution refers to... 223 The solution is obtained by mixing an aqueous solution of RaCl2 with an oily solvent and removing the water. Preferably, a method known in the art can be used to remove the water, for example, in a rotary evaporator, exemplarily, at a rotary evaporation temperature of 40–90°C for 0.5–2 hours.

[0040] According to an embodiment of the present invention, the oily solvent is selected from at least one of oleic acid, oleylamine and octadecene, preferably oleic acid.

[0041] According to an embodiment of the present invention, the exception 223 Core materials other than Ra include Ln b trifluoroacetate, M a Trifluoroacetate.

[0042] According to an embodiment of the present invention, in step S2, the high-temperature pyrolysis method specifically includes: containing... 223 The Ra solution, core material, and first solvent are added to the reaction vessel and mixed until dissolved. The temperature is then raised to the first reaction temperature and reacted for a period of time. After cooling, the precipitate obtained is the aforementioned rare earth luminescent nucleus.

[0043] According to an embodiment of the present invention, in step S2, the Ln b trifluoroacetate and M a M in trifluoroacetates (e.g., calcium trifluoroacetate) a 、Ln b The molar ratio is (0.1-1) mmol:(0.1-1) mmol, preferably (0.1-0.5) mmol:(0.1-0.5) mmol.

[0044] According to an embodiment of the present invention, in step S2, the Ln b Trifluoroacetate and containing 223 The molar volume ratio of Ra solution is, for example, (0.1-1) mmol:(1-20) ml, preferably (0.1-0.5) mmol:(1-10) ml.

[0045] According to an embodiment of the present invention, in step S2, the high-temperature pyrolysis method is carried out under vacuum conditions and / or an inert atmosphere.

[0046] According to an embodiment of the present invention, in step S2, the first reaction temperature is 200-400°C, preferably 280-340°C, for example 300°C.

[0047] According to an embodiment of the present invention, in step S2, after heating to the first reaction temperature, the reaction time can be any time known in the art, for example, a reaction time of 0.5 to 2 hours.

[0048] According to an embodiment of the present invention, in step S3, the layer-by-layer epitaxial growth method refers to: adding the nuclide rare earth luminescent core, shell material and second solvent into the reaction vessel and mixing until dissolved, continuing to heat to the second reaction temperature, reacting for a period of time, and cooling down to obtain the precipitate, which is the nanoparticle with the core-shell structure.

[0049] According to an embodiment of the present invention, in step S3, the shell material includes M c Trifluoroacetates, such as calcium trifluoroacetate.

[0050] According to an embodiment of the present invention, in step S3, the ratio of the amount of the rare earth luminescent nucleus core and the shell material is not specifically limited, as long as a shell with the definition described above can be obtained.

[0051] According to an embodiment of the present invention, in step S3, the layer-by-layer epitaxial growth method is carried out under vacuum conditions and / or an inert atmosphere.

[0052] According to an embodiment of the present invention, in step S3, the second reaction temperature is 200-400°C, preferably 280-340°C, for example 300°C.

[0053] According to an embodiment of the present invention, in step S3, after heating to the second reaction temperature, the reaction time can be any time known in the art, for example, a reaction time of 0.5 to 2 hours.

[0054] According to an embodiment of the present invention, the first solvent and the second solvent may be the same or different, and are independently selected from at least one of oleic acid, oleylamine and octadecene, preferably a mixture of oleic acid, oleylamine and octadecene; for example, by molar amount, the first solvent comprises 1 part octadecene, (0.5 to 1) parts oleylamine and (0.5 to 1) parts oleic acid.

[0055] According to an embodiment of the present invention, in steps S2 and S3, the heating rate is 10-20 °C / min.

[0056] According to an embodiment of the present invention, in steps S2 and S3, the cooling refers to natural cooling to room temperature.

[0057] According to an embodiment of the present invention, in steps S2 and S3, the precipitate may also be centrifuged and washed using methods known in the art.

[0058] According to an embodiment of the present invention, step S4, the step of modifying the functional biomolecular layer, includes: dispersing the core-shell structured nanoparticles in an organic solvent, adding the material of the functional biomolecular layer and stirring, removing the organic solvent (e.g., allowing the organic solvent to evaporate naturally under stirring conditions), and optionally adding water and mixing with ultrasound to obtain the rare earth nanotherapeutic formulation. Preferably, the material of the functional biomolecular layer has the meaning as described above, such as distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and antibody Anti-CEA McAb. Preferably, stirring is stopped when the core-shell structured nanoparticles change from oil-soluble to water-soluble after adding the material of the functional biomolecular layer.

