A manganese ion-synergistic rare earth radioactive diagnosis and treatment integrated preparation and its preparation method and application

Abstract

The present invention discloses a manganese ion-synergized rare earth radioactive diagnostic and therapeutic integrated preparation, as well as its preparation method and application. The rare earth radioactive diagnostic and therapeutic integrated preparation of the present invention comprises rare earth-doped inorganic nanocrystals, a radioactive medical isotope R, and a functional tumor-targeting biomolecule LI, wherein the functional tumor-targeting biomolecule LI is loaded on the surface of the rare earth inorganic nanocrystals; the radioactive medical isotope R is at least distributed within the rare earth-doped inorganic nanocrystals and / or coupled to the functional tumor-targeting biomolecule LI; the rare earth-doped inorganic nanocrystals have a core-shell-shell-shell structure and include at least manganese element Mn and rare earth luminescent ions Em. The rare earth radioactive diagnostic and therapeutic integrated preparation prepared by the present invention exhibits excellent radiochemical stability and good biosafety, can be used safely in vivo, and has thus led to many applications related to clinical medicine, while possessing both therapeutic and diagnostic functions.

Description

Technical Field

[0001] The present invention belongs to the field of nano-biomedical diagnosis and treatment integration technology, and relates to a manganese ion-synergistic rare earth radioactive diagnosis and treatment integrated preparation and its preparation method and application, and in particular to a manganese ion-synergistic rare earth radioactive diagnosis and treatment integrated preparation labeled with radioactive medical isotopes and its preparation method and application. Background Art

[0002] Malignant tumors are a major threat to public health, causing millions of deaths worldwide each year. Currently, the main treatments for malignant tumors include radiotherapy, chemotherapy, and surgery. Radionuclide therapy, a commonly used radiotherapy method in clinical practice, primarily utilizes the decay of radionuclides to emit high-energy rays, which can effectively overcome the physical barriers of tumors and thus better kill cancer cells. Currently, in order to reduce the side effects of radionuclide therapy, the efficient use of radionuclides has become a hot topic in radionuclide therapy research. Precision delivery and synergistic therapy are two effective ways to improve the utilization rate of radionuclide drugs.

[0003] As a new generation of biological imaging fluorescent probes, rare earth doped inorganic nanofluorescent materials have great prospects in the field of non-invasive imaging due to their low biological toxicity, no background fluorescence, good stability, and high imaging resolution. More importantly, rare earth inorganic nanofluorescent materials are easily doped with rare earth medical radioisotopes (such as 177 Lu, 169 Second, 153 Sm, 86 Y. 90 Y, etc.), can serve as a good radionuclide carrier. Moreover, after its surface is modified with multifunctional targeting groups, it can precisely deliver radionuclide drugs to the tumor site, achieving accurate diagnosis and treatment of tumor lesions.

[0004] Manganese is one of the essential trace elements for the human body. It is the core element of many metalloenzymes in the body and also has biocatalytic function. 2+ In the tumor microenvironment, it can catalyze the decomposition of endogenous hydrogen peroxide to generate oxygen, alleviate the hypoxia inside the tumor, transfer electrons to oxygen to generate superoxide free radicals, induce tumor cell apoptosis, and then kill tumor cells. 2+ The synergistic therapeutic effect between drugs and radionuclides can achieve better therapeutic effects with lower doses of drugs.

[0005] Therefore, the development of a manganese ion-doped rare earth radioactive diagnostic and therapeutic integrated preparation that can simultaneously achieve stable loading of radioactive isotopes and precise diagnosis and treatment of tumor lesions has important practical significance and clinical value in the application of integrated diagnosis and treatment of diseases. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A rare earth radioactive diagnostic and therapeutic integrated preparation comprising rare earth-doped inorganic nanocrystals, a radioactive medical isotope R, and a functional tumor-targeting biomolecule LI, wherein the functional tumor-targeting biomolecule LI is loaded on the surface of the rare earth inorganic nanocrystals; the radioactive medical isotope R is at least distributed within the rare earth-doped inorganic nanocrystals and / or coupled to the functional tumor-targeting biomolecule LI;

[0008] The rare earth-doped inorganic nanocrystal has a core-shell-shell-shell structure, which includes, from the inside to the outside, a rare earth ion core, a rare earth ion energy transfer layer, a rare earth ion energy absorption layer, and a rare earth ion inert protective layer; the rare earth-doped inorganic nanocrystal includes at least manganese element Mn and rare earth luminescent ions Em.

[0009] According to an embodiment of the present invention, Em is a rare earth luminescent ion selected from one or more of Pr, Nd, Eu, Dy, Ho, Er, Tm, Yb, and Ce; preferably Yb, Er, and Ce.

[0010] According to an embodiment of the present invention, the rare earth luminescent ion Em is distributed in the core-shell-shell-shell structure of the rare earth-doped inorganic nanocrystal. Preferably, the rare earth luminescent ion Em is distributed in at least one or more layers of the rare earth ion core, rare earth ion energy transfer layer, rare earth ion energy absorption layer, and rare earth ion inert protective layer of the rare earth-doped inorganic nanocrystal, and is preferably distributed in the rare earth ion core and / or the rare earth ion energy absorption layer.

[0011] According to an embodiment of the present invention, the rare earth ion core, the rare earth ion energy transfer layer and the rare earth ion inert protective layer may be composed of the same or different materials and are independently selected from MLnF4; the rare earth ion energy absorption layer is selected from MLnF4:Mn / Em; wherein,

[0012] M is selected from one or more of the alkali metal elements Li, Na, K, Rb or Cs;

[0013] Ln is a rare earth element selected from one or more of Y, Sc, La, Ce, Pr, Nd, Po, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu, preferably one or more of Yb, Nd, Er, Tm, Ho, Gd, Y, Ce and Lu, exemplified by one or more of Gd, Yb, Nd, Y, Er and Ce.

[0014] According to an embodiment of the present invention, the radioactive medical isotope R is selected from 177 Lu, 169 Second, 153Sm, 90 Y. 86 Y. 44 Sc, 47 One or more of Sc; preferably 177 Lu and 90 One or more of Y, exemplified by 177 Lu.

[0015] According to an embodiment of the present invention, the radioactive medical isotope R is distributed in at least one or more than two layers of the rare earth ion core, the rare earth ion energy transfer layer, the rare earth ion energy absorption layer and the rare earth ion inert protection layer.

[0016] According to an embodiment of the present invention, the functional tumor-targeting biomolecule is used to enhance the biocompatibility of the rare earth radiotherapy integrated diagnostic and therapeutic preparation, while also providing the preparation with tumor targeting properties. Those skilled in the art will appreciate that the functional tumor-targeting molecule can be selected based on the specific needs of different tumor types.

[0017] According to a preferred embodiment of the present invention, the functional tumor-targeting biomolecule is partially or completely coated on the surface of the rare-earth-doped inorganic nanocrystal. Preferably, the coating method for the functional tumor-targeting biomolecule includes at least one of ligand oxidation, layer-by-layer deposition, acid washing, and ligand exchange, such as a ligand exchange method. Preferably, the ligand exchange method includes: modifying the surface of the rare-earth-doped inorganic nanocrystal with the functional tumor-targeting biomolecule to make it water-soluble, and then partially or completely coating the surface of the rare-earth-doped inorganic nanocrystal with the functional tumor-targeting biomolecule.

[0018] According to an embodiment of the present invention, the functional tumor-targeting biomolecule LI is prepared from an amphiphilic ligand without tumor-specific recognition ability and a biomolecule with tumor-specific recognition ability. Preferably, the mass ratio of the water-soluble ligand without tumor-specific recognition ability to the biomolecule with tumor-specific recognition ability is (1-50):1; preferably (1-20):1. For example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, or any range therebetween.

