Organic nanoparticle vaccine for targeting cytoplasm as well as preparation method and application of organic nanoparticle vaccine

Abstract

The invention provides a cytoplasm-targeted organic nanoparticle vaccine as well as a preparation method and application thereof. The organic nanoparticle vaccine comprises organic nanoparticles prepared from an organic high-molecular polymer, an immunologic adjuvant and an antigen, and the organic nanoparticle vaccine is good in morphology, uniform in particle and simple to prepare, has excellent natural immune stimulation performance and immunogenicity, and has wide application prospects in the fields of pathogen infection prevention and the like. The organic nanoparticles contain cyclic amino and boric acid group coordinate bonds, have a proton sponge effect, can mediate escape of immunologic adjuvant lysosome, and can load protein by using electrostatic adsorption. The organic high-molecular polymer is safe and degradable, can realize the effects of signal amplification, targeted delivery, precise release, synergistic interaction and the like, and can effectively improve the biotoxicity problem and improve the safety; the one-step synthesis method is simple in preparation process and can be stably stored; functional groups can be flexibly modified, and functional design is diversified.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a cytoplasm-targeting organic nanoparticle vaccine and a preparation method and application thereof. Background Art

[0002] Nanotechnology provides a promising approach for vaccine delivery. A wide variety of nanoparticles have emerged, including polymer nanoparticles, inorganic nanoparticles, and lipid-based nanoparticles. However, most nanoparticles currently require complex preparation processes, exhibit poor stability, and are unable to degrade in the body. Long-term retention in the body can lead to potential biotoxicity risks.

[0003] Bacillus anthracis is an aerobic or facultatively anaerobic Gram-positive bacterium belonging to the genus Bacillus. Anthrax, caused by infection with Bacillus anthracis, is an extremely dangerous zoonosis. Vaccination is one of the most important means of preventing pulmonary anthrax. However, currently approved human anthrax vaccines are primarily live attenuated spore-based vaccines, which have limitations such as age restrictions, adverse reactions, poor protection against pulmonary anthrax, short-lived efficacy, complex immunization procedures, adverse reactions, and difficulty in widespread adoption. Therefore, the development of a safe and effective protective vaccine against Bacillus anthracis is of great significance. Summary of the Invention

[0004] In view of this, in order to make up for the deficiencies of the prior art, the present invention is proposed.

[0005] The first aspect of the present invention provides an organic high molecular polymer, the organic high molecular polymer comprising MP1 and MP2, wherein the structure of MP1 is shown in formula (1), and the structure of MP2 is shown in formula (2);

[0006]

[0007] Formula (1);

[0008]

[0009] Formula (2);

[0010] Furthermore, the mass ratio of MP1 to MP2 is 2:1-2:5.

[0011] Furthermore, the mass ratio of MP1 to MP2 is 2:3.

[0012] Furthermore, in the formula (1)-formula (2), m is an integer of 2-500.

[0013] Furthermore, in the formula (1) and the formula (2), m is an integer of 50-200.

[0014] Furthermore, in the formula (1)-formula (2), m is 113.

[0015] Furthermore, in the formula (1), x is an integer of 1-20.

[0016] Furthermore, in the formula (1), x is 1.

[0017] Furthermore, in the formula (1), y is an integer of 1-20.

[0018] Furthermore, in the formula (1), y is 1.

[0019] Furthermore, in the formula (2), n is an integer of 2-20.

[0020] Furthermore, in the formula (2), n is 10.

[0021] The second aspect of the present invention provides an organic nanoparticle, which comprises the organic high molecular polymer described in the first aspect of the present invention and an immune adjuvant.

[0022] The "nanoparticles" mentioned in the present invention refer to ultrafine particles that are between microscopic and macroscopic systems and have a size between 1-1000 nm.

[0023] Furthermore, the mass ratio of the organic high molecular polymer to the immune adjuvant is 5:1-50:1.

[0024] Furthermore, the mass ratio of the organic high molecular polymer to the immune adjuvant is 25:1.

[0025] Furthermore, the immune adjuvant includes but is not limited to aluminum salt adjuvant, STING agonist, water-in-oil emulsion, saponin adjuvant, Toll-like receptor agonist, liposome / nanoparticle adjuvant, and cytokine adjuvant.

[0026] Furthermore, the immune adjuvant is selected from STING agonists.

[0027] Furthermore, the STING agonists include but are not limited to cGAMP, MSA-2, ADU-S100, MK-1454, SR-717, diABZI, and SNX281.

[0028] Furthermore, the STING agonist is selected from MSA-2.

[0029] The third aspect of the present invention provides an organic nanoparticle vaccine, which comprises the organic nanoparticles described in the second aspect of the present invention and an antigen.

[0030] In the present invention, a vaccine is a biological composition that stimulates the immune system and enables it to fight infection or disease. These vaccines include preventive vaccines and therapeutic vaccines. Preventive vaccines are used in healthy individuals or those who have not yet been infected with a specific pathogen to prevent future infection or disease by activating the immune system. Therapeutic vaccines are used in individuals who are already ill to help control disease progression and eliminate pathogens or abnormal cells (such as cancer cells) by regulating or enhancing the immune system.

[0031] Furthermore, the vaccine is selected from preventive vaccines.

[0032] In the present invention, the antigen refers to a substance that induces a specific immune response in a host animal. Antigens include, but are not limited to, nucleic acids, proteins, polypeptides, bacteria, fungi, viruses, or toxoids.

[0033] Based on the type of antigen, vaccines can be categorized as including but not limited to live attenuated vaccines, inactivated vaccines, polysaccharide and polysaccharide conjugate vaccines, subunit vaccines, and nucleic acid vaccines. In the context of the present invention, live attenuated vaccines refer to live viruses or bacteria, which undergo chemical or physical treatment to alter their structure, significantly reducing their toxicity while retaining their antigenicity. These vaccines typically require a longer development cycle, but can induce strong immune responses, making them crucial for combating pandemics of highly pathogenic and potentially lethal infectious diseases. Inactivated vaccines are vaccines in which pathogens are treated physically or chemically to completely eliminate their infectivity while retaining their immunogenicity. Compared to live attenuated vaccines, inactivated vaccines are relatively safer but have lower immunogenicity. Given their lower efficacy and immunogenicity, they are often used with adjuvants to assist in activating the immune system. Subunit vaccines are composed of specific components of the pathogen that can elicit an immune response, typically proteins or peptides. Compared to attenuated or inactivated vaccines, subunit vaccines retain only the antigenic components of the pathogen that can elicit an immune response, effectively improving vaccine safety. A nucleic acid vaccine refers to a vaccine that uses a plasmid as a medium to inject an exogenous gene sequence that can encode a specific protein into the body and express the corresponding protein antigen in the host cells, thereby inducing the host to produce a corresponding immune response and exerting the effect of the vaccine.

[0034] Furthermore, the antigen is selected from proteins.

[0035] Furthermore, the vaccine is selected from subunit vaccines.

[0036] Based on disease classification, vaccines can be divided into vaccines targeting tumors, vaccines targeting infectious diseases, and vaccines targeting chronic diseases. Among them, vaccines targeting tumors include, but are not limited to, messenger RNA tumor vaccines, viral vector tumor vaccines, and tumor peptide vaccines. Messenger RNA tumor vaccines are prepared by in vitro transcription to obtain mRNA sequences encoding tumor-specific antigens or tumor-associated antigens. After being prepared into vaccines, they are injected into the human body and translated into antigenic proteins, thereby inducing the body to produce a specific immune response. Viral vector tumor vaccines use modified replication-deficient or attenuated viruses as vectors to deliver genetic information encoding tumor antigens to host cells, prompting them to express these antigens and thus activating a specific anti-tumor immune response. Tumor peptide vaccines are vaccines produced by chemically synthesizing peptides designed based on the amino acid sequence of tumor antigen epitopes. Vaccines targeting infectious diseases include, but are not limited to, vaccines against diseases caused by pathogens such as bacteria and viruses. Such bacteria include, but are not limited to, Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Spirochaetes, and Chlamydia. Proteobacteria include, but are not limited to, Enterobacteriaceae, Vibrio, Pseudomonas, and Yersinia; Firmicutes include, but are not limited to, Bacillus, Staphylococcus, and Clostridium; Actinobacteria include, but are not limited to, Mycobacterium and Streptomyces; Bacteroidetes include, but are not limited to, Bacteroides and Prevotella; Spirochetes include, but are not limited to, Leptospira and Treponema; and Chlamydia include, but are not limited to, Chlamydia. Viruses include, but are not limited to, the following families: Retroviridae, Togaviridae, Coronaviridae, Filoviridae, Rhabdoviridae, Bunyaviridae, Orthomyxoviridae, Paramyxoviridae, Arenaviridae, Hepadnaviridae, Herpesviridae, Flaviviridae, Baculoviridae, Poxviridae, or Picornaviridae. Vaccines targeting chronic diseases can be used to treat chronic allergic diseases, diabetes, hypertension, obesity, Alzheimer's disease, rheumatoid arthritis, and other conditions.