[0059] According to an embodiment of the present invention, in step S4, the organic solvent is one or more mixed solvents selected from cyclohexane, dichloromethane, trichloromethane, tetrahydrofuran, and N,N-dimethylformamide, for example, a mixture of cyclohexane and trichloromethane.

[0060] According to an embodiment of the present invention, in step S4, after loading the functional biomolecule layer, the reaction solution is further subjected to precipitation and washing; the precipitation and washing can be performed using methods known in the art, such as high-speed centrifugation.

[0061] According to an embodiment of the present invention, in step S4, after precipitation and washing, further dispersion or sterile filtration may be performed. Exemplarily, the dispersion refers to dispersion in physiological saline.

[0062] For example, in step S2, the core raw material is rare earth trifluoroacetate and calcium trifluoroacetate, and the first solvent is a mixture of oleylamine, oleic acid, and 1-octadecene; rare earth trifluoroacetate, calcium trifluoroacetate, oleylamine, oleic acid, 1-octadecene, and containing 223 The molar volume ratio of Ra solution is (0.1-1) mmol:(0.1-1) mmol:(1-20) ml:(1-20) ml:(1-20) ml:(1-20) ml; preferably (0.1-0.5) mmol:(0.1-0.5) mmol:(2-10) ml:(2-10) ml:(2-10) ml:(2-10) ml.

[0063] For example, in step S4, the material of the functional biomolecular layer is distearylphosphatidylethanolamine-polyethylene glycol and antibody Anti-CEA McAb; the organic solvent is cyclohexane and chloroform; the molar volume ratio of the core-shell structured nanoparticles, distearylphosphatidylethanolamine-polyethylene glycol, antibody Anti-CEA McAb, cyclohexane and chloroform is (0.01-0.1) mmol:(0.2-0.8) mmol:(1-10) ml:(2.5-10) ml, preferably (0.05-0.1) mmol:(0.3-0.5) mmol:(0.01-0.5) mmol:(1-3) ml:(8-10) ml.

[0064] According to an embodiment of the present invention, in the rare earth nano-therapeutic agents prepared by the above preparation method, 223 The Ra loading rate is greater than 50%, for example, 60%, 70%, or 80%. In this invention, 223 Ra loading rate refers to the rare earth nano-therapeutic agent in step S4. 223 The radiation dose of Ra and the raw materials 223 The ratio of Ra to the total radiation dose.

[0065] The present invention also provides the application of the above-mentioned rare earth nanotherapeutic agents in the pharmaceutical field, such as in the preparation of drugs for treating and / or diagnosing tumors, such as in the preparation of integrated tumor diagnosis and treatment agents.

[0066] According to embodiments of the present invention, the tumor refers to tumors known in the art, such as at least one of a variety of tumors including ovarian cancer, cervical cancer, endometrial cancer, melanoma, prostate cancer, pancreatic cancer, lung cancer, liver cancer, gastric cancer, lymphoma, head and neck cancer, breast cancer, and colorectal cancer, and for example, human ovarian cancer, cervical cancer, and endometrial cancer.

[0067] According to an embodiment of the present invention, the drug for treating and / or diagnosing tumors includes the above-mentioned rare earth nanotherapeutic agents.

[0068] According to an embodiment of the present invention, the integrated tumor diagnosis and treatment formulation includes the above-mentioned rare earth nano-therapeutic formulation.

[0069] Beneficial effects

[0070] (1) In the rare earth nano-therapeutic formulation stably labeled with the radioactive medical isotope Ra-223 of the present invention, the similarity properties caused by the similar electronic structure between elements of the same group are utilized, and M, which belongs to the alkaline earth metal group as the radioactive isotope Ra-223, is selected. aThe elemental core, acting as a carrier, can significantly improve the loading rate of Ra-223 in the preparation of rare earth nanotherapeutic agents and further enhance their therapeutic effects.

[0071] (2) In this invention, by constructing a dual protection mechanism of shell and functional biomolecular layer, Ra-223 is effectively encapsulated, so that its recoil nucleus is retained in the tumor site, preventing its recoil nucleus from escaping into the surrounding environment of the tumor. This can enhance the safety of the Ra-223 treatment process, ensure that the radioactive energy is accurately applied to the lesion, and minimize the risk of damage to healthy tissues.

