Injectable aluminum-based MOFs biomimetic mineralized tumor vaccine, compound, method, pharmaceutical composition and application

Abstract

The invention belongs to the technical field of tumor vaccines, and discloses a 6F-TF-BSA-MIL-53 (Al)-FA compound, and the compound comprises a metal organic framework material MIL-53 (Al)-FA and a fluoroglycoprotein antigen 6F-TF-BSA loaded in the MIL-53 (Al)-FA; wherein an organic ligand in the MIL-53 (Al)-FA is fumaric acid, and a metal ion in the MIL-53 (Al)-FA is Al < 3 + >; and the loading capacity of the 6F-TF-BSA is 0.1 to 0.5 g / g. The therapeutic biomimetic mineralized tumor vaccine is injected into a tumor-bearing mouse for experimental determination, and the result shows that the therapeutic biomimetic mineralized tumor vaccine can induce generation of an effective IgG antibody titer level in the mouse, can effectively inhibit tumor growth, is good in safety and has potential application value in therapeutic tumor vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor vaccines, and in particular relates to a co-loading synthesis method, a complex, a method, a pharmaceutical composition and an immunological application of a fluorinated Thomsen-Friedenreich (TF) antigen and an aluminum-based metal-organic framework (MOFs). Background Art

[0002] In the past few decades, the importance of tumor vaccines in cancer immunotherapy has been increasing gradually. By utilizing the patient's own immune system, the cascade antigen-specific reaction of the immune system is activated, and ultimately the killing effect on tumor cells is achieved. As the cornerstone of therapeutic cancer vaccines, tumor antigens have also made great progress in recent years. It can be simply divided into the following categories: Tumor-Associated Antigens (TAA), Tumor-Specific Antigens (TSA), and whole tumor cell antigens that have not been further identified and overcome tumor antigen heterogeneity. Thomsen-Friedenreich (TF) antigen (TF or T antigen, Galβ1,3GalNAcαSer / Thr) is one of the most common tumor-associated glycoproteins (TAA), and is overexpressed on the surface of 90% of tumor cells, such as breast cancer, prostate cancer, ovarian cancer and lung cancer. The TF antigen belongs to a self-antigen, has low immunogenicity, is easily hydrolyzed by glycosidases in the body, and has poor metabolic stability. It is reported that the introduction of fluorine atoms not only enhances the immunogenicity of the antigen, but also effectively improves its metabolic stability, liposolubility and bioavailability. The serum antibodies produced after immunizing mice with the fluorinated vaccine can specifically recognize and attach to MCF-7 human breast cancer cells, and kill tumor cells through complement-dependent cytotoxicity. At present, it has been reported that fluorinated tumor-associated glycoproteins have achieved good results in anti-tumor experiments in mice. Therefore, based on these studies, the synthesis of fluorinated derivatives of natural tumor-associated glycoproteins is a promising approach to obtain relatively good immune effects.

[0003] Currently, the research and development of tumor vaccines mainly focus on the structural design of vaccines, novel adjuvants, optimization of vaccine delivery systems, and combination therapy with other immunotherapies or chemotherapy to overcome the immune evasion of cancer cells. As an important component of vaccines, adjuvants can generate rapid and persistent immune responses, improve the immunogenicity and effectiveness of vaccines, and reduce the dosage of antigens and the number of immunizations. Among them, inorganic metal adjuvants, as temporary antigen depots, can prolong the antigen exposure time, contribute to the recruitment and maturation of immune cells, and promote the processes of antigen uptake, presentation, and cross-presentation; they can also selectively induce different types of immune responses. Aluminum adjuvant, as the only adjuvant currently approved for vaccines in China, has heterogeneous physicochemical properties and induces weak cellular immune responses, which limits its immune effect and application scope. Metal-organic frameworks (MOFs) materials, as a new type of organic-inorganic composite material, have excellent properties that cannot be compared with traditional porous materials, such as high specific surface area, well-defined crystal structure, adjustable pore size and shape, variable structure, and post-modifiability. Currently, there are reports on the application of immobilizing biomolecules (such as enzymes, etc.) in the MOF structure for biocatalysis, sensing, separation, and imaging. However, due to the certain cytotoxicity of metal ions and organic ligands in MOFs and their strong adaptability and specificity for biomolecules with different molecular weights and spatial structures, there are currently few types of MOF materials suitable for the pharmaceutical field.

[0004] In recent years, tumor vaccines, as a new treatment strategy, have brought hope for cancer treatment. After one or more subcutaneous injections, tumor vaccines can induce systemic tumor antigen-specific immune responses and establish immune memory effects, thereby clearing metastatic tumor cells and preventing tumor recurrence, providing a new treatment method for the postoperative management of breast cancer. However, the current immune response to therapeutic tumor vaccines is still low. One of the most important reasons is the lack of carriers that can effectively deliver antigens and adjuvants that can stimulate immune responses. To achieve this goal, precise control in both the spatial and temporal dimensions of the vaccine is required. In terms of spatial control, antigens must be delivered to the paracortical area of lymph nodes for DCs to present antigens to T cells. In terms of temporal control, continuous antigen stimulation is considered to be more effective for vaccine potency.

[0005] To enhance the immunization effect of synthetic vaccines, the present invention designs and explores the co - loading of synthetic tumor vaccines and aluminum - based metal - organic frameworks (MOFs), and combines them with thermosensitive gels for tumor immunotherapy. Aluminum - based metal - organic frameworks (MOFs) are crystalline porous hybrid materials composed of metal nodes (metal ions or clusters) and organic ligands, with ordered pores and a periodic structural framework. It can protect antigens within the framework, improve stability, and when taken up by APCs, it disintegrates in the intracellular fluid and exerts an immune adjuvant effect, effectively enhancing the immune effect. First, using fluorinated galactose and galactosamine hydrochloride as raw materials, the required sugar antigen is synthesized through an enzymatic reaction; then, the sugar antigen is conjugated with a carrier protein to prepare a glycoprotein conjugate; after that, through in - situ encapsulation, the glycoprotein conjugate and a new adjuvant based on an aluminum - based crystalline cluster are co - loaded to prepare a complex. Finally, the complex is combined with a thermosensitive hydrogel for application. The purpose of this combined application is to extend the release time of the antigens loaded in the hydrogel through the slow - release effect of the hydrogel. The evaluation of the vaccine immunization effect is completed by constructing and immunizing a tumor - bearing mouse model. This injectable biomimetic mineralized tumor vaccine can overcome the obstacles in the time dimension and space dimension encountered in traditional vaccine delivery and effectively trigger a systemic antigen - specific cellular immune response.

[0006] Through retrieval, no patent publication documents related to the present invention patent application have been found. Summary of the Invention

[0007] The object of the present invention is to overcome the deficiencies in the prior art and provide a co - loading synthesis method, complex, method, pharmaceutical composition, and immunological application containing fluorinated Thomsen - Friedenreich (TF) antigen and aluminum - based metal - organic frameworks (MOFs).