[0019] Preferably, the biomolecule with tumor-specific recognition ability is selected from one or more of a small biomolecule, a macromolecule, an antibody, and a polypeptide, with polypeptides being an example. Furthermore, the small biomolecule is, for example, an oligopeptide, vitamin C, folic acid, or adenosine triphosphate. Furthermore, the biomacromolecule is, for example, a nucleic acid or a protein. Furthermore, the antibody is, for example, a monoclonal antibody. Furthermore, the polypeptide is, for example, a pancreatic cancer-targeting peptide.

[0020] Preferably, the amphiphilic ligand does not have tumor-specific recognition ability. Preferably, the amphiphilic ligand includes a hydrophilic group, and the hydrophilic group is at least one or more of ethanolamine, polyethylene glycol (PEG), carboxyl polyethylene glycol, amino polyethylene glycol, polyvinyl pyrrolidone (PVP), and polyvinyl alcohol (PVA). Further, the amphiphilic ligand can be selected from at least one of distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), distearoylphosphatidylethanolamine polyethylene glycol carboxyl (DSPE-PEG-COOH), distearoylphosphatidylethanolamine-polyethylene glycol-amino copolymer (DSPE-PEG-NH2), etc. Exemplarily, the amphiphilic ligand is distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

[0021] Furthermore, the number average molecular weight of the polyethylene glycol is preferably 500-30,000, more preferably 1,000-10,000, for example, 1,000, 2,000, 3,000, 4,000, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, or any range between any two values.

[0022] According to an embodiment of the present invention, the mass ratio of the rare earth-doped inorganic nanocrystals to the functional tumor-targeting biomolecule LI is 1:(2-10), exemplarily 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any range between any of the above values.

[0023] According to an exemplary embodiment of the present invention, the integrated rare earth radioactive diagnosis and treatment preparation includes rare earth-doped inorganic nanocrystals, radioactive medical isotopes R and functional tumor-targeting biological molecules LI; the rare earth-doped inorganic nanocrystals are NaGdF4:Em@NaYbF4@NaNdF4:Mn / Em@NaYF4, wherein Em is selected from at least one of Yb, Er, and Ce; the radioactive medical isotope R is distributed at least in the core-shell-shell-shell structure of the rare earth-doped inorganic nanocrystals, and the functional tumor-targeting biological molecules LI are loaded on the surface and / or core-shell-shell-shell structure of the rare earth-doped inorganic nanocrystals.

[0024] According to an embodiment of the present invention, the functional tumor-targeting biomolecule is located on the surface of the rare earth-doped inorganic nanocrystal, for example, partially covers or completely covers the surface of the rare earth-doped inorganic nanocrystal.

[0025] According to an embodiment of the present invention, the thickness of the functional tumor-targeting biomolecules on the surface of the rare earth-doped inorganic nanocrystals is 5-150 nm, preferably 5-40 nm.

[0026] According to an embodiment of the present invention, the size of the rare earth-doped inorganic nanocrystals is 5 to 70 nm, preferably 20 to 50 nm, such as 20 nm, 30 nm, 40 nm, 50 nm or any range between the above values.

[0027] According to an embodiment of the present invention, the size of the rare earth radioactive diagnostic and therapeutic integrated preparation is 20 to 300 nm, preferably 20 to 150 nm, for example, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 150 nm, or any range therebetween.

[0028] The present invention also provides a method for preparing the above-mentioned rare earth radioactive diagnosis and treatment integrated preparation, the preparation method comprising:

[0029] (1) preparing the rare earth ion core;

[0030] (2) sequentially growing a rare earth ion energy transfer layer, a rare earth ion energy absorption layer, and a rare earth ion inert protective layer on the surface of the rare earth ion core to obtain a core-shell-shell-shell structured nanocrystal;

[0031] (3) loading functional tumor-targeting biomolecules onto the surface of the core-shell-shell-shell structured nanocrystals through water-soluble modification to obtain the rare earth radioactive diagnosis and treatment integrated preparation;

[0032] In step 1 and / or step 2, a rare earth luminescent ion Em is further added, wherein the rare earth luminescent ion Em has the meaning as described above;

[0033] In at least one of step 1, step 2 and step 3, a radioactive medical isotope R is also added, and the radioactive medical isotope R has the meaning as described above.

[0034] According to an embodiment of the present invention, the rare earth ion core, rare earth ion energy transfer layer, rare earth ion energy absorption layer and rare earth ion inert protective layer can be prepared by methods known in the art, such as high-temperature co-precipitation, high-temperature thermal decomposition, hydrothermal method or sol-gel method, for example, by high-temperature thermal decomposition method.

[0035] According to an embodiment of the present invention, in step 1 and step 2, the rare earth ion core, rare earth ion energy transfer layer, rare earth ion energy absorption layer and rare earth ion inert protective layer are prepared under an inert atmosphere, and the inert atmosphere can be selected from inert gases known in the art, such as nitrogen and / or argon.

[0036] According to an embodiment of the present invention, the method for preparing the rare earth radioactive diagnosis and treatment integrated preparation comprises the following steps:

[0037] (a) adding a stable rare earth source and an alkali metal source into a mixed solvent, mixing and dissolving them, and reacting to obtain a rare earth ion core;

[0038] (b) adding the rare earth ion core, the stable rare earth source, and the alkali metal source obtained in step (a) into a mixed solvent, mixing and dissolving them, and reacting to obtain core-shell structured nanocrystals;

[0039] (c) adding the core-shell structured nanocrystals obtained in step (b), a stable rare earth source, an alkali metal source, and a manganese source into a mixed solvent, mixing and dissolving them, and reacting to obtain core-shell structured nanocrystals;

[0040] (d) adding the core-shell-shell structured nanocrystals obtained in step (c), a stable rare earth source, and an alkali metal source into a mixed solvent, mixing and dissolving them, and reacting to obtain core-shell-shell-shell structured nanocrystals;

[0041] (e) modifying the surface of the core-shell-shell-shell nanocrystals obtained in step (d) with functional tumor-targeting biomolecules to obtain the rare earth radioactive diagnosis and treatment integrated preparation;

[0042] Adding a radioactive medical isotope R in any one of the steps (a), (b), (c), (d) and (e);

[0043] Rare earth luminescent ions Em are added in at least one of the steps (a), (b), (c) and (d), and preferably rare earth luminescent ions Em are added in step (a).

[0044] According to an embodiment of the present invention, the dissolution temperature in steps (a), (b), (c) and (d) is 80-160°C, for example, 110°C.

[0045] According to an embodiment of the present invention, in step (a), step (b), step (c) and step (d), the reaction temperature is 250-350°C, for example, 310°C; the reaction time is 0.5-1.5 hours, for example, 0.5 hour or 1 hour.

[0046] According to an embodiment of the present invention, in steps (a), (b), (c), and (d), the reaction is carried out in a reactor, preferably in an inert atmosphere free of water and oxygen. Further, the conditions known in the art may be used for the anhydrous and oxygen-free reaction, and the present invention is not particularly limited thereto.

[0047] According to an embodiment of the present invention, in step (a), step (b) and step (c), the reaction is a constant temperature reaction, and the fluctuation of the reaction temperature is within ±5°C.

[0048] According to an embodiment of the present invention, the heating rate in step (a), step (b) and step (c) is 1 to 20° C. / min, for example, 10° C. / min.

[0049] According to an embodiment of the present invention, the stable rare earth source is selected from at least one of a trifluoroacetate of the rare earth ion Ln, an acetate of the rare earth ion Ln, a chloride of the rare earth ion Ln, and a nitrate of the rare earth ion Ln, preferably a trifluoroacetate of the rare earth ion Ln and a chloride of the rare earth ion Ln, wherein Ln has the meaning as described above. Furthermore, the stable rare earth source is exemplified by at least one of gadolinium trifluoroacetate, ytterbium trifluoroacetate, erbium trifluoroacetate, cerium trifluoroacetate, neodymium trifluoroacetate, and yttrium trifluoroacetate.