[0037] Furthermore, the vaccine is selected from vaccines against infectious diseases.

[0038] In the present invention, the infectious disease is a disease caused by bacteria.

[0039] Furthermore, the bacteria are selected from the phylum Firmicutes.

[0040] Furthermore, the bacteria is selected from the genus Bacillus.

[0041] Furthermore, the bacteria is selected from Bacillus anthracis.

[0042] Bacillus anthracis is an aerobic or facultatively anaerobic Gram-positive bacterium belonging to the genus Bacillus. It exists in two forms: vegetative bodies and spores. The spores are highly resilient and can survive for decades in a dry, room-temperature environment. Anthrax, caused by Bacillus anthracis, is an extremely dangerous zoonosis. Anthrax is primarily classified into three types based on the route of infection and clinical manifestations: cutaneous anthrax, pulmonary anthrax, and intestinal anthrax.

[0043] Furthermore, the anthrax is selected from pulmonary anthrax.

[0044] In the present invention, the protein can be a recombinant protein or a natural protein.

[0045] Furthermore, the protein is selected from recombinant proteins.

[0046] Furthermore, the protein is the recombinant protective antigen rPA of Bacillus anthracis.

[0047] Furthermore, the mass ratio of the antigen to the organic high molecular polymer in the organic nanoparticles is 10:1-50:1.

[0048] Furthermore, the mass ratio of the antigen contained in the organic nanoparticles to the organic high molecular polymer is 40:1.

[0049] Furthermore, the administration methods of the organic nanoparticle vaccine include but are not limited to inhalation, injection, oral administration, nasal spray, and transdermal administration.

[0050] Furthermore, the administration method of the organic nanoparticle vaccine is selected from inhalation.

[0051] The fourth aspect of the present invention provides a composition comprising the organic high molecular polymer described in the first aspect of the present invention, the organic nanoparticles described in the second aspect of the present invention, or the organic nanoparticle vaccine described in the third aspect of the present invention.

[0052] Furthermore, the composition also includes pharmaceutically acceptable carriers and / or excipients.

[0053] Furthermore, the pharmaceutically acceptable carriers and / or excipients include but are not limited to diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, lubricants, surfactants, coating materials, colorants, pH regulators, antioxidants or antibacterial agents.

[0054] Furthermore, the diluent includes but is not limited to lactose, sodium chloride, glucose, urea, starch, water, etc. The binder includes but is not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, etc. The surfactant includes but is not limited to sodium lauryl sulfate, monoglyceride stearate, cetyl alcohol, etc. The lubricant includes but is not limited to zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauric sucrose, magnesium lauryl sulfate, etc. The filler includes but is not limited to mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, starch, etc. The disintegrant includes but is not limited to cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soy polysaccharide, etc.

[0055] The fifth aspect of the present invention provides a method for preparing the organic high molecular weight polymer according to the first aspect of the present invention, the method comprising dissolving MP1 and MP2 in an organic solvent and removing the organic solvent by dialysis.

[0056] Furthermore, the organic solvent includes but is not limited to methanol, ethanol, isopropanol, formalin, chloroform, acetone, hydrogen sulfide, and DMSO.

[0057] Furthermore, the organic solvent is selected from DMSO.

[0058] Furthermore, the concentration of the MP1 after being dissolved in an organic solvent is 1-10 mg / mL.

[0059] Furthermore, the concentration of the MP1 after being dissolved in an organic solvent is 5 mg / mL.

[0060] Furthermore, the concentration of the MP2 after being dissolved in an organic solvent is 1-10 mg / mL.

[0061] Furthermore, the concentration of the MP2 after being dissolved in an organic solvent is 7.5 mg / mL.

[0062] Furthermore, the method also includes a method for preparing MP1, comprising the following steps:

[0063] L-lysine diisocyanate, 2,2'-(4,4'-(propane-1,3-diyl)bis(piperidin-4,1-diyl))diethanol and bis(2-hydroxyethyl)disulfide are dissolved in anhydrous N,N-dimethylformamide and reacted to obtain a reaction system;

[0064] After dissolving polyethylene glycol in anhydrous N,N-dimethylformamide, add it into the reaction system and continue the reaction;

[0065] N,N-dimethylformamide was removed and the product was dried to obtain MP1.

[0066] Furthermore, the equivalent ratio of L-lysine diisocyanate, 2,2'-(4,4'-(propane-1,3-diyl)bis(piperidin-4,1-diyl))diethanol, bis(2-hydroxyethyl)disulfide and polyethylene glycol is (5-15):(1-10):(1-10):(1-5).

[0067] Furthermore, the equivalent ratio of L-lysine diisocyanate, 2,2'-(4,4'-(propane-1,3-diyl)bis(piperidine-4,1-diyl))diethanol, bis(2-hydroxyethyl)disulfide and polyethylene glycol is 11:5:5:2.

[0068] Furthermore, the concentration of the L-lysine diisocyanate after being dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 eq / mL.

[0069] Furthermore, the concentration of the L-lysine diisocyanate after being dissolved in anhydrous N,N-dimethylformamide is 0.11 eq / mL.

[0070] Furthermore, the concentration of the 2,2'-(4,4'-(propane-1,3-diyl)bis(piperidine-4,1-diyl))diethanol after being dissolved in anhydrous N,N-dimethylformamide is 0.01-0.1 eq / mL.

[0071] Furthermore, the concentration of the 2,2'-(4,4'-(propane-1,3-diyl)bis(piperidine-4,1-diyl))diethanol after being dissolved in anhydrous N,N-dimethylformamide is 0.05 eq / mL.

[0072] Furthermore, the concentration of the bis(2-hydroxyethyl) disulfide after being dissolved in anhydrous N,N-dimethylformamide is 0.01-0.1 eq / mL.

[0073] Furthermore, the concentration of the bis(2-hydroxyethyl) disulfide after being dissolved in anhydrous N,N-dimethylformamide is 0.05 eq / mL.

[0074] Furthermore, the concentration of the polyethylene glycol after being dissolved in anhydrous N,N-dimethylformamide is 0.01-0.05 eq / mL.

[0075] Furthermore, the concentration of the polyethylene glycol after being dissolved in anhydrous N,N-dimethylformamide is 0.02 eq / mL.

[0076] Furthermore, the polyethylene glycol is polyethylene glycol 5000.

[0077] Furthermore, the drying includes but is not limited to freeze drying, reduced pressure distillation, and spray drying.

[0078] Furthermore, the removal method includes dialysis.

[0079] Furthermore, the drying is selected from freeze-drying.

[0080] Furthermore, the method also includes a method for preparing MP2, comprising the following steps:

[0081] Dissolving 1,2,4,5-cyclohexanetetracarboxylic dianhydride and bis(2-hydroxyethyl)disulfide in anhydrous N,N-dimethylformamide, and reacting to obtain a reaction system;

[0082] After dissolving polyethylene glycol in anhydrous N,N-dimethylformamide, add it into the reaction system and continue the reaction;

[0083] After removing N,N-dimethylformamide and drying, a white polymer was obtained;

[0084] The white polymer was dissolved in anhydrous N,N-dimethylformamide, followed by the addition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine. After activation, 4-hydroxymethylphenylboronic acid was added and the reaction continued.

[0085] N,N-dimethylformamide was removed and MP2 was obtained by drying.

[0086] Furthermore, the equivalent ratio of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl)disulfide, and polyethylene glycol is (5-15):(5-15):(1-5).

[0087] Furthermore, the equivalent ratio of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl)disulfide, and polyethylene glycol is 11:10:2.

[0088] Furthermore, the concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride after being dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 eq / mL.

[0089] Furthermore, the concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride after being dissolved in anhydrous N,N-dimethylformamide is 0.11 eq / mL.

[0090] Furthermore, the concentration of the bis(2-hydroxyethyl) disulfide after being dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 eq / mL.