[0072] (3) The rare earth nano-therapeutic formulation of the present invention exhibits good luminescence performance in both the visible and near-infrared regions, especially in the 1400-1700nm wavelength range of the near-infrared II region. This characteristic makes it extremely promising for application in the field of real-time fluorescence imaging of deep tissue-penetrating biological organisms, enabling real-time monitoring of the Ra-223 radiotherapy process. Attached Figure Description

[0073] Figure 1 This is an X-ray powder diffraction pattern of the oil-soluble rare earth nano-therapeutic agent prepared in Example 1 of this invention.

[0074] Figure 2-1 This is a transmission electron microscope image of the rare earth nano-therapeutic agent prepared in Example 1 of the present invention.

[0075] Figure 2-2 This is a hydrated particle size diagram of the rare earth nano-therapeutic agent prepared in Example 1 of the present invention.

[0076] Figure 3 These are the rare-earth nano-therapeutic agents prepared in Example 1 and Comparative Example 1 of this invention. 223 Ra load rate comparison chart.

[0077] Figure 4 This is the fluorescence emission spectrum of the rare earth nano-therapeutic agent prepared in Example 1 of this invention.

[0078] Figure 5 This is a graph showing the cytotoxicity results of the rare earth nano-therapeutic agent prepared in Example 1 of this invention 48 hours after administration.

[0079] Figure 6 This is a diagram showing the effect of the rare earth nano-therapeutic agent prepared in Example 1 of this invention on the mitochondrial morphology of tumor cells under a bioelectron microscope.

[0080] Figure 7 This is a near-infrared fluorescence imaging effect of mouse tumors after subcutaneous administration of the rare earth nano-therapeutic agent prepared in Example 1 of this invention.

[0081] Figure 8 This is an anatomical diagram of tumor tissue in a nude mouse subcutaneous tumor model of human ovarian cancer.

[0082] Figure 9 This is a diagram showing the pathological examination results of the main organs and tissues in a nude mouse subcutaneous tumor model of human ovarian cancer.

[0083] Figure 10 The image shows the treatment results of the rare earth nano-therapeutic agents prepared in Example 1 of this invention, the Ra-223 group, and Comparative Example 2 on a human ovarian cancer subcutaneous tumor model in nude mice. Detailed Implementation

[0084] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0085] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0086] X-ray powder diffractometer (MiniFlex2, Rigaku), transmission electron microscope (TECNAI G2 F20, FEI), spectrometer (FLS980, Edinburgh), laser particle size analyzer (Zetasizer Ultra, Malvern), visible-near-infrared micro-area confocal imaging microscope (A1RHD25, Nikon), and full-spectrum small animal in vivo imaging system (NirVivo-Mix, RayLight).

[0087] Example 1: CaF2:Yb / Er / Ce / 223 Preparation of Ra@CaF2@LIG rare earth nano-therapeutic agents

[0088] The preparation method can be any of the known methods for preparing nanomaterials in the art, such as hot injection, high-temperature co-precipitation, high-temperature thermal decomposition, hydrothermal method, or sol-gel method. In this embodiment, the high-temperature thermal decomposition method is used, including the following steps:

[0089] S101. Add 5 mL at room temperature 223 The aqueous solution of RaCl2 (raw material) and 3 mL of oleic acid were mixed and evaporated in a rotary evaporator at 80 °C to remove the aqueous solution, yielding... 223 An oleic acid solution of Ra, denoted as 223 Ra-oleic acid solvent.

[0090] S102. Weigh 0.0680 g of calcium trifluoroacetate, 0.0800 g of ytterbium trifluoroacetate, 0.0040 g of erbium trifluoroacetate, and 0.0060 g of cerium trifluoroacetate into a three-necked flask at room temperature, and add 3 mL of [unclear text - possibly a typo, should be "3 mL"]. 223 Ra-oleic acid solvent, 3 mL oleylamine and 6.4 mL octadecene were used as a mixed solvent; the mixture was heated under vacuum until the above trifluoroacetate dissolved, and then heated to 300 °C under an inert atmosphere. After reacting for 0.5 hours, it was naturally cooled to room temperature, precipitated and washed to obtain CaF2:Yb / Er / Ce / 223 Ra-based rare-earth luminescent nuclei, in which... 223 The abundance of Ra is at least 0.1 μCi / g.

