Al-Mn composite nanocrystals and their manufacturing method and use

Abstract

Provided is an Al-Mn composite nanocrystal and its preparation method and use. The preparation method of the Al-Mn composite nanocrystal includes step 1 of mixing an aluminum salt solution, a manganese salt solution and an anionic auxiliary solution to obtain a mixture, and adjusting the pH of the mixture to 5.5-8.5, and step 2 of heating the mixture to react, and washing the obtained solid product to obtain Al-Mn composite nanocrystal. If the Al-Mn composite nanocrystal obtained by the preparation method and the Al-Mn composite nanocrystal are used in the preparation of a vaccine adjuvant, a pharmaceutical composition, a drug delivery carrier, or an immunogenic composition, the technical problem that conventional aluminum adjuvants cannot simultaneously activate humoral immunity and cellular immunity can be effectively solved.

Description

[Technical Field]

[0001] This application claims priority from China Patent Application No. 202111633153.6, filed with the State Intellectual Property Administration of China on December 28, 2021, entitled "Al-Mn composite nanocrystals and their manufacturing method and use," the entire contents of which are incorporated herein by reference.

[0002] The present application relates to the field of vaccine technology, and in particular to Al-Mn composite nanocrystals and their preparation and use. [Background technology]

[0003] Aluminum salt adjuvants are the most widely used and safest of traditional vaccine adjuvants. Examples include aluminum potassium sulfate, aluminum phosphate, and aluminum hydroxide. Among them, aluminum hydroxide adjuvants are the most widely used. The immune-stimulating mechanisms of aluminum adjuvants are currently mainly explained by a "reservoir" effect, promotion of phagocytosis, and activation of the pro-inflammatory pathway NLRP3. Commercially available aluminum hydroxide adjuvants are typically low-crystalline boehmite with a size of 1-10 μm, which generally activates humoral immunity with little T cell-mediated immunity. This is related to the presentation mode of antigens adsorbed by aluminum hydroxide adjuvants. After micron-sized aluminum hydroxide adjuvants adsorb antigens, they typically bind to the cell membrane of antigen-presenting cells, such as dendritic cells (DCs), inducing lipid raft formation and activating downstream pathways, without affecting the fate of the presented antigen in the cell. However, antigens are presented via the major histocompatibility complex II (MHC-II) after processing through the lysosomal pathway, and are not cross-presented via the major histocompatibility complex I (MHC-I). However, recent research has shown that addressing the needs of more common pathologies requires not only the generation of humoral immunity to produce antibodies that neutralize pathogenic microorganisms, but also the generation of T cell-mediated immunity to eliminate viruses, bacteria, or abnormal physiological responses, and the generation of memory T cells for prevention and protection. Therefore, new aluminum adjuvant formulations or composite adjuvants are needed to meet the demands of preventive vaccine protection.

[0004] Because aluminum adjuvants have the advantages of safety and convenience during long-term use, as well as their usefulness as carriers for antigens and molecular adjuvants, novel adjuvants formed by combining aluminum salts with molecular adjuvants have become the main trend in the development of new adjuvants. For example, composite adjuvants formed from aluminum salts with 3M-052, CpG, and TLR agonists have shown further advantages in improving vaccine antibody titers and cellular immune responses. However, the preparation of molecular adjuvants and their compounding with aluminum adjuvants require numerous steps, which can significantly affect the efficacy of new adjuvants. Therefore, the development of aluminum adjuvants that are easy to manufacture, have sustained release properties for carriers and antigens, and are highly safe can address the shortcomings of aluminum adjuvants in cellular immunity. Furthermore, new composite adjuvants that are easy to manufacture and produce are of greater practical value. Summary of the Invention [Problem to be solved by the invention]

[0005] The present application has been made in view of the above circumstances, and provides an Al-Mn composite nanocrystal that can effectively solve the technical problem that conventional aluminum adjuvants cannot simultaneously activate humoral immunity and cellular immunity, as well as a method for producing and using the same. [Means for solving the problem]

[0006] One aspect of the present application provides a method for producing Al-Mn composite nanocrystals, including: Step 1: mixing an aluminum salt solution, a manganese salt solution, and an anionic auxiliary solution to obtain a mixture, and adjusting the pH of the mixture to 5.5 to 8.5; and Step 2: heating the mixture to cause a reaction, and washing the obtained solid product to obtain Al-Mn composite nanocrystals.

[0007] Specifically, in step 1, the pH of the mixture is adjusted with an acidity / alkali adjuster. The acidity / alkali adjuster may be an alkaline solution and / or an acid solution. The alkaline solution and the acid solution are ordinary pH adjusting solutions, and the concentration of the alkaline solution is 0.01 to 1 mol / L, preferably 0.05 to 0.6 mol / L. The alkaline solution is one or more of an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, an aqueous ammonia solution, an aqueous sodium bicarbonate solution, and an aqueous sodium phosphate solution. The concentration of the aqueous sodium hydroxide solution is 0.05 to 1 mol / L, preferably 0.3 to 0.6 mol / L.

[0008] Specifically, the pH of the mixture is adjusted because the Al-Mn composite nanocrystals obtained within this range are so stable that their crystal form does not change even after sterilization. Specifically, the phosphate salt of the aqueous sodium phosphate solution is Na2HPO4 and / or Na3PO4.

[0009] Specifically, in step 1, the molar ratio of the total solutes of the aluminum salt solution, the manganese salt solution, and the anionic auxiliary solution to the solutes of the alkaline solution is 1:(1 to 6). For example, the molar ratio of the total solutes of the aluminum salt solution, the manganese salt solution, and the anionic auxiliary solution to the NaOH alkaline solution is 1:(1 to 6). Preferably, the molar ratio of the total solutes of the aluminum salt solution, the manganese salt solution, and the anionic auxiliary solution to the solutes of the alkaline solution is 1:(2 to 4). For example, the molar ratio of the total solutes of the aluminum salt solution, the manganese salt solution, and the anionic auxiliary solution to the NaOH alkaline solution is 1:(2 to 4). Specifically, in step 1, the pH value of the mixture is 5.5 to 8.0. Preferably, the pH value of the mixture is 6.0 to 7.0.

[0010] Specifically, in step 1, the aluminum salt solution, the manganese salt solution, and the anionic auxiliary solution are mixed by stirring while dropping the solutions. The dropping speed of the aluminum salt solution, the manganese salt solution, and the anionic auxiliary solution is 2 to 15 mL / min, preferably 3 to 8 mL / min. The stirring speed during the mixing is 50 to 1000 rpm, preferably 300 to 800 rpm. The stirring time during the mixing is 1.5 to 10 hours, preferably 1 to 5 hours.

[0011] Specifically, when adjusting the pH value of the mixture with an alkaline solution, the dropping rate of the alkaline solution is 0.5 to 10 mL / min, and preferably 1 to 4 mL / min.

[0012] Specifically, in step 2, the mixture is centrifuged with NaCl solution to remove unreacted free ions and desorbed anionic auxiliary agents, and then redissolved and dispersed in NaCl solution. After sterilization by moist heat at 121°C for 30 to 60 minutes, the mixture is dispensed to obtain Al-Mn composite nanocrystals. The Al-Mn composite nanocrystals can be stored in a refrigerator (2 to 8°C). The centrifugal washing is performed at a rotation speed of 2,000 to 10,000 g, for a centrifugation time of 1 to 100 minutes, and for two to five cycles. Preferably, the centrifugal washing is performed at a rotation speed of 4,000 to 8,000 g, for a centrifugation time of 5 to 20 minutes, and for two to three cycles.

