A rare earth nanodiagnostic agent for radioactive-photodynamic synergistic therapy and its preparation method and application

Abstract

The present invention discloses a rare earth nano-diagnostic agent for radioactive-photodynamic synergistic therapy, its preparation method, and application. The nano-diagnostic agent of the present invention comprises a nano-material and a radionuclide, wherein the radionuclide is distributed on the nano-material; the nano-material has a core-shell nanostructure; the nano-material comprises, from the inside to the outside, a rare earth ion luminescent core ALn1F4:Ln2 3+ , rare earth ion inert layer ALn1F4 and rare earth ion energy transfer layer ALn1F4:Ln3 3+ , and its surface also carries photosensitizers and modified water-soluble molecules. The rare earth nano-diagnostic agent of the present invention can be excited by near-infrared light waves and perform fluorescence imaging at NIR-IIb (1525nm); under 808nm excitation, the rare earth nano-diagnostic agent of the present invention can perform photodynamic therapy; at the same time, the rare earth nano-diagnostic agent of the present invention is loaded with radioactive nuclides 177 Lu, can achieve radio-photodynamic therapy for tumors under the guidance of fluorescence imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-biomaterials, and in particular relates to a rare earth nano-diagnostic agent for radioactive-photodynamic synergistic therapy, a preparation method thereof, and an application thereof. Background Art

[0002] In recent years, the incidence of cancer has continued to rise, becoming a major threat to human health. Tumor progression is a complex physiological process involving multiple factors. Tumors can utilize various regulatory mechanisms to evade apoptosis, making them difficult to cure and prone to recurrence. Current cancer treatments include surgery, radiotherapy, chemotherapy, and immunotherapy. However, single treatments often have limitations, and cancer treatment often requires a multifaceted, multidisciplinary approach.

[0003] Photodynamic therapy is a new type of tumor treatment method that relies on the activation of photosensitizers in tumor tissues by irradiating light sources of specific wavelengths to produce biotoxic singlet oxygen and other reactive oxygen species, which then oxidize and damage tumor cells to achieve the therapeutic purpose. However, most photosensitizers currently require the use of ultraviolet-visible light (200-700nm) for excitation. Light sources of this wavelength have limited tissue penetration and cannot exert their diagnostic and therapeutic performance. Near-infrared light has a strong tissue penetration depth and spatial resolution, so the use of near-infrared light for photosensitization to produce singlet oxygen has broad application prospects. However, existing near-infrared photosensitizers / photosensitive materials have some problems: (1) The molecular synthesis process of near-infrared photosensitizers is complex, and the efficiency of singlet oxygen generation cannot be guaranteed; (2) Based on the upconversion process, photosensitizers are sensitized by fluorescence resonance energy transfer, but the extremely low upconversion efficiency limits the application of this method; (3) It is difficult to visualize, and the integration of diagnosis and treatment cannot be achieved.

[0004] Radiation therapy for tumors uses high-energy radiation (such as α, β, γ, and X-rays) generated by the decay of radionuclides to irradiate localized tumor lesions and remove them. It has a wide range of indications, is non-invasive, and can precisely kill tumor cells while minimizing damage to normal tissue, making it a promising treatment option. Combining radiation therapy with photodynamic therapy, which works synergistically, offers enhanced tumor treatment efficacy.

[0005] Therefore, a rare earth nano-diagnostic and therapeutic agent for radioactive-photodynamic synergistic therapy was designed, that is, fluorescence imaging was performed at NIR-IIb (1525nm) under 980nm laser irradiation, and photodynamic therapy was performed under 808nm excitation. At the same time, it was loaded with radionuclides, which can achieve high signal-to-noise ratio imaging of tumors in the body and produce efficient therapeutic effects, realizing integrated diagnosis and treatment. 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 nano-diagnostic agent, comprising a nano-material and a radioactive nuclide, wherein the radioactive nuclide is distributed on the nano-material; the nano-material has a core-shell nano-structure; the nano-material comprises, from the inside to the outside, a rare earth ion luminescent core ALn1F4:Ln2 3+ , rare earth ion inert layer ALn1F4 and rare earth ion energy transfer layer ALn1F4:Ln3 3+ ;in,

[0008] A is a metal element selected from one or more of Li, Na, K, Rb, Cs, Mg, Ca, Sr or Ba;

[0009] Ln1, Ln2, and Ln3 are selected from stable rare earth elements, and Ln1, Ln2, and Ln3 are the same or different and are independently selected from one or more of Y, Sc, Yb, Er, Tm, Ho, Gd, Ce, Eu, Tb, Sm, Dy, Ce, Nd, La, Pr, and Lu, preferably one or more of Yb, Er, Tm, Ho, Eu, Nd, Y, and Lu, for example, Yb and Er are co-doped.

[0010] According to an embodiment of the present invention, the distribution means that the radioactive nuclides can be doped in at least one of the rare earth ion luminescent core, the rare earth ion inert layer and the rare earth ion energy transfer layer in the nanomaterial, for example, doped in the rare earth ion luminescent core, and / or the rare earth ion inert layer, and / or the rare earth ion energy transfer layer, for example, doped in the rare earth ion inert layer.

[0011] According to an embodiment of the present invention, the radionuclide is selected from radioactive medical isotopes R, for example, selected from 90 Y. 177 Lu, 153 Sm, 111 In and 89 One or more of Zr, preferably 90 Y. 177 Lu, 111 In and 89 One or more of Zr, exemplified by 177 Lu.

[0012] According to an embodiment of the present invention, a photosensitizer is present on the surface of the rare earth ion energy transport layer.