[0091] S103. The CaF2:Yb / Er / Ce / 223 Ra rare-earth luminescent nuclei were added to a mixed solvent of 6.4 mL oleic acid and 6.4 mL octadecene, followed by 0.0680 g of calcium trifluoroacetate. The mixture was heated under vacuum until the trifluoroacetate dissolved, then heated to 300 °C under an inert atmosphere. After reacting for 0.5 hours, the mixture was allowed to cool naturally to room temperature, precipitated, and washed to obtain CaF2:Yb / Er / Ce / 223 Ra@CaF2 core-shell structured nanoparticles, wherein the particle size of the rare earth nuclide luminescent core is 5 nm and the thickness of the CaF2 shell is 2 nm.

[0092] S104. At room temperature, 10 mg of CaF2:Yb / Er / Ce / 223 Ra@CaF2 core-shell nanoparticles were dispersed in a mixed solution of 2 mL cyclohexane and 10 mL chloroform. While stirring, 10 times the mass of the nanoparticles of DSPE-PEG2000 and 0.1 mg of the antibody Anti-CEA McAb were rapidly added until all the solvents, cyclohexane and chloroform, evaporated naturally, resulting in the modified functional biomolecular layer LIG. The obtained solid was redispersed with an appropriate amount of pure water. The reaction solution was centrifuged at high speed to precipitate and washed. The precipitate obtained was the rare earth nanotherapeutic agent, dispersed and stored in physiological saline. The hydrated particle size of the rare earth nanotherapeutic agent in this embodiment was 50 nm. Figure 2-2 ).

[0093] See Figure 1 As shown, this is the CaF2:Yb / Er / Ce / prepared in this embodiment. 223 X-ray powder diffraction pattern of Ra@CaF2@LIG rare earth nano-therapeutic formulation. Figure 2-1 The image shows a transmission electron microscope image. As can be seen from the image, the rare earth nano-therapeutic agent prepared in this embodiment has a crystal structure, and its basic morphology is a cubic phase crystal structure with good crystallinity.

[0094] See Figure 4 As shown, this is the CaF2:Yb / Er / Ce / prepared in this embodiment. 223 The near-infrared II emission spectrum of Ra@CaF2 rare earth nano-therapeutic formulation under 980nm excitation. As shown in the figure, the CaF2:Yb / Er / Ce / ... prepared in this embodiment... 223 Ra@CaF2 rare earth nano-therapeutic agents exhibit good near-infrared luminescence.

[0095] Comparative Example 1: Preparation of NaLuF4:Yb / Er / Ce / 223 Ra-earth nanotherapeutic agents using Ra@NaLuF4@LIG nanoparticles

[0096] The NaLuF4:Yb / Er / Ce / prepared in this comparative example 223 The Ra@NaLuF4 nanoparticles were prepared according to the method in Example 1, except that calcium trifluoroacetate in steps S102 and S103 was replaced with lutetium trifluoroacetate, and the amount of lutetium trifluoroacetate added was 0.6800 g; the other conditions were the same as in Example 1, and NaLuF4:Yb / Er / Ce / was prepared. 223 Ra@NaLuF4@LIG, denoted as the comparative rare earth nano-therapeutic agent 1.

[0097] Comparative Example 2: Preparation of CaF2:Yb / Er / Ce@CaF2: 223 Ra@LIG nanoparticles for rare-earth nanotherapeutic applications

[0098] The NaLuF4:Yb / Er / Ce / prepared in this comparative example 223 The Ra@NaLuF4 nanoparticles were prepared using the same method as in Example 1, except that no additives were added to S102. 223 Ra-oleic acid solvent, added to S103 223 Ra-oleic acid solvent, and 223 The amount of Ra-oleic acid solvent added was the same as in Example 1; all other conditions were the same as in Example 1, and CaF2:Yb / Er / Ce@CaF2: was prepared. 223 Ra@LIG, denoted as the comparative rare earth nanotherapeutic agent 2.

[0099] like Figure 3 As shown, compared to the comparative rare-earth nanotherapeutic formulation 1 of Comparative Example 1, the rare-earth nanotherapeutic formulation prepared in Example 1 has... 223 Ra loading rate can reach 80%; Comparative Example 2 shows the difference between rare earth nano-therapeutic agents 2 and those of the previous example. 223 The Ra load rate is basically the same as that in Example 1.