[0008] The technical solution adopted by the present invention to solve its technical problems is:

[0009] A method for preparing and applying a tumor vaccine co-loaded with a fluorinated TF antigen and an aluminum-based metal-organic framework (MOFs). That is, a method of in-situ encapsulating the 6F-TF-BSA antigen with the aluminum-based MOFs material MIL-53(Al)-FA. Under mild conditions of room temperature aqueous phase, through the precise assembly of MIL-53(Al)-FA and effectively wrapping the 6F-TF-BSA antigen to form a stable 6F-TF-BSA@MIL-53(Al)-FA biomimetic mineralized tumor vaccine. This method can achieve an encapsulation rate of 6F-TF-BSA above 90%, and the loading amount of 6F-TF-BSA in the complex is in the range of 0.1 - 0.5 g / g. Among them, MIL-53(Al)-FA is synthesized from disodium fumarate and aluminum chloride in an aqueous solution at room temperature. Before immunization, the prepared 6F-TF-BSA@MIL-53(Al)-FA is mixed evenly in a thermosensitive hydrogel system to obtain an injectable biomimetic mineralized tumor vaccine.

[0010] In the first aspect, the present invention provides a 6F-TF-BSA@MIL-53(Al)-FA complex (1), which includes a metal-organic framework material MIL-53(Al)-FA and a fluorinated glycoprotein antigen 6F-TF-BSA loaded in MIL-53(Al)-FA; wherein, the organic ligand in the MIL-53(Al)-FA is fumaric acid (FA), and the metal ion is Al 3+ ; the loading amount of 6F-TF-BSA is 0.1 - 0.5 g / g. The structure of this complex is as Figure 16 shown.

[0011] In the second aspect, the present invention provides a method for preparing the above 6F-TF-BSA@MIL-53(Al)-FA complex, including the following steps:

[0012]

[0013] Step a: In the presence of an alkaline buffer solution (100 mM) and a divalent metal ion (30 mM), make D-(+)-galactose 2 undergo an enzymatic reaction with adenosine triphosphate ATP. Under the condition of a 37°C water bath for 3 hours, the dosage of adenosine triphosphate ATP is equivalent to the molar equivalent of D-(+)-galactose 2, and compound 3 can be obtained;

[0014] Step b: Adjust the pH value of the reaction solution in step a to weakly acidic. Then, add the receptor compound 4 to the system and dropwise add the BiGalHexNAcP enzyme solution, and continue to react in a 37°C water bath for 24 hours to obtain compound 5.

[0015] Step c: Compound 5 undergoes a Pd / C-catalyzed hydrogenation reaction to obtain compound 6.

[0016] Step d: Under alkaline conditions and in a mixed solvent (V DMF :V PBS 4:1), couple compound 6 with bifunctional reagent 7 at room temperature for 4 - 8 hours, and obtain compound 8 after purification;

[0017] Step e: In PBS buffer (0.01 M, pH = 8.0), add compound 8 and carrier protein BSA, where the molar mass ratio of compound 8 to BSA is 20:1 - 50:1, oscillate at 30 °C and 180 rpm for 72 hours, and obtain glycoprotein conjugate 9 after purification and lyophilization.

[0018] Step f: Weigh solid aluminum salt and dissolve it in a mixed solution of water and absolute ethanol (V 水 :V 无水乙醇 = 1:1) to prepare 0.1 M aluminum chloride solution; prepare an aqueous solution of glycoprotein conjugate 9 (1 - 10 mg / mL), add 3 parts of disodium fumarate solution (0.1 M) to it, mix evenly, then add 1 part of the prepared aluminum chloride solution (0.1 M) to it, quickly pipette and mix evenly, and let it stand for 30 minutes. Finally, centrifuge the reaction solution at 9000 rpm, wash the precipitate three times with distilled water, and dry it to obtain complex 6F-TF-BSA@MIL-53(Al)-FA(1).

[0019] Furthermore, in step a, the solvent is selected from an alkaline buffer solution system; or, in step a, the reaction temperature is 10 - 40 °C.

[0020] Furthermore, in step b, the acidic condition is created using a dilute hydrochloric acid solution; or, in step b, the solvent is one or more selected from water, methanol, ethanol, and an acidic buffer solution; or, in step b, the reaction temperature is 10 - 40 °C.

[0021] Furthermore, in step c, the solvent is selected from one or more of water, tetrahydrofuran, dichloromethane, methanol, ethyl acetate, and an alkaline buffer solution; or, in step c, the reaction temperature is 10 - 30 °C.

[0022] Furthermore, in step f, the aluminum salt is one or more of aluminum nitrate, aluminum chloride, aluminum sulfate, aluminum acetate, aluminum phosphate, or aluminosilicate.

[0023] Furthermore, in step f, the solvent is selected from one or more of water, methanol, ethanol, and an alkaline buffer solution; or, in step f, the reaction temperature is 10 - 30 °C.

[0024] In a third aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned 6F-TF-BSA@MIL-53(Al)-FA complex and Pluronic F127 thermosensitive gel solution.

[0025] In a fourth aspect, the present invention provides the use of the above-mentioned 6F-TF-BSA@MIL-53(Al)-FA complex or the 6F-TF-BSA@MIL-53(Al)-FA complex prepared by the above-mentioned preparation method or the above-mentioned pharmaceutical composition in the field of tumor vaccines.

[0026] The advantages and positive effects achieved by the present invention are as follows:

[0027] 1. For the therapeutic biomimetic mineralized tumor vaccine of the present invention, through the experiment of injecting tumor-bearing mice, the results show that it can induce an effective IgG antibody titer level in mice, can effectively inhibit tumor growth and has good safety, showing potential application value in therapeutic tumor vaccines.

[0028] 2. The preparation method of the present invention has a short synthetic route, mild reaction conditions, high yield and convenient operation.

[0029] 3. The pharmaceutical composition provided by the present invention is injectable, with higher application convenience and patient compliance.

[0030] 4. The 6F-TF-BSA@MIL-53(Al)-FA complex provided by the present invention comprises the metal-organic framework material MIL-53(Al)-FA and the fluorinated glycoprotein antigen 6F-TF-BSA loaded in MIL-53(Al)-FA; wherein, the organic ligand in MIL-53(Al)-FA is fumaric acid and the metal ion is Al 3+ . The preparation process is as follows: Add the disodium fumarate solution to the 6F-TF-BSA aqueous solution, then add the aluminum chloride solution, and let it stand for reaction; then wash and dry the solid product obtained from the reaction to obtain it.