[0013] Specifically, the concentration of the NaCl solution is 0.1% to 1.5%, and preferably 0.15% to 0.8%.

[0014] In another embodiment, the solute of the aluminum salt solution is one or more selected from the group consisting of aluminum chloride, aluminum sulfate, aluminum nitrate, and aluminum acetate. The solvent of the aluminum salt solution is one or more selected from the group consisting of sodium acetate solution, physiological saline, water, and ethanol. Specifically, the concentration of the solvent of the aluminum salt solution is 0.01 to 1 mol / L, and the concentration of the sodium acetate solution is 0.03 to 0.07 mol / L.

[0015] In another embodiment, the solute of the manganese salt solution is one or more selected from the group consisting of manganese chloride, manganese sulfate, manganese nitrate, and manganese acetate, and the solvent of the manganese salt solution is one or more selected from the group consisting of water, saline, and ethanol. In another embodiment, the solute of the anionic auxiliary solution is an organic acid salt and / or an amino acid, and the solvent of the anionic auxiliary solution is one or more selected from the group consisting of water, saline, and ethanol.

[0016] Specifically, the anionic auxiliary solution can fully realize the combined effect of the anionic auxiliary and the metal ions (aluminum and manganese). The pre-precipitate in the mixture is subjected to heat treatment to promote uniform growth and crystal formation of the pre-precipitate in the mixture, improving sterilization stability. After centrifugal sedimentation, washing, and sterilization, Al-Mn composite nanocrystals are obtained.

[0017] Specifically, the anionic auxiliary is an organic substance containing a polyhydroxyl group, a carboxyl structure, and a phosphorus element. The anionic auxiliary in this application can form a stable compound with both aluminum ions and manganese ions, making it an ideal auxiliary for stabilizing and controlling the particle size and distribution of Al-Mn composite nanocrystals.

[0018] In another embodiment, the organic acid salt is citric acid and / or salicylic acid, and the amino acid is one or more selected from the group consisting of cysteine, cystine, tyrosine, aspartic acid, and glutamic acid.

[0019] In another embodiment, the molar ratio of aluminum element in the aluminum salt solution to manganese element in the manganese salt solution in the mixture is 1:(0.05-1). Preferably, the molar ratio of aluminum element in the aluminum salt solution to manganese element in the manganese salt solution is 1:(0.1-0.5). The ratio of the concentration of the anionic auxiliary to the total concentration of metal ions is (0.1-10):1. Preferably, the ratio of the concentration of the anionic auxiliary to the total concentration of metal ions is (0.3-3):1. Here, the total concentration of metal ions is the sum of the concentrations of the aluminum salt solution and the manganese salt solution.

[0020] Specifically, the concentration of the aluminum salt solution is 0.01 to 1 mol / L, preferably 0.03 to 0.07 mol / L, the concentration of the manganese salt solution is 0.001 to 1 mol / L, preferably 0.003 to 0.05 mol / L, and the concentration of the anionic auxiliary solution is 0.01 to 0.5 mol / L, preferably 0.05 to 0.5 mol / L.

[0021] In another embodiment, in step 2, the heating reaction is carried out at a temperature of 60° C. to 130° C., and the heating reaction time is 0.5 to 20 hours.

[0022] A second aspect of the present application provides Al—Mn composite nanocrystals including Al—Mn composite nanocrystals produced by the above-described production method.

[0023] Specifically, the pH value of the Al-Mn composite nanocrystals before and after sterilization is 5.5 to 8.5, the particle size of the Al-Mn composite nanocrystals is stable at 20 to 5,000 nm, the adsorption rate of the Al-Mn composite nanocrystals to bovine serum albumin exceeds 1.6 mg, and the obtained Al-Mn composite nanocrystal product has excellent dispersibility and stability.

[0024] A third aspect of the present application discloses Al-Mn composite nanocrystals obtained by the above-mentioned preparation method, and the use of the Al-Mn composite nanocrystals in the preparation of vaccine adjuvants, immune enhancers, pharmaceutical compositions, drug delivery carriers, or immunogenic compositions.

[0025] Specifically, the Al-Mn composite nanocrystals provided herein are used in the manufacture of vaccine adjuvants and immune enhancers for improving innate and / or adaptive immunity. In some embodiments, the Al-Mn composite nanocrystals stimulate the expression of type I interferon and gamma interferon, thereby improving innate and / or adaptive immunity and regulating immune balance. In other embodiments, the Al-Mn composite nanocrystals can promote antigen presentation and increase antibody production levels, thereby improving innate and / or adaptive immunity. In other embodiments, the Al-Mn composite nanocrystals can be administered in combination with, in combination with, or sequentially administered with an immunomodulator to promote the activation of multiple immune pathways and improve innate and / or adaptive immunity. The Al-Mn composite nanocrystals of the present application can also be used as immune enhancers for tumor prevention and / or treatment of diseases such as bacterial infections, fungal infections, viral infections, parasitic infections, tumors, and autoimmune diseases.

[0026] The Al-Mn composite nanocrystals of the present application can also be used as a pharmaceutical composition to treat autoimmune diseases, such as rheumatoid arthritis, type 1 diabetes, psoriasis, systemic lupus erythematosus, and multiple sclerosis. More specifically, when the Al-Mn composite nanocrystals of the present application are used as components of a pharmaceutical composition, the pharmaceutical composition contains the Al-Mn composite nanocrystals and a prophylactic / therapeutic agent, both of which are administered to a subject in need thereof. The composition can also be combined with small molecules, immunomodulators such as polypeptides and antibodies, antibody drugs, or polypeptide drugs by mixing, sequential administration, or modification.

[0027] Specifically, the drug delivery carrier includes a composition of Al-Mn nanocrystals combined with one or more liquid, solid, or commonly used pharmaceutical auxiliaries and / or optional auxiliary ingredients, including pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, inert diluents, wetting agents, emulsifiers, and dispersing agents.

[0028] A fourth aspect of the present application provides an antitumor pharmaceutical composition comprising the Al-Mn composite nanocrystals and a therapeutic agent for antitumor diseases.

[0029] Specifically, the present invention provides applications of antitumor pharmaceutical compositions in the treatment of solid tumors and non-solid tumors, including lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, bladder cancer, kidney cancer, colorectal cancer, placental choriocarcinoma, cervical cancer, ovarian cancer, testicular cancer, uterine cancer, and leukemia. Non-solid tumors include glioma and hematoma.

[0030] Specifically, the anti-tumor disease therapeutic agent is an anti-tumor small molecule, polypeptide, or antibody-based drug, such as one or more of PD-L1 antibody, trastuzumab, metformin (Met), paclitaxel, doxorubicin, cisplatin, asparaginase, rituximab, gefitinib, triptorelin, docetaxel, doxorubicin, and fluorouracil.

[0031] A fifth aspect of the present application provides a vaccine composition comprising an antigen and the Al—Mn composite nanocrystals.

[0032] Specifically, the vaccine composition is a protein vaccine and / or an inactivated vaccine, such as an influenza vaccine strain recombinant antigen or a hepatitis B recombinant antigen. The vaccine composition may also be a varicella-zoster virus recombinant protein vaccine, a novel coronavirus subunit protein vaccine, or a novel coronavirus inactivated vaccine.