[0100] Experimental Example 1: Evaluation of the antitumor effect of rare earth nanotherapeutic agents at the in vitro cellular level

[0101] Human ovarian cancer (SKOV-3, procured from Fenghui Biotechnology) was treated with 3×10 4 The rare earth nanoparticles were seeded at a concentration of [number] per mL in 96-well plates and, after adhesion, co-cultured with different drugs for 48 hours. The drugs were: the water-soluble rare earth nanoparticle therapeutic agent prepared in Example 1, with concentrations of 0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL; and the Ra group consisted of pure radionuclides with radiation doses corresponding to the various concentration groups of the rare earth nanoparticle therapeutic agent in Example 1. 223 Ra; The cytotoxicity of the above rare-earth nanotherapeutic agents was detected using the Cell Counting Kit-8 formulation (CCK8), and the results are as follows: Figure 5 As shown, the rare earth nano-therapeutic agent of Example 1 has a good killing effect on tumors, with an IC50 of approximately 100 μg / mL and 12.5 nCi / mL.

[0102] like Figure 6 As shown, bioelectron microscopy revealed that the 100 μg / mL rare earth nanotherapeutic agent prepared in Example 1 affected the mitochondrial morphology of SKOV-3 cells, including severe damage to the mitochondrial double membrane, mitochondrial swelling and vacuolation, and a significant reduction in cristae.

[0103] The results of the above in vitro cell-level experiments show that the rare earth nanotherapeutic agents prepared in Example 1 can exert anti-tumor effects by acting on the mitochondria of tumor cells.

[0104] Experimental Example 2: Fluorescence Imaging Effect of Rare Earth Nanoparticle Therapeutic Agents on Mouse Tumors

[0105] The following animal experiments were approved by the Laboratory Animal Ethics Committee of Fujian Medical University.

[0106] A human ovarian cancer (SKOV-3) subcutaneous tumor model was constructed in nude mice (using the method described in the reference, Nano Today, 55, 2024, 102214), with tumor masses of approximately 100 mm² selected. 3 SKOV-3 nude mice with human ovarian cancer.

[0107] Nude mice with subcutaneous tumors of human ovarian cancer SKOV-3 were injected subcutaneously with the rare-earth nanotherapeutic agent prepared in Example 1 at a dose of 150 mg / kg, and then immediately subjected to fluorescence imaging to observe its in vivo fluorescence imaging effect.

[0108] Imaging was performed using a 980nm laser excitation system in a small animal in vivo fluorescence imaging system, and the results are as follows: Figure 7As shown, rare earth nano-therapeutic agents and 223 The distribution of Ra at the tumor site is where fluorescence imaging has an advantage.

[0109] Experimental Example 3: The evaluation of the anti-tumor effect of rare earth nanotherapeutic agents in animals is as follows:

[0110] This was achieved by constructing a human ovarian cancer (SKOV-3) subcutaneous tumor model in nude mice, with tumor masses approximately 100 mm in volume selected. 3 Nude mice with human ovarian cancer SKOV-3 subcutaneous tumors were divided into groups of 6 mice each and injected with the following drugs:

[0111] The first group received normal saline injections; this was the control group.

[0112] The second group received injections of pure radionuclides. 223 Ra is a pure nuclide. 223 Group Ra, denoted as Ra-223;

[0113] The third group was injected with the rare earth nano-therapeutic agent of Example 1, which is Example 1 group;

[0114] The fourth group was injected with the control rare earth nano-therapeutic agent 1 of control example 2, which is control example 2.

[0115] The mice were administered once via in situ injection at a dose of 20 mg / kg. One mouse from each of the above groups was used for pathological analysis, and the remaining five mice were subjected to the following procedures: the weight of the nude mice was measured weekly, the mice were observed daily, and the time of death was recorded on a daily basis.

[0116] In the control group, Ra-223 group, and comparative group 2, when the tumor size of a mouse in each group increased to the mortality standard (1500 mm), 3 The mice were considered dead and dissected. In Example 1, due to the small size of the tumor, the mice were dissected on the 36th day after administration. Important organs such as the heart, liver, spleen, lungs, and kidneys were taken. The organs were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and examined for histopathology.