[0050] According to an embodiment of the present invention, the alkali metal source is selected from at least one or more of trifluoroacetates containing an alkali metal element M, oleates containing an alkali metal element M, acetates containing an alkali metal element M, hydroxides containing an alkali metal element M, fluorides containing an alkali metal element M, and hydrofluorides containing an alkali metal element M, preferably hydroxides containing an alkali metal element M, hydrofluorides containing an alkali metal element M, or acetates containing an alkali metal element M, exemplified by sodium trifluoroacetate. Preferably, the alkali metal source provides the element M for the core-shell-shell structured nanocrystals.

[0051] According to an embodiment of the present invention, the manganese source is selected from one or more of the following salts: one or more of manganese nitrate, manganese sulfate, manganese carbonate, manganese acetate, and manganese chloride, preferably manganese acetate and manganese chloride, exemplified by manganese acetate.

[0052] According to an embodiment of the present invention, the mixed solvent comprises a long alkane chain organic acid and a long alkane chain olefin. Preferably, the volume ratio of the long alkane chain organic acid to the long alkane chain olefin is (3-30) mL:(3-30) mL.

[0053] Preferably, the long alkane chain organic acid is selected from at least one of octanoic acid, dodecanoic acid and oleic acid, exemplified by oleic acid.

[0054] Preferably, the long alkane chain olefin is selected from 1-dodecene and / or 1-octadecene, exemplified by 1-octadecene.

[0055] According to an embodiment of the present invention, the radioactive medical isotope R is provided by the oxalate of the radioisotope and / or the chloride of the radioisotope, exemplified by the chloride of the radioisotope. 177 LuCl3 provided.

[0056] According to an embodiment of the present invention, the molar volume ratio of the rare earth element Ln and the mixed solvent in the stable rare earth source is (0.01-1) mmol: (6-60) mL; preferably (0.01-0.3) mmol: (6-30) mL.

[0057] According to an embodiment of the present invention, the molar ratio of the radioactive medical isotope R to the rare earth element Ln in the stable rare earth source is (0.01-200) mCi: (0.01-0.5) mmol; preferably (1-100) mCi: (0.01-0.2) mmol.

[0058] According to an embodiment of the present invention, in steps (a)-(d), a washing solvent is added after the reaction to precipitate the product and then separate it, and the washing solvent is selected from one or more mixed solvents of methanol, ethanol, and acetone. Preferably, the washing solvent is ethanol or acetone, exemplarily selected from ethanol.

[0059] According to an embodiment of the present invention, in step (e), the method for modifying the functional tumor-targeting biological molecule comprises: dispersing the core-shell-shell-shell structured nanocrystals of step (d) in a volatile solvent, adding the functional tumor-targeting biological molecule and stirring for a period of time so that the nanocrystals are converted from oil-soluble to water-soluble, and the modification is completed after the volatile solvent evaporates naturally. Preferably, after the modification is completed, an appropriate amount of water can be optionally added for mixing, filtering, or further dispersed in water or physiological saline. Preferably, the functional tumor-targeting biological molecule has the meaning as described above; for example, it is prepared from distearoylphosphatidylethanolamine-polyethylene glycol and a polypeptide, wherein the mass ratio of distearoylphosphatidylethanolamine-polyethylene glycol to the polypeptide is 10:1.

[0060] According to an embodiment of the present invention, in step (e), the volatile solvent is a mixed solvent of one or more of cyclohexane, dichloromethane, chloroform, tetrahydrofuran and N,N-dimethylformamide. Preferably, the volatile solvent is at least two of cyclohexane, dichloromethane and chloroform; exemplarily, cyclohexane and chloroform are selected from the group consisting of cyclohexane and chloroform, and the volume ratio of cyclohexane to chloroform is 1:(5-20) mL, preferably 1:(5-10) mL.

[0061] According to an embodiment of the present invention, the mass ratio of the core-shell-shell-shell structured nanocrystals to the functional tumor-targeting biological molecules is (1-4):(4-16), for example, 1:4.

[0062] The present invention also provides the use of the above-mentioned rare earth radioactive diagnostic and therapeutic integrated preparation in the field of fluorescent medical imaging. Preferably, the rare earth radioactive diagnostic and therapeutic integrated preparation is used as a developer, such as a rare earth nanofluorescent tumor medical imaging agent or a nuclear medicine tumor imaging agent.

[0063] The present invention also provides a developer, which includes the above-mentioned rare earth radioactive diagnosis and treatment integrated preparation.

[0064] The present invention also provides the use of the above-mentioned rare earth radioactive diagnosis and treatment integrated preparation in rare earth nanofluorescence-nuclear medicine multimodal medical imaging, preferably as a tumor radioactive therapeutic agent.

[0065] The present invention also provides a tumor radioactive therapeutic agent, which includes the above-mentioned rare earth radioactive diagnosis and treatment integrated preparation.

[0066] Beneficial effects

[0067] The present invention uses lanthanide rare earths as matrix materials and incorporates rare earth radioactive medical nuclides into their crystal lattices using a modified high-temperature thermal decomposition method to obtain a precursor for a rare earth radioactive diagnosis and treatment integrated preparation. The precursor is then functionally modified to prepare a rare earth radioactive diagnosis and treatment integrated preparation having the following advantages:

[0068] 1) The synthesis method is easy to control, has good reproducibility, and is highly universal. The prepared rare earth radioactive diagnostic and therapeutic integrated preparation has good dispersibility, maintains good uniformity in morphology and size, and can produce luminescence from the ultraviolet to the near-infrared region under near-infrared light excitation. For example, it can simultaneously achieve 808nm and 980nm near-infrared light excitation to produce rare earth ion fluorescence emission in the visible region of 400-1700nm to the near-infrared region II.

[0069] Lanthanide rare earth-based nanofluorescent materials are easily doped with rare earth medical isotopes. Lattice-doped medical isotopes can be stably present in the crystalline material, resulting in improved stability, reduced off-target effects, and enhanced biosafety. The integrated rare earth radioactive diagnostic and therapeutic preparation of this invention not only stably loads the medical isotope but also enables precise rare earth medical fluorescence imaging and localization of tumor lesions, enabling combined tumor treatment based on precise diagnosis.

[0070] 3) The surface of the precursor of the rare earth radioactive diagnostic and therapeutic integrated preparation is easy to modify and can be selectively covered with various functional tumor-targeting biological molecules, which not only gives the rare earth radioactive diagnostic and therapeutic integrated preparation good biocompatibility, but also gives it broad clinical transformation prospects.

[0071] 4) Mn 2+ The doping of Mn can achieve synergistic treatment with radionuclides, and achieve better therapeutic effects with lower doses of drugs. The rare earth radioactive diagnosis and treatment integrated preparation of the present invention can accurately deliver radionuclides; the rare earth radioactive diagnosis and treatment integrated preparation of the present invention can also effectively overcome the physical barriers of tumors by emitting high-energy rays from the decay of radionuclides, and can effectively overcome the physical barriers of tumors by emitting high-energy rays from the decay of radionuclides. 2+ It works in the tumor microenvironment to achieve combined therapy and better kill cancer cells.

[0072] 5) The rare earth radioactive diagnostic and therapeutic integrated preparation prepared by the present invention exhibits excellent radiochemical stability and good biosafety, and can be used safely in vivo. As a result, many applications related to clinical medicine have emerged, and it has both therapeutic and diagnostic functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is the X-ray powder diffraction pattern of the rare earth radioactive diagnosis and treatment integrated preparation in Example 1 of the present invention.

[0074] Figure 2 The rare earth radioactive diagnosis and treatment integrated preparation in Example 1 of the present invention is doped with different proportions of Mn 2+ Spectrum diagram of .

[0075] Figure 3 This is a transmission electron micrograph of the rare earth radioactive diagnosis and treatment integrated preparation in Example 1 of the present invention.

[0076] Figure 4 This is a transmission electron micrograph of the rare earth radioactive diagnosis and treatment integrated preparation in Example 1 of the present invention.

[0077] Figure 5 This is a diagram of the hydrated particle size of the rare earth radioactive diagnosis and treatment integrated preparation in Example 1 of the present invention.