[0033] Specifically, the antigen in the vaccine composition is derived from a virus, a bacterium, a parasite, or a tumor neoantigen. For example, the virus is selected from DNA viruses and RNA viruses. Preferably, the virus is selected from the group consisting of Coronaviridae, Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Hepadnaviridae, Flaviviridae, Papillomaviridae, Poxviridae, and Retroviridae. More preferably, the virus is selected from the group consisting of novel coronavirus, influenza virus, herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV, and HBV. For example, the bacterium is selected from Gram-positive and Gram-negative bacteria. Preferably, the bacterium is selected from the group consisting of Streptococcus pneumoniae, Haemophilus influenzae, Salmonella enterica, Neisseria meningitidis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Acinetobacter baumannii, Mycobacterium tuberculosis, and Helicobacter pylori.

[0034] More specifically, the antigens in the vaccine composition are derived from influenza virus, alpha or beta coronavirus (e.g., novel coronavirus and variants), hepatitis virus (e.g., hepatitis A virus, hepatitis B virus), poliovirus, rabies virus, HPV virus, encephalitis virus (e.g., Japanese encephalitis virus), mumps virus, rubella virus, Clostridium tetani, Bordetella pertussis, Corynebacterium diphtheriae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria meningitidis, Streptococcus pneumoniae, and various forms of antigens thereof, such as activated viral / bacterial forms, live attenuated viral / bacterial forms, and subunit proteins, recombinant proteins, or chimeric proteins.

[0035] Specifically, the water used in the present application may be ultrapure water or / and water for injection. Manganese is a trace element essential for human growth, metabolism, neuronal function, and antioxidant defense processes. Combining the two inorganic elements aluminum and manganese to form a composite adjuvant preserves the "reservoir" effect, promotion of phagocytosis, and activation of the pro-inflammatory pathway NLRP3, which are inherent to aluminum adjuvants. 2+ The present invention complements natural and adaptive immunity through the use of α- and β-glucan, and satisfies the need for both humoral and cellular immunity to resist infectious pathogens. Furthermore, the present invention makes it possible to prepare Al-Mn composite nanocrystals with uniform size and controllable morphology.

[0036] Thus, this application discloses aluminum-manganese composite nanocrystals that can be used as vaccine adjuvants, which are simple to process, uniform in composition, stable in structure, can be stored for a long time after sterilization, and are convenient to use. The Al-Mn composite nanocrystals can simultaneously activate humoral and cellular immunity, providing a better immune barrier for preventing infectious bacteria or viruses. [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 is a transmission electron microscope image of the Al—Mn composite nanocrystals (AlMn-001) provided in Example 1 of the present application. [Figure 2] FIG. 2 is a transmission electron microscope image of the Al—Mn composite nanocrystals (AlMn-002) provided in Example 2 of the present application. [Figure 3] FIG. 3 is a transmission electron microscope image of the Al—Mn composite nanocrystals (AlMn-003) provided in Example 3 of the present application. [Figure 4] FIG. 4 shows the XRD results of the Al—Mn composite nanocrystals provided in Examples 1 to 3 of the present application. [Figure 5] FIG. 5 is a diagram showing the DC activation effect of different proportions of Al-Mn composite nanocrystals provided in Examples 1 to 3 of the present application. [Figure 6]FIG. 6 shows fluorescent imaging images of DC cell endocytosis after binding of the Al-Mn composite nanocrystals (AlMn-003) provided in Example 3 of the present application or a commercially available aluminum adjuvant with an antigen. [Figure 7] FIG. 7 is a graph showing the experimental results of antibody titers after intramuscular injection of the Al-Mn composite nanocrystals (AlMn-003) provided in Example 3 of the present application or a commercially available aluminum adjuvant in combination with OVA antigen into mice. [Figure 8] Figure 8 shows the results of antibody titers of mice immunized with the Al-Mn composite nanocrystals provided in Examples 1 to 3 of the present application combined with a novel coronavirus RBD recombinant protein antigen. [Figure 9] FIG. 9 shows the experimental results of antibody titers after intramuscular injection of mice with H1N1-HA antigen combined with the Al-Mn composite nanocrystal (AlMn-003) provided in Example 3 of the present application or the commercially available aluminum adjuvant Alum. [Figure 10] FIG. 10 shows the results of IFN-γ expression in mouse splenocytes on day 56 after immunization with the Al-Mn composite nanocrystals AlMn-003 provided in Example 3 of the present application in combination with H1N1 HA antigen. [Figure 11] FIG. 11 shows the results of cytokines in mouse splenocytes on day 56 after immunization with Al-Mn composite nanocrystals AlMn-003 provided in Example 3 of the present application in combination with H1N1-HA antigen. [Figure 12] FIG. 12 shows the percentage of memory B cells in the spleen and lung of mice on day 56 after immunization with the Al-Mn composite nanocrystals AlMn-003 provided in Example 3 of the present application in combination with H1N1-HA antigen. [Figure 13] FIG. 13 shows the percentage of memory T cells in the spleens of mice on day 56 after immunization with the Al-Mn composite nanocrystals AlMn-003 provided in Example 3 of the present application in combination with H1N1-HA antigen. [Figure 14]Figure 14 shows graphs showing the results of enhancing the antitumor effect of the living body using the Al-Mn composite nanocrystal AlMn-001 provided in Example 1 of the present application as an immunopotentiator. The three figures, from left to right, are Figure 14A, Figure 14B, and Figure 14C, respectively. [Figure 15] FIG. 15 is a graph showing the results of enhancing the anti-distant tumor effect of the living body using the Al-Mn composite nanocrystal AlMn-002 provided in Example 2 of the present application as an immunopotentiator. [Figure 16] FIG. 16 is a graph showing the results of enhancing the anti-liver cancer effect in vivo using the Al-Mn composite nanocrystal AlMn-003 provided in Example 3 of the present application as an immunopotentiator. [Figure 17] FIG. 17 is a graph showing the results of enhancing the anti-metastatic tumor effect in vivo using the Al-Mn composite nanocrystal AlMn-001 provided in Example 1 of the present application as an immunopotentiator. DETAILED DESCRIPTION OF THE INVENTION

[0038] In this application, to solve the technical defect that conventional aluminum adjuvants cannot simultaneously activate humoral immunity and cellular immunity, Al-Mn composite nanocrystals and their preparation method and use are provided.

[0039] The technical solutions of the present invention will be described more clearly and comprehensively below with reference to examples of the present invention, but it is clear that the examples described are only a part of the embodiments of the present invention, and do not represent all the embodiments. All other examples that those skilled in the art can obtain based on the embodiments of the present invention without creative effort shall fall within the scope of protection of the present invention. The raw materials or reagents used in the following examples are either commercially available or home-made. [Example]

[0040] Example 1 In the examples of the present application, Al-Mn composite nanocrystal AlMn-001 was provided, which was produced by a specific production method including the following steps.

[0041] (1) AlCl3·6H2O was weighed and dissolved in 0.01 mol / L NaAc to prepare an aluminum salt solution with an aluminum ion content of 1 mol / L. MnCl2·4H2O was weighed and dissolved in water for injection to prepare a manganese salt solution with a manganese ion content of 0.1 mol / L. Salicylic acid was weighed and dissolved in pure water to prepare an anionic auxiliary solution with a content of 0.05 mol / L. NaOH was weighed and dissolved in water for injection to prepare an alkaline solution with a content of 0.1 mol / L.