[0013] According to an embodiment of the present invention, the photosensitizer is one or more of tetrakis(4-carboxyphenyl)porphyrin, phthalocyanine, and dihydrochlorin (Ce6), for example, Ce6.

[0014] According to an embodiment of the present invention, the surface of the rare earth ion energy transfer layer further comprises a modifier, preferably, the modifier is, for example, a water-soluble molecule DPG.

[0015] According to an embodiment of the present invention, the water-soluble molecule DPG can be at least one selected from polyvinyl pyrrolidone, chitosan, polyethyleneimine, polyacrylic acid, phospholipid-polyethylene glycol, etc., for example, phospholipid-polyethylene glycol.

[0016] According to the embodiment of the present invention, the structural formula of the rare earth nano-diagnostic agent is R-ALn1F4:Ln2 3+ @ALn1F4@ALn1F4:Ln3 3+ @S-DPG.

[0017] According to an embodiment of the present invention, the size of the nanomaterial is 10-150 nm, preferably the size of the nanomaterial is 15-100 nm, for example, 20-50 nm.

[0018] According to an embodiment of the present invention, the amount of the modifier in the nano-theranostic agent is not particularly limited and can be adjusted based on the requirements for the water solubility and / or biocompatibility of the rare earth-doped core-shell nanoparticles. Exemplarily, the mass ratio of the modifier to the nanomaterial is 5:1.

[0019] According to an embodiment of the present invention, in the nano-diagnostic and therapeutic agent, the loading rate of the photosensitizer is 0.1wt%-10wt% (i.e., mass percentage), for example, 1wt%-7wt%. In the present invention, the loading rate of the photosensitizer refers to the mass percentage of the photosensitizer in the nano-diagnostic and therapeutic agent.

[0020] According to an embodiment of the present invention, the rare earth nano-theranostic agent has a core-shell-shell nanostructure.

[0021] According to an embodiment of the present invention, the hydrated particle size of the nano-theranostic agent is larger than the size of the nano-material, for example, 10-150 nm, such as 20-90 nm.

[0022] According to an embodiment of the present invention, under the excitation of near-infrared light (for example, 808 nm), the photosensitizer in the nano-diagnostic and therapeutic agent is activated, and the activated photosensitizer further reacts with the surrounding oxygen to produce a large amount of reactive oxygen species (ROS), thereby rapidly oxidizing biological molecules such as proteins and nucleic acids, thereby effectively killing target cancer cells.

[0023] According to an embodiment of the present invention, under the excitation of near-infrared light (eg, 980 nm), the nano-theranostic agent can generate NIR-IIb emission, thereby realizing diagnosis and monitoring of the treatment process.

[0024] According to an embodiment of the present invention, the radioactive nuclides in the nano-diagnostic and therapeutic agent irradiate the diseased tissue at close range with the rays emitted by the decay of the radioactive nuclides, thereby achieving a therapeutic effect.

[0025] According to an embodiment of the present invention, each layer of the nanomaterial in the rare earth nano-diagnostic and therapeutic agent can be synthesized by at least one of the following methods: hydrothermal / solvent method, sol-gel method, high-temperature thermal decomposition method or high-temperature co-precipitation method, for example, high-temperature co-precipitation method.

[0026] According to an exemplary embodiment of the present invention, the nanomaterial is prepared by a step-by-step layer-by-layer epitaxial high-temperature co-precipitation method.

[0027] The present invention also provides a method for preparing the above-mentioned nano-diagnostic and therapeutic agent, which comprises the following steps:

[0028] (1) Preparation of rare earth ion luminescent core ALn1F4:Ln2 3+ ;

[0029] (2) Through the shell epitaxial growth method, the rare earth ion luminescent core ALn1F4:Ln2 in step (1) 3+ The rare earth ion inert shell ALn1F4 and the rare earth ion energy transfer shell ALn1F4:Ln3 are grown on the surface of 3+ , obtaining the nanomaterial;

[0030] (3) reacting the nanomaterial of step (2) with a modifier and / or a photosensitizer to obtain the nanodiagnostic and therapeutic agent;

[0031] In step (1) and / or step (2), a radionuclide is also added, said radionuclide having the meaning as described above.

[0032] According to an embodiment of the present invention, in step (1), preparing the rare earth ion luminescent core specifically comprises the following steps:

[0033] (1a) mixing and dissolving a non-radioactive rare earth source, a surfactant, and a high-boiling-point organic solvent to obtain a solution a;

[0034] (1b) Adding lithium source and fluorine source to solution a of step (1a) to undergo coprecipitation reaction to obtain rare earth ion luminescent core ALn1F4:Ln2 3+ .

[0035] According to an embodiment of the present invention, the molar volume ratio of the non-radioactive rare earth source, the surfactant, and the long alkane chain organic olefin is (0.01-0.5) mmol: (5-10) mL: (5-10) mL. Preferably, the molar volume ratio of the non-radioactive rare earth source, the surfactant, and the long alkane chain organic olefin is (0.01-0.3) mmol: (6-12) mL: (6-12) mL.

[0036] According to an embodiment of the present invention, in step (1a), the mixing is performed under stirring conditions at a temperature of 80-120° C. (preferably 110-120° C.) in an inert atmosphere. Preferably, the inert atmosphere can be an inert gas known in the art, such as N 2 .

[0037] Preferably, in step (1a), the mixing time is 0.2-1 h, for example 0.3-0.6 h.

[0038] Preferably, in step (1a), after mixing until dissolved, solution a is optionally cooled to room temperature.

[0039] Preferably, the solution a is a light yellow clear solution.

[0040] According to an embodiment of the present invention, in step (1b), the lithium source is one or more of lithium hydroxide, lithium oleate, lithium acetate and lithium fluoride, preferably lithium hydroxide or lithium acetate.