[0078] Figure 6 This is a schematic diagram of the radiochemical stability of the rare earth radioactive diagnosis and treatment integrated preparation in Example 1 dispersed in physiological saline and fetal bovine serum in Experimental Example 1 of the present invention.

[0079] Figure 7 This is a diagram showing the effect of tumor-targeted fluorescence imaging of tumor cells in vitro using the rare earth radioactive diagnosis and treatment integrated preparation of Example 1 in Experimental Example 2 of the present invention.

[0080] Figure 8This is a biological electron microscopic image of tumor cells in vitro using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1 in Experimental Example 2 of the present invention.

[0081] Figure 9 This is a diagram showing the killing effect of the nanomaterials in Comparative Examples 1 and 2 on PANC-1 tumor cells in vitro in Experimental Example 2 of the present invention.

[0082] Figure 10 The present invention is experimental example 2 using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1, the nanomaterial in Comparative Example 1, 177 Figure 3 shows the killing effect of LuCl3 on PANC-1 tumor cells in vitro.

[0083] Figure 11 The present invention is experimental example 3 using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1, the nanomaterial in Comparative Example 1, 177 A diagram showing the effect of LuCl3 on the invasion ability of tumor cells.

[0084] Figure 12 The present invention is experimental example 4 using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1, the nanomaterial in Comparative Example 1, 177 Diagram of the effects of LuCl3 on tumor cell proliferation and cell population dependence.

[0085] Figure 13 The present invention is experimental example 5 using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1, the nanomaterial in Comparative Example 1, 177 Diagram of apoptosis in LuCl3 tumor cells.

[0086] Figure 14 The present invention is experimental example 6 using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1, the nanomaterial in Comparative Example 1, 177 Diagram of LuCl3 tumor cell pyroptosis.

[0087] Figure 15 This is a schematic diagram of the fluorescence imaging effect in nude mice 1, 24, 48, and 72 hours after administration of the rare earth radioactive diagnosis and treatment integrated preparation in Example 1 through the tail vein in Experimental Example 7 of the present invention.

[0088] Figure 16 The present invention is experimental example 8 using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1, the nanomaterial in Comparative Example 1, 177 Volume change curves of tumors in each group during the treatment of nude mice with LuCl3 and normal saline.

[0089] Figure 17 The present invention is experimental example 8 using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1, the nanomaterial in Comparative Example 1, 177Representative photos of nude mice in each group on the 1st, 7th and 15th days during the treatment of LuCl3 and normal saline.

[0090] Figure 18 The present invention is experimental example 8 using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1, the nanomaterial in Comparative Example 1, 177 Representative HE staining images of tumor tissues in each group after the treatment of nude mice with LuCl3 and normal saline.

[0091] Figure 19 The present invention is experimental example 8 using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1, the nanomaterial in Comparative Example 1, 177 Representative TUNEL results of tumor tissues in each group after the treatment of nude mice with LuCl3 and normal saline.

[0092] Figure 20 The present invention is experimental example 9 using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1, the nanomaterial in Comparative Example 1, 177 Body weight change curves of nude mice in each group during the treatment with LuCl3 and normal saline.

[0093] Figure 21 The present invention is experimental example 9 using the rare earth radioactive diagnosis and treatment integrated preparation in Example 1, the nanomaterial in Comparative Example 1, 177 Representative HE staining images of heart, liver, spleen, lung and kidney tissues of each group after the treatment of nude mice with LuCl3 and normal saline. DETAILED DESCRIPTION

[0094] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations 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 encompassed within the scope of protection that the present invention is intended to protect.

[0095] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0096] The instruments involved in the following embodiments are as follows:

[0097] X-ray powder diffractometer (instrument model MiniFlex2, manufacturer Rigaku), laser particle size analyzer (instrument model Zetasizer Ultra, manufacturer Malvern), transmission electron microscope (instrument model TECNAI G2 F20, manufacturer FEI), intelligent calibrator (instrument model Zhonghe FH463B, manufacturer Guangzhou Yitongxing Instrument Co., Ltd.), confocal microscope (instrument model A1+, manufacturer Nikon), small animal living fluorescence imaging system (instrument model Ninox 640SU, manufacturer Raptor Photonics).

[0098] Example 1:

[0099] (1)NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Preparation of Lu nanomaterials

[0100] Preparation of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4 based on high temperature thermal decomposition method: 177 Lu, specifically as follows:

[0101] (a) In a 100-mL three-necked flask, 0.136 g of sodium fluoroacetate, 0.3671 g of gadolinium trifluoroacetate, 0.0101 g of erbium trifluoroacetate, 0.0127 g of cerium trifluoroacetate, and 0.1024 g of ytterbium trifluoroacetate were added to a mixture of 3.17 mL of oleic acid, 3.29 mL of oleylamine, and 6.4 mL of octadecene. The mixture was evacuated and heated to 110°C to remove impurities such as adsorbed water and oxygen. After bubbling disappeared, the mixture was backfilled with high-purity nitrogen. Under an inert atmosphere, the temperature was continued to 310°C. After reacting for 0.5 hour, the heat source was removed. After cooling the reaction solution to below 70°C, ethanol was added for precipitation. The mixture was then centrifuged for 10 minutes, and the precipitate was dispersed in 10 mL of cyclohexane to obtain α-phase NaGdF4:Yb / Er / Ce nanocrystals.

[0102] (b) In a 100 mL three-necked flask, α-phase NaGdF4:Yb / Er / Ce nanocrystals, 0.068 g sodium trifluoroacetate, 0.1835 g gadolinium trifluoroacetate, 0.005 g erbium trifluoroacetate, 0.0063 g cerium trifluoroacetate, and 0.0512 g ytterbium trifluoroacetate were added to a mixture of 6.4 mL of oleic acid and 6.4 mL of octadecene. The mixture was evacuated and heated to 110°C to remove impurities such as adsorbed water and oxygen. After bubbling disappeared, the mixture was backfilled with high-purity nitrogen. Under an inert atmosphere, the temperature was continued to 310°C. After reacting for 0.5 hour, the heat source was removed. After cooling the reaction solution to below 70°C, ethanol was added for precipitation. The mixture was then centrifuged for 10 minutes, and the precipitate was dispersed in 10 mL of cyclohexane to obtain β-phase NaGdF4:Yb / Er / Ce nanocrystals.

[0103] (c) In a 100-mL three-necked flask, β-phase NaGdF4:Yb / Er / Ce nanocrystals, 0.136 g of sodium trifluoroacetate, and 0.512 g of ytterbium trifluoroacetate were added to a mixed solvent of 6.4 mL of oleic acid and 6.4 mL of octadecene. The mixture was evacuated and heated to 110°C to remove impurities such as adsorbed water and oxygen. After bubbling disappeared, the mixture was backfilled with high-purity nitrogen. Under an inert atmosphere, the temperature was raised to 310°C. After reacting for 0.5 hour, the heat source was removed. After cooling the reaction solution to below 70°C, ethanol was added for precipitation. The mixture was then centrifuged for 10 minutes, and the precipitate was dispersed in 10 mL of cyclohexane to obtain NaGdF4:Yb / Er / Ce@NaYbF4 nanocrystals.

[0104] (d) In a 100-mL three-necked flask, NaGdF4:Yb / Er / Ce@NaYbF4 nanocrystals, 0.136 g sodium trifluoroacetate, 0.3864 g neodymium trifluoroacetate, 0.0512 g ytterbium trifluoroacetate, and 0.024 g manganese acetate were added to a mixture of 6.4 mL of oleic acid and 6.4 mL of octadecene. The mixture was evacuated and heated to 110°C to remove impurities such as adsorbed water and oxygen. After bubbling disappeared, the mixture was backfilled with high-purity nitrogen. Under an inert atmosphere, the temperature was further raised to 310°C. After reacting for 0.5 h, the heat source was removed. After cooling the reaction solution to below 70°C, ethanol was added for precipitation. The mixture was then centrifuged for 10 min, and the precipitate was dispersed in 10 mL of cyclohexane to obtain NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb nanocrystals.