[0031] 5. The pharmaceutical composition 6F-TF-BSA@MIL-53(Al)-FA Gel provided by the present invention has higher immunogenicity compared with 6F-TF-BSA / Alum Gel or the TF-BSA@MIL-53(Al)-FA complex, has a significant tumor suppression effect and good safety. Its preparation method is simple, the encapsulation rate of 6F-TF-BSA is high, and the reaction conditions are mild. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the 1H NMR spectrum of compound 5 in deuterated water in the present invention;

[0033] Figure 219F NMR spectrum of compound 5 in deuterated water in the present invention;

[0034] Figure 3 1H NMR spectrum of compound 6 in deuterated water in the present invention;

[0035] Figure 4 19F NMR spectrum of compound 6 in deuterated water in the present invention;

[0036] Figure 5 1H NMR spectrum of compound 8 in deuterated water in the present invention;

[0037] Figure 6 13C NMR spectrum of compound 8 in deuterated water in the present invention;

[0038] Figure 7 19F NMR spectrum of compound 8 in deuterated water in the present invention;

[0039] Figure 8 MALDI-TOF-MS mass spectrum of compound 9 in the present invention;

[0040] Figure 9 TEM image of compound 1 in the present invention;

[0041] Figure 10 SEM image of compound 1 in the present invention;

[0042] Figure 11 PXRD pattern of compound 1 in the present invention;

[0043] Figure 12 Graph of IgG antibody titer levels induced by the vaccine in the present invention;

[0044] Figure 13 Graph of IgM antibody titer levels induced by the vaccine in the present invention;

[0045] Figure 14 Graph of the change in tumor volume of mice after vaccination with the vaccine in the present invention;

[0046] Figure 15 Graph of the change in body weight monitoring of mice during the immunization vaccine process;

[0047] Figure 16 Structural diagram of the 6F-TF-BSA@MIL-53(Al)-FA(1) complex in the present invention. Detailed implementation manners

[0048] The present invention will be further described below in conjunction with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0049] All the various experimental operations involved in the specific embodiments are conventional techniques in the art. For the parts not specifically annotated in this article, those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literature, or relevant specifications, manuals, etc. before the filing date of this invention application for implementation.

[0050] Synthesis of 6F-TF-BSA@MIL-53(Al)-FA complex:

[0051] A biomimetic mineralized tumor vaccine 6F-TF-BSA@MIL-53(Al)-FA containing a fluorinated TF antigen, and the biomimetic mineralized tumor vaccine is (1), and its structure is as Figure 16 shown.

[0052] A synthesis method of the synthetic tumor vaccine as described above, and its synthetic route is as follows:

[0053]

[0054] Preferably, the steps are as follows:

[0055] Step a: In the presence of an alkaline buffer solution and divalent metal ions, make D-(+)-galactose 2 undergo an enzymatic reaction with adenosine triphosphate ATP. The reaction is carried out in a 37°C water bath for 3 hours, and the dosage of adenosine triphosphate ATP is equivalent to the molar equivalent of galactose 2, and compound 3 can be obtained;

[0056] Step b: Adjust the pH value of the solution after the reaction in step a to weakly acidic. Then, add the receptor compound 4 to the system, and dropwise add the BiGalHexNAcP enzyme solution, and continue to react in a 37°C water bath for 24 hours to obtain compound 5;

[0057] Step c: Dissolve compound 5 in anhydrous methanol, add Pd / C powder, where m 化合物5 :V 无水甲醇 :m Pd / C = 100 mg: 15 mL: 100 mg, and react in a hydrogen environment for 3 - 5 hours to obtain compound 6;

[0058] Step d: Under alkaline conditions and in a mixed solvent (V DMF :V PBS 4:1), couple compound 6 with the bifunctional reagent adipic acid 1,6-bis(2,5-dioxo-1-pyrrolidinyl) ester 7. The reaction temperature is room temperature, and the reaction time is 4 - 8 hours. After purification, compound 8 is obtained;

[0059] Step e: In PBS buffer (0.01M, pH = 8.0), add compound 8 and carrier protein BSA, where the molar mass ratio of compound 8 to BSA is 20:1 - 50:1. Oscillate at 30 °C and 180 rpm for 72 hours, and obtain glycoprotein conjugate 9 after purification and lyophilization.

[0060] Step f: Weigh solid aluminum salt and dissolve it in a mixed solution of water and absolute ethanol (V 水 :V 无水乙醇 = 1:1) to prepare 0.1M aluminum chloride solution; prepare an aqueous solution of glycoprotein conjugate 9 (1 - 10 mg / mL), add 3 parts of disodium fumarate solution (0.1M) to it, mix well, then add 1 part of the prepared aluminum chloride solution (0.1M) to it, quickly pipette and mix well, and let it stand for 30 minutes. Finally, centrifuge the reaction solution at 9000 rpm, wash the precipitate three times with distilled water and then dry it to obtain complex 6F-TF-BSA@MIL-53(Al)-FA(1).

[0061] Preferably, in step a, the solvent is selected from an alkaline buffer solution system; or, in step a, the reaction temperature is 10 - 40 °C.

[0062] Preferably, in step b, the acidic condition is created using a dilute hydrochloric acid solution; or, in step b, the solvent is one or more selected from water, methanol, ethanol, and acidic buffer solution; or, in step b, the reaction temperature is 10 - 40 °C.

[0063] Preferably, in step c, the solvent is one or more selected from water, tetrahydrofuran, dichloromethane, methanol, ethyl acetate, and alkaline buffer solution; or, in step c, the reaction temperature is 10 - 30 °C.

[0064] Preferably, in step d, the solvent is one or more selected from water, methanol, ethanol, and alkaline buffer solution; or, in step b, the reaction temperature is 10 - 30 °C.

[0065] Preferably, in step f, the aluminum salt is one or more selected from aluminum nitrate, aluminum chloride, aluminum sulfate, aluminum acetate, aluminum phosphate, or aluminosilicate.

[0066] Use of the intermediate and product as described above in the preparation or as a tumor vaccine.

[0067] As a preferred embodiment of the preparation method of the present invention, compound 3 is obtained by reacting under the action of a kinase, the enzyme is galactokinase, the alkaline buffer solution is Tris-HCl buffer solution, the divalent metal ion is magnesium ion, and the reaction temperature is 37 °C.

[0068] As a preferred embodiment of the preparation method of the present invention, for the synthesis of compound 5, the reaction solution is Tris-HCl buffer solution, and the pH value of the system is between 4.0 and 7.0.

[0069] As a preferred embodiment of the preparation method of the present invention, for the synthesis of compound 8, the organic solvent is a mixed solution of N, N-dimethylformamide and phosphate buffer solution.

[0070] As a preferred embodiment of the preparation method of the present invention, for the preparation of 0.1M aluminum chloride solution, aluminum chloride hexahydrate is dissolved in a mixed solution of water and absolute ethanol, and the volume ratio of water to absolute ethanol is 1:1.

[0071] As a preferred embodiment of the preparation method of the present invention, in the synthesis of complex 1, the volume ratio of the aqueous solution of glycoprotein conjugate 9 (1 - 10 mg / mL), 0.1M disodium fumarate solution and 0.1M aluminum chloride solution is 1:3:1.