[0041] According to an embodiment of the present invention, in step (1b), the fluorine source is one or more selected from ammonium fluoride, sodium fluoride, potassium fluoride, sodium bifluoride and trifluoroacetate, preferably ammonium fluoride, sodium fluoride, potassium fluoride, sodium bifluoride or potassium hydroxide.

[0042] According to an embodiment of the present invention, in step (1b), the lithium source and the fluorine source are first dissolved in a solvent and then added to solution a. Preferably, the solvent is ethanol or methanol, for example, methanol. Preferably, the ratio of the lithium source, the fluorine source and the solvent is (0.1-5) mmol: (0.2-5) mmol: (3-12) mL, and preferably, the ratio of the alkali metal source, the fluorine source and the solvent is (0.6-4) mmol: (2-4) mmol: (5-12) mL.

[0043] According to an embodiment of the present invention, in step (1b), after the alkali metal source and the fluorine source are added to solution a for co-precipitation reaction, the step of removing the solvent is also included; preferably, the solvent is removed by evaporation under an inert atmosphere of N2, for example, the temperature is raised to 40-60°C under the protection of inert gas N2 and kept warm for 60 minutes to remove methanol.

[0044] Preferably, in step (1b), the temperature is raised to the reaction temperature after the solvent is removed.

[0045] According to an embodiment of the present invention, in step (1b), the coprecipitation reaction temperature is 260-340°C, and the coprecipitation reaction time is 0.3-1h; preferably, the coprecipitation reaction temperature is 260-280°C, and the coprecipitation reaction time is 0.5-1h.

[0046] Preferably, the coprecipitation reaction in step (1b) is carried out under an inert atmosphere and vigorous stirring.

[0047] Preferably, after the coprecipitation reaction in step (1b) is completed, the process further comprises the steps of naturally cooling to room temperature, precipitation, and washing. Preferably, after washing, the process further comprises the steps of centrifugal separation and purification.

[0048] According to an embodiment of the present invention, step (2) specifically includes:

[0049] (2a) Preparation of rare earth ion inert shell ALn1F4;

[0050] (2b) Preparation of rare earth ion energy transfer shell ALn1F4:Ln3 3+ .

[0051] According to an embodiment of the present invention, the method for preparing the rare earth ion inert shell ALn1F4 in step (2a) comprises the following steps: 3+ , a non-radioactive rare earth source, a surfactant and a high boiling point organic solvent are mixed and dissolved to obtain a solution b; then a lithium source and a fluorine source are added to the solution b to undergo a coprecipitation reaction to obtain ALn1F4:Ln2 3+ @ALn1F4.

[0052] According to an embodiment of the present invention, in step (2a), the molar volume ratio of the non-radioactive rare earth source, the surfactant and octadecene is 0.5 mmol: (6-12) mL: (6-12) mL.

[0053] According to an embodiment of the present invention, in step (2a), the lithium source and the fluorine source are first dissolved in a solvent and then added to solution b, wherein the solvent has the meaning as described above. Preferably, the ratio of the lithium source, the fluorine source, and the solvent is (0.6-4) mmol: (2-4) mmol: (5-12) mL.

[0054] According to an embodiment of the present invention, in step (2b), the rare earth ion energy transfer shell ALn1F4:Ln3 3+ The synthesis method comprises: ALn1F4:Ln2 3+@ALn1F4, a non-radioactive rare earth source, a surfactant and a high-boiling point organic solvent are mixed and dissolved to obtain a solution c; then a lithium source and a fluorine source are added to the solution c to undergo a coprecipitation reaction to obtain the nanomaterial ALn1F4:Ln2 3+ @ALn1F4@R-ALn1F4:Ln3 3+ .

[0055] According to an embodiment of the present invention, the molar ratio of the radioactive nuclide to the total molar amount of the non-radioactive rare earth source in the nanomaterial is (0.01-10) mCi: (0.1-0.5) mmol, preferably (4-8) mCi: (0.1-0.4) mmol.

[0056] According to an embodiment of the present invention, the non-radioactive rare earth source is selected from one or more of the following substances: acetates of stable rare earth elements, chlorides of stable rare earth elements, and nitrates of stable rare earth elements, preferably acetates of stable isotopes of rare earth ions and / or chlorides of stable rare earth elements. The stable rare earth element has the meaning as described above, preferably at least one of Yb, Er, Tm, Ho, Eu, Ce, Nd, Y, and Lu. Preferably, the non-radioactive rare earth source is, for example, at least one of Yb acetate, Yb chloride, Er acetate, and / or Er chloride.

[0057] According to an embodiment of the present invention, the surfactant is selected from at least one of octanoic acid, lauric acid and oleic acid, and is preferably octanoic acid or oleic acid.

[0058] According to an embodiment of the present invention, the high boiling point organic solvent is selected from 1-dodecene and / or 1-octadecene, exemplified by 1-octadecene.

[0059] According to an embodiment of the present invention, in step (2b), the lithium source and the fluorine source are first dissolved in a solvent and then added to solution c, wherein the solvent has the meaning as described above. Preferably, the ratio of the lithium source, the fluorine source, and the solvent is (0.6-4) mmol: (2-4) mmol: (5-12) mL.

[0060] According to an embodiment of the present invention, in step (3), the mass ratio of the nanomaterial, the modifier and the photosensitizer can be (0.1-1) mg: (3-15) mg: (0.001-0.01) mg, for example (0.2-0.6) mg: (5-10) mg: (0.005-0.009) mg.

[0061] According to an embodiment of the present invention, in step (3), the reaction can be carried out at room temperature. Preferably, the reaction time can be 6-12 hours, for example 7 hours.