[0105] (e) In a 100 mL three-necked flask, NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb, 0.136 g sodium trifluoroacetate, 0.4279 g yttrium trifluoroacetate, 3 mCi 177LuCl3 was added to a mixed solvent of 6.4 mL of oleic acid and 6.4 mL of octadecene. The mixture was evacuated and heated to 110°C to remove impurities such as adsorbed water and oxygen. High-purity nitrogen was then backfilled until no bubbles appeared in the solution. The temperature was then raised to 310°C under an inert atmosphere. After reacting for 0.5 hours, the heat source was removed. After the reaction solution cooled to below 70°C, ethanol was added for precipitation. The mixture was then centrifuged for 10 minutes. The precipitate was dispersed in 10 mL of cyclohexane to obtain NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu nanocrystals, spare.

[0106] (2) For NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu nanocrystals coated with functional tumor-targeting biomolecules:

[0107] Take 1mL NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu nanocrystal cyclohexane dispersion was dispersed in a mixed solution consisting of 2 mL of cyclohexane and 10 mL of chloroform. Distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and its peptide derivative (10 times the mass of the nanocrystals, where the hydrophilic monomer is DSPE-PEG and the peptide derivative is a pancreatic cancer-targeting peptide, with a mass ratio of 10:1) were rapidly added while stirring until the solvent evaporated naturally. An appropriate amount of pure water was added, and the resulting solid was dispersed in water by sonication. The reaction solution was subjected to high-speed centrifugation and washed to obtain a core-shell-shell-shell structured rare earth radioactive diagnostic and therapeutic integrated preparation, which was dispersed and stored in normal saline.

[0108] Depend on Figure 1 It can be seen that the rare earth radioactive diagnosis and treatment integrated preparation prepared in this embodiment is a hexagonal phase. Figure 2 It can be seen that Mn 2+ Doping significantly enhances the luminescence properties of nanoparticles. Figure 3-4 It can be seen that the rare earth radioactive diagnosis and treatment integrated preparation prepared in this embodiment has good dispersibility and maintains good uniformity in morphology and size. The size of the nanoparticles of the rare earth radioactive diagnosis and treatment integrated preparation is about 40nm (e.g. Figure 4 As shown), the hydrated particle size is 86nm (as Figure 5 shown).

[0109] Comparative Example 1:

[0110] Preparation of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI nanomaterials

[0111] The preparation method of the nanomaterial in this comparative example is the same as that in Example 1, except that no 177 LuCl3; to prepare the nanomaterial NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI of this comparative example.

[0112] Comparative Example 2

[0113] Preparation of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Yb@NaYF4@LI nanomaterials

[0114] The preparation method of the nanomaterial in this comparative example is the same as that in Example 1, the only difference is that manganese acetate is not added in step (d), and step (e) is not added. 177 LuCl3; Prepare the nanomaterial of this comparative example

[0115] NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Yb@NaYF4@LI.

[0116] Experimental Example 1

[0117] For NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4 prepared in Example 1: 177 Testing of the radiological stability of Lu@LI rare earth radioactive diagnostic and therapeutic integrated preparations:

[0118] 1.5MBq of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu@LI was mixed with equal volumes of physiological saline and fetal bovine serum, incubated at 37°C, and the supernatant was removed by ultrafiltration tube centrifugation at 0.5, 1, 2, 4, 8, 12, 24, and 48 hours, respectively. The radiostability of the material was analyzed using an intelligent calibrator.

[0119] like Figure 6 As shown, it can be seen that the rare earth radioactive diagnosis and treatment integrated preparation prepared in Example 1 of the present invention has good stability in both normal saline and fetal bovine serum, and the activities after incubation at 37°C for 48 hours are 95.83% and 95.99% of the initial values, respectively.

[0120] Experimental Example 2

[0121] Tumor targeting effect of rare earth radioactive diagnostic and therapeutic integrated preparations:

[0122] (1)NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu@LI cell fluorescence imaging to detect tumor targeting of nanomaterials

[0123] PANC-1 cells were cultured at 2×10 4 / well density in the culture dish, place in the incubator to culture for a period of time, after they are completely attached to the wall, aspirate and discard the original DMEM complete medium, wash once with PBS, and add complete medium to each dish. Add 10μL of 20mg / mL NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu@LI, shake well, incubate at 37℃ for a period of time, discard the old DMEM complete medium, wash twice with PBS, add 1 mL of 4% paraformaldehyde to fix in the dark for 10 minutes, wash once with PBS, then stain the nucleus with DAPI for 5 minutes, wash twice with PBS on a shaker for 3 minutes each time, and observe the cell fluorescence under a confocal microscope.

[0124] from Figure 7 It can be seen that the rare earth radioactive diagnosis and treatment integrated preparation has a good targeting ability for PANC-1 tumor cells, and the rare earth radioactive diagnosis and treatment integrated preparation can be targeted and enter the cytoplasm in a short time.

[0125] (2)NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu@LI biological electron microscopy detects tumor targeting of nanomaterials

[0126] Take cells in the logarithmic growth phase, digest with trypsin, and disperse into single cell suspension with DMEM complete medium. Inoculate into 10 cm culture dish, place incubator at 37℃, 5% CO2 to incubate.

[0127] NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu@LI, with a concentration of 150 μg / mL, was incubated for 0 h, 4 h, 8 h, and 24 h. The cells were collected by centrifugation after trypsin digestion, and electron microscopy fixative was added to disperse the cells. The cells were placed in a 4°C refrigerator for 5 minutes, centrifuged, and electron microscopy fixative was added. After sample preparation, the entry of the nanomaterial into the cells was observed using a transmission electron microscope.

[0128] like Figure 8 As shown, it can be seen that the nanoparticles entered the cytoplasm after incubation for 4 hours, 8 hours, and 24 hours, further illustrating that the NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4 of Example 1:177 Lu@LI has good targeting ability to PANC-1 cells.

[0129] (3)NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu@LI in vitro cell killing assay

[0130] a. The cell cytotoxicity of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI (denoted as the NPs group) and NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Yb@NaYF4@LI (denoted as the NPs without Mn group) was determined using the CCK8 assay. The specific steps are as follows:

[0131] PANC-1 cells were cultured at 3×10 4 / well of a 96-well plate and incubated overnight at 37°C under 5% CO2. After the cells were completely attached, 10 μL of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI (NPs) and NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI (NPs without Mn) were added respectively. After incubation at 37°C under 5% CO2 for 24 h, the old DMEM complete medium was discarded and replaced with fresh DMEM blank medium. After that, 10 μL of CCK-8 working solution was added to each well and the cells were incubated in the incubator for another 2 h. The absorbance of each group was measured at 450 nm using a microplate reader and recorded as the OD value of the drug-treated group. The untreated cells were used as the control group, and the cell viability was recorded as 100%. The cell proliferation activity of each group was calculated as follows: cell viability (%) = [(OD value of the drug-treated group - OD value of the blank group) / (OD value of the control group - OD value of the blank group)] × 100%.

[0132] Under the same dosage conditions, Figure 9 It is known that rare earth inorganic nanocrystals doped with Mn 2+ Later it has a stronger killing power.