[0091] Furthermore, the concentration of the bis(2-hydroxyethyl) disulfide after being dissolved in anhydrous N,N-dimethylformamide is 0.1 eq / mL.

[0092] Furthermore, the concentration of the polyethylene glycol after being dissolved in anhydrous N,N-dimethylformamide is 0.01-0.05 eq / mL.

[0093] Furthermore, the concentration of the polyethylene glycol after being dissolved in anhydrous N,N-dimethylformamide is 0.02 eq / mL.

[0094] Furthermore, the polyethylene glycol is HO-PEG 5000-COOH.

[0095] Furthermore, the equivalent ratio of the white polymer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and 4-hydroxymethylphenylboronic acid is (0.5-2):(1-5):(1-5).

[0096] Furthermore, the equivalent ratio of the white polymer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and 4-hydroxymethylphenylboronic acid is 1:2:2:2.

[0097] Furthermore, the concentration of the white polymer after being dissolved in anhydrous N,N-dimethylformamide is 0.005-0.02 eq / mL.

[0098] Furthermore, the concentration of the white polymer after being dissolved in anhydrous N,N-dimethylformamide is 0.01 eq / mL.

[0099] Furthermore, the drying includes but is not limited to freeze drying, reduced pressure distillation, and spray drying.

[0100] Furthermore, the removal method includes dialysis. Furthermore, the drying is selected from freeze-drying.

[0101] The sixth aspect of the present invention provides a method for preparing the organic nanoparticles described in the second aspect of the present invention, the method comprising dissolving the organic high molecular weight polymer described in the first aspect of the present invention and an immune adjuvant in an organic solvent, and dialyzing to remove the organic solvent.

[0102] Furthermore, the organic solvent is selected from DMSO.

[0103] Furthermore, the organic high molecular polymer is dissolved in an organic solvent at a concentration of 1-20 mg / mL.

[0104] Furthermore, the organic high molecular polymer is dissolved in an organic solvent at a concentration of 12.5 mg / mL.

[0105] Furthermore, the concentration of the immune adjuvant dissolved in the organic solvent is 100-1000 μg / mL.

[0106] Furthermore, the concentration of the immune adjuvant dissolved in the organic solvent is 500 μg / mL.

[0107] The seventh aspect of the present invention provides a method for preparing the organic nanoparticle vaccine described in the third aspect of the present invention, which comprises mixing the organic nanoparticles described in the second aspect of the present invention with an antigen.

[0108] Furthermore, the antigen is used at a concentration of 1-50 mg / μL.

[0109] Furthermore, the antigen is used at a concentration of 10 mg / μL.

[0110] An eighth aspect of the present invention provides any of the following methods:

[0111] (1) A method for promoting dendritic cell maturation, comprising administering the organic high molecular weight polymer described in the first aspect of the present invention, the organic nanoparticles described in the second aspect of the present invention, the organic nanoparticle vaccine described in the third aspect of the present invention, or the composition described in the fourth aspect of the present invention;

[0112] (2) A method for activating an innate immune signaling pathway, the method comprising administering the organic high molecular weight polymer of the first aspect of the present invention, the organic nanoparticle of the second aspect of the present invention, the organic nanoparticle vaccine of the third aspect of the present invention, or the composition of the fourth aspect of the present invention;

[0113] (3) A method for promoting the secretion of type I interferon, the method comprising administering the organic high molecular weight polymer described in the first aspect of the present invention, the organic nanoparticles described in the second aspect of the present invention, the organic nanoparticle vaccine described in the third aspect of the present invention, or the composition described in the fourth aspect of the present invention;

[0114] Preferably, the type I interferon is IFN-β;

[0115] (4) A method for targeting cytoplasm, comprising administering the organic high molecular polymer described in the first aspect of the present invention, the organic nanoparticles described in the second aspect of the present invention, the organic nanoparticle vaccine described in the third aspect of the present invention, or the composition described in the fourth aspect of the present invention.

[0116] A ninth aspect of the present invention provides any one of the following applications:

[0117] (1) Use of the organic high molecular weight polymer described in the first aspect of the present invention in loading an immune adjuvant;

[0118] Furthermore, the immune adjuvant includes but is not limited to aluminum salt adjuvant, STING agonist, water-in-oil emulsion, saponin adjuvant, Toll-like receptor agonist, liposome / nanoparticle adjuvant, and cytokine adjuvant.

[0119] Furthermore, the immune adjuvant is selected from STING agonists.

[0120] Furthermore, the STING agonists include but are not limited to cGAMP, MSA-2, ADU-S100, MK-1454, SR-717, diABZI, and SNX281.

[0121] Furthermore, the STING agonist is selected from MSA-2.

[0122] (2) Use of the organic high molecular weight polymer described in the first aspect of the present invention or the organic nanoparticles described in the second aspect of the present invention in the preparation of vaccines;

[0123] Furthermore, the organic high molecular polymer can be used as a high molecular skeleton to deliver adjuvants and / or antigens.

[0124] (3) Use of the organic high molecular weight polymer described in the first aspect of the present invention or the organic nanoparticles described in the second aspect of the present invention as a drug carrier;

[0125] Organic polymers or organic nanoparticles can be used as drug carriers to deliver antibacterial, antiviral or anticancer drugs, while enhancing immune stimulation and exerting antibacterial, antiviral or anticancer effects.

[0126] (4) Use of the organic high molecular weight polymer described in the first aspect of the present invention or the organic nanoparticles described in the second aspect of the present invention as a drug dressing;

[0127] Organic polymers or organic nanoparticles can be used as drug dressings to accelerate wound healing.

[0128] (5) Use of the organic high molecular polymer described in the first aspect of the present invention, the organic nanoparticles described in the second aspect of the present invention, the organic nanoparticle vaccine described in the third aspect of the present invention, or the composition described in the fourth aspect of the present invention in the preparation of a drug for preventing pathogen infection;

[0129] Furthermore, the pathogens include but are not limited to bacteria, viruses, fungi, parasites, and prions.

[0130] Furthermore, the pathogen is selected from bacteria.

[0131] Furthermore, the bacteria include Bacillus anthracis of mice.

[0132] (6) Use of the organic high molecular polymer described in the first aspect of the present invention, the organic nanoparticles described in the second aspect of the present invention, the organic nanoparticle vaccine described in the third aspect of the present invention, or the composition described in the fourth aspect of the present invention in promoting dendritic cell maturation;

[0133] (7) Use of the organic high molecular polymer described in the first aspect of the present invention, the organic nanoparticles described in the second aspect of the present invention, the organic nanoparticle vaccine described in the third aspect of the present invention, or the composition described in the fourth aspect of the present invention in activating innate immune signaling pathways;

[0134] (8) Use of the organic high molecular polymer described in the first aspect of the present invention, the organic nanoparticles described in the second aspect of the present invention, the organic nanoparticle vaccine described in the third aspect of the present invention, or the composition described in the fourth aspect of the present invention in promoting the secretion of type I interferon;

[0135] Furthermore, the type I interferon is IFN-β.

[0136] (9) Use of the organic high molecular polymer described in the first aspect of the present invention, the organic nanoparticles described in the second aspect of the present invention, the organic nanoparticle vaccine described in the third aspect of the present invention, or the composition described in the fourth aspect of the present invention in targeting cytoplasm.

[0137] The present invention has the following advantages and beneficial effects:

[0138] The present invention provides a cytoplasm-targeted organic nanoparticle vaccine, and its preparation method and application. The organic nanoparticle vaccine includes organic nanoparticles made of organic high molecular polymers, immune adjuvants, and antigens. The organic nanoparticle vaccine has good morphology, uniform particles, simple preparation, excellent natural immune stimulation and immunogenicity, and has broad application prospects in the fields of preventing pathogen infection. The organic nanoparticles contain cyclic amino groups and boronic acid group coordination bonds, have a proton sponge effect, can mediate the lysosomal escape of immune adjuvants, and can use electrostatic adsorption to load proteins. The organic high molecular polymer is safe and degradable, and can achieve signal amplification, targeted delivery, precise release, and synergistic enhancement, and can effectively improve biological toxicity problems and enhance safety; the preparation process using a one-step synthesis method is simple and can be stably stored; functional groups can also be flexibly modified, and the functional design is diverse. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1 This is the H-NMR spectrum of organic polymer MP1.

[0140] Figure 2 This is the H-NMR spectrum of organic polymer MP2.