[0062] According to an embodiment of the present invention, in step (3), the reaction can be carried out in a reaction solvent, such as at least one selected from dimethyl sulfoxide, chloroform, or tetrahydrofuran. Exemplarily, the nanomaterial, the modifier, the photosensitizer, and chloroform are mixed and then reacted.

[0063] According to an embodiment of the present invention, in step (3), the reaction can be carried out under stirring conditions, and the stirring can be carried out using a magnetic stirrer.

[0064] According to an embodiment of the present invention, in step (3), the modifier has the meaning as described above, for example, it can be selected from one of polyvinyl pyrrolidone, chitosan, polyethylene imine, polyacrylic acid, phospholipid-polyethylene glycol, etc.

[0065] According to an embodiment of the present invention, the photosensitizer has the meaning as described above, for example, it can be selected from one of tetrakis(4-carboxyphenyl)porphyrin, phthalocyanine, and dihydrochlorin (Ce6), such as Ce6.

[0066] According to an embodiment of the present invention, in step (3), after the reaction is completed, at least one of rotary evaporation, ultrasound, separation and water washing may be optionally included.

[0067] According to an embodiment of the present invention, step (3) further comprises washing the nano-diagnostic and therapeutic agent and then redispersing it, for example, in physiological saline. Preferably, the dispersed solution can also be sterile filtered.

[0068] The present invention also provides use of the nano-diagnostic and therapeutic agent in the field of medical imaging.

[0069] According to an embodiment of the present invention, the medical imaging is fluorescence imaging.

[0070] According to an embodiment of the present invention, the nano-diagnostic and therapeutic agent achieves fluorescence imaging after being administered via intratumoral injection or tail vein injection.

[0071] The present invention also provides a fluorescence imaging method, which includes: after the above-mentioned nano-diagnostic and therapeutic agent is administered by intratumoral injection or tail vein injection, under the excitation of near-infrared light waves (wavelength, for example, 980nm), emission light in the visible light region of 400-700nm to the near-infrared IIb region of 1400-1700nm is generated, thereby enabling real-time positioning of the tumor.

[0072] Beneficial effects of the present invention

[0073] (1) The rare earth nanodiagnostic and therapeutic agent of the present invention has a core-shell-shell micro-nanostructure, which is easy to be doped with radioactive nuclides. The outer surface of the rare earth nanodiagnostic and therapeutic agent also includes a modifier, which improves the biocompatibility of the material.

[0074] (2) The rare earth nano-diagnostic agent of the present invention can be excited by near-infrared light waves (for example, simultaneously or separately by 808nm and 980nm excitation light). For example, under 980nm laser irradiation, the rare earth nano-diagnostic agent of the present invention performs fluorescence imaging at NIR-IIb (1525nm), and under 808nm excitation, the rare earth nano-diagnostic agent of the present invention can perform photodynamic therapy; at the same time, the rare earth nano-diagnostic agent of the present invention is loaded with radioactive nuclides. 177 Lu, can achieve radio-photodynamic synergistic therapy of tumors under the guidance of fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 1 is the X-ray powder diffraction pattern of the tetragonal rare earth-doped LiLuF4 core nanocrystals, core-shell nanocrystals, and core-shell-shell nanocrystals in Example 1 of the present invention.

[0076] Figure 2 1 is a transmission electron microscope image of tetragonal rare earth-doped LiLuF4 core nanocrystals, core-shell nanocrystals, and core-shell-shell nanocrystals in Example 1 of the present invention.

[0077] Figure 3 This is a high-resolution transmission electron microscopy image of tetragonal rare earth-doped LiLuF4 core-shell-shell nanocrystals in Example 1 of the present invention.

[0078] Figure 4 These are the photoluminescence spectra (excitation wavelength is 980 nm) of tetragonal rare earth-doped LiLuF4 core nanocrystals, core-shell nanocrystals, and core-shell-shell nanocrystals in Example 1 of the present invention and the corresponding luminescence photos.

[0079] Figure 5 This is a Fourier transform infrared spectrum of the photosensitizer Ce6 and the LiLuF4 core-shell nanocrystals before and after modification in Example 1 of the present invention.

[0080] Figure 6 This is the ultraviolet absorption spectrum of the photosensitizer Ce6 and the LiLuF4 core-shell nanocrystals before and after modification in Example 1 of the present invention.

[0081] Figure 7 It is the hydrated particle size of NPs-Ce6 dispersed in different media such as physiological saline, PBS and DMEM culture medium in Example 1 of the present invention.

[0082] Figure 8 This is a down-conversion emission spectrum of LiLuF4 core-shell nanocrystals before and after modification with photosensitizer Ce6 in Example 1 of the present invention (excitation wavelength is 808 nm).

[0083] Figure 9It is the time-resolved luminescence spectrum (excitation wavelength is 808 nm) of LiLuF4 core-shell nanocrystals before and after modification with photosensitizer Ce6 in Example 1 of the present invention.

[0084] Figure 10 These are microscopic images of the brain, left leg, and right leg of a nude mouse 1 minute after administration of the nanodiagnostic and therapeutic agent in Example 1 through the tail vein in Experimental Example 2 of the present invention (at a concentration of 150 mg / kg).

[0085] Figure 11 This is a near-infrared imaging image of a nude mouse 1 minute after the nanodiagnostic and therapeutic agent in Example 1 was administered through the tail vein in Example 2 of the present invention (at a concentration of 150 mg / kg).

[0086] Figure 12 This is a curve chart of the relative absorption (410 nm) of DPBF (denoted as DPBF), DPBF under 808 nm laser irradiation (denoted as DPBF+808 nm laser), and a mixture of DPBF and NPs-Ce6 under 808 nm laser irradiation (denoted as DPBF+NPs-Ce6+808 nm laser) as a function of irradiation time in Example 3 of the present invention.