[0117] Tumor tissues of human ovarian cancer SKOV-3 were taken from nude mice dissected above and arranged from left to right in descending order of size, as shown below. Figure 8 As shown in the figure, the rare earth nano-therapeutic agent prepared in Example 1 can significantly inhibit the growth of subcutaneous tumors of human ovarian cancer in nude mice, improve the condition of nude mice with ovarian cancer, delay weight loss, and prolong the survival time of nude mice. The CaF2:Yb / Er / Ce / prepared in Example 1 can be obtained. 223The tumor inhibition rate of Ra@CaF2@LIG rare earth nanotherapeutic agents was 39.5%, and that of Ra-223 was 32.8%. The radiation dose of Comparative Example 2 was basically the same as that of Example 1, and the tumor inhibition rate was comparable.

[0118] In this invention, the tumor inhibition rate = (average tumor weight in the treatment group - average tumor weight in the control group) / average tumor weight in the control group, wherein the treatment group corresponds to Example 1 and Comparative Example 2, respectively.

[0119] like Figure 9 As shown, mice treated with the rare earth nano-therapeutic agents prepared in Example 1 showed no obvious toxic pathological changes in major organs such as the heart, liver, spleen, lungs, and kidneys after dissection.

[0120] like Figure 10 As shown, mice in the Ra-223 group and Comparative Example 2 group experienced varying degrees of bleeding in the tumor and organ sites after treatment, which may be due to bleeding caused by leakage of the Ra-223 recoil nucleus; while mice in Example 1 group had scabs formed after tumor healing, indicating that the drug was safer.

[0121] The results of the above in vivo animal experiments show that the rare earth nanotherapeutic agents prepared in Example 1 have good in vivo antitumor efficacy and biosafety.

[0122] The exemplary embodiments of the present invention have been described above. However, the scope of protection of this application is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A rare earth nanodiagnostic and nanotherapeutic formulation, characterized in that, The rare earth nanodiagnosis and nanotherapy preparation comprises a radionuclide rare earth luminescent inner core, a shell layer and a functional biomolecule layer; the shell layer is located on the surface of the radionuclide rare earth luminescent inner core, and the functional biomolecule layer is located on the surface of the shell layer. The rare earth element luminescent core comprises an alkaline earth metal fluoride doped with a radioactive medical isotope Ra-223 and rare earth luminescent ions; the structural general formula of the material of the rare earth element luminescent core is M a F2:Ln b , 223 Ra; wherein, M a is an alkaline earth metal element; Ln b is a rare earth luminescent ion; The material of the shell layer has a general structure of M c F2, wherein M c is an alkaline earth metal element.

2. The rare earth nanodiagnostic and nanotherapeutic formulation as claimed in claim 1, wherein, The M a at least one or more selected from the group consisting of Mg, Ca, Sr, and Ba. Preferably, said Ln b at least one or more selected from the group consisting of Y, Sc, Yb, Er, Tm, Ho, Gd, Eu, Tb, Sm, Dy, Ce, Nd, La, Pr and Lu. Preferably, said M c at least one or more selected from the group consisting of Mg, Ca, Sr, and Ba. Preferably, the radionuclide rare earth luminescent inner core has a crystal structure. Preferably, the rare earth element luminescent core has an atomic number of at least 57 and an atomic weight of at most 200 g / mol, and the rare earth element luminescent core has a luminescence wavelength of at most 500 nm. 223 The abundance of Ra is at least 0.1 μ 3.The rare earth nanodiagnostic and nanotherapeutic formulation of claim 1 or 2, characterized in that, The radionuclide rare earth luminescent inner core is a nanocrystal particle with a size of 1-50 nm. Preferably, the thickness of the shell layer is 1-50 nm. Preferably, the thickness of the shell layer is less than 50% of the size of the radionuclide rare earth luminescent inner core. Preferably, the radionuclide rare earth luminescent inner core and the shell layer form a nanometer particle with a core-shell structure. Preferably, the nanometer particle with the core-shell structure has a size of 2-200 nm. Preferably, the rare earth luminescent ions are distributed in the interior of the radionuclide rare earth luminescent inner core by being doped in an alkaline earth metal fluoride. Preferably, the radioactive medical isotope Ra-223 is distributed in the interior of the radionuclide rare earth luminescent inner core by being doped in an alkaline earth metal fluoride.