[0141] Figure 3 Figures 2 and 3 are transmission electron micrographs of the nanoparticles prepared in Example 1 of the present invention. Figures 2 and 3 are transmission electron micrographs of YIMIAO-1.4, YIMIAO-1.5, YIMIAO-1.6, and YIMIAO-1.7 nanoparticles, respectively. Scale bar: 200 nm.

[0142] Figure 4 This is a Zeta potential diagram of the nanoparticles during the preparation of YIMIAO-1.7 in Example 1 of the present invention.

[0143] Figure 5 The particle size distribution of YIMIAO-1.7 prepared in Example 1 of the present invention.

[0144] Figure 6 This is a diagram showing the Coomassie Brilliant Blue staining verification result of YIMIAO-1.7 prepared in Example 1 of the present invention.

[0145] Figure 7 This is a graph showing the evaluation results of the release effect of YIMIAO-1.7 on the agonist MSA-2 under the action of glutathione prepared in Example 1 of the present invention.

[0146] Figure 8 This is a diagram showing the in vitro biocompatibility evaluation results of YIMIAO-1.7 prepared in Example 1 of the present invention.

[0147] Figure 9 This is the in vivo biosafety evaluation result of YIMIAO-1.7 prepared in Example 1 of the present invention. Magnification: 200 times.

[0148] Figure 10 This is a diagram showing the effect of YIMIAO-1.7 prepared in Example 1 on promoting the maturation of dendritic cells isolated from mouse bone marrow.

[0149] Figure 11 This is a molecular effect diagram of YIMIAO-1.7 prepared in Example 1 activating the innate immune signaling pathways STING, TBK1, and IRF3.

[0150] Figure 12 This is a diagram showing the effect of YIMIAO-1.7 prepared in Example 1 on promoting the secretion of type I interferon.

[0151] Figure 13 These are diagrams showing the effects of YIMIAO-1.7 prepared in Example 1 on promoting antibody production, wherein a is a diagram showing the effects of YIMIAO-1.7 prepared in Example 1 on promoting the production of IgG antibodies in mice, b is a diagram showing the effects of YIMIAO-1.7 on promoting the production of IgG1 antibodies in mice, and c is a diagram showing the effects of YIMIAO-1.7 on promoting the production of IgG2c antibodies in mice.

[0152] Figure 14 80×LD 50 Survival curve of YIMIAO-1.7-immunized mice after challenge with Bacillus anthracis Pasteur II strain.

[0153] Figure 15 80×LD 50Figure 3. Body weight changes of YIMIAO-1.7-immunized mice after challenge with Pasteur II strain of Bacillus anthracis.

[0154] Figure 16 80×LD 50 Bacillus anthracis Pasteur II strain infects the organs of YIMIAO-1.7-immunized mice.

[0155] Figure 17 80×LD 50 Pathological changes in organs of YIMIAO-1.7-immunized mice after challenge with Bacillus anthracis Pasteur II strain. Magnification: 200x. DETAILED DESCRIPTION

[0156] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0157] Example 1 Preparation and characterization of inhalable cytoplasm-targeted organic nanoparticle vaccines

[0158] First, organic polymers MP1 and MP2 were synthesized by polymerization condensation reaction (NMR hydrogen spectrum as shown in Figure 1 、 2 ).

[0159] MP1 synthesis steps: L-lysine diisocyanate (1.1 eq), 2,2'-(4,4'-(propane-1,3-diyl)bis(piperidin-4,1-diyl))diethanol (0.5 eq), and bis(2-hydroxyethyl) disulfide (0.5 eq) were weighed into a 50 mL round-bottom flask, dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF), and reacted with magnetic stirring at room temperature for 24 hours. Polyethylene glycol (PEG5k, 0.2 eq) was dissolved in 10 mL of anhydrous DMF and added to the reaction system, and the reaction continued for 24 hours. The solution was transferred to a dialysis bag (8-10 kDa) and dialyzed for 48 hours to completely remove the DMF. The solution was then transferred to a lyophilizer and lyophilized to obtain MP1 as a white solid. MP1 contains cyclic amino groups and exhibits a proton sponge effect, which may mediate the lysosomal escape of immune adjuvants.

[0160] MP2 synthesis steps: 1,2,4,5-cyclohexanetetracarboxylic dianhydride (1.1 eq) and bis(2-hydroxyethyl) disulfide (1.0 eq) were weighed and added to a 50 mL round-bottom flask. The mixture was dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF) and reacted with magnetic stirring at room temperature for 24 hours. Polyethylene glycol (HO-PEG5k-COOH, 0.2 eq) was dissolved in 10 mL of anhydrous DMF and added to the reaction system. The reaction was continued for another 24 hours. The mixture was transferred to a dialysis bag (8-10 kDa) and dialyzed for 48 hours to completely remove the DMF. The solution was then transferred to a lyophilizer and lyophilized to obtain a white solid product. A white polymer (1.0 eq) was weighed and dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF). 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.0 eq) and 4-dimethylaminopyridine (DMAP, 2.0 eq) were then added. After activation for 30 minutes, 4-hydroxymethylphenylboronic acid (2.0 eq) was added and the reaction continued for 24 hours. The product was transferred to a dialysis bag (8-10 kDa) and dialyzed for 48 hours to completely remove the DMF. The solution was then transferred to a lyophilizer and lyophilized to obtain the white solid product MP2. The product contains a boronic acid group coordination bond, allowing it to be loaded with proteins via electrostatic adsorption.

[0161] Preparation of YIMIAO-1.4: MP1 (20 mg) and MP2 (30 mg) were dissolved in 0.4 mL of dimethyl sulfoxide (DMSO) using water bath sonication. The solution was then quickly added to PBS (3.6 mL) and dialyzed (molecular weight cutoff 3.5 kDa) for 24 h to remove DMSO.

[0162] Preparation of YIMIAO-1.5: MP1 (20 mg), MP2 (30 mg), and MSA-2 (2 mg) were dissolved in 0.4 mL of dimethyl sulfoxide (DMSO) using water bath sonication. The solution was then quickly added to PBS (3.6 mL) and dialyzed (molecular weight cutoff 3.5 kDa) for 24 h to remove DMSO.

[0163] Preparation of YIMIAO-1.6: 40 μL of rPA protein (10 mg / μL), 800 μL of YIMIAO-1.4 (12.5 mg / mL), and 160 μL of PBS were mixed and reacted with magnetic stirring at room temperature for 3 h. The mixture was then dialyzed (molecular weight cutoff 100 kDa) for 3 h.

[0164] Preparation of YIMIAO-1.7: 40 μL of rPA protein (10 mg / μL), 800 μL of YIMIAO-1.5 (polymer concentration 12.5 mg / mL, MSA-2 concentration 500 μg / mL), and 160 μL of PBS were mixed and reacted with magnetic stirring at room temperature for 3 h. The mixture was dialyzed (molecular weight cutoff 100 kDa) for 3 h to obtain YIMIAO-1.7.

[0165] All groups of the present invention are:

[0166] (1) Mock (PBS);

[0167] (2) YIMIAO-1.1:MSA-2 (free immune adjuvant);

[0168] (3) YIMIAO-1.2: rPA (free antigenic protein);

[0169] (4) YIMIAO-1.3: rPA+MSA-2 (a mixture of free immune adjuvant and antigen protein);

[0170] (5) YIMIAO-1.4: polymer skeleton (pure polymer skeleton nanoparticles);

[0171] (6) YIMIAO-1.5: polymer skeleton + MSA-2 (nanoparticles loaded with immune adjuvant);

[0172] (7) YIMIAO-1.6: rPA + polymer skeleton (nanoparticles loaded with antigen protein);

[0173] (8) YIMIAO-1.7: rPA+polymer skeleton+MSA-2 (nanoparticles loaded with immune adjuvants and antigen proteins).

[0174] Transmission electron microscopy results show that the YIMIAO-1.4, YIMIAO-1.5, YIMIAO-1.6, and YIMIAO-1.7 nanoparticles are uniformly spherical with rough surfaces and similar morphologies ( Figure 3 Zeta potential results showed that YIMIAO-1.4 was positively charged, which was conducive to protein loading, while YIMIAO-1.7 after loading rPA protein was negatively charged ( Figure 4 ); DLS results show that the hydrodynamic size of YIMIAO-1.7 is about 176.8 nm ( Figure 5 ); Coomassie Brilliant Blue staining results showed that rPA protein was successfully loaded on YIMIAO-1.7 ( Figure 6To verify the effectiveness of nanoparticle vaccines in targeting the cytoplasm to release MSA-2, we placed YIMIAO-1.5 and YIMIAO-1.7 nanoparticles in PBS and GSH (glutathione) environments, respectively, and found that YIMIAO-1.5 and YIMIAO-1.7 could successfully release MSA-2 under the action of GSH ( Figure 7 In summary, the cytoplasm-targeted biodegradable organic nanoparticle vaccine YIMIAO-1.7 loaded with Bacillus anthracis rPA protein was successfully prepared.