[0072] As a preferred embodiment of the preparation method of the present invention, 6F-TF-BSA@MIL-53(Al)-FA powder is mixed with Pluronic F127 thermosensitive hydrogel to prepare an injectable biomimetic mineralized tumor vaccine 6F-TF-BSA@MOF / Gel.

[0073] As a preferred embodiment of the application of the present invention, the cancers are colon cancer, prostate cancer, breast cancer and ovarian cancer that can overexpress TF antigen.

[0074] Specifically, the relevant preparation processes and test results are as follows:

[0075] A biomimetic mineralized tumor vaccine 6F-TF-BSA@MIL-53(Al)-FA containing fluorinated TF antigen, the biomimetic mineralized tumor vaccine is (1), and its structure is as Figure 16 shown.

[0076] A synthesis method of the above-mentioned synthetic tumor vaccine, and its synthesis route is as follows:

[0077]

[0078] Specifically, the steps are as follows:

[0079] Step a: In the presence of 100 mM Tris-HCl buffer solution (pH 8.5) and divalent metal ions, substrate galactose 2 (23 mM) reacts with adenosine triphosphate ATP (23 mM) under the action of galactokinase, and the reaction time is 3 hours under the condition of 37 °C water bath to obtain compound 3;

[0080] Step b: Adjust the pH of the solution after the reaction in step a to 6.0. Then, add the receptor compound 6F-GalNAcProN34 (15 mM) to the system, and add dropwise 100 μL of BiGalHexNAcP enzyme solution. Continue the reaction in a water bath at 37 °C for 24 hours to obtain compound 5;

[0081] Step c: Dissolve compound 5 in anhydrous methanol, add Pd / C powder, where m 化合物5 :V 无水甲醇 :m Pd / C = 100 mg: 15 mL: 100 mg, and react under a hydrogen atmosphere for 3 - 5 hours to obtain compound 6;

[0082] Step d: Under the conditions of pH 8.0 and a mixed solvent (V DMF :V PBS = 4:1, volume ratio), couple compound 6 with the bifunctional reagent adipic acid 1,6-bis(2,5-dioxo-1-pyrrolidinyl) ester 7. The reaction temperature is at room temperature, and the reaction time is 4 - 8 hours. After purification, compound 8 is obtained;

[0083] Step e: In a PBS buffer solution (0.01 M, pH = 8.0), add compound 8 and the carrier protein BSA, where the molar mass ratio of compound 8 to BSA is 20:1 - 50:1. Oscillate at 30 °C and 180 rpm for 72 hours. After purification and lyophilization, the glycoprotein conjugate 9 is obtained.

[0084] Step f: Weigh aluminum chloride hexahydrate and dissolve it in a mixed solution of water and anhydrous ethanol (V 水 :V 无水乙醇 = 1:1, volume ratio) to prepare a 0.1 M aluminum chloride solution; Prepare an aqueous solution of the glycoprotein conjugate 9 with a weight of 1 part (1 - 10 mg / mL), add 3 parts by weight of disodium fumarate solution (0.1 M) to it, mix well, then add 1 part by weight of the prepared aluminum chloride solution (0.1 M) to it, quickly pipette and mix well, and let it stand for 30 minutes. Finally, centrifuge the reaction solution at 9000 rpm, wash the precipitate three times with distilled water and then dry it to obtain the complex 6F-TF-BSA@MIL-53(Al)-FA(1).

[0085] A pharmaceutical combination of the above complex is prepared as follows:

[0086] Dissolve the thermosensitive hydrogel such as Pluronic F127 (PF127) in deionized water and keep it at 4 °C until completely dissolved. Record the gelation temperature using the tube inversion method. When in use, mix the 6F-TF / TF-BSA@MIL-53(Al)-FA powder with the thermosensitive hydrogel. The dosage ratio of the 6F-TF-BSA@MIL-53(Al)-FA powder to the thermosensitive hydrogel solution is 10 - 250 mg:mL, and the injectable biomimetic mineralized tumor vaccine 6F-TF-BSA@MOF / Gel is obtained.

[0087] The synthesis method of the above compound is as follows in more specific steps:

[0088] Preparation of compound 5:

[0089] Add the weighed glycosyl acceptor compound 3-azidopropyl β-D-6-deoxy-6-fluoro-2-acetamido-2-deoxy-α-D-galactopyranoside (6FGalNAcProN3,4) (100 mg, 327 μmol), D-(+)-galactose (125 mg, 694 μmol), adenosine triphosphate ATP (350 mg, 690 μmol), and anhydrous magnesium chloride (100 mg, 1.05 mmol) into 30 mL Tris-HCl buffer (100 mmol, pH = 8.5), and shake up and down until all components are dissolved. Then, aliquot it into 2 mL EP tubes, with each tube having a 1 mL reaction system. Add 100 μL of GalK enzyme (galactokinase) and place it in a 37 °C water bath for a constant-temperature reaction for 3 h. Monitor the progress of the reaction using TLC (EA:MeOH:H2O:AcOH = 4:2:1:0.5, volume ratio). After developing with anisaldehyde, if the reaction is shown to be complete, adjust the reaction solution to pH 6.0 with 0.1 M hydrochloric acid, and then introduce 100 μL of BiGalHexNAcP enzyme and continue the reaction in a 37 °C water bath for 24 hours. Track and monitor the reaction process using TLC analysis (EA:MeOH:H2O:AcOH = 4:2:1:0.5, volume ratio). After the reaction is completed, transfer the entire reaction system to a 50 mL centrifuge tube. Then add an equal volume of anhydrous methanol, stir the solution vigorously, and then store it at 4 °C for 30 minutes. Subsequently, centrifuge the reaction mixture at 4 °C and 8000 rpm for 30 minutes. The supernatant is concentrated and freeze-dried to obtain the crude product. Continue to separate and purify the crude product using silica gel column chromatography (EA:MeOH = 5:1, volume ratio), and compound 5 (117 mg, 263 μmol, 89%) can be obtained after purification by a BioGel P-2 gel column. The NMR detection results of compound 5 are as Figure 1 and Figure 2 shown, and the specific NMR assignment results are as follows: 11H NMR (400 MHz, D2O) δ 4.92 (d, J = 4.0 Hz, 1H), 4.76 - 4.69 (m, 1H), 4.62 - 4.54 (m, 1H), 4.45 (d, J = 8.0 Hz, 1H), 4.34 (dd, J = 8.0, 4.0 Hz, 1H), 4.30 - 4.24 (m, 2H), 4.04 (dd, J = 12.0, 8.0 Hz, 1H), 3.90 (d, J = 4.0 Hz, 1H), 3.80 - 3.73 (m, 3H), 3.66 - 3.60 (m, 2H), 3.53 - 3.45 (m, 3H), 3.24 (s, 1H), 2.02 (s, 3H), 1.90 (t, J = 4.0, 4.0 Hz, 2H). 13 13C NMR (100 MHz, D2O) δ 174.58, 104.77, 97.38, 84.41, 82.77, 76.84, 75.02, 72.52, 70.63, 68.62, 65.13, 61.03, 59.35, 48.54, 48.17, 27.98, 22.03. 19 19F NMR (376 MHz, D2O) δ -229.48.