[0133] b. Determination of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4 by CCK8 method: 177 Lu@LI (denoted as NPs- 177Lu group), NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI (denoted as NPs group), 177 LuCl3 (denoted as 177 Lu group) cell killing ability, the specific steps are as follows:

[0134] PANC-1 cells were cultured at 3×10 4 The cells were seeded at a density of 10 μg / well in a 96-well plate and incubated overnight at 37°C under 5% CO2. After the cells were completely attached, 10 μL of different doses (0 μCi, 0.17 μCi, 0.34 μCi, 0.68 μCi, 1.35 μCi, 2.7 μCi and 5.4 μCi) of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu@LI(NPs- 177 Lu), 10 μL of different doses (0 μCi, 0.17 μCi, 0.34 μCi, 0.68 μCi, 1.35 μCi, 2.7 μCi and 5.4 μCi) 177 LuCl3( 177 Lu), 10 μL of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI(NPs) at different concentrations (0 μg / ml, 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 400 μg / ml), incubated at 37°C, 5% CO2 for 24 h, discarded the old DMEM complete medium, replaced with fresh DMEM blank medium, added 10 μL CCK-8 working solution to each well, and continued incubation in the incubator for 2 h. The absorbance of each group at 450 nm was measured using a microplate reader and recorded as the OD value of the drug-treated group. The untreated cells were used as the control group, and their cell viability was recorded as 100%. The cell proliferation activity of each group was calculated as follows: cell viability (%) = [(OD value of the drug-treated group - OD value of the blank group) / (OD value of the control group - OD value of the control blank group)] × 100%. The control blank group refers to the group in which only DMEM culture medium was added; the blank group refers to the corresponding drug-treated group in which no PANC-1 cells were added.

[0135] The blank groups corresponding to the NPS-177Lu-doped groups were prepared by adding different doses (0 μCi, 0.17 μCi, 0.34 μCi, 0.68 μCi, 1.35 μCi, 2.7 μCi and 5.4 μCi) of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4 into DMEM culture medium. 177 Lu@LI;

[0136] 177 The blank groups corresponding to the Lu group were prepared by adding 10 μL of 177LuCl3 of different concentrations (0 μCi, 0.17 μCi, 0.34 μCi, 0.68 μCi, 1.35 μCi, 2.7 μCi, and 5.4 μCi) into DMEM medium;

[0137] The blank group corresponding to the NPs-doped group was composed of 10 μL of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI (NPs) with different concentrations (0 μg / ml, 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml) added to the DMEM culture medium.

[0138] Under the same dosage conditions, NPs without radionuclide (400 μg / mL) and pure radionuclide (5.4 μCi) had almost no killing effect on PANC-1 cells, and the cell survival rates of both were above 80% ( Figure 10 ), while NPs- 177 Lu (400 μg / mL, 5.4 μCi) reduced the survival rate of PANC-1 cells to 59.7% after 24 hours of action. Therefore, it can be seen that the rare earth radioactive diagnosis and treatment integrated preparation of the present invention has a strong killing ability on PANC-1 cells.

[0139] Experimental Example 3

[0140] Rare earth radioactive diagnosis and treatment integrated preparation NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 The effect of Lu@LI on the invasion ability of tumor cells was tested as follows:

[0141] Starve the cells in advance with serum-free medium. Dilute Matrigel:DMEM in a ratio of 1:8 on ice, take 50 μL and add it to the upper chamber. Incubate overnight in a 37°C, 5% CO2 incubator. The next day, discard the liquid in the upper chamber and add 100 μL of serum-free medium to hydrate at 37°C for 30 minutes. Aspirate and discard before adding the cell solution. Digest the starved cells, resuspend them in serum-free medium, and count to 2×10 5 / ml, add 600μL complete culture medium to the lower chamber and place it in the upper chamber. Drop 200μL cell suspension into the upper chamber, evenly tap the four sides of the culture plate to allow the liquid to spread completely, let it stand for 15 minutes and then place it in the incubator. 200μg / mL of drug was administered respectively, and a blank group (denoted as Con) and

[0142] NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI group (denoted as NPs group), 177 LuCl3 group (denoted as 177 Lu group), NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu@LI group (denoted as NPs- 177 Lu group), each well was added with the same dose of different nanocomplex solutions (i.e., 200 μg / mL NPs solution, 2.7 μCi 177 LuCl3 solution and 200 μg / mL, 2.7 μCi NPs- 177 Lu solution) for 12-48 hours. After incubation, aspirate the liquid from the upper and lower chambers, wash twice with PBS, add 1 ml of paraformaldehyde to a clean air chamber, place the chamber in the chamber, and fix for 10 minutes. The fixative is aspirated and the chamber is rinsed with PBS. Add 500 μL of 0.1% crystal violet stain to a 24-well plate and place the chamber in it. Immerse the chamber bottom in the crystal violet stain for 20 minutes. After rinsing with ultrapure water, wipe the cells on the upper side of the upper chamber with a cotton swab. Add a small amount of PBS to a glass slide, place the chamber on it, and photograph and count five fields of view (top, bottom, left, right, and center) on the diameter.

[0143] Under the same dosage conditions, NPs group, 177 LuCl3 group, and NPs- 177 Compared with the blank group, the Lu group had a negative effect on the cell invasion ability, and NPs- 177 The number of cells invading in the Lu group was the least ( Figure 11 ). It can be seen that the rare earth radioactive diagnosis and treatment integrated preparation of the present invention significantly reduced the invasive ability of PANC-1 cells.

[0144] Experimental Example 4

[0145] Rare earth radioactive diagnosis and treatment integrated preparation NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 The effect of Lu@LI on tumor cell proliferation and cell population dependence was tested as follows:

[0146] PANC-1 cells in the exponential growth phase were obtained, digested with 0.25% trypsin and blown into single cells, and the cells were suspended in DMEM culture medium containing 10% fetal bovine serum for later use. The cell suspension was diluted in gradient multiples, and each group of cells was inoculated into a six-well plate containing 2mL complete culture medium at a gradient density of 500 cells per dish, and gently rotated to disperse the cells evenly. The cells were cultured in a cell culture incubator at 37℃, 5% CO2 and saturated humidity to allow the cells to adhere to the wall. After the cells adhered to the wall, the medium was changed to set up a blank control group, a NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI group (referred to as the NPs group), 177 LuCl3 group ( 177 Lu group), NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu@LI group (NPs- 177 Lu group), each well was added with the same dose of different nanocomplex solutions (i.e., 200 μg / mL NPs solution, 2.7 μCi 177 LuCl3 solution and 200 μg / mL, 2.7 μCi NPs- 177 Lu's solution) and culture in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 2 to 3 weeks. Change the medium every three days, and administer the drug again after the medium change. Terminate the culture when visible colonies appear in the culture dish. Discard the supernatant and carefully rinse the cells twice with PBS. Fix the cells with 1 mL of 4% paraformaldehyde for 10 minutes. Then remove the fixative and add 1 mL of 0.1% crystal violet stain for 20 minutes. Then slowly wash away the stain with PBS and air dry. Count colonies containing 50 or more cells under a microscope.

[0147] like Figure 12 As shown in the figure on the left, the vertical direction is three parallel replicates of the experiment, which shows that under the same dose (200ug / ml, 2.7μCi), NPs- 177 The number of clones in the Lu group was significantly lower than that in the blank control group, NPs group, 177 It can be seen from this that the rare earth radioactive diagnosis and treatment integrated preparation of the present invention significantly reduces the cell proliferation ability and cell population dependence.

[0148] Experimental Example 5

[0149] Rare earth radioactive diagnosis and treatment integrated preparation NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 The effect of Lu@LI on tumor cell apoptosis was tested as follows:

[0150] PANC-1 cells in logarithmic growth phase were taken, washed twice with PBS, digested with trypsin, and dispersed into single cell suspension in complete medium. Each cell line was cultured at 1.5×10 5 Cells were seeded into 6-well plates at a density of 100 cells / well, with 2 mL of culture medium per well, and cultured in a 37°C, 5% CO2 incubator for 24 h. The original culture medium was aspirated and 2 mL of fresh complete culture medium was added to each well.