[0175] Example 2 Biosafety Verification of YIMIAO-1.7 Prepared in Example 1 of the Present Invention

[0176] Mouse erythrocytes were used to evaluate the in vitro hematotoxicity of YIMIAO-1.7. YIMIAO-1.7 was dispersed in PBS at concentrations of 50, 100, 250, and 500 μg mL -1 , and then added to red blood cells. Meanwhile, blood cells dispersed in deionized water and PBS served as positive and negative controls, respectively. All samples were then allowed to stand at room temperature for 1 hour, centrifuged at 3000 rpm for 10 minutes, and the supernatant collected. The absorbance of the collected supernatant was measured at 570 nm.

[0177] The formula for calculating the hemolysis rate is:

[0178] Hemolysis rate (%) = (sample absorption - negative control absorption) / (positive control absorption - negative control absorption) × 100%.

[0179] The in vivo biosafety of YIMIAO-1.7 was evaluated in C57BL / 6J mice. YIMIAO-1.7 (10 μg rPA, 250 μg YIMIAO-1.4) was administered via liquid aerosol pulmonary immunization at a dose of 50 μL. Seven days after the first immunization, five C57BL / 6J mice were sacrificed by CO2. Organ tissues, including the lungs, spleen, liver, kidneys, and heart, were removed and fixed in 4% paraformaldehyde for at least 48 hours. Pathological sections were obtained by washing, dehydration, clearing, paraffin embedding, sectioning, and HE staining at Wuhan Sewell Biotechnology Co., Ltd. The sections were then examined under a light microscope and scored for histological pathology.

[0180] The in vitro safety of YIMIAO-1.7 was preliminarily verified by hemolysis test. -1 The hemolysis rate remained below 5% at a concentration of Figure 8), indicating that YIMIAO-1.7 has good biosafety in vitro. In vivo pathological tissue sections showed that no obvious histopathological changes were observed at any time point after immunization ( Figure 9 ), indicating that YIMIAO-1.7 has good biosafety in vivo.

[0181] Example 3 Determination of the effect of YIMIAO-1.7 on activating natural immunity prepared in Example 1 of the present invention

[0182] Using STING deletion (Sting1 - / - ) C57BL / 6J mice and wild-type (WT) C57BL / 6J mice bone marrow dendritic cells (BMDCs) were set up in five experimental groups, and the Mock group was the negative control group. -1 )、YIMIAO-1.4(125 μg mL -1 )、YIMIAO-1.5(5 μg mL -1 MSA-2, 125 μg mL -1 )、YIMIAO-1.7(5 μg mL -1 rPA, 5 μg mL -1 MSA-2, 125 μg mL -1 YIMIAO-1.4 (YIMIAO-1.4) was mixed with BMDCs in triplicate and placed in a 37°C, 5% CO2 incubator. BMDC maturation was assessed by flow cytometry, STING, TBK1, and IRF3 phosphorylation by Western blot, and type I interferon secretion by ELISA. Specific assay methods are as follows.

[0183] 1. Detection of BMDCs maturity by flow cytometry.

[0184] (1) In 2 mL 1×10 6 cells mL -1 BMDCs were added to the corresponding experimental groups and stimulated for 12 hours;

[0185] (2) Aspirate the cells into a flow cytometry tube, centrifuge at 400 × g for 5 minutes, discard the supernatant, add 1 mL of DPBS, centrifuge at 400 × g for 5 minutes, discard the supernatant, and repeat this step twice.

[0186] (3) Staining scheme

[0187] Table 1 BMDCs flow cytometry antibody protocol

[0188]

[0189] A single staining tube was set up for each antibody as compensation.

[0190] (4) Dyeing

[0191] 1) Add 3.9 μL of staining antibody to each flow cytometry tube and stain for 25 minutes at room temperature in the dark.

[0192] 2) Add 2 mL of DPBS and centrifuge at 400 × g for 5 minutes. Repeat twice.

[0193] 3) Discard the solution, add 50 μL DPBS to resuspend, and analyze the sample using a BDVerse flow cytometer.

[0194] 2. Western Blot Detection of STING, TBK1, and IRF3 Phosphorylation Expression

[0195] (1) Sample preparation: 1×10 6 cells mL -1 BMDCs were added to the corresponding experimental groups and stimulated for 2 hours and 6 hours respectively. The supernatant was discarded, 200 μL cell lysis buffer was added, and the cells were lysed on ice for 30 minutes. Then, the cells were scraped off with a cell scraper and transferred to an EP tube. The cells were centrifuged at 12000 rpm and 4°C for 10 minutes. The precipitate was picked out and the corresponding volume of 5× protein loading buffer was added. The cells were incubated in a boiling water bath for 10 minutes, cooled on ice, and stored at -80°C for later use.

[0196] (2) Preparation of protein gel: Prepare 12.5% SDS-PAGE protein gel using a PAGE gel rapid preparation kit according to the corresponding formula;

[0197] (3) Electrophoresis: Add the prepared sample and protein marker to the gel wells, then align the electrophoresis tank with the positive and negative electrodes, run the sample through the concentrated gel at 80 V, and then switch to 120 V to run the gel;

[0198] (4) Transfer: First, soak the cut PVDF membrane in methanol solution to activate it, and then transfer it to the transfer solution for use. Take out the protein gel and cut the gel according to the size of the target band. Place the cut gel on the transfer clip with the corresponding sponge pad and filter paper. Then place the PVDF membrane, filter paper and sponge pad on the gel in sequence. Then close the transfer clip and place it in the transfer tank. Add pre-cooled transfer solution and ice box, and start transfer. Generally, the transfer time is controlled according to the molecular weight of the protein.

[0199] (5) Blocking: After the transfer is completed, immerse the PVDF membrane in a blocking solution containing 5% skim milk powder and place it on a shaker at room temperature for 1 hour;

[0200] (6) Primary antibody: After blocking, dilute the primary antibody p-STING, STING, p-TBK1, TBK1, IRF-3, and p-IRF3 at 1:1000, and place the PVDF membrane in a box containing the primary antibody and incubate at room temperature for 1 hour;

[0201] (7) Secondary antibody: After the primary antibody incubation is completed, the PVDF membrane is placed in 1×TBST for washing for 5 minutes each time, for a total of 3 times. After the washing is completed, the membrane is placed in a box containing secondary antibody and incubated for 30 minutes;

[0202] (8) Exposure: After the incubation of the secondary antibody, the membrane is washed three times in 1×TBST, each time for 5 minutes. Then, the luminescent solution A and solution B are mixed in a ratio of 1:1 to prepare the developing solution. The membrane is dried on absorbent paper and then immersed in the developing solution. The membrane is then placed in the chemiluminescence imaging system for automatic exposure.

[0203] 3. ELISA detection of cytokine secretion

[0204] (1) Sample preparation: After the BMDCs were added to the corresponding experimental groups and stimulated for 12 hours, the supernatant was aspirated and centrifuged at 2200 rpm for 5 minutes. The supernatant was aspirated and stored at -80℃ for later use.

[0205] (2) ELISA kit preparation:

[0206] 1) Reagents warming: 30 minutes before the experiment, place the test kit and the sample to be tested at room temperature. If crystals appear in the concentrated washing solution, place it in a 37°C warm bath until all crystals dissolve.

[0207] 2) Dissolve the standard: Add 1 mL of standard / sample diluent to the lyophilized standard tube, let it stand for 15 minutes to fully dissolve, and mix well before use;

[0208] 3) Dilute the standard: Take 6 EP tubes, mark them, and dilute the standard in 2-fold ratios. Then take the standard / sample dilution solution as a blank control.

[0209] 4) Washing buffer: Dilute 20× Washing Buffer to 1× with double-distilled water;

[0210] 5) Biotinylated Antibody: Pre-calculate the required amount for the assay and dilute 100× antibody concentrate to 1× working solution using assay diluent (mix thoroughly before dilution).