[0042] (2) First, the aluminum salt solution and the manganese salt solution were mixed in equal volumes to obtain a mixed solution. 30 mL of the mixed solution was added to 50 mL of salicylic acid solution at a rate of 10 mL / min. Then, the mixture was stirred at 1000 rpm for 3 hours to allow the mixing reaction to occur sufficiently, and a mixture was obtained.

[0043] (3) The mixture was stirred at room temperature at 600 rpm, and 30 mL of ammonia solution was added at a rate of 12 mL / min. After the dropwise addition, the pH of the mixture was adjusted to 7.6, and the temperature was raised to 100°C and heated for 1 hour.

[0044] (4) The mixture was cooled to room temperature and centrifuged at 6000 g for 20 minutes. The resulting solid product was washed twice with 0.5% sodium chloride solution. It was then centrifuged at 6000 g for 10 minutes and redissolved in 0.5% sodium chloride solution to obtain Al-Mn composite nanocrystals, designated AlMn-001. The mixture was then sterilized at 121°C for 30 minutes and packaged separately.

[0045] (5) The Al-Mn composite nanocrystals were stored refrigerated (2–8°C) for a long period of time. Referring to Figure 1, Figure 1 shows the size and morphology of the Al-Mn composite nanocrystals prepared in Example 1 of the present application, as measured by a transmission electron microscope (FEI Company, model number Tecnai G2 20S-TWIN). Figure 1 shows that the Al-Mn composite nanocrystals prepared in Example 1 of the present application are composite particles composed of thin sheet-like nanosheets with a width of 30 to 120 nm and a length of 50 to 300 nm. Example 2

[0046] In the examples of the present application, Al-Mn composite nanocrystals AlMn-002 were provided, which were produced by a specific production method including the following steps.

[0047] (1) Al(NO3)3·6H2O was weighed and dissolved in 0.01 mol / L NaAC to prepare an aluminum salt solution with an aluminum ion content of 0.4 mol / L. Mn(SO4)2·4H2O was weighed and dissolved in water for injection to prepare a manganese salt solution with a manganese ion content of 0.1 mol / L. Glutamic acid was weighed and dissolved in pure water to prepare an anionic auxiliary solution with a concentration of 0.20 mol / L. KOH was weighed and dissolved in water for injection to prepare an alkaline solution with a KOH content of 0.5 mol / L.

[0048] (2) First, the aluminum salt solution and the manganese salt solution were mixed in equal volumes to obtain a mixed solution. 40 mL of the mixed solution was added to a container containing 30 mL of glutamic acid solution, and the mixture was reacted at room temperature with a stirring speed of 800 rpm for 7 hours to allow the complexation reaction to occur sufficiently, obtaining a mixture.

[0049] (3) The rotation speed was adjusted to 500 rpm for stirring, and then potassium hydroxide solution was added at a rate of 6.9 mL / min so that the pH of the solution became 8.5, the temperature was adjusted to 80°C, and the reaction was maintained for 7 hours.

[0050] (4) After cooling to room temperature, the mixture was centrifuged at 6000 g for 20 minutes, and the resulting solid product was washed twice with 0.5% sodium chloride solution. It was then centrifuged at 6000 g for 10 minutes and redissolved in 0.5% sodium chloride solution to obtain Al-Mn composite nanocrystals, designated AlMn-002.

[0051] (5) After that, they were sterilized with moist heat at 121°C for 30 minutes and then packaged separately. The Al-Mn composite nanocrystals were stored refrigerated (2–8°C) for a long period of time. Referring to Figure 2, Figure 2 shows the size and morphology of the Al-Mn composite nanocrystals prepared in Example 2 of the present application, as measured by a transmission electron microscope (FEI Company, model number Tecnai G2 20S-TWIN). Figure 2 shows that the Al-Mn composite nanocrystals prepared in Example 2 of the present application are composite particles composed of thin sheet-like nanosheets with a width of 20 to 70 nm and a length of 30 to 100 nm. Example 3

[0052] In the examples of the present application, Al-Mn composite nanocrystals AlMn-003 were provided by a specific manufacturing method including the following steps.

[0053] (1) Aluminum acetate was weighed and dissolved in 0.01 mol / L NaAC to prepare an aluminum salt solution with an aluminum ion content of 0.5 mol / L. MnCl2·4H2O was weighed and dissolved in water for injection to prepare a manganese salt solution with a manganese ion content of 0.1875 mol / L. Citric acid was weighed and an anionic auxiliary solution with a concentration of 0.1 mol / L was prepared. Sodium hydroxide was weighed and dissolved in water for injection to prepare an alkaline solution with a content of 0.2 mol / L.

[0054] (2) First, an aluminum salt solution and a manganese salt solution were mixed in equal volumes to obtain a mixed solution. 10 mL of the mixed solution was added to a reactor containing 50 mL of citric acid solution, and the mixture was mixed at room temperature at 1,200 rpm for 1 hour to allow the mixing reaction to occur sufficiently, obtaining a mixture.

[0055] (3) Next, 10 mL of aqueous sodium hydroxide solution was added dropwise at a rate of 16.9 mL / min to adjust the pH of the solution to 9, and the temperature was raised to 60°C, and the reaction was maintained for 12 hours.

[0056] (4) After cooling to room temperature, the mixture was centrifuged at 8000 g for 10 minutes, and the resulting solid product was washed three times with 0.5% sodium chloride solution. It was then centrifuged at 8000 g for 10 minutes and redissolved in 0.5% sodium chloride solution to obtain Al-Mn composite nanocrystals, designated AlMn-003.

[0057] (5) After that, they were sterilized with moist heat at 121°C for 30 minutes and then packaged separately. The Al-Mn composite nanocrystals were stored refrigerated (2–8°C) for a long period of time. Referring to Figure 3, Figure 3 shows the size and morphology of the Al-Mn composite nanocrystals prepared in Example 3 of the present application, as measured by a transmission electron microscope (FEI Company, model number Tecnai G2 20S-TWIN). Figure 3 shows that the Al-Mn composite nanocrystals prepared in Example 3 of the present application are composite particles composed of thin sheet-like nanosheets with a width of 20 to 100 nm and a length of 30 to 200 nm. Example 4

[0058] The examples of the present application provide the measurement results of the physicochemical properties of the Al—Mn composite nanocrystals obtained in Examples 1 to 3. Specifically, the following measurement tests are included. The following detection method can be applied to measure the physicochemical properties of Al-Mn composite nanocrystals at any Mn:Al molar ratio.

[0059] (1) Characterization of hydrated particle size and potential The Al-Mn composite nanocrystals prepared in Examples 1, 2, and 3 were diluted 100 to 200 times with ultrapure water until they became transparent and colorless, and 800 μL of the diluted solution was placed in a Zeta potential sample cell for testing.

[0060] (2) X-ray diffraction (XRD) characterization The Al-Mn composite nanocrystals were placed in a centrifuge tube and freeze-dried. The solid powder was then evenly packed into the window of the glass sample holder and pressed down with a slide to flatten the convex part so that the powder filled the entire concave window and was flush with the top surface of the glass slide. The sample holder was then placed in the X-ray diffraction instrument, and the parameters were set in the software to scan. The results are shown in Figure 4. From Figure 4, it was found that the manganese contained in the Al-Mn composite nanocrystal samples obtained in Examples 1 to 3 of the present application was in a trimanganese tetroxide crystalline form, and the aluminum was in an AlO(OH) crystalline form.