[0090] Preparation of Compound 6:

[0091] Compound 5 (117 mg, 263.32 μmol) was placed in a 50 mL round-bottom flask and dissolved in 18 mL of anhydrous methanol. Subsequently, 100 mg of Pd / C was added to the reaction mixture under a hydrogen atmosphere, and the reaction was carried out for about 4 hours. After the reaction was completed, thin-layer chromatography analysis (EA:H2O:MeOH:AcOH = 4:1:2:0.5, volume ratio) was performed, and anisaldehyde was used for color development to detect whether the reaction had ended. Then, the reaction solution was filtered to remove the Pd / C catalyst, and the product was purified and separated using a Sephadex LH-20 gel chromatography column. After lyophilization, a white powder, namely Compound 6 (116 mg), was obtained. The NMR detection results of Compound 6 are as Figure 3 and Figure 4 shown below. The specific NMR assignment results are as follows: 11H NMR (400 MHz, D2O) δ 4.92 (d, J = 4.0 Hz, 1H), 4.72 (d, J = 4.0 Hz, 1H), 4.60 - 4.56 (m, 1H), 4.45 (d, J = 8.0 Hz, 1H), 4.35 - 4.29 (m, 3H), 4.23 (dd, J = 8.0, 4.0 Hz, 1H), 4.04 (d, J = 4.0 Hz, 1H), 3.90 (d, J = 4.0 Hz, 3H), 3.74 - 3.60 (m, 2H), 3.52 (d, J = 4.0 Hz, 2H), 2.75 (s, 1H), 2.02 (s, 3H), 1.78 (d, J = 4.0 Hz, 2H). 13 13C NMR (100 MHz, D2O) δ 174.57, 104.76, 97.37, 84.40, 82.76, 76.83, 75.01, 72.49, 70.59, 68.59, 68.46, 66.04, 61.01, 48.52, 37.64, 30.70, 21.95. 19 19F NMR (376 MHz, D2O) δ -229.31.

[0092] Preparation of Compound 8:

[0093] Prepare 10 mL of a mixed solvent of DMF:PBS (pH = 8.0) (v:v = 4:1, volume ratio). Add compound 6 (20 mg) to this solvent, and then add adipic acid 1,6 - bis(2,5 - dioxo - 1 - pyrrolidinyl) ester 7 (40 mg) as the linker. Stir until dissolved and react at room temperature for 6 hours. Monitor the reaction progress by TLC analysis (EA:MeOH:H2O:AcOH = 4:2:1:0.5, volume ratio) and detect using anisaldehyde color development; after determining the reaction is complete, remove the solvent under reduced pressure using a rotary evaporator. Then, add a small amount of deionized water and filter off the insoluble matter with filter paper or a filter membrane. The filtrate is dried to obtain a white powdery compound, namely the activated ester 8 (30 mg, 98%). The purified 8 can be directly used for protein binding. The NMR test results of compound 8 are as shown in Figure 5 、 Figure 6 and Figure 7 shown below. The specific NMR assignment results are as follows: 1HNMR(400MHz, D2O) δ 4.90 (d, J = 4.0 Hz, 1H), 4.71 - 4.68 (m, 1H), 4.59 - 4.56 (m, 1H), 4.46 (d, J = 8.0 Hz, 1H), 4.34 (dd, J = 12.0, 8.0 Hz, 1H), 4.29 - 4.19 (m, 2H), 4.05 (dd, J = 12.0, 8.0 Hz, 1H), 3.91 (d, J = 4.0 Hz, 1H), 3.76 - 3.71 (m, 6H), 3.69 - 3.61 (m, 2H), 3.53 - 3.49 (m, 2H), 2.67 (s, 5H), 2.25 - 2.18 (m, 5H), 2.02 (s, 3H), 1.83 (dd, J = 12.0, 16.0 Hz, 1H), 1.57 - 1.54 (m, 3H). 13 CNMR(100MHz, D2O) δ 176.87, 174.59, 104.78, 97.37, 84.37, 83.72, 76.89, 74.99, 72.50, 70.61, 69.22, 69.02, 68.30, 68.40, 65.01, 60.99, 60.58, 59.43, 48.49, 36.08, 35.63, 28.05, 25.83, 25.28, 24.95, 22.01. 19 FNMR(376MHz, D2O) δ -229.35.

[0094] Preparation of Compound 9:

[0095] According to the molar mass ratio of sugar chain to protein of 50:1, carrier protein BSA and activated ester 8 were weighed and dissolved in 5 mL of PBS buffer (0.01 M, pH = 8.0), and shaken at 30 °C and 180 rpm for 72 hours. Then the solution was diluted with 10 mL of deionized water, and centrifuged at 6000 rpm for 20 minutes at 4 °C using a 50 KD ultrafiltration tube (Amicon Ultra, Millipore) to remove the uncoupled sugar chains. Subsequently, the glycoprotein conjugate was concentrated and collected, and after freeze-drying, a white fluffy glycoprotein conjugate 6F-TF-BSA9 was obtained. The average molecular weight of the protein conjugate was determined to be 70.464 KDa by MALDI-TOF-MS. The molecular weight detection results of the conjugate 6F-TF-BSA9 are as Figure 8 shown.

[0096] Meanwhile, according to the same method as above, a conjugate TF-BSA of natural TF antigen and carrier protein BSA was prepared as a reference for subsequent immune evaluation. The specific preparation method is as follows:

[0097]

[0098] Weigh 10 of TF antigen activated ester and carrier protein BSA according to the molar mass ratio of sugar chain to protein of 50:1, dissolve them in 5 mL of PBS buffer (0.01 M, pH = 8.0), and oscillate at 30 °C and 180 rpm for 72 hours. Then dilute the solution with 10 mL of deionized water, and use a 50 KD ultrafiltration tube (AmiconUltra, Millipore) to centrifuge at 4 °C at a speed of 6000 rpm for 20 minutes to remove the uncoupled sugar chains. Subsequently, concentrate and collect the glycoprotein conjugate, and after freeze-drying, obtain a white and fluffy glycoprotein conjugate TF-BSA.