[0087] Figure 13 This is a confocal fluorescence image of A549 lung cancer cells after incubation of the NPs-Ce6-PEG2000 nano-diagnostic and therapeutic agent for 24 hours in Example 3 of the present invention, staining the cells with DAPI and DCFH-DA, and irradiating them with 808 nm laser for 3 minutes.

[0088] Figure 14 This is a CCK-8 cytotoxicity experiment in which the NPs-Ce6-PEG2000 nano-diagnostic and therapeutic agent at different dosage concentrations in Example 4 of the present invention was co-incubated with A549 lung cancer cells for 24 hours.

[0089] Figure 15 This is a CCK-8 cytotoxicity experiment in which the NPs-Ce6-PEG2000 nano-diagnostic and therapeutic agent at different dosages in Example 5 of the present invention was co-incubated with SW480, HeLa, IOSE and SKOV3 cells for 24 hours at different radionuclide doses.

[0090] Figure 16 This is a graph showing the relative volume changes of tumors in tumor-bearing nude mice in Example 5 of the present invention.

[0091] Figure 17 This is a graph showing changes in body weight of tumor-bearing nude mice in Example 5 of the present invention.

[0092] Figure 18 These are photos of tumor-bearing nude mice in different treatment groups after 12 days of treatment in Example 5 of the present invention. DETAILED DESCRIPTION

[0093] 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.

[0094] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0095] X-ray powder diffractometer (instrument model MiniFlex2, manufacturer Rigaku), transmission electron microscope (instrument model TECNAI G2 F20, manufacturer FEI), spectrometer (instrument model FLS980, manufacturer Edinburgh), camera (instrument model EOS 5D, manufacturer Canon), infrared spectrometer (instrument model Vertex70, manufacturer Bruker), UV-visible-near-infrared spectrophotometer (instrument model Lambda 950, manufacturer Perkin-Elmer), laser particle size analyzer (instrument model Zetasizer Ultra, manufacturer Malvern Panalytical), X-ray source (model TUB00146-W06, manufacturer Magpro), in vivo imager (model NirVivo-Mix, manufacturer RayLight), and microscopic imaging system (model NirVivo-Pro, manufacturer RayLight).

[0096] Example 1: LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd / 177 Lu-Ce6-

[0097] PEG2000 (NPs-Ce6-PEG2000) rare earth nanodiagnostic agent and its preparation

[0098] (S1) Preparation of LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd / 177 Lu tetragonal core-shell nanocrystals:

[0099] (a) LiLuF4:Yb / Er / Ce core nanocrystals were prepared as follows: 0.72 mmol of lutetium acetate, 0.11 mmol of ytterbium acetate, 0.10 mmol of erbium acetate, and 0.07 mmol of cerium acetate were placed in a three-necked flask at room temperature in a stoichiometric ratio. 10 mL of oleic acid and 10 mL of octadecene were then added as solvents. The mixture was heated to 110-120°C under an inert atmosphere, maintained at this temperature for 25-35 min, and cooled to room temperature to yield a bright yellow solution a. A methanol solution containing 3.00 mmol of lithium hydroxide and 3.50 mmol of ammonium fluoride was added to the bright yellow solution a, and the temperature was raised to 40-60°C under inert gas protection. The temperature was kept for 1 hour to remove the methanol, and then the temperature was continued to rise to 280-290°C. After vigorous stirring and reacting for 0.5-1 hour, the solution was naturally cooled to room temperature, precipitated and washed, and dispersed in 5 mL of cyclohexane to obtain a LiLuF4:Yb / Er / Ce cyclohexane solution. The tetragonal LiLuF4:Yb / Er / Ce nanoparticles were recorded as core nanocrystals, and their X-ray powder diffraction pattern was shown in FIG. Figure 1 The core is about 9 nm in size (see Figure 2 );

[0100] (b) The synthesis steps of LiLuF4:Yb / Er / Ce@LiLuF4 core-shell nanocrystals are as follows: The synthesis steps of core-shell nanocrystals are basically the same as those for preparing LiLuF4:Yb / Er / Ce core nanocrystals in step (a), except that lutetium acetate, ytterbium acetate, erbium acetate and cerium acetate are replaced with 1.00 mmol of lutetium acetate, the LiLuF4:Yb / Er / Ce cyclohexane solution of step (a) is added, and then 10 mL of oleic acid and 10 mL of octadecene are added to react to obtain a bright yellow solution b; the treatment method of the bright yellow solution b is the same as that of the bright yellow solution a to obtain LiLuF4:Yb / Er / Ce@LiLuF4 core-shell nanocrystals, which are dispersed in 5 mL of cyclohexane to obtain a LiLuF4:Yb / Er / Ce@LiLuF4 cyclohexane solution;

[0101] (c)LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd / 177 The synthesis steps of Lu core-shell nanocrystals are as follows: The synthesis steps of core-shell nanocrystals are basically the same as those for preparing LiLuF4:Yb / Er / Ce core nanocrystals in step (a) above, except that 0.95mmol of lutetium acetate and 0.05mmol of neodymium acetate are replaced by 0.5mCi 177Lu and the LiLuF4:Yb / Er / Ce@LiLuF4 cyclohexane solution of step (b) were mixed, and 10 mL of oleic acid and 10 mL of octadecene were added to react to obtain a bright yellow solution c; the treatment method of the bright yellow solution c was the same as that of the bright yellow solution a to obtain LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd / 177 Lu core-shell nanocrystals were dispersed in 5 mL of cyclohexane to obtain LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd / 177 Lu cyclohexane solution.