[0151] A negative control group, a NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI group (NPs group), 177 LuCl3 group ( 177 Lu group), NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu@LI group (NPs- 177 Lu group), each well was added with the same dose of different nanocomplex solutions (i.e., 200 μg / mL NPs solution, 2.7 μCi 177 LuCl3 solution and 200 μg / mL, 2.7 μCi NPs- 177 Lu's solution), and continue to culture in the incubator for 24 hours. Collect the old cell culture fluid into a clean centrifuge tube for later use. Wash once with PBS, digest the cells with trypsin, add the collected cell culture fluid, gently blow off the cells, and collect them into the centrifuge tube again. Centrifuge the precipitated cells. Carefully aspirate the supernatant, add 1mL of pre-cooled PBS, resuspend the cells, and transfer to a 1.5mL centrifuge tube and centrifuge for 5 minutes. Wash the cells 1-2 times with PBS solution, dilute Bing Buffer 1:4 with deionized water, add 100μL of the above solution to the centrifuged cells and blow them away to obtain a cell suspension. Transfer the above cell suspension to a flow tube, stain, gently mix and resuspend the cells, incubate at 37℃ in the dark for 30 minutes, and detect cell apoptosis by flow cytometry.

[0152] like Figure 13 As shown, under the same dosage conditions, the apoptosis rate of the blank control group was 10.33%, and the apoptosis rate of the NPs group was 14.57%. 177 The apoptosis rate in the Lu group was 13.22%, and that in the NPs- 177 The apoptosis rate of the Lu group was 22.52%. Therefore, it can be seen that the rare earth radioactive diagnosis and treatment integrated preparation of the present invention significantly promotes the apoptosis of tumor cells.

[0153] Experimental Example 6

[0154] Rare earth radioactive diagnosis and treatment integrated preparation NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177The effect of Lu@LI on tumor cell pyroptosis was tested as follows:

[0155] Add 20 μL of culture medium to a 12-well plate, pick up the slide with tweezers, put it into the well after burning it with an alcohol burner, digest the cells with trypsin, resuspend the cells in complete culture medium, pipette to a single cell suspension, inoculate cells at a certain density in a 12-well plate, culture them in a 37°C, 5% CO2 incubator overnight to adhere to the wall, discard the supernatant, add

[0156] NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4@LI (denoted as NPs group), 177 LuCl3 (denoted as 177 Lu group), NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Lu@LI (denoted as NPs- 177 Lu group) culture medium, set up a blank control group (i.e., only culture medium was added), continue incubation for a certain time (24h, 48h), discard the supernatant, wash twice with PBS, add electron microscopy fixative, store at 4℃, and observe the cell morphology with scanning electron microscopy after sample preparation.

[0157] like Figure 14 As shown, under the same dosage conditions, the blank control group, NPs group, 177 The cell morphology of Lu group was normal, while that of NPs- 177 In the Lu group, cells formed holes in the cell membrane, swelled until the cell membrane ruptured, and formed inflammasomes. This indicates that the rare earth radioactive diagnosis and treatment integrated preparation of the present invention significantly promoted pyroptosis of tumor cells.

[0158] Experimental Example 7

[0159] NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 The fluorescence imaging effect of Lu@LI rare earth radioactive diagnosis and treatment integrated preparation, the testing process is as follows:

[0160] Nude mice bearing PANC-1 subcutaneous tumors were injected with 100 mg / kg of the rare earth radioactive diagnostic and therapeutic integrated preparation prepared in Example 1 through the tail vein. Fluorescence imaging was performed at 1, 24, 48, and 72 hours to observe its in vivo fluorescence imaging effect, distribution, and metabolism:

[0161] In the small animal in vivo fluorescence imaging system, 808nm and 980nm laser excitation imaging was used, and the results were as follows Figure 15As shown, within the first hour of injection, the capillaries throughout the mouse body could be clearly observed with a resolution of tens of microns. The fluorescent signal from the imaging revealed that the blood was primarily distributed throughout the nude mouse. 24 hours after injection, the fluorescent signal was essentially non-existent in the blood, primarily distributed in organs like the liver and spleen, indicating that metabolism had begun outside the body. After 72 hours, the signal was virtually invisible, indicating that most of the rare earth radioactive diagnostic and therapeutic agent had been metabolized out of the body.

[0162] Experimental Example 8

[0163] NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 In vivo evaluation of the anti-tumor effect of Lu@LI rare earth radiotherapy integrated preparation:

[0164] Nude mice bearing PANC-1 subcutaneous tumors were randomly divided into 4 groups (6 mice in each group) and treated with NS (0.9% NaCl), 177 LuCl3 (100 μCi), NPs (150 mg / kg) and NPs- 177 The mice were treated with Lu (100 μCi, 150 mg / kg) by intratumoral injection and were designated as NS group, 177 LuCl3 (100 μCi) group, NPs group and NPs- 177 Lu group. During the treatment period, the tumor size and weight of nude mice were measured every 2 days. 3 Alternatively, nude mice were sacrificed if their body weight decreased by more than 20%. At the end of the observation period, nude mice were euthanized and vital organs, including the heart, liver, spleen, lungs, kidneys, and tumors, were carefully isolated and completely immersed in 4% paraformaldehyde fixative. The fixative was then stored at 4°C. After the fixation was completely depleted of radioactivity, tumor tissue was collected for HE and TUNEL analysis.

[0165] According to the tumor growth curve during treatment ( Figure 16 ) and the tumor treatment effect diagram of each group ( Figure 17 ), NS group, NPs group and 177 The average tumor volume of the LuCl3 (100 μCi) group continued to increase. In the early stage of treatment, NPs- 177 The average tumor volume of the Lu group also showed a slow increasing trend, while after the 7th day, the average tumor volume of the NS group, NPs group and 177 The average tumor volume of the LuCl3 (100 μCi) group still showed a rapid growth trend, and the NPs- 177 The Lu group began to slowly decrease, accompanied by the phenomenon of scab shedding, and by the end of the treatment period, the tumor had almost disappeared. Figure 18) The results show that: the nuclear morphology of tumor cells in the normal saline group was intact and the cell density was high; in the NPs group, a small area had vacuoles formed by cell apoptosis, and the tumor cell density was also slightly lower; 177 The tumor cell density in the LuCl3 group was slightly lower, and there were also vacuoles formed by apoptosis in a small part of the area. 177 There were large areas of vacuoles formed by apoptosis of tumor cells in the tumor site of the Lu group. TUNEL test of tumor tissue ( Figure 19 ) can show the apoptosis of tumor cells. The TUNEL method can perform in situ staining of intact single apoptotic cell nuclei or apoptotic bodies, accurately reflecting the most typical biochemical and morphological characteristics of apoptotic cells, and can detect very small amounts of apoptotic cells. Figure 19 It can be seen that there are a few apoptotic cells in the saline group, which is caused by normal cell apoptosis. 177 The LuCl3 group also showed only a small amount of apoptosis, in sharp contrast to the NPs- 177 The Lu group showed a large area of ​​green fluorescence, indicating a large number of apoptotic cells.

[0166] Therefore, the above experimental results can show that the rare earth radioactive diagnosis and treatment integrated preparation prepared by the present invention has good in vivo anti-tumor ability.

[0167] Experimental Example 9

[0168] NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 The biosafety of Lu@LI rare earth radioactive diagnostic and therapeutic integrated preparations is tested as follows:

[0169] In Experimental Example 8, when evaluating the in vivo anti-tumor ability, the tumor volume and the weight change of the nude mice were recorded to evaluate the effect of NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4: 177 Biosafety of Lu@LI: After the treatment period, vital organs such as the heart, liver, spleen, lungs, and kidneys were carefully isolated and fixed with 4% paraformaldehyde for histopathological examination.

[0170] Figure 20 The figure shows the weight change trend of nude mice in each group during the treatment. Weight is an important indicator of the health status of nude mice. As can be seen from the figure, the weight of all nude mice showed an upward trend during the treatment, which shows that our drug did not cause significant harm to the nude mice. Figure 21 In order to select representative pathological sections of nude mice tissues from each group, we observed the main organs of nude mice in each group and found that the NPs group, 177 LuCl3 group and NPs-177 There was no significant difference between the Lu group and the negative control group, which proved that the rare earth radioactive diagnosis and treatment integrated preparation prepared by the present invention has good biosafety.

[0171] The above describes exemplary embodiments of the present invention. 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 in the scope of protection of the present invention.