[0099] At the same time, according to the same method as above, a conjugate of natural TF antigen and ovalbumin OVA (TF-OVA) was prepared and used as a coating antigen for ELISA assay. The specific preparation method is as follows:

[0100]

[0101] Weigh 10 of carrier TF antigen activated ester and ovalbumin OVA according to the molar mass ratio of sugar chain to protein of 50:1, dissolve them in 5 mL of PBS buffer (0.01 M, pH = 8.0), and oscillate at 30 °C and 180 rpm for 72 hours. Then dilute the solution with 10 mL of deionized water, and use a 50 KD ultrafiltration tube (AmiconUltra, Millipore) to centrifuge at 4 °C at a speed of 6000 rpm for 20 minutes to remove the uncoupled sugar chains. Subsequently, concentrate and collect the glycoprotein conjugate, and after freeze-drying, obtain a white and fluffy glycoprotein conjugate TF-OVA.

[0102] Preparation of Compound 1:

[0103] Weigh 24.1 mg of aluminum chloride hexahydrate and dissolve it in a 1 mL mixed solution of water and absolute ethanol (water: absolute ethanol = 1:1, volume ratio) to prepare a 0.1 M aluminum chloride solution. Weigh 5 mg of glycoprotein conjugate 6F-TF-BSA9 and dissolve it in 1 mL of deionized water to prepare a 5 mg / mL protein solution. Then measure 200 μL of the protein solution and add it to 600 μL of disodium fumarate solution (0.1 M), mix well, and finally add 200 μL of the prepared aluminum chloride solution thereto, quickly pipette to mix evenly, and let it stand at room temperature (25 °C ± 2 °C) for 30 min. Centrifuge at 8000 rpm and room temperature for 5 min, collect the precipitate, wash it three times with deionized water, and dry it. Collect the supernatant for measuring the amount of protein loaded on the MOFs material. The protein encapsulation efficiency (LE%) (Formula 1) and loading capacity (LC, g / g) (Formula 2) of 6F-TF-BSA@MIL-53(Al)-FA(1) are determined by analyzing the amount of remaining free protein in the supernatant using a Bradford protein quantification kit. LE and LC are calculated by the following formulas.

[0104]

[0105] m 总蛋白 Refers to the total amount of protein input into the reaction system / mg; m 上清蛋白 Refers to the mass of the remaining protein in the solution after the reaction / mg.

[0106]

[0107] m 蛋白 Refers to the mass of the protein loaded onto the MOFs material / mg; m 材料 Refers to the mass of the MOFs material / mg.

[0108] Meanwhile, prepare the MOFs material MIL-53(Al)-FA coated with natural TF-BSA conjugate, namely TF-BSA@MIL-53(Al)-FA, according to the above method for subsequent immune evaluation. The specific preparation method is as follows:

[0109] Weigh 24.1 mg of aluminum chloride hexahydrate and dissolve it in a 1 mL mixed solution of water and absolute ethanol (water: absolute ethanol = 1:1, volume ratio) to prepare a 0.1 M aluminum chloride solution. Weigh 5 mg of glycoprotein conjugate TF-BSA and dissolve it in 1 mL of deionized water to prepare a 5 mg / mL protein solution. Then measure 200 μL of the protein solution and add it to 600 μL of disodium fumarate solution (0.1 M), mix well, and finally add 200 μL of the prepared aluminum chloride solution thereto, quickly pipette to mix evenly, and let it stand at room temperature (25 °C ± 2 °C) for 30 min. Centrifuge at 8000 rpm and room temperature for 5 min, collect the precipitate, wash it three times with deionized water, and dry it.

[0110] Meanwhile, the blank MOF material MIL-53(Al)-FA was prepared according to the above method for subsequent immune evaluation.

[0111] Preparation method of the blank MOF material MIL-53(Al)-FA: Weigh 24.1 mg of aluminum chloride hexahydrate and dissolve it in a mixed solution of 1 mL of water and anhydrous ethanol (water:anhydrous ethanol = 1:1, volume ratio) to prepare a 0.1 M aluminum chloride solution. Add 600 μL of disodium fumarate solution (0.1 M) to 200 μL of deionized water, mix well, then add 200 μL of the prepared aluminum chloride solution, quickly pipette and mix evenly, and let it stand at room temperature (25 °C ± 2 °C) for 30 min. Centrifuge at 8000 rpm and room temperature for 5 min, collect the precipitate, wash it three times with deionized water, and dry it.

[0112] TEM image, SEM image, PXRD of Compound 1 Figures 9 to 11 As shown. After Bradford protein quantification, the experimental result shows that the encapsulation efficiency (LE%) of 6F-TF-BSA@MIL-53(Al)-FA(1) is 95%, demonstrating the superiority of the in-situ encapsulation preparation method and enabling efficient encapsulation of the antigen. Moreover, each gram of the MOF material can effectively load 0.37 grams of protein, that is, the loading capacity (LC, g / g) is 0.37 g / g, showing a high loading capacity of the MOF material for the antigen.

[0113] Preparation of the injectable biomimetic mineralized tumor vaccine 6F-TF-BSA@MIL-53(Al)-FA Gel:

[0114] Dissolve 2.1 g of the thermosensitive hydrogel Pluronic F127 (PF127) in 10 mL of deionized water and keep it at 4 °C until completely dissolved. Record the gelation temperature using the tube inversion method. When in use, mix the 6F-TF-BSA@MIL-53(Al)-FA powder with the thermosensitive hydrogel Pluronic F127 (7.5 mg / mL) according to the dosage to obtain the injectable biomimetic mineralized tumor vaccine 6F-TF-BSA@MOF / Gel.

[0115] Meanwhile, TF-BSA@MOF / Gel was prepared according to the above method. When in use, mix the TF-BSA@MIL-53(Al)-FA powder with the thermosensitive hydrogel Pluronic F127 (7.5 mg / mL) according to the dosage for subsequent immune evaluation. The difference between TF-BSA@MOF / Gel and 6F-TF-BSA@MOF / Gel is that the sugar antigen loaded on the MOF material is the natural TF antigen rather than the fluorinated TF antigen.

[0116] Evaluation of the anti-tumor immune activity of the vaccine:

[0117] Fifty-four female Balb / c mice aged 6 - 8 weeks were selected as experimental subjects. The tumor cells used were 4T1 murine breast cancer cells to establish a tumor-bearing mouse model. The 4T1 tumor cells were cultured in vitro, and 1×10 6 tumor cells were subcutaneously implanted into each mouse. When the tumor size of the mice reached 50 mm 3 , immunization began. On day 0, day 5, and day 10, the mice were grouped and injected with the vaccine.