[0211] 6) Streptavidin-enzyme conjugate: Prepare the required amount for each test by diluting 100× concentrated enzyme conjugate into 1× working solution with enzyme conjugate diluent (centrifuge before dilution).

[0212] (3) Testing:

[0213] 1) Remove the desired strips from the sealed bag that has been equilibrated to room temperature, wash them three times, and spin dry before use;

[0214] 2) Add standards and samples: Add 100 μL of standards and test samples to the reaction wells, seal the plate, and incubate at 37°C for 90 minutes.

[0215] 3) Washing: Discard the liquid in the wells and add 300 μL of washing solution to each well. Let it stand for 30 seconds and then spin dry. Repeat 5 times. The last time, tap dry on filter paper or absorbent paper.

[0216] 4) Add biotinylated antibody: Add 100 μL of biotinylated antibody working solution to each well, seal the membrane, and incubate at 37°C for 60 minutes;

[0217] 5) Washing: Repeat step 3;

[0218] 6) Add streptavidin-enzyme conjugate: Add 100 μL of enzyme conjugate working solution to each well, seal the membrane, and incubate at 37°C in the dark for 30 minutes;

[0219] 7) Washing: Repeat step 3;

[0220] 8) Add chromogenic substrate (3,3',5,5'-Tetramethylbenzidine, TMB): add 100 μL TMB to each well and incubate at 37°C in the dark for 15 minutes;

[0221] 9) Add stop solution: Add 50 μL of stop solution to each well;

[0222] 10) Detection results: After the reaction is terminated, the OD value is measured using a microplate reader at a wavelength of 450-630 nm and the data is analyzed.

[0223] The results showed that rPA stimulation of WT BMDCs significantly increased the expression of cell surface maturity markers CD80, CD40, and CD86 ( Figure 10 ), the phosphorylation levels of key signaling pathway molecules STING, TBK-1, and IRF-3 proteins were significantly increased ( Figure 11 ), the secretion level of cytokine beta interferon increased significantly, and the immune adjuvant and the polymer skeleton played a synergistic role ( Figure 12 ), indicating that YIMIAO-1.5 and YIMIAO-1.7 can promote cell maturation, cGAS-STING signaling pathway activation and cytokine secretion, which has the effect of activating innate immune stimulation.

[0224] Example 4 Determination of the immune protection effect of YIMIAO-1.7 prepared in Example 1 of the present invention

[0225] Four experimental groups were set up, namely Mock, YIMIAO-1.2 (10 μg), YIMIAO-1.3 (10 μg rPA, 10 μg MSA-2), YIMIAO-1.6 (10 μg rPA, 250 μg YIMIAO-1.4), and YIMIAO-1.7 (10 μg rPA, 10 μg MSA-2, 250 μg YIMIAO-1.4). Mice were immunized twice with liquid aerosol lung delivery 14 days apart, and 80×LD was administered 14 days after the last immunization. 50 Anthrax Pasteur II strain liquid aerosol was delivered to the lungs for challenge. Changes in antibody titers in mice following immunization were assessed using ELISA. Protective efficacy of the vaccine was evaluated through survival analysis, histopathological analysis, and organ bacterial load measurements. Specific implementation methods are as follows.

[0226] 1. Mouse Immunization and Virus Challenge Protocol

[0227] The mice were immunized three times with a liquid aerosol pulmonary delivery, every 14 days. 50 μL of the drug liquid aerosol was delivered to the lungs of the mice.

[0228] On day 14 after the third immunization, 2 × 10 5 CFU (80×LD 50 All immunized mice were challenged with liquid aerosol lung delivery of Bacillus anthracis Pasteur II strain. Mice were injected intraperitoneally at 100 mg / kg -1 A dose of 1% sodium pentobarbital was injected to achieve the anesthesia effect. After the mouse was successfully anesthetized, it was fixed on the operating table in a supine position. Subsequently, the operating table was adjusted so that its tilt direction formed a suitable angle with the horizontal direction. Next, the tracheal opening of the mouse was fully exposed with the help of a laryngoscope. At this time, the generating head of the handheld liquid aerosol lung delivery device was inserted into the mouse trachea in a direction parallel to the trachea, with an insertion depth of about 2 cm. Afterwards, the push handle of the generator was quickly pushed to deliver 80×LD 50 The Pasteur II strain of Bacillus anthracis was delivered to the lungs of mice to complete the infection operation.

[0229] 2. Serum antibody level testing

[0230] (1) Sample collection and preparation: Six mice were randomly selected from each immunization group and marked. On the 7th and 14th day after each immunization, blood was collected from the marked mice in each group using the tail blood collection method, for a total of six blood collections. Each collected sample was placed in a clean EP tube and allowed to stand at room temperature for 4 hours. The sample was then centrifuged at 4000 rpm and 4°C for 10 minutes to separate the serum. The serum was then re-packed into new sterilized EP tubes and stored at -80°C for later use. ELISA was subsequently used to detect the levels of specific IgG, IgG1, and IgG2c antibodies in the serum.

[0231] (2) Coating: Dilute the protein rPA to 1 μg mL with coating buffer. -1 , pipette the diluted rPA and add 100 μL per well to the 96-well ELISA plate of each immune group, and place it at 4°C for coating overnight;

[0232] (3) Blocking: After shaking off the coating solution, gently pat the 96-well ELISA plate dry on absorbent paper, then use a pipette to draw up the blocking solution, add 200 μL per well to the plate, and incubate in a 37°C constant temperature incubator for 2 hours;

[0233] (4) Primary antibody incubation: discard the blocking solution and pat the plate dry on absorbent paper. The serum of each immune group after each immunization was diluted with diluent in 2-fold ratio in 8-12 steps (the serum 7 and 14 days after the first immunization was 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600; the serum 7 and 14 days after the second immunization was 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600). The serum was diluted as follows: 1:204800, 1:409600, 1:819200, 1:1638400, 1:3276800, and 1:6553600; the serum 7 and 14 days after the third immunization was diluted as follows: 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600, 1:819200, 1:1638400, 1:3276800, 1:6553600, 1:13107200, and 1:26214400). After dilution, 100 μL of the diluted serum was pipetted out and added to the coated and blocked 96-well ELISA enzyme-linked immunosorbent assay plate in order from low to high concentration. Then, sera from 6 blank mice were diluted 1:10 and added to the control wells in a volume of 100 μL and incubated at 37°C for 30 minutes;

[0234] (5) Washing the plate: remove the diluted serum primary antibody liquid, add 200 μL of washing solution to each well, shake for about 1 minute and then discard. Wash the plate 5 times according to this step and gently pat the 96-well ELISA enzyme-linked immunosorbent plate dry on absorbent paper;

[0235] (6) Secondary antibody incubation: Add 1:10000 diluted HRP-labeled goat anti-mouse IgG, IgG1, and IgG2c antibodies to a 96-well ELISA plate using a dispenser, 100 μL per well, and incubate at 37°C for 20 minutes.

[0236] (7) Washing: Remove the secondary antibody solution, add 200 μL of washing solution to each well, shake for about 1 minute and then discard. Wash the plate 5 times according to this step and gently pat the 96-well ELISA enzyme-linked immunosorbent plate dry on absorbent paper;

[0237] (8) Color development: Add TMB color development solution to a 96-well ELISA plate at 100 μL / well using a syringe gun and incubate at 37°C in the dark for 5-10 minutes;

[0238] (9) Termination: Use a pipette to draw up the stop solution and add 100 μL of stop solution to each well to terminate the reaction;

[0239] (10) Detection: Use a microplate reader to detect the absorbance of each well of a 96-well ELISA plate at wavelengths of 450 nm and 630 nm. A positive result is recorded when the ratio of the absorbance of the test sample well to the absorbance of the normal mouse serum sample well is greater than 2. The maximum dilution gradient corresponding to the positive value is the serum IgG, IgG1, and IgG2c antibody level;

[0240] 3. Cultivation of Bacillus anthracis spores

[0241] (1) The spore seeds were inoculated into 20 mL of liquid LB medium (the medium was pre-equilibrated at 37°C), placed in a 37°C shaker, and cultured at 220 r / min for 12-16 h until it reached the plateau phase (OD600≈2.5).

[0242] (2) Transfer the entire bacterial suspension into 200 mL of liquid LB medium and culture at 26°C with shaking at 220 rpm for 6 days. All operations must be performed at room temperature to avoid low-temperature shock.