[0061] (3) Digestion quantification of Al-Mn composite nanocrystals 100 μL of the Al-Mn composite nanocrystals obtained in Examples 1-3 of this application, diluted 10-fold with ultrapure water, was taken and placed in a 50 mL polytetrafluoroethylene digestion bottle. 2-3 mL of MOS-grade concentrated nitric acid was added, the temperature was raised to 90 °C on an electric heating plate, and 2 mL of MOS-grade hydrogen peroxide was added dropwise to clarify the solution. The temperature was then increased to 160 °C in 10 °C increments and held for 20 minutes. The solution was slowly evaporated until 0.5 mL of solution remained, then removed, cooled to room temperature, and quantified to 3 g. A 3% cleaning solution was prepared with MOS-grade nitric acid, and a calibration curve was created using standard samples. Detection and quantification were performed using an inductively coupled plasma mass spectrometer.

[0062] (4) Adsorption rate: The adsorption rate was calculated by the adsorption effect of Al-Mn composite nanocrystals on bovine serum albumin. Preparation of test sample solutions: An appropriate amount of the test sample was diluted with 0.85%-0.90% sodium chloride solution to an aluminum content of 1 mg / mL, and the pH was adjusted to 6.0-7.0 to prepare the test sample solution. Note that the pH of different batches of the same type of Al-Mn adjuvant must be adjusted to a fixed value to ensure consistency between different test batches. Bovine serum albumin (also simply called "BSA") solution: An appropriate amount of bovine serum albumin was taken and a 10 mg / mL solution was prepared using 0.85% to 0.90% sodium chloride solution, and the pH value was adjusted to match the pH value of the test sample solution. Measurement method: Five 15 mL centrifuge tubes were prepared, and 0.08 mL, 0.16 mL, 0.4 mL, 0.8 mL, and 1.2 mL of bovine serum albumin solution (10 mg / mL) was added to each tube. 0.85% to 0.90% sodium chloride solution was added to each tube to make the total volume 4.0 mL. After mixing well, 1.0 mL of the test sample solution was added to each tube and mixed well. The amount of bovine serum albumin in each tube was 0.8 mg, 1.6 mg, 4 mg, 8 mg, and 12 mg, respectively, and the aluminum content in each tube was 1 mg. Each tube was left at room temperature for 1 hour (with vigorous shaking every 10 minutes), then centrifuged at 5000 g for 10 minutes to collect the supernatant. The free bovine serum albumin content in each tube was measured by the Lowry method (General Rule 0731, Method 2) or other appropriate method, and the absorbance value of each tube was recorded to calculate the protein content. The adsorption rate (A) of each tube was calculated based on the free bovine serum albumin content in the supernatant of each tube corresponding to the total amount of bovine serum albumin. For calculations, the volume of the supernatant was set at 5 ml. Adsorption rate = (total amount of BSA - amount of supernatant BSA) ÷ total amount of BSA × 100. Results evaluation: The supernatant protein in the two tubes containing 0.8 mg and 1.6 mg BSA should be undetectable, meaning the adsorption rate should not fall below 90%. The absorbance values ​​in the tubes containing 4 mg, 8 mg, and 12 mg BSA should show an overall increasing trend (if the absorbance value cannot be determined, the supernatant protein content should be detected to determine the overall increasing trend), and the adsorption rate will be evaluated as passing.

[0063] (5) 24-hour sedimentation rate: The pH of the test sample was adjusted to 6.0-7.0 with dilute hydrochloric acid or sodium hydroxide solution, and the test sample was diluted with water to an aluminum concentration of 5 mg / mL. If the aluminum content of the test sample was lower than 5 mg / mL, it was diluted to 1 mg / mL with 9 g / L sodium chloride solution after adjusting the pH. If the aluminum content of the test sample was lower than 1 mg / mL, it was left for an appropriate period of time, and the supernatant was discarded to adjust the aluminum content to 1 mg / mL. After shaking for at least 30 seconds, 25 mL of the solution was transferred to a measuring cylinder or graduated colorimetric tube and left for 24 hours. The sedimentation rate of the test sample was calculated according to the volume of the precipitated supernatant using the following formula: Sedimentation rate (%) = Supernatant volume / 25 × 100%.

[0064] In the above examples, the hydrated particle size and surface charge of aluminum hydroxide adjuvants were measured using a nanoparticle size and Zeta potential analyzer (purchased from Malvern, model number Zetasizer Nano ZS). The aluminum and manganese contents of Al-Mn composite adjuvants were measured using an inductively coupled plasma optical emission spectroscopy (ICP-OES, purchased from PerkinElmer, model number PerkinElmer Optima 5300DV). The adsorption and precipitation states were measured in accordance with the Chinese Pharmacopoeia (2020 edition). The experimental results are shown in Table 1.

[0065] [Table 1] Example 5

[0066] In the examples of the present application, a comparison of the BMDC activation abilities of the Al-Mn composite nanocrystals prepared in Examples 1 to 3 is provided as follows.

[0067] The BMDC activation effects of Al-Mn composite nanocrystals AlMn-001, Al-Mn composite nanocrystals AlMn-002, and Al-Mn composite nanocrystals AlMn-003 prepared in Examples 1 to 3 of the present application were evaluated.

[0068] Immature BMDCs were plated in a 6-well plate at 5 x 105 BMDCs were seeded at 1000 cells / well and cultured in parallel with three groups of the commercially available aluminum adjuvant Alum (purchased from Invivogen, CAS: 21645-51-2) at a total metal ion concentration of 50 μg / ml, the Al-Mn composite nanocrystals AlMn-001 obtained in Example 1, the Al-Mn composite nanocrystals AlMn-002 obtained in Example 2, and the Al-Mn composite nanocrystals AlMn-003 obtained in Example 3 for 24 hours. After 24 hours of culture, the cells were harvested and stained with anti-CD11c, anti-CD80, and anti-CD86 flow stains. The expression levels of the costimulatory factors CD80 and CD86 on the surface of BMDCs were measured by flow cytometry.

[0069] 5, it can be seen that the dendritic cell activation ability of the Al-Mn composite nanocrystals obtained by implementing the present invention increases with increasing Mn ratio, and the dendritic cell activation ability of the Al-Mn composite nanocrystal AlMn-003 is approximately twice that of commercially available aluminum adjuvants, indicating that the Al-Mn composite nanocrystals obtained in this application have a superior dendritic cell activation ability. Example 6

[0070] In the examples of the present application, the cell uptake effect after the Al-Mn composite nanocrystals adsorbed the antigen is provided as follows. DC2.4 cells were cultured in RPMI 1640 medium containing 10% (v / v) FBS in a humidified atmosphere at 37°C with 5% CO2. The cells and the constructed nanovaccines (a mixture of 10 mg / L FITC-labeled antigen molecules and 25 mg / L Al-Mn composite nanocrystals AlMn-003 or 100 mg / L commercial aluminum adjuvant Alum (purchased from Invivogen, CAS: 21645-51-2)) were incubated for 8 and 24 hours, respectively. The cells were then washed, labeled with Lysotracker Red, fixed, and observed under a fluorescence microscope (Perkin Elmer Spinning Disc confocal microscope, 60x oil immersion lens). Images were captured using UltraVIEW VoX software.

[0071] As shown in Figure 6, Figure 6 shows the fluorescence imaging images of DC cell entosis after binding of antigen with Al-Mn composite nanocrystals AlMn-003 prepared in Example 3 of the present application or aluminum adjuvant Alum.