4. The rare earth nanodiagnostic and nanotherapeutic formulation according to any one of claims 1 to 3, wherein, The material of the functional biomolecule layer is selected from at least one of an organic material, an inorganic material and an organic / inorganic hybrid material. Preferably, the functional biomolecule layer is partially or entirely coated on the surface of the shell layer. Preferably, the thickness of the functional biomolecule layer is 1-100 nm. Preferably, the rare earth nanodiagnosis and nanotherapy preparation has a hydration particle size of 3-1000 nm.

5. The method of claim 1-4, wherein the method is characterized by, The preparation method adopts at least one of a hot injection method, a high-temperature co-precipitation method, a high-temperature thermal decomposition method, a hydrothermal method and a sol-gel method.

6. The preparation method according to claim 5, characterized in that, The preparation method comprises: S1, preparing a solution containing 223 Ra; S2, adding the solution of S1 to a solution of 223 Ra and removing the solution of Ra 223 The alkali earth metal fluoride doped with radioactive medical isotope Ra-223 and rare earth luminescent ions, i.e. the radionuclide rare earth luminescent kernel, is obtained by high temperature thermal decomposition method. S3, using the radionuclide rare earth luminescent inner core as a seed, growing an alkaline earth metal fluoride, i.e. a shell layer, on the surface of the radionuclide rare earth luminescent inner core by a layer-by-layer epitaxial growth method, so that the radionuclide rare earth luminescent inner core and the shell layer form a nanometer particle with a core-shell structure; S4, loading a functional biomolecule layer on the surface of the nanometer particle of step S3 to obtain the rare earth nanodiagnosis and nanotherapy preparation.

7. The preparation method according to claim 6, characterized in that, In step S1, the solution containing 223 A solution of Ra means that 223 An aqueous solution of RaCl2is mixed with an oily solvent, and the water is removed to obtain. Preferably, the oily solvent is selected from at least one of oleic acid, oleylamine and octadecene. Preferably, the removal of 223 The core material other than Ra includes Ln b trifluoroacetate of M a trifluoroacetate of M Preferably, in step S2, the high-temperature thermal decomposition method specifically comprises: adding the solution containing Ra, the core material and the first solvent into a reaction container and mixing to dissolve, continuously heating to a first reaction temperature, reacting for a period of time, and obtaining the precipitate after cooling, which is the rare earth luminescent core. 223 The solution of Ra, the core material and the first solvent are added into a reaction container and mixed to dissolve, the temperature is continuously increased to a first reaction temperature, the reaction is continued for a period of time, and the precipitate obtained after cooling is the rare earth luminescent core. Preferably, in step S2, the Ln b trifluoroacetate and M a trifluoroacetate is M a , Ln b in a molar ratio of (0.1-1) mmol : (0.1-1) mmol. Preferably, in step S2, the Ln b trifluoroacetate salt and the solution containing 223 Ra have a molar volume ratio of (0.1-1) mmol : (1-20) ml. Preferably, in step S2, the high-temperature thermal decomposition method is performed under vacuum and / or an inert atmosphere. Preferably, in step S2, the first reaction temperature is 200-400 ℃.

8. The method of any one of claims 5-7, wherein, In step S3, the layer-by-layer epitaxial growth method refers to: adding the radionuclide rare earth luminescent inner core, shell layer raw materials and a second solvent into a reaction container, mixing until dissolution, continuously heating to a second reaction temperature, reacting for a period of time, and cooling to obtain a precipitate, i.e. the nanometer particle with the core-shell structure. Preferably, in step S3, the shell precursor comprises M c trifluoroacetate. Preferably, in step S3, the layer-by-layer epitaxial growth method is performed under vacuum and / or an inert atmosphere. Preferably, in step S3, the second reaction temperature is 200-400 ℃. Preferably, the first solvent and the second solvent are the same or different and are independently selected from at least one of oleic acid, oleylamine and octadecene. Preferably, in steps S2 and S3, the heating rate is 10-20 ℃ / min. Preferably, in steps S2 and S3, the cooling refers to natural cooling to room temperature.

9. The method of any one of claims 5-8, wherein, The step S4 of modifying the functional biomolecule layer comprises: dispersing the nanoparticles with core-shell structure in an organic solvent, stirring after adding the material of the functional biomolecule layer, removing the organic solvent, and optionally adding water and mixing by ultrasonic to obtain the rare earth nanodiagnostic and nanotherapeutic preparation.

10. Use of the rare earth nanodiagnostic and nanotherapeutic preparation according to any one of claims 1-4 in the field of medicine.

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