[0243] (3) Transfer 200 mL of bacterial solution to a 250 mL centrifuge bottle and centrifuge at 3000 × g for 10 min at 4°C. Discard the supernatant and retain the spore pellet. If necessary, seal and freeze at -80°C for short-term storage.

[0244] (4) Add 20 mL of sterile water containing 0.05% poloxamer, and use a capillary pipette to fully suspend the spores. Incubate in a 65°C water bath for 40 min, centrifuge at 3000 × g for 10 min at 4°C, and discard the supernatant.

[0245] (5) Add 20 mL of sterile water containing 0.05% poloxamer and pipette to fully suspend the spores. Centrifuge at 3000 × g for 10 min at 4°C and discard the supernatant. Add an appropriate amount of sterile water containing 0.05% poloxamer to resuspend the spores. Observe under a phase contrast microscope and sonicate for 2 min before counting on a plate (the stock solution is diluted 10-fold first, and three dilutions ranging from 5 to 8 are counted, for a total of three replicates).

[0246] (6) Centrifuge at 3000 × g for 10 min at 4°C, discard the supernatant, and freeze at -80°C.

[0247] 4. Observation of clinical symptoms after challenge with Bacillus anthracis liquid aerosol

[0248] On day 14 after the third immunization, 2 × 10 5 CFU (80× LD 50 All immunized mice were challenged with Pasteur II strain of Bacillus anthracis by liquid aerosol lung delivery, and the survival and body weight of the mice were observed within 14 days after challenge, and the survival curve and body weight curve were drawn.

[0249] 5. Detection of Organ Bacterial Loads After Lung Challenge with Liquid Aerosol of Bacillus Anthrax

[0250] 42 hours after the anthrax liquid aerosol lung delivery challenge, 5 mice were randomly selected from each group and killed with CO2. The mice's eyeballs were removed in a biosafety cabinet to obtain whole blood. The lungs, spleen, and liver were then dissected and placed in sterile dishes. Organ tissues of appropriate size were then cut, weighed, and placed in homogenizer tubes containing 800 μL of sterile PBS. The mixture was homogenized at 5200 rpm. -1 Homogenize for 90 seconds, then dilute the homogenate 5-fold with PBS, and pipette 10 μL of each dilution onto a blood agar plate. Incubate the plate upside down at 37°C for 48 hours, and count the number of colonies in each organ at different dilution gradients.

[0251] 6. Organ Tissue Pathology Detection After Lung Challenge with Bacillus Anthrax Liquid Aerosol

[0252] (1) Sampling and fixing tissues: 42 hours after the anthrax liquid aerosol lung delivery challenge, 5 mice were randomly selected from each group and killed with CO2. The liver, spleen, and lungs were removed after dissection, and the mouse organs were fixed in 4% paraformaldehyde solution.

[0253] (2) Dehydration and transparency: After fixation for 48 hours, remove the organs and dehydrate them with 80%, 90%, 95%, and 100% ethanol in sequence, each for 2 hours. Then, place the organs in xylene and let them stand for 1 hour.

[0254] (3) Wax embedding: Place the organ in 55°C liquid paraffin and wait for the paraffin to solidify naturally to form a wax block;

[0255] (4) Sectioning and staining: Cut the wax block into 4-6 μm thick wax strips. Use tweezers to gently pick up the wax strips and place them on a 40℃~45℃ water surface. After the slices are completely unfolded naturally, gently pick them up and place them on a clean glass slide. Pour off the remaining water on the glass slide and bake them in a 60℃-65℃ constant temperature box for 30 minutes. Stain with HE and observe the changes in tissue cells under a microscope.

[0256] The results showed that mice immunized with YIMIAO-1.7 vaccine could produce a strong humoral immune response, and the titers of serum antibodies IgG, IgG1, and IgG2c increased with the progress of immunization and were higher than those in other immunization groups ( Figure 13 ). At 80×LD 50 At the same level of challenge, YIMIAO-1.7 immunization still had a complete protective effect on mice, which was significantly higher than the 40% (4 / 10) survival rate of the YIMIAO-1.3 group ( Figure 14 、 Figure 15 At 42 hours after challenge, the bacterial load in the spleen and liver of mice in the YIMIAO-1.7 group was significantly reduced, and the lung pathology score was significantly reduced ( Figure 16 、 Figure 17 In conclusion, YIMIAO-1.7 immunization enhanced the protection of mice against pulmonary anthrax.

[0257] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. An organic high molecular polymer, characterized in that The organic high molecular polymer includes MP1 and MP2, the structure of MP1 is shown in formula (1), and the structure of MP2 is shown in formula (2); Formula (1); Formula (2).

2. The organic high molecular polymer according to claim 1, characterized in that The mass ratio of MP1 to MP2 is 2:1-2:5; Preferably, the mass ratio of MP1 to MP2 is 2:3; Preferably, m in the formula (1)-formula (2) is an integer of 2-500; Preferably, m in the formula (1)-formula (2) is an integer of 50-200; Preferably, m in the formula (1)-formula (2) is 113; Preferably, in the formula (1), x is an integer of 1-20; Preferably, in the formula (1), x is 1; Preferably, in the formula (1), y is an integer of 1-20; Preferably, in the formula (1), y is 1; Preferably, n in the formula (2) is an integer of 2-20; Preferably, n in the formula (2) is 10.

3. An organic nanoparticle, characterized in that: The organic nanoparticles include the organic high molecular polymer according to any one of claims 1 to 2 and an immune adjuvant; Preferably, the mass ratio of the organic high molecular polymer to the immune adjuvant is 5:1-50:1; Preferably, the mass ratio of the organic high molecular polymer to the immune adjuvant is 25:1; Preferably, the immune adjuvant includes aluminum salt adjuvant, STING agonist, water-in-oil emulsion, saponin adjuvant, Toll-like receptor agonist, liposome / nanoparticle adjuvant, cytokine adjuvant; Preferably, the immune adjuvant is selected from STING agonists; Preferably, the STING agonist includes cGAMP, MSA-2, ADU-S100, MK-1454, SR-717, diABZI, SNX281; Preferably, the STING agonist is selected from MSA-2.

4. An organic nanoparticle vaccine, characterized in that The organic nanoparticle vaccine comprises the organic nanoparticles according to claim 3 and an antigen; Preferably, the antigen comprises a nucleic acid, protein, polypeptide, bacteria, fungus, virus or toxoid; Preferably, the antigen is selected from proteins; Preferably, the protein is the recombinant protective antigen rPA of Bacillus anthracis; Preferably, the mass ratio of the antigen to the organic high molecular polymer in the organic nanoparticles is 10:1-50:1; Preferably, the mass ratio of the antigen contained in the organic nanoparticles to the organic high molecular polymer is 40:1; Preferably, the administration of the organic nanoparticle vaccine includes inhalation, injection, oral administration, nasal spray, and transdermal administration; Preferably, the administration method of the organic nanoparticle vaccine is selected from inhalation.

5. A composition, characterized in that The composition comprises the organic high molecular polymer according to any one of claims 1 to 2, the organic nanoparticles according to claim 3, or the organic nanoparticle vaccine according to claim 4; Preferably, the composition further comprises a pharmaceutically acceptable carrier and / or excipient.