[0072] As shown in Figure 6, the Al-Mn composite nanocrystals obtained in Example 3 of the present application were able to transport more antigens into cells than the commercially available aluminum adjuvant Alum, even when used in a quarter of the amount of the commercially available aluminum adjuvant. This indicates that the Al-Mn composite nanocrystals obtained in the present application have a superior vaccine carrier effect. Example 7

[0073] In the examples of the present application, the following verification tests are provided for the immune enhancing effect of the Al-Mn composite nanocrystals obtained in Examples 1 to 3. Specific tests include the following:

[0074] The Al-Mn composite nanocrystals obtained in Examples 1 to 3 had different Mn:Al molar ratios. Al-Mn composite nanocrystals with different Mn / Al molar ratios were each injected into mice in combination with OVA antigen, and the titers of the specific antibodies produced were measured. The specific method is as follows.

[0075] Experimental animals: Balb / C mice, 6-8 weeks old, 5 mice / group, female. Control group: Alum, a commercially available aluminum adjuvant (purchased from Invivogen, CAS: 21645-51-2). Dosage: OVA antigen (ovalbumin OVA purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS: 9006-59-1) was 10 μg / mouse, Al-Mn composite adjuvant was 50 μg / 100 μL / mouse, and aluminum adjuvant was 50 μg / 100 μL / mouse. Experimental groups: (1) AlMn-001 (Mn / Al = 0.1), (2) AlMn-002 (Mn / Al = 0.25), (3) AlMn-003 (Mn / Al = 0.375). Immunization regimen: The above drugs were mixed with the antigen in the same volume and injected intramuscularly into mice three times at 3-week intervals. After immunization, mice were divided into groups and orbital blood was collected on days 19, 35, and 56. Serum antibody titers were measured by ELISA.

[0076] As shown in Figure 7, the Al-Mn composite nanocrystals with different Mn / Al molar ratios obtained in Examples 1 to 3 not only showed immediate efficacy after a single injection of the compound, but also had a clear positive correlation between the dose and effect. The dose-effect relationship remained clear even after two immunizations. This indicates that the Al-Mn composite nanocrystals have a clear effect on enhancing humoral immunity of the OVA vaccine. Example 8

[0077] The examples of this application provide tests on the effectiveness of Al-Mn composite nanocrystals obtained in Examples 1 to 3 in enhancing the immune protective effect of the novel coronavirus SARS-CoV-2 RBD recombinant subunit protein antigen, as follows: Experimental animals: Balb / C mice, 6-8 weeks old, 5 mice / group, female. Dosage: The antigen was 10 μg / mouse, the commercially available aluminum adjuvant Alum (purchased from Invivogen, CAS: 21645-51-2) was 100 μg / 100 μl / mouse, and the AlMn-003Al-Mn composite nanocrystals were 100 μg / 100 μl / mouse. Experimental groups: (1) the spike protein RBD of the novel coronavirus (also referred to simply as "RBD antigen"; purchased from Sino Biological Company, product number 40592-V08H4); (2) commercially available aluminum adjuvant Alum + RBD; (3) Al-Mn composite nanocrystals obtained in Examples 1 to 3 + RBD, namely AlMn-001 + RBD, AlMn-002 + RBD, and AlMn-003 + RBD. Immunization regimen: Mice were immunized using group (1). The other two groups were immunized with a commercially available aluminum adjuvant, Alum, or the Al-Mn composite nanocrystal AlMn-003 obtained in embodiment 3, mixed in the same volume as the antigen. The mice were boosted according to the immunization group three weeks after the first immunization, and blood was collected via the orbit on day 14 after the second immunization, and the antibody titer in the serum was measured.

[0078] The experimental results, as shown in Figure 8, showed that the antibody titer still increased with increasing manganese content in the Al-Mn composite nanocrystals and was higher than that of commercially available aluminum adjuvants. The Al-Mn composite nanocrystals also had good immune-enhancing ability against the novel coronavirus subunit protein antigen. Example 9

[0079] The examples of the present application provide tests on the enhancement of the protective effect of influenza subunit vaccines by the Al-Mn composite nanocrystals obtained in Example 3. Specific tests include the following: The prepared Al-Mn composite nanocrystals, AlMn-003, were used as an adjuvant and combined with influenza H1N1 HA antigen and injected into mice. The titers of specific IgG and IgM antibodies produced were measured. The specific method is as follows:

[0080] Experimental animals: Balb / C mice, 6-8 weeks old, 10 mice / group, female. Dosage: H1N1 HA (purchased from Sino Biological Company, product number 40731-V07H) antigen was 5 μg / mouse, commercially available aluminum adjuvant Alum (purchased from Invivogen, CAS: 21645-51-2) was 100 μg / 100 μl / mouse, and AlMn-003Al-Mn composite nanocrystals was 100 μg / 100 μl / mouse. Experimental groups: (1) H1N1 HA antigen; (2) commercial aluminum adjuvant Alum + H1N1 HA; (3) Al-Mn composite nanocrystal AlMn-003 obtained in Example 3 + H1N1 HA; Immunization regimen: Mice were immunized using group (1). The other two groups were immunized with a commercially available aluminum adjuvant, Alum, or the Al-Mn composite nanocrystal AlMn-003 obtained in embodiment 3, mixed in the same volume as the antigen. The mice were immunized according to the group assignment three weeks after the first immunization, and blood was collected from the orbit two weeks after the second immunization to measure the antibody titer in the serum.

[0081] Serum antibody titers were measured by enzyme-linked immunosorbent assay (ELISA) to assess vaccine-induced IgG and IgM levels in mouse serum. Briefly, 96-well microtitration plates were precoated with H1N1 HA, incubated overnight at 4°C, and blocked with 2% BSA for 2 hours at 37°C. Serially diluted mouse sera were then added to the 96-well plates, incubated for 1 hour at 37°C, and washed four times with PBS. Bound antibodies were reacted with goat anti-mouse IgG conjugated to HRP for 1 hour at 37°C. After washing four times with PBS, 3,3',5,5'-tetramethylbenzidine (TMB; Sigma) was added as a substrate to the plates, and the reaction was terminated by adding 0.05% H2SO4. Absorbance at 450 nm and 630 nm was measured by ELISA (Tecan, San Jose, CA).

[0082] FIG. 9 shows the results of analyzing the titers of specific antibodies produced two weeks after the second immunization when mice were intramuscularly injected with either the Al-Mn composite nanocrystal AlMn-003 prepared in Example 3 of the present application or the commercially available aluminum adjuvant Alum in combination with an H1N1 HA antigen at three-week intervals.

[0083] As shown in Figure 9A, the experimental results showed that the Al-Mn composite nanocrystals obtained in Example 3 of the present application enhanced immune responses better than commercially available aluminum adjuvants, and the antibody titer generated by the Al-Mn composite nanocrystals was 104 times higher than that of the aluminum adjuvant.

[0084] As a result of the experiment, as shown in Figure 9B, the Al-Mn composite nanocrystals obtained in Example 3 of the invention had a higher IgM level than commercially available aluminum adjuvants, and the antibody titer induced by the Al-Mn composite nanocrystals was three times higher than that of the aluminum adjuvant.