[0102] (S2) Preparation of LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd / loaded with photosensitizer Ce6 177 The nanocrystalline composite of Lu (denoted as NPs-Ce6) is as follows:

[0103] (a) 1 mL of the LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd / 177 Lu cyclohexane solution, add ethanol, centrifuge to obtain a precipitate, and put it into an oven to dry. The precipitate was redispersed in a mixed solution of 6mL acetone and 50uL dilute hydrochloric acid, ultrasonicated for 20-40min to remove excess oleic acid on the surface, and centrifuged again to collect the precipitate, washed three times with acetone, and dispersed in dimethylformamide solution to obtain

[0104] LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd / 177 Lu solution in dimethylformamide.

[0105] (b) 1 mL of the LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd / 177 The dimethylformamide solution of Lu was mixed with the chloroform solution of 838 umol / L photosensitizer Ce6, stirred at room temperature for 6 h, and ethanol was added to obtain a precipitate. The precipitate was collected by centrifugation to obtain the NPs-Ce6 nanocrystal complex.

[0106] (S3) Preparation of LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd / 177 Lu-Ce6-PEG2000 (denoted as NPs-Ce6-PEG2000) nanodiagnostic and therapeutic agent

[0107] The NPs-Ce6 nanocrystal complex obtained in step (S2) was redispersed in a chloroform solution, and 80 mg of a water-soluble functional ligand PEG2000 was added. The mixture was stirred overnight at room temperature and the chloroform was removed by rotary evaporation. 10 mL of deionized water was added for ultrasonic dispersion. The precipitate was discarded after centrifugation to remove possible large aggregates. The supernatant was filtered through a 0.2 μm filter and the filtrate was centrifuged to obtain a precipitate, which was a rare earth nanodiagnostic agent. The precipitate was dispersed in 1 mL of normal saline to obtain a dispersion of a rare earth nanodiagnostic agent with a mass concentration of 25 mg / mL for later use.

[0108] Example 2: In vivo fluorescence effect of rare earth nanodiagnostic agents

[0109] (1) Microscopic imaging test: Figure 10 As shown, nude mice (animals provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were injected with 200 μL of the dispersion of the rare earth nanodiagnostic agent in Example 1 (25 mg / mL) through the tail vein, and imaging was performed using a 980 nm excitation light under a microscopic imaging system. Figure 10 , you can see the tiny blood vessels in the brain and legs of the nude mouse.

[0110] (2) In vivo imaging test: Figure 11 As shown, nude mice (animals provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were injected with 200 μL of the dispersion of the rare earth nanodiagnostic agent in Example 1 (25 mg / mL) through the tail vein. Under the in vivo imaging system, the imaging was excited using the 980 nm channel. Figure 11 , the blood vessel distribution of nude mice can be clearly seen.

[0111] Example 3: Evaluation of the active oxygen effect of rare earth nanodiagnostic agents

[0112] (1) Detection of singlet oxygen produced under 808 nm laser illumination: Figure 12 As shown, the rare earth nanodiagnostic agent prepared in Example 1 and the probe 1,3-diphenylisobenzofuran (DPBF) were mixed and the power was 1W / cm 2 After irradiation with 808nm laser for 14 minutes, the singlet oxygen produced can reduce the probe 1,3-diphenylisobenzofuran (DPBF) by 90%, indicating that rare earth nano-diagnostic agents can efficiently generate singlet oxygen.

[0113] (2) The specific test for detecting intracellular reactive oxygen species is as follows: A549 cells were cultured at 2×10 4The concentration of cells / mL was inoculated into confocal microplates and incubated at 37°C for 24h in a 5% CO2 atmosphere. The F12K culture medium was discarded and the cells were washed once with PBS. At the same time, the rare earth nano-therapeutic agent NPs-Ce6-PEG2000 of Example 1 was dispersed into the F12K culture medium to obtain F12K culture medium containing rare earth nano-therapeutic agents at different concentrations (0, 50, 100 μg / mL). The above-mentioned F12K culture medium containing rare earth nano-therapeutic agents at different concentrations was then added to the confocal microplates (0.5mL per well). A549 cells were further incubated at 37°C and 5% CO2 for 2h. After the incubation with the rare earth nano-therapeutic agent, the cells were washed three times with PBS (1mL); then irradiated with 808nm laser for 3min to obtain irradiated cell culture medium containing rare earth nano-therapeutic agents and cells; before irradiation, a near-infrared detection card was placed on each well to ensure that the light source was located in the center of the cover.

[0114] After irradiation, 2',7'-dichlorofluorescein (DCFH-DA) was added as a ROS probe to the cell culture medium containing the irradiated rare earth nano-diagnostic agent and cells, and the generation of singlet oxygen was monitored by chemiluminescence, specifically comprising: mixing DCFH-DA (10mM, 1μL) with fresh F12K culture medium (1mL), incubating with the cell culture medium containing the irradiated rare earth nano-diagnostic agent and cells at 37°C for 30min under a 5% CO2 atmosphere. Subsequently, the cells were fixed and stained with 4',6-diamino-2-phenylindole (DAPI) for 10min at room temperature to obtain a sample until the sample was imaged using a laser scanning confocal microscope (Nikon Eclipse Ti A1, Japan). During imaging, DCFH-DA was excited with a 488nm laser, and fluorescence emission was collected in the green channel to represent the concentration level of singlet oxygen. DAPI was excited with a 365nm laser, and fluorescence emission was collected in the blue channel to locate the position of the cells. The superposition channel is the result of the superposition of the green channel and the blue channel, such as Figure 13 shown.