[0118] Group V1 in the first group was the negative control group, and the mice were injected with PBS (0.1M, pH = 7.4) solution; Group V2 in the second group was the hydrogel group, and the mice were injected with sterile thermosensitive hydrogel Pluronic F127 solution; Group V3 in the third group was the blank MOFs group, and the mice were injected with MIL-53(Al)-FA solution (concentration 5 mg / mL, dosage 100 μL / mouse); Group V4 in the fourth group was the TF-BSAGel group (dissolving TF-BSA powder in the above thermosensitive gel Pluronic F127, concentration 2 mg / mL, dosage 100 μL / mouse); Group V5 in the fifth group was the 6F-TF-BSAGel group (dissolving 6F-TF-BSA powder in the above thermosensitive gel Pluronic F127, concentration 2 mg / mL, dosage 100 μL / mouse); Group V6 in the sixth group was the TF-BSA@MIL-53(Al)-FAGel group (dissolving TF-BSA powder in the above thermosensitive gel Pluronic F127, concentration 2 mg / mL, dosage 100 μL / mouse); Group V7 in the seventh group was the 6F-TF-BSA@MIL-53(Al)-FAGel group (dissolving 6F-TF-BSA@MIL-53(Al)-FA in the above thermosensitive gel Pluronic F127, concentration 7.5 mg / mL, dosage 100 μL / mouse); Group V8 in the eighth group was the TF-BSA / Alum Gel group (dissolving TF-BSA and aluminum adjuvant in the above thermosensitive gel Pluronic F127, where the TF-BSA concentration was 2 mg / mL and the aluminum adjuvant concentration was 1 mg / mL, dosage 100 μL / mouse), and Group V9 in the ninth group was the 6F-TF-BSA / Alum Gel group (dissolving 6F-TF-BSA and aluminum adjuvant in the above thermosensitive gel Pluronic F127, where the 6F-TF-BSA concentration was 2 mg / mL and the aluminum adjuvant concentration was 1 mg / mL, dosage 100 μL / mouse).

[0119] The vaccination dose per injection is 100 μL. Among them, glycoprotein antigen is used, and the content of TF sugar antigen in each dose is 10 μg; when using commercially available aluminum adjuvant, the content of aluminum ions in each dose is 100 μg. After the last immunization, the feeding time of the mice is extended by 5 days, which can enhance the stability of the activation of the immune response in Balb / c mice. After the immunization period, the mice are euthanized and blood is drawn from the femoral artery (0.5 mL / mouse). The collected blood is allowed to stand at room temperature for 2 hours, then centrifuged at 3000 rpm at 4 °C, and the serum is collected and stored in a -80 °C refrigerator for subsequent immunological activity detection. During the immunization cycle, the changes in tumor volume and body weight of the mice are recorded.

[0120] Preparation of solutions:

[0121] Coating solution: Dissolve 8.4 g of NaHCO3 in 1 L of distilled water, adjust the pH value to 9.6 with 1 M NaOH solution, and store at 4 °C for use.

[0122] Washing solution: Dissolve 0.5 mL of Tween-20 in 1 L of 0.01 M PBS solution and let it stand at room temperature.

[0123] Blocking solution: Dissolve 2.5 g of bovine serum albumin (BSA) in 125 mL of 0.01 M phosphate buffer solution (PBS) and store at 4 °C for use.

[0124] Antibody dilution solution: Dissolve 2.5 g of bovine serum albumin (BSA) in 250 mL of 0.01 M phosphate buffer solution (PBS), then add 1.25 mL of 20-fold diluted Tween-20 and store at 4 °C for use.

[0125] Chromogenic solution: 0.1 M citric acid: Add 960 mg of citric acid to 50 mL of distilled water (A); 0.2 M disodium hydrogen phosphate: Dissolve 3.582 g of disodium hydrogen phosphate dodecahydrate in 50 mL of distilled water (B); Take 24.3 mL of solution (A), 25.7 mL of solution (B), and then add 50 mL of distilled water. Immediately add 50 mg of OPD (o-phenylenediamine) and 150 μL of 30% H2O2 before use.

[0126] Stop solution: It is 2 M H2SO4: Accurately measure 55.5 mL of concentrated sulfuric acid with a measuring cylinder and add deionized water to 500 mL.

[0127] The ELISA method is used to determine the antibody titer in the serum of mice:

[0128] (1) Preparation of substrate-coated plates: First, completely dissolve the glycoprotein conjugate TF-OVA in the coating solution. Second, calculate the loading amount of the glycoprotein conjugate to ensure that the relative content of the glycoprotein in the solution is always maintained at 20 μg / mL. Finally, add 100 μL of glycoprotein solution evenly to each 96-well plate, then coat at room temperature for 1 hour, and finally coat overnight at 4°C.

[0129] (2) Blocking the substrate plate: spin dry the coating solution, add 100 μL / well of blocking solution, and incubate at the optimal temperature (37°C) for 2 h.

[0130] (3) Drying the substrate plate: Use a plate spinner to spin off the blocking solution, then incubate in an incubator (37°C) for 1 to 2 hours. Once the liquid on the bottom of the plate is completely dry, proceed to the next step.

[0131] (4) Quantitative detection of antibody binding: We diluted the mouse serum 50 times, took 10 μL of serum and mixed it with 490 μL of Solebol antibody diluent, and added 250 μL of the prepared antibody diluent to each of the remaining 11 EP tubes. Pipette 250 μL of liquid from the first tube and mix it with the liquid in the next EP tube, and dilute it to 102400 times in this way. Add the diluted serum sample to a 96-well plate, 100 μL per well, and incubate at 37°C for 1.5 hours. After drying the liquid, add 300 μL of washing solution to each well and shake and wash for 60 seconds. Repeat the washing several times, add HRP-rabbit anti-mouse IgG / IgM antibody diluted 1:2000, 100 μL per well, and incubate at 37°C for another 1 hour. Dry the liquid in the dark, wash the plate several times, add the color developing solution in the dark, 100 μL per well, react at room temperature for about 17 minutes, and add 50 μL of stop solution. Finally, an ELISA reader was used to detect the absorbance value at a wavelength of 490 nm.

[0132] Using PBS as the negative control group, the IgG and IgM antibody titers produced by each vaccine group were calculated. The statistical results are as follows: Figure 12 and Figure 13As shown. Specifically, the TF-BSA Gel group (2437), 6F-TF-BSA Gel group (5744), TF-BSA@MIL-53(Al)-FA Gel group (29446), 6F-TF-BSA@MIL-53(Al)-FA Gel group (42678), TF-BSA / Alum Gel group (15446), 6F-TF-BSA / Alum Gel group (20146). The OD values of the blank thermosensitive Gel group and the MIL-53(Al)-FA group were close to those of the negative control, indicating that the adjuvant and the delivery system alone could not produce an immune effect. At the same time, when the glycoprotein conjugate was used for immunization alone, the IgG antibody titer level was low. When immunized with the composite thermosensitive gel, the IgG antibody titer could be increased by 1 fold. Interestingly, the 6F-TF-BSA@MIL-53(Al)-FA Gel group had higher immunogenicity than the 6F-TF-BSA Gel group, and the titer was also higher than that of the 6F-TF-BSA / Alum Gel group. Therefore, it can be known that the metal-organic framework material MIL-53(Al)-FA not only has an adjuvant effect, but also has a better effect than the commercially available aluminum hydroxide adjuvant, showing excellent advantages. Compared with the TF-BSA@MIL-53(Al)-FA Gel (29446) group, the specific IgG antibody titer produced by the 6F-TF-BSA@MIL-53(Al)-FA Gel group reached 42678, and the induced specific antibody titer increased by 1.4 times. This indicates that the introduction of fluorine atoms successfully stimulated a stronger level of antigen-specific response in mice, fully demonstrating the advantages of fluorinated antigens.