Claims

1. A manganese ion-synergistic rare earth radioactive diagnosis and treatment integrated preparation, characterized in that: The method comprises rare earth-doped inorganic nanocrystals, radioactive medical isotopes R and functional tumor-targeting biomolecules LI, wherein the functional tumor-targeting biomolecules LI are loaded on the surface of the rare earth inorganic nanocrystals; The rare earth-doped inorganic nanocrystal has a core-shell-shell-shell structure, which includes, from the inside to the outside, a rare earth ion core, a rare earth ion energy transfer layer, a rare earth ion energy absorption layer, and a rare earth ion inert protective layer; the rare earth-doped inorganic nanocrystal includes at least manganese element Mn and rare earth luminescent ions Em; Em is a rare earth luminescent ion, including one or more of Er, Yb, and Ce; The rare earth luminescent ions Em are distributed in the rare earth ion core rare earth ion energy absorption layer of the rare earth doped inorganic nanocrystal; The rare earth doped inorganic nanocrystals are NaGdF4:Yb / Er / Ce@NaYbF4@NaNdF4:Mn / Yb@NaYF4; Radioactive medical isotopes R are selected from 177 Lu; The radioactive medical isotope R is distributed in the rare earth ion inert protective layer.

2. The manganese ion-synergistic rare earth radioactive diagnosis and treatment integrated preparation according to claim 1, characterized in that The functional tumor-targeting biomolecule is used to enhance the biocompatibility of the manganese ion-synergistic rare earth radioactive diagnosis and treatment integrated preparation, while making the rare earth radioactive diagnosis and treatment integrated preparation have tumor targeting properties; The functional tumor-targeting biological molecules are partially or completely coated on the surface of the rare earth-doped inorganic nanocrystals.

3. The manganese ion-synergistic rare earth radioactive diagnosis and treatment integrated preparation according to claim 1, characterized in that: The functional tumor-targeting biomolecule LI is prepared from an amphiphilic ligand without tumor-specific recognition ability and a biomolecule with tumor-specific recognition ability; the mass ratio of the water-soluble ligand without tumor-specific recognition ability to the biomolecule with tumor-specific recognition ability is 1-50:1; The biomolecules having tumor-specific recognition capabilities are selected from one or more of biosmall molecules, antibodies, and polypeptides; The amphiphilic ligand without tumor-specific recognition ability; The amphiphilic ligand includes a hydrophilic group, and the hydrophilic group is at least one or more of ethanolamine, polyethylene glycol, polyvinyl pyrrolidone, and polyvinyl alcohol; The mass ratio of the rare earth-doped inorganic nanocrystals to the functional tumor-targeting biomolecule LI is 1:2-10; The functional tumor-targeting biomolecule is located on the surface of the rare earth-doped inorganic nanocrystal; The thickness of the functional tumor-targeting biomolecules on the surface of the rare earth-doped inorganic nanocrystals is 5-150 nm; The rare earth doped inorganic nanocrystals have a size of 5 to 70 nm; The size of the rare earth radioactive diagnosis and treatment integrated preparation is 20-300 nm.

4. The method for preparing the manganese ion-synergistic rare earth radioactive diagnosis and treatment integrated preparation according to any one of claims 1 to 3, characterized in that: The preparation method comprises: (1) preparing the rare earth ion core; (2) A rare earth ion energy transfer layer, a rare earth ion energy absorption layer, and a rare earth ion inert protective layer are sequentially grown on the surface of the rare earth ion core to obtain core-shell-shell-shell structured nanocrystals; (3) loading functional tumor-targeting biomolecules onto the surface of the core-shell-shell-shell structured nanocrystals through water-soluble modification to obtain the rare earth radioactive diagnosis and treatment integrated preparation; In step (1), rare earth luminescent ions Em are also added; In step (3), a radioactive medical isotope R is also added.

5. The preparation method according to claim 4, characterized in that The method for preparing the manganese ion-synergistic rare earth radioactive diagnosis and treatment integrated preparation comprises the following steps: (a) adding a stable rare earth source and an alkali metal source to a mixed solvent, mixing and dissolving them, and reacting to obtain a rare earth ion core; (b) adding the rare earth ion core, the stable rare earth source, and the alkali metal source obtained in step (a) into a mixed solvent, mixing and dissolving them, and reacting to obtain core-shell structured nanocrystals; (c) adding the core-shell structured nanocrystals obtained in step (b), a stable rare earth source, an alkali metal source, and a manganese source into a mixed solvent, mixing and dissolving them, and reacting to obtain core-shell structured nanocrystals; (d) adding the core-shell-shell structured nanocrystals obtained in step (c), a stable rare earth source, and an alkali metal source into a mixed solvent, mixing and dissolving them, and reacting to obtain core-shell-shell-shell structured nanocrystals; (e) modifying the surface of the core-shell-shell-shell structured nanocrystals obtained in step (d) with functional tumor-targeting biomolecules to obtain the rare earth radioactive diagnosis and treatment integrated preparation; Adding radioactive medical isotope R in step (d); Rare earth luminescent ions Em are added in the steps (a) and (d).

6. The preparation method according to claim 5, characterized in that The dissolution temperature in steps (a), (b), (c) and (d) is 80-160°C; In steps (a), (b), (c) and (d), the reaction temperature is 250-350° C. and the reaction time is 0.5-1.5 hours; The steps (a), (b), (c) and (d) are carried out in an inert atmosphere free of water and oxygen; In step (a), step (b) and step (c), the reaction is a constant temperature reaction, and the reaction temperature fluctuation is within ±5°C; The heating rate in step (a), step (b) and step (c) is 1-20°C / min.

7. The preparation method according to claim 5, characterized in that The stable rare earth source is selected from at least one of trifluoroacetate of rare earth ion Ln, acetate of rare earth ion Ln, chloride of rare earth ion Ln and nitrate of rare earth ion Ln; the rare earth element Ln is selected from one or more of Y, Nd, Gd and Yb; The alkali metal source is selected from at least one or more of trifluoroacetate containing alkali metal element Na, oleate containing alkali metal element Na, acetate containing alkali metal element Na, hydroxide containing alkali metal element Na, fluoride containing alkali metal element Na and hydrofluoride containing alkali metal element Na; The manganese source is selected from one or more of the following salts: one or more of manganese nitrate, manganese sulfate, manganese acetate, and manganese chloride; The mixed solvent comprises a long alkane chain organic acid and a long alkane chain olefin; the volume ratio of the long alkane chain organic acid to the long alkane chain olefin is 3-30 mL:3-30 mL; The long alkane chain organic acid is selected from at least one of octanoic acid, dodecanoic acid and oleic acid; The long alkane chain olefin is selected from 1-dodecene and / or 1-octadecene; The radioactive medical isotope R is provided by the oxalate of the radioisotope and / or the chloride of the radioisotope; The molar volume ratio of the rare earth element Ln and the mixed solvent in the stable rare earth source is 0.01-1 mmol: 6-60 mL; The radioactivity molar ratio of the radioactive medical isotope R to the rare earth element Ln in the stable rare earth source is 0.01-200 mCi: 0.01-0.5 mmol; In steps (a) to (d), a washing solvent is added after the reaction to precipitate the product and then separate it, and the washing solvent is selected from one or more mixed solvents of methanol, ethanol, and acetone; The mass ratio of the core-shell-shell-shell structured nanocrystals to the functional tumor-targeting biological molecules is 1-4:4-16.

8. Use of the manganese ion-synergistic rare earth radioactive diagnosis and treatment integrated preparation according to any one of claims 1 to 3 in the preparation of fluorescent medical imaging agents.

9. A developer, comprising the manganese ion-synergistic rare earth radioactive diagnosis and treatment integrated preparation according to any one of claims 1 to 3.

10. A tumor radiotherapy agent, comprising the manganese ion-synergistic rare earth radiotherapy integrated diagnosis and treatment preparation according to any one of claims 1 to 3.

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