[0115] Example 4: Evaluation of the in vitro cell-killing effect of rare earth nanodiagnostic agents

[0116] The in vitro cell killing effect was determined using a standard CCK-8 cell activity assay kit, specifically including: A549 cells were plated at 5×10 3 The cells were seeded into 96-well cell culture plates at a density of 100 μg / well until adhered to the wall, and then incubated with different concentrations (0, 25, 50, 100, and 200 μg / mL) of NPs-Ce6-PEG2000 for 24 h. 2The cells were irradiated with 808nm laser (Changchun Optoelectronics MDL-N-10W, China) for 3 minutes and incubated at 37°C for 24 hours. Then, the CCK-8 cytotoxicity test was performed to determine the survival rate of the cells relative to the untreated control cells. The 808nm group was irradiated with 808nm laser only, the NPs-Ce6 group was treated with nano-therapeutic agents only, and the NPs-Ce6+808nm group was incubated with nano-therapeutic agents and irradiated with 808nm laser at the same time. The test results are shown in Figure 5. Figure 14 The cck-8 cytotoxicity experiment of NPs-Ce6-PEG2000 nano-diagnostic agents with different dosages and different radionuclide doses was conducted by co-incubating SW480, HeLa, IOSE and SKOV3 cells for 24 hours. The experimental process was the same as above, and the test results were shown in Figure 2. Figure 15 shown.

[0117] Example 5: Study on the treatment of nude mice bearing human lung cancer subcutaneous tumors with rare earth nanodiagnostic agents

[0118] Unexcited saline, saline excited by 808nm excitation light, pure radionuclide excited by 808nm excitation light 177 Comparative Example 1 excited by Lu and 808 nm excitation light without loading radionuclides

[0119] LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd-Ce6-PEG2000, and 808nm excitation light excited Example 1 rare earth nano diagnostic and therapeutic agent LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd- 177 Lu-Ce6-PEG2000 was used as a drug for radio-photodynamic therapy; the tumor volume was about 100 mm 3 Nude mice bearing subcutaneous tumors of human lung cancer cells A549 (hereinafter referred to as tumor-bearing nude mice) were divided into groups (8 nude mice in each group), and each group was injected intratumorally with the above drugs.

[0120] Among them, the radioactive dose of the rare earth nano-diagnostic agent in Example 1 is 0.06mCi, the mass concentration is 7mg / ml, and the injection volume is about 100ul; the radioactive dose and volume of pure radionuclide 177Lu are the same as those of the rare earth nano-diagnostic agent; the mass concentration and volume of other groups are the same as those of the rare earth nano-diagnostic agent in Example 1; the volume of unexcited saline and saline excited by 808nm excitation light is the same as that of the rare earth nano-diagnostic agent in Example 1; the power density of 808nm excitation light is 0.5W / cm 2 The irradiation time was 30 min (1 min rest after every 5 min of irradiation).

[0121] After drug injection, the body weight and tumor volume of nude mice in each group were recorded every two days.

[0122] Figure 16 The graph is a graph showing the average tumor volume changes in each group of tumor-bearing nude mice. It can be seen from the graph that as time goes by, the nano-diagnostic and therapeutic agent prepared in Example 1 has a significant inhibitory effect on tumor growth, and the inhibitory effect is significantly higher than that of the pure radionuclide and the nanomaterial of the comparative example.

[0123] Figure 17 The figure is a graph showing the average weight change of tumor-bearing nude mice in each group. The weight of the nude mice in each group increased steadily, which shows that the nano-diagnostic and therapeutic agent prepared by the present invention has no adverse effect on body weight.

[0124] 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 rare earth nanodiagnostic agent, characterized in that: The rare earth nano diagnostic and therapeutic agent comprises a nano material and a radioactive nuclide, wherein the radioactive nuclide is distributed on the nano material; the nano material has a core-shell-shell nanostructure; the nano material comprises, from the inside to the outside, a rare earth ion luminescent core LiLuF4:Yb / Er / Ce, a rare earth ion inert layer LiLuF4 and a rare earth ion energy transfer layer LiLuF4:Nd / 177 Lu; the nanomaterial is prepared by shell epitaxial growth method; Radionuclides 177 The radioactivity molar ratio of Lu to the total molar amount of the non-radioactive rare earth source in the nanomaterial is 0.01-10 mCi: 0.1-0.5 mmol; the stable rare earth element in the non-radioactive rare earth source is selected from Yb, Er, Ce, Nd, and Lu; The rare earth ion energy transfer layer has a photosensitizer and a modifier on its surface; the photosensitizer is dihydrochlorin, and the loading rate of the photosensitizer is 0.1wt%-10wt%; the modifier is a water-soluble molecule DPG; The water-soluble molecule DPG is phospholipid-polyethylene glycol; Under the excitation of near-infrared light, the rare earth nano-diagnostic and therapeutic agent generates NIR-IIb emission; the radioactive nuclides in the rare earth nano-diagnostic and therapeutic agent emit radiation at close range through radioactive nuclide decay.

2. The rare earth nanodiagnostic agent according to claim 1, characterized in that The size of the nanomaterial is 10-150 nm; The mass ratio of the modifier to the nanomaterial is 5:1; In the rare earth nano-diagnostic and therapeutic agent, the loading rate of the photosensitizer is 1 wt%-7 wt%.

3. The rare earth nanodiagnostic agent according to claim 1, characterized in that The hydrated particle size of the rare earth nano-diagnostic and therapeutic agent is larger than the size of the nano-material; Under the excitation of near-infrared light, the photosensitizer in the rare earth nano-diagnostic and therapeutic agent is activated, and the activated photosensitizer further reacts with the surrounding oxygen to produce reactive oxygen species, oxidizing proteins and nucleic acid biomolecules.