6. The method for preparing an organic high molecular polymer according to claim 1, characterized in that: The method comprises dissolving MP1 and MP2 in an organic solvent and removing the organic solvent by dialysis; Preferably, the organic solvent is selected from DMSO; Preferably, the concentration of the MP1 after being dissolved in an organic solvent is 1-10 mg / mL; Preferably, the concentration of MP1 after dissolving in an organic solvent is 5 mg / mL; Preferably, the concentration of the MP2 after being dissolved in an organic solvent is 1-10 mg / mL; Preferably, the concentration of the MP2 after dissolving in an organic solvent is 7.5 mg / mL; Preferably, the method further comprises a method for preparing MP1, comprising the following steps: L-lysine diisocyanate, 2,2'-(4,4'-(propane-1,3-diyl)bis(piperidin-4,1-diyl))diethanol and bis(2-hydroxyethyl)disulfide are dissolved in anhydrous N,N-dimethylformamide and reacted to obtain a reaction system; After dissolving polyethylene glycol in anhydrous N,N-dimethylformamide, add it into the reaction system and continue the reaction; Remove N,N-dimethylformamide and dry to obtain MP1; Preferably, the equivalent ratio of L-lysine diisocyanate, 2,2'-(4,4'-(propane-1,3-diyl)bis(piperidin-4,1-diyl))diethanol, bis(2-hydroxyethyl)disulfide and polyethylene glycol is (5-15):(1-10):(1-10):(1-5); Preferably, the equivalent ratio of L-lysine diisocyanate, 2,2'-(4,4'-(propane-1,3-diyl)bis(piperidin-4,1-diyl))diethanol, bis(2-hydroxyethyl)disulfide and polyethylene glycol is 11:5:5:2; Preferably, the concentration of the L-lysine diisocyanate after being dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15eq / mL; Preferably, the concentration of the L-lysine diisocyanate after being dissolved in anhydrous N,N-dimethylformamide is 0.11eq / mL; Preferably, the concentration of the 2,2'-(4,4'-(propane-1,3-diyl)bis(piperidine-4,1-diyl))diethanol after dissolving in anhydrous N,N-dimethylformamide is 0.01-0.1eq / mL; Preferably, the concentration of the 2,2'-(4,4'-(propane-1,3-diyl)bis(piperidine-4,1-diyl))diethanol after dissolving in anhydrous N,N-dimethylformamide is 0.05 eq / mL; Preferably, the concentration of the bis(2-hydroxyethyl) disulfide after being dissolved in anhydrous N,N-dimethylformamide is 0.01-0.1eq / mL; Preferably, the concentration of the bis(2-hydroxyethyl) disulfide after being dissolved in anhydrous N,N-dimethylformamide is 0.05eq / mL; Preferably, the concentration of the polyethylene glycol after being dissolved in anhydrous N,N-dimethylformamide is 0.01-0.05eq / mL; Preferably, the concentration of the polyethylene glycol after dissolving in anhydrous N,N-dimethylformamide is 0.02eq / mL; Preferably, the polyethylene glycol is polyethylene glycol 5000; Preferably, the method of removal comprises dialysis; Preferably, the drying comprises freeze-drying; Preferably, the method further comprises a method for preparing MP2, comprising the following steps: Dissolving 1,2,4,5-cyclohexanetetracarboxylic dianhydride and bis(2-hydroxyethyl)disulfide in anhydrous N,N-dimethylformamide, and reacting to obtain a reaction system; After dissolving polyethylene glycol in anhydrous N,N-dimethylformamide, add it into the reaction system and continue the reaction; After removing N,N-dimethylformamide and drying, a white polymer was obtained; The white polymer was dissolved in anhydrous N,N-dimethylformamide, followed by the addition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine. After activation, 4-hydroxymethylphenylboronic acid was added and the reaction continued. Remove N,N-dimethylformamide and dry to obtain MP2; Preferably, the equivalent ratio of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl) disulfide, and polyethylene glycol is (5-15):(5-15):(1-5); Preferably, the equivalent ratio of 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl) disulfide, and polyethylene glycol is 11:10:2; Preferably, the concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride after being dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15eq / mL; Preferably, the concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride after dissolving in anhydrous N,N-dimethylformamide is 0.11eq / mL; Preferably, the concentration of the bis(2-hydroxyethyl) disulfide after being dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15eq / mL; Preferably, the concentration of the bis(2-hydroxyethyl) disulfide after being dissolved in anhydrous N,N-dimethylformamide is 0.1 eq / mL; Preferably, the concentration of the polyethylene glycol after being dissolved in anhydrous N,N-dimethylformamide is 0.01-0.05eq / mL; Preferably, the concentration of the polyethylene glycol after being dissolved in anhydrous N,N-dimethylformamide is 0.02eq / mL; Preferably, the polyethylene glycol is HO-PEG 5000-COOH; Preferably, the equivalent ratio of the white polymer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and 4-hydroxymethylphenylboronic acid is (0.5-2):(1-5):(1-5); Preferably, the equivalent ratio of the white polymer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and 4-hydroxymethylphenylboronic acid is 1:2:2:2; Preferably, the concentration of the white polymer after being dissolved in anhydrous N,N-dimethylformamide is 0.005-0.02eq / mL; Preferably, the concentration of the white polymer after dissolving in anhydrous N,N-dimethylformamide is 0.01eq / mL; Preferably, the method of removal comprises dialysis; Preferably, the drying comprises freeze-drying.

7. The method for preparing organic nanoparticles according to claim 3, characterized in that: The method comprises dissolving the organic high molecular weight polymer according to any one of claims 1 to 2 and an immune adjuvant in an organic solvent, and dialyzing to remove the organic solvent; Preferably, the organic solvent is selected from DMSO; Preferably, the organic high molecular weight polymer is dissolved in an organic solvent at a concentration of 1-20 mg / mL; Preferably, the organic high molecular weight polymer is dissolved in an organic solvent at a concentration of 12.5 mg / mL.

8. The method for preparing the organic nanoparticle vaccine according to claim 4, characterized in that: The method comprises mixing the organic nanoparticles of claim 3 with an antigen; Preferably, the antigen is used at a concentration of 1-50 mg / μL; Preferably, the antigen is used at a concentration of 10 mg / μL.

9. Any of the following methods: (1) A method for promoting dendritic cell maturation, characterized in that: The method comprises administering the organic high molecular polymer according to any one of claims 1 to 2, the organic nanoparticle according to claim 3, the organic nanoparticle vaccine according to claim 4, or the composition according to claim 5; (2) A method for activating an innate immune signaling pathway, characterized in that the method comprises administering the organic high molecular weight polymer according to any one of claims 1 to 2, the organic nanoparticle according to claim 3, the organic nanoparticle vaccine according to claim 4, or the composition according to claim 5; (3) A method for promoting the secretion of type I interferon, characterized in that the method comprises administering the organic high molecular polymer according to any one of claims 1 to 2, the organic nanoparticle according to claim 3, the organic nanoparticle vaccine according to claim 4, or the composition according to claim 5; Preferably, the type I interferon is IFN-β; (4) A method for targeting cytoplasm, characterized in that the method comprises administering the organic high molecular polymer according to any one of claims 1 to 2, the organic nanoparticle according to claim 3, the organic nanoparticle vaccine according to claim 4, or the composition according to claim 5.

10. Any of the following applications: (1) Use of the organic high molecular weight polymer according to any one of claims 1 to 2 in loading an immune adjuvant; Preferably, the immune adjuvant includes aluminum salt adjuvant, STING agonist, water-in-oil emulsion, saponin adjuvant, Toll-like receptor agonist, liposome / nanoparticle adjuvant, cytokine adjuvant; Preferably, the immune adjuvant is selected from STING agonists; Preferably, the STING agonist includes cGAMP, MSA-2, ADU-S100, MK-1454, SR-717, diABZI, SNX281; Preferably, the STING agonist is selected from MSA-2; (2) Use of the organic high molecular weight polymer according to any one of claims 1 to 2 or the organic nanoparticles according to claim 3 in the preparation of vaccines; (3) Use of the organic high molecular weight polymer according to any one of claims 1 to 2 or the organic nanoparticles according to claim 3 as a drug carrier; (4) Use of the organic high molecular polymer according to any one of claims 1 to 2 or the organic nanoparticles according to claim 3 as a drug dressing; (5) Use of the organic high molecular polymer according to any one of claims 1 to 2, the organic nanoparticle according to claim 3, the organic nanoparticle vaccine according to claim 4, or the composition according to claim 5 in the preparation of a drug for preventing pathogen infection; Preferably, the pathogens include bacteria, viruses, fungi, parasites, and prions; Preferably, the pathogen is selected from bacteria; Preferably, the bacteria include Bacillus anthracis; (6) Use of the organic high molecular polymer according to any one of claims 1 to 2, the organic nanoparticle according to claim 3, the organic nanoparticle vaccine according to claim 4, or the composition according to claim 5 in promoting the maturation of dendritic cells; (7) Use of the organic high molecular polymer according to any one of claims 1 to 2, the organic nanoparticle according to claim 3, the organic nanoparticle vaccine according to claim 4, or the composition according to claim 5 in activating innate immune signaling pathways; (8) Use of the organic high molecular polymer according to any one of claims 1 to 2, the organic nanoparticle according to claim 3, the organic nanoparticle vaccine according to claim 4, or the composition according to claim 5 in promoting the secretion of type I interferon; Preferably, the type I interferon is IFN-β; (9) Use of the organic high molecular polymer according to any one of claims 1 to 2, the organic nanoparticle according to claim 3, the organic nanoparticle vaccine according to claim 4, or the composition according to claim 5 in targeting the cytoplasm.

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