[0085] Mice immunized with different vaccines in Example 9 were used for the subsequent measurements in Examples 10 to 13. 56 days after immunization, five mice were euthanized, spleen and lung tissues were collected, and cell suspensions were prepared for testing to evaluate the immunological efficacy of the vaccines. Example 10

[0086] The examples of this application provide antigen-specific IFN-γ T cell tests by ELISPOT after immunizing mice with the Al-Mn composite nanocrystals obtained in Example 3 and the commercially available aluminum adjuvant Alum. Specific tests include: 1) ELISPOT plates were precoated with anti-IFN-γ antibody (5 μg / ml) overnight at 4°C. 2) RPMI1640 medium containing 10% FBS was sealed at room temperature for 2 hours. 3) In Example 9, the spleens of the mice on day 56 after immunization were crushed with 200-mesh gauze to prepare single cell suspensions, and 5 × 10 cells were added per well. 5 The cells were added and incubated in a 5% CO2, 37°C incubator for 24 hours. 4) Biotinylated anti-IFN-γ antibody was added and incubated at room temperature for 2 hours. 5) HRP-crosslinked avidin was added and incubated at room temperature for 1 hour. 6) Using an Immunospot Analyzer ELISPOT reader board, the number of IFN-γ secreting cells (IFN-γ spot-forming cells, SFC) was read using Immunospot software (v. 3.0).

[0087] As shown in Figure 10, the experimental results showed that AlMn-003Al-Mn composite nanocrystals promoted γ-IFN secretion by T cells, and the amount of γ-IFN secreted was greater than that of the commercially available aluminum adjuvant Alum. The commercially available aluminum adjuvant Alum group promoted γ-IFN+ secretion by T cells, with a PBMC rate of 43.5% compared to the H1N1 HA antigen group. The AlMn-003Al-Mn composite nanocrystal group was 73% of the H1N1 HA antigen group. This indicates that AlMn-003Al-Mn composite nanocrystals have a superior effect in stimulating cellular immune responses. Example 11

[0088] The examples of the present application provide tests that measured intracellular factors in splenocytes extracted from the spleen tissue of mice 56 days after immunization with different vaccines, as collected in Example 9. Specific tests include the following: Flow cytometry was performed using a multicolor intracellular cytokine staining assay using splenocytes from mice stimulated with 2 μg / ml protein (H1N1 HA protein). DMSO was used as a negative control (background). Ten million cells were incubated for 6 hours at 37°C and 5% CO2 in the presence of Golgistop (BD Biosciences) and Brefeldin A (Sigma). Cell viability was assessed by measuring intracellular cytokine staining using DAPI-live / dead as an activity dye (Invitrogen) and anti-CD3, anti-CD4, anti-CD8, anti-CD69, anti-CD134, and anti-CD137 antibodies (Biolegend) for surface staining. Cells were permeabilized and stained with anti-IL-2, anti-IL-4, anti-IFN-γ, and anti-TNF-α antibodies (Biolegend). At least 100,000–200,000 lymphocytes were collected using a BD LSR Fortessa (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR). Samples were defined as positive if the antigen-specific response was at least three times greater than background and at least 0.05% higher than background.

[0089] As shown in Figure 11, among the cell factors in the spleen, the levels of IL-2, IL-4, TNF-α, and IFN-γ were high, and the level of IL-4 was higher than that of IL-2. IL-2 belongs to Th1 factors, and IL-4 belongs to Th2 factors. This indicates that the Al-Mn composite nanocrystal AlMn-003 according to the present application can promote the secretion of IFN-γ factors, which can improve the phagocytic ability of macrophages, and that the Al-Mn composite nanocrystal AlMn-003 can also promote the differentiation of T cells and the maturation of CD8 CTLs. Example 12

[0090] The examples of this application provide tests of splenic and lung memory B cell levels in mice 56 days after immunization with different vaccines in Example 9. Specific tests include the following:

[0091] B cells produce high-affinity antibodies, generate immunological memory, function as antigen-presenting cells, and secrete cytokines (including CCL22, CCL17, IL-2, IL-4, IL-6, IFN-γ, TNF-α, GM-CSF, IL-10, TGF-β1, and IL-35). Memory B cells and plasma B cells can produce antibodies, such as immunoglobulins (Ig), IgM, IgG, and IgE. Memory B cells are long-lived and remain dormant until they encounter an antigen again, at which point they can produce antibodies against that antigen. Upon reexposure to a foreign antigen, memory B cells reactivate and differentiate into plasma cells capable of secreting antibodies. IgG homoantibodies are secreted by these plasma cells and have high affinity for specific foreign antigens. These IgG antibodies effectively and rapidly neutralize viral and bacterial antigens and become part of a secondary immune response that includes immunological memory (particularly from memory B cells and memory T lymphocytes).

[0092] Therefore, in this application, the memory B cell levels on day 56 were evaluated for mice immunized with a combination of the Al-Mn composite nanocrystal AlMn-003 adjuvant obtained in Example 3 and H1N1 HA antigen.

[0093] The memory B cell levels of mice immunized with the Al-Mn composite nanocrystals AlMn-003 obtained in Example 3 combined with H1N1 HA antigen were measured on day 56, and the results are shown in Figure 12. The memory B cell flow results showed that the Al-Mn composite nanocrystal group induced more memory B cells in the spleen and lungs, and had better anti-reinfection capabilities. Example 13

[0094] The examples of this application provide a study of splenic memory T cell levels in mice 56 days after immunization with different vaccines in Example 9. Specific studies include the following:

[0095] In this application, we examined the phenotype of memory T cells in mice 56 days after immunization with the H1N1 HA vaccine, considering that memory CD4+ and CD8+ T cells are important components of protective immunity and that effectively inducing memory T cell responses is a primary goal of vaccines against chronic infections.

[0096] Mice were immunized with the Al-Mn composite nanocrystals AlMn-003 obtained in Example 3 combined with H1N1 HA antigen, and the CD4+ T and CD8+ T cell levels were measured on day 56. The experimental results are shown in Figure 13. The flow chart showed that memory T cells (Tcm) among CD4+ T and CD8+ T cells in the spleen were increased in the Al-Mn composite nanocrystal group. Example 14

[0097] The examples of the present application provide the antitumor effects of the Al-Mn composite nanocrystal AlMn-001 obtained in Example 1 as an immunopotentiator, specifically including the following:

[0098] Mice were randomly divided into four groups according to body weight: a blank control group (Ctrl), an OVA control group, OVA and AlMn-001 (ALMN-001-OVA), and an OVA and commercial aluminum adjuvant group (Alum-OVA). According to the experimental design, mice were immunized three times (once every 7 days) and then subcutaneously inoculated with melanoma B16-OVA to establish an orthotopic tumor model. As shown in Figures 14A to 14C, Figure 14A shows the changes in mouse body weight in each group, Figure 14B shows the changes in tumor volume in each group, and Figure 14C shows the survival rate of mice in each group. As shown in Figure 14A, there was little change in mouse body weight in each group. As shown in Figure 14B, on day 20, the average tumor size in the control group was 1982 mm. 3 In the group immunized with commercially available aluminum adjuvant, tumor growth was suppressed to some extent, with the average tumor size being 734.9 mm 3 However, the use of AlMn-001 according to the present application effectively suppressed tumor growth, reducing the average tumor size of immunized mice to 201.7 mm 3 As shown in Figure 14C, the longest survival time of mice in the control group was 26 days, and the longest survival time of mice immunized with the commercial aluminum adjuvant group was 43 days. However, some mice in the AlMn-001-immunized group were still alive at day 65, and the survival time of other mice in the group was also significantly extended. These results indicate that pre-administration of aluminum adjuvant slightly inhibited tumor growth and slightly improved mouse survival, whereas pre-administration of AlMn-001 significantly inhibited tumor growth, significantly improved mouse survival, and significantly extended survival. AlMn-001 can be used as a tumor immunopotentiator to enhance the antigenic tumor response in vivo. Example 15