[0133] After the first administration, the tumor sizes of mice in each group were measured every 2 days for a total of 2 weeks to determine the association between the immune response induced after inoculation with the candidate vaccine and the tumor growth rate. The results showed ( Figure 14 ), on the 14th day, the tumors in the group without antigen grew rapidly, exceeding 250 mm 3 , while the tumor growth in the group containing the 6F-TF / TF-BSA antigen was significantly inhibited. It is worth noting that the tumor suppression effect of the 6F-TF-BSA@MIL-53(Al)-FA Gel group was stronger than that of the 6F-TF-BSA / Alum Gel group. During the treatment period, the tumor volume remained at about 50 mm 3 and did not increase. This may be due to the fact that MIL-53(Al)-FA can slowly release 6F-TF-BSA and 6F-TF-BSA@MIL-53(Al)-FA can protect 6F-TF-BSA from degradation by glycosidases in the body, resulting in a longer presence time and a longer immune stimulation time of 6F-TF-BSA in the body. The safety of the vaccine can also be evaluated by the broken line graph of the mouse body weight, such as Figure 15As shown. It can be seen from the line graph of mouse body weight that during the vaccination period, the body weights of mice in each group increased steadily, proving that the tumor vaccines injected in this study had no obvious toxicity.

[0134] In the present invention, through in-situ encapsulation, a conjugate of glycoprotein and a new adjuvant based on an aluminum-based crystalline cluster are co-loaded to prepare a complex. And the complex is combined with a thermosensitive hydrogel for application. This combined application can overcome the obstacles in the time dimension and space dimension encountered in traditional vaccine delivery and effectively trigger a systemic antigen-specific cellular immune response. There has been no similar technical report so far.

[0135] Although embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that various substitutions, changes, and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A 6F-TF-BSA@MIL-53(Al)-FA complex, characterized in that: The complex includes metal-organic framework material MIL-53(Al)-FA and fluorinated glycoprotein antigen 6F-TF-BSA loaded in MIL-53(Al)-FA; wherein, the organic ligand in MIL-53(Al)-FA is fumaric acid and the metal ion is Al 3+ ; the loading amount of 6F-TF-BSA is 0.1 to 0.5 g / g.

2. The preparation method of the 6F-TF-BSA@MIL-53(Al)-FA complex according to claim 1, characterized in that: It includes the following steps:

3. The synthesis method of the 6F-TF-BSA@MIL-53(Al)-FA complex according to claim 2, characterized in that: Step a: In the presence of 100 mM alkaline buffer solution and 30 mM divalent metal ions, an enzymatic reaction is carried out between D-(+)-galactose 2 and adenosine triphosphate ATP at 37 °C in a water bath for 3 hours. The dosage of adenosine triphosphate ATP is equivalent to the molar equivalent of D-(+)-galactose 2 to obtain compound 3; Step b: Adjust the solution after the reaction in step a to pH 6.

0. Then, add the receptor compound 4 to the system and dropwise add the BiGalHexNAcP enzyme solution, and continue to react in a 37 °C water bath for 24 hours to obtain compound 5; Step c: Compound 5 undergoes Pd / C-catalyzed hydrogenation reaction to obtain compound 6; Step d: Under alkaline conditions and in a mixed solvent, the mixed solvent is obtained by mixing DMF and PBS with a volume ratio of V DMF :V PBS of 4:1, coupling compound 6 with bifunctional reagent 7 at room temperature for 4 - 8 hours, and obtaining compound 8 through purification; Step e: In a 0.01 M, pH = 8.0 PBS buffer solution, add compound 7 and the carrier protein BSA, where the molar mass ratio of compound 8 to BSA is 20:1 to 50:

1. Oscillate at 30 °C and 180 rpm for 72 hours, and obtain the glycoprotein conjugate 9 through purification and lyophilization; Step f: Weigh the aluminum salt solid and dissolve it in a mixed solution of water and absolute ethanol, where the volume ratio of water to absolute ethanol V 水 :V 无水乙醇 is 1:1 to 1:3 to prepare a 0.1 M aluminum chloride solution; prepare an aqueous solution of 1 to 10 mg / mL of 1 mass portion of the glycoprotein conjugate 9, add 3 mass portions of a 0.1 M disodium fumarate solution thereto, mix well, then add 1 mass portion of the prepared 0.1 M aluminum chloride solution thereto, quickly pipette evenly, and let stand for 20 to 60 min; finally, centrifuge the reaction solution at 9000 rpm, wash the precipitate three times with distilled water, and then freeze-dry to obtain the complex 6F-TF-BSA@MIL-53(Al)-FA(1).

4. The preparation method according to claim 3, characterized in that: The aluminum salt is selected from one or more of aluminum nitrate, aluminum chloride, aluminum sulfate, aluminum acetate, aluminum phosphate or aluminosilicate.

5. The preparation method according to any one of claims 2 to 4, characterized in that: The time for the static reaction is 30 minutes, and the temperature for the static reaction is 10 - 40 °C.

6. An injectable biomimetic mineralized tumor vaccine 6F-TF-BSA@MOF / Gel loaded with the 6F-TF-BSA@MIL-53(Al)-FA complex as claimed in claim 1, characterized in that: Dissolve the thermosensitive hydrogel Pluronic F127 in deionized water to prepare a thermosensitive gel solution; when in use, mix the 6F-TF-BSA@MIL-53(Al)-FA powder with the thermosensitive hydrogel solution immediately before use. The concentration of 6F-TF-BSA@MIL-53(Al)-FA after mixing is 7.5 mg / mL to obtain the injectable biomimetic mineralized tumor vaccine 6F-TF-BSA@MOF / Gel.

7. The injectable biomimetic mineralized tumor vaccine 6F-TF-BSA@MOF / Gel according to claim 6, characterized in that: The dosage ratio of the 6F-TF-BSA@MIL-53(Al)-FA powder to the thermosensitive hydrogel solution is 10 - 250:1 in mg:mL.

8. A pharmaceutical composition, characterized in that: The composition includes the 6F-TF-BSA@MIL-53(Al)-FA complex according to claim 1 or the Pluronic F127 thermosensitive gel; the preparation method of the Pluronic F127 thermosensitive gel is as follows: Dissolve the thermosensitive hydrogel Pluronic F127 in deionized water to prepare a 210 mg / mL gel solution.

9. The pharmaceutical composition according to claim 8, characterized in that: The administration route of the pharmaceutical composition is injection.

10. The application of the 6F-TF-BSA@MIL-53(Al)-FA complex according to claim 1 or the pharmaceutical composition according to claim 8 or 9 in the preparation of a tumor vaccine.

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