4. The method for preparing the rare earth nanodiagnostic agent according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) Preparation of rare earth ion luminescent core LiLuF4:Yb / Er / Ce; (2) Through the shell epitaxial growth method, the rare earth ion luminescent core LiLuF4:Yb / Er / Ce in step (1) is sequentially grown on the surface to obtain the rare earth ion inert shell LiLuF4 and the rare earth ion energy transfer shell LiLuF4:Nd / 177 Lu, obtain the nanomaterial; (3) reacting the nanomaterial of step (2) with a modifier and a photosensitizer to obtain the rare earth nanodiagnostic agent.

5. The preparation method according to claim 4, characterized in that In step (1), the preparation of the rare earth ion luminescent core specifically includes the following steps: (1a) mixing and dissolving a non-radioactive rare earth source with a surfactant and a high-boiling-point organic solvent to obtain a solution a; (1b) Adding a lithium source and a fluorine source to the solution a of step (1a) to undergo a coprecipitation reaction to obtain a rare earth ion luminescent core LiLuF4:Yb / Er / Ce.

6. The preparation method according to claim 5, characterized in that The molar volume ratio of the non-radioactive rare earth source, the surfactant and the high-boiling-point organic solvent is 0.01-0.5 mmol: 5-10 mL: 5-10 mL; In step (1a), the mixing is carried out under stirring conditions in an inert atmosphere at a temperature of 80-120°C; In step (1a), the mixing time is 0.2-1 h; In step (1a), after mixing until dissolved, solution a is optionally cooled to room temperature; The solution a is a light yellow clear solution; In step (1b), the lithium source is one or more of lithium hydroxide, lithium oleate, lithium acetate and lithium fluoride; In step (1b), the fluorine source is selected from one or more of ammonium fluoride, sodium fluoride, potassium fluoride, sodium bifluoride and trifluoroacetate; In step (1b), the lithium source and the fluorine source are first dissolved in a solvent and then added to solution a; the solvent is ethanol or methanol; the ratio of the lithium source, the fluorine source and the solvent is 0.1-5 mmol: 0.2-5 mmol: 3-12 mL; In step (1b), after adding the lithium source and the fluorine source to solution a for co-precipitation reaction, the step further includes removing the solvent; In the step (1b), the temperature is raised to the reaction temperature after the solvent is removed; In step (1b), the coprecipitation reaction temperature is 260-340°C, and the coprecipitation reaction time is 0.3-1 h; the coprecipitation reaction temperature is 260-280°C, and the coprecipitation reaction time is 0.5-1 h; The coprecipitation reaction in step (1b) is carried out under an inert atmosphere and vigorous stirring.

7. The preparation method according to claim 5, characterized in that In step (1b), the ratio of the lithium source, the fluorine source and the solvent is (0.6-4) mmol: (2-4) mmol: (5-12) mL.

8. The preparation method according to claim 4, characterized in that Step (2) specifically includes: (2a) Preparation of rare earth ion inert shell LiLuF4; (2b) Preparation of rare earth ion energy transfer shell LiLuF4:Nd / 177 Lu.

9. The preparation method according to claim 8, characterized in that The method for preparing the rare earth ion inert shell LiLuF4 in step (2a) comprises the following steps: mixing and dissolving a rare earth ion luminescent core LiLuF4:Yb / Er / Ce, a non-radioactive rare earth source, a surfactant, and a high-boiling-point organic solvent to obtain a solution b; then adding a lithium source and a fluorine source to the solution b to undergo a coprecipitation reaction to obtain LiLuF4:Yb / Er / Ce@LiLuF4; In step (2a), the molar volume ratio of the non-radioactive rare earth source, surfactant and high-boiling point organic solvent is 0.5 mmol:6-12 mL:6-12 mL; In step (2a), the lithium source and the fluorine source are first dissolved in a solvent and then added to solution b; the ratio of the lithium source, the fluorine source and the solvent is 0.6-4 mmol: 2-4 mmol: 5-12 mL.

10. The preparation method according to claim 8, characterized in that In step (2b), the rare earth ion energy transfer shell LiLuF4:Nd / 177 The synthesis method of Lu comprises: mixing and dissolving LiLuF4:Yb / Er / Ce@LiLuF4, a non-radioactive rare earth source, a surfactant and a high-boiling point organic solvent to obtain a solution c; then adding a lithium source and a fluorine source to the solution c for coprecipitation reaction to obtain the nanomaterial LiLuF4:Yb / Er / Ce@LiLuF4@LiLuF4:Nd / 177 Lu; The radioactivity molar ratio of the radioactive nuclide to the total molar amount of the non-radioactive rare earth source in the nanomaterial is 0.01-10 mCi: 0.1-0.5 mmol; The non-radioactive rare earth source is selected from one or more of the following substances: acetates of stable rare earth elements, chlorides of stable rare earth elements and nitrates of stable rare earth elements; The surfactant is selected from at least one of octanoic acid, lauric acid and oleic acid; The high boiling point organic solvent is selected from 1-dodecene and / or 1-octadecene; In step (2b), the lithium source and the fluorine source are first dissolved in a solvent and then added to solution c; the ratio of the lithium source, the fluorine source and the solvent is 0.6-4 mmol: 2-4 mmol: 5-12 mL.

11. The preparation method according to claim 4, characterized in that In step (3), the mass ratio of the nanomaterial, the modifier and the photosensitizer is 0.1-1 mg: 3-15 mg: 0.001-0.01 mg; In step (3), the reaction is carried out at room temperature; the reaction time is 6-12 h; In step (3), the reaction is carried out in a reaction solvent, and the reaction solvent is selected from at least one of dimethyl sulfoxide, chloroform or tetrahydrofuran.

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