[0099] The examples of this application provide a test to combat breast cancer distal tumors using the Al-Mn composite nanocrystals AlMn-002 obtained in Example 2 in combination with PD-L1 antibody. Specific tests include the following:

[0100] 100 μL of 4T1 cells (1 × 105 ) was inoculated into the right hind leg of Balb / c mice and marked as an orthotopic tumor. The average tumor volume was 100 mm 3 When tumor size reached 100 μg / day, mice were randomly divided into four groups: control (Ctrl), AlMn-002, PD-L1 antibody (Biolegend, USA, catalog: 124329, clone 10F.9G2), and AlMn-002-PD-L1 antibody combination group. AlMn-002 (100 μg / injection / mouse) was injected via the tail vein. Day 0 was recorded, and mice in each group received the corresponding treatment on days 1, 3, and 5, respectively. Tumor volume in each group was recorded every two days. On day 10, 5×10 4 4T1 cells were inoculated into the left hind limb of each mouse, and the tumor volume was recorded as a distal tumor. The tumor volume on both sides was recorded until day 25.

[0101] The results are shown in Figure 15. Figure 15 shows the therapeutic effect of the combination of Al-Mn composite nanocrystals AlMn-002 and PD-L1 antibody on distal breast cancer tumors. Figure 15 shows that the combination of Al-Mn composite nanocrystals AlMn-002 and PD-L1 antibody not only significantly inhibited the growth of existing tumors, but also significantly inhibited the growth of new tumors. Example 16

[0102] The examples of the present application provide anti-liver cancer tumor tests using the Al-Mn composite nanocrystals AlMn-003 obtained in Example 3 in combination with metformin (Met). Specific tests include the following:

[0103] 100 μL of HepG2 cells (1 × 10 5 ) was inoculated into the right hind leg of Balb / c mice. The average tumor volume was 100 mm 3When the tumor size reached 100 μg / day, the mice were randomly divided into four groups: control (Ctrl), AlMn-003, metformin (Met, purchased from Meilunbio, CAS: 657-24-9), and AlMn-003-Met combination group. AlMn-003 (100 μg / mouse) was injected via the tail vein. Day 0 was recorded, and mice in each group received the corresponding treatment on days 1, 3, and 5, respectively. Tumor volume in each group was recorded every two days.

[0104] The results are shown in Figure 16. Figure 16 shows the therapeutic effect of the combination of Al-Mn composite nanocrystals AlMn-003 and Met antibody on a liver cancer tumor model. Figure 16 shows that the combination of Al-Mn composite nanocrystals AlMn-003 and Met antibody has a clear inhibitory effect on tumor growth in the liver cancer tumor model. Example 17

[0105] The examples of the present application provide tests on the inhibition of metastatic tumor growth by the Al-Mn composite nanocrystals AlMn-001 obtained in Example 1. Specific tests include the following:

[0106] A mouse 4T1 breast cancer model was established. Tumor-bearing mice were randomly divided into three groups. Each group was injected intratumorally with (1) saline, (2) paclitaxel (purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., CAS: 33069-62-4), and (3) AlMn-001-paclitaxel. Each group received one treatment every two days for a total of three treatments. On day 10, 4 × 10 mice were injected into the tumor to induce lung metastasis. 5100 μL of PBS containing 4T1 cells was injected into the tail vein. On day 28, the mice were sacrificed and lung tissue was removed. The results are shown in Figure 17. Figure 17 shows the effect of treating liver cancer metastasis with AlMn-001 in combination with paclitaxel (leftmost: blank control group, middle: paclitaxel only group, rightmost: AlMn-001 and paclitaxel combination treatment group). The results showed that the lungs of mice in the blank control group were filled with metastatic tumor lesions, while the lungs of mice treated with paclitaxel alone showed a certain number of metastatic tumor lesions, whereas the lungs of mice treated with AlMn-001 showed almost no tumors. These results indicate that AlMn-001 can be used as an immune enhancer to enhance the anti-metastatic effect in vivo.

[0107] Thus, the Al-Mn composite nanocrystals of the present application are as easy to use as commercially available aluminum adjuvants, but at lower concentrations than commercially available aluminum adjuvants, have better DC cell activation effects, better antigen presentation effects, better promotion of antigen cellular uptake, and lysosomal escape effects, and can simultaneously activate humoral and cellular immunity. Furthermore, they have the ability to strongly enhance the effects of tumor immunotherapy, and can be used as a delivery system for combined immunotherapeutic agents to improve the effects of immunotherapy.

[0108] It should be noted that the above description is only a preferred embodiment of the present invention, and that those skilled in the art may make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications are also deemed to fall within the protection scope of the present invention.

Claims

1. Step 1: mixing an aluminum salt solution, a manganese salt solution, and an anionic auxiliary solution to obtain a mixture, and adjusting the pH of the mixture to 5.5 to 8.5; and step 2, wherein the mixture is heated to react with the mixture and the resulting solid product is washed to obtain Al-Mn composite nanocrystals, the solute of the anionic auxiliary solution is an organic acid salt and / or an amino acid; The method for producing the anionic auxiliary solution is characterized in that the solvent for the anionic auxiliary solution is one or more selected from the group consisting of water, physiological saline, and ethanol.

2. the solute of the aluminum salt solution is one or more selected from the group consisting of aluminum chloride, aluminum sulfate, aluminum nitrate, and aluminum acetate; the solvent of the aluminum salt solution is one or more selected from the group consisting of sodium acetate solution, physiological saline, water, and ethanol; the solute of the manganese salt solution is one or more selected from the group consisting of manganese chloride, manganese sulfate, manganese nitrate, and manganese acetate; 2. The method according to claim 1, wherein the solvent of the manganese salt solution is one or more selected from the group consisting of water, saline, and ethanol.

3. the organic acid salt is citric acid and / or salicylic acid, 2. The method according to claim 1, wherein the amino acid is one or more selected from the group consisting of cysteine, cystine, tyrosine, aspartic acid, and glutamic acid.

4. 2. The method according to claim 1, wherein the mixture has a molar ratio of aluminum element in the aluminum salt solution to manganese element in the manganese salt solution of 1:(0.05 to 1), and a ratio of the concentration of the anionic auxiliary to the total metal ion concentration (where the total metal ion concentration is the sum of the concentrations of the aluminum salt solution and the manganese salt solution) of (0.1 to 10):

1.

5. 2. The method according to claim 1, wherein in step 2, the heating reaction is carried out at a temperature of 60° C. to 130° C. for a time of 0.5 to 20 hours.

6. An Al—Mn composite nanocrystal, characterized by comprising an Al—Mn composite nanocrystal produced by the production method according to any one of claims 1 to 5.

7. Use of the Al-Mn composite nanocrystals according to claim 6 in the manufacture of a vaccine adjuvant, an immune enhancer, a pharmaceutical composition, a drug delivery carrier, or an immunogenic composition.

8. 10. An antitumor pharmaceutical composition comprising the Al-Mn composite nanocrystal of claim 6 and a therapeutic agent for antitumor diseases.

9. A vaccine composition comprising an antigen and the Al-Mn composite nanocrystal of claim 6.

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