Manganese-based delivery system with adjuvant-like function and application of manganese-based delivery system in vaccine preparation

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a manganese-based delivery system with an adjuvant-like function and application of the manganese-based delivery system in vaccine preparation. The invention firstly provides two novel vaccine vectors, namely PMn and YC-coated PMn. PMn is a novel manganese oxide-polyetherimide complex and is obtained by carrying out redox reaction on potassium permanganate and polyetherimide. PMn can be used for loading antigens, loading adjuvants or can be used as adjuvants per se. PMn is loaded into a hollow yeast microcapsule, and a vaccine delivery system which is more stable and has an adjuvant-like function and a slow-release effect is obtained. The vaccine delivery system can be further loaded with protein antigens and / or other molecular adjuvants. Animal experiments prove that the novel sustained-release vaccine delivery system with the adjuvant-like function can be used for remarkably inducing high-level antigen-specific IgG, IgG1 and IgG2a antibodies in animal bodies.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine technology, and particularly relates to a manganese-based delivery system with adjuvant-like function and its application in the preparation of vaccines. Background Art

[0002] Vaccines, as one of the most important means for humans to combat diseases, have been used to control various infectious diseases and made invaluable contributions to the cause of human health. With the development of biotechnology, the varieties and types of vaccines are increasing. Currently, the vaccines on the market include live attenuated vaccines, inactivated vaccines, subunit vaccines, polysaccharide vaccines, polysaccharide-protein conjugate vaccines, combined vaccines, viral vector vaccines, mRNA vaccines, etc. Existing vaccines have played an important role in preventing and controlling diseases. However, with the development of biotechnology, people have put forward higher requirements for the effects and uses of vaccines.

[0003] Subunit vaccines refer to vaccines prepared by extracting immunologically active fragments of pathogens through means such as chemical decomposition and recombinant expression. Subunit vaccines have the characteristics of higher safety, clear composition, good stability, and easy large-scale production compared with inactivated vaccines and live attenuated vaccines, showing great clinical potential. However, the immunogenicity of subunit antigens is low, and the induced immune response is weak. Adjuvants are often needed to be used in combination to enhance the response intensity of the body. However, most of the existing marketed adjuvants cannot effectively protect antigens, cannot achieve co-delivery of antigens and adjuvants, and cannot target the delivery of antigens to antigen-presenting cells (APCs). In addition, the existing adjuvants usually can only be simply physically mixed with antigens, and the delivery efficiency is low, which limits the applications with higher requirements for targeted delivery of vaccines, such as the application of preparing tumor vaccines. Therefore, the development of an efficient antigen delivery (adjuvant) system is crucial for expanding the application of subunit vaccines.

[0004] Manganese (Mn) is one of the essential trace inorganic elements in the body. Manganese (especially Mn 2+ ) can stimulate the immune system of the body and is a metal adjuvant that can effectively activate the cyclic guanosine monophosphate-adenosine synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. However, due to the influence of rapid metabolism and non-specific targeting, soluble Mn 2+ cannot target the delivery of antigens to APCs and fully exert the adjuvant effect. Therefore, a more suitable manganese-based delivery system needs to be developed. On the other hand, for the preparation methods of manganese-based nanoparticles, there are currently various methods to prepare manganese-based nanoparticles with different morphologies. The preparation process usually involves using an oxidant to oxidize Mn 2+ or using a reducing agent to reduce MnO4 - , but most of the reaction conditions are relatively harsh, such as the need for organic solvents, high temperature, catalysts, strong acids or strong bases, which is not conducive to further production applications.

[0005] In summary, it is necessary to propose new strategies and methods to improve the deficiencies of the existing technology. SUMMARY OF THE INVENTION

[0006] The object of the present invention is to provide a manganese-based delivery system with adjuvant-like function and its application in the preparation of vaccines, which partially solves or alleviates the above deficiencies in the existing technology. The present invention specifically adopts the following technical solutions.

[0007] Application of a novel manganese-based nanoparticle in the preparation of a vaccine, wherein the novel manganese-based nanoparticle is obtained by an oxidation-reduction reaction between potassium permanganate and polyetherimide; the novel manganese-based nanoparticle is a manganese oxide-polyetherimide complex; the surface of the novel manganese-based nanoparticle is positively charged; the novel manganese-based nanoparticle is used to load protein antigens, load adjuvants or act as an adjuvant.

[0008] Furthermore, the protein antigen includes one or more selected from hepatitis B surface antigen HBsAg, recombinant PreS1, PreS2, recombinant core protein, hepatitis C virus antigen, hepatitis E virus antigen, anthrax toxin-binding protein, pneumococcal protein, group A M protein, streptococcal C5a peptidase, streptococcal enolase-binding protein, serine carboxylesterase, outer membrane protein OMP of Neisseria meningitidis serogroup B, tumor cell lysate protein, bacterial lysate protein, chicken ovalbumin, Treponema pallidum surface lipoprotein, bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myoalbumin, legumelin, gliadin, myoglobin, collagen, fibronectin or polypeptide antigen; the polypeptide antigen includes one or more selected from group A streptococcal M peptide, TRP2, HGP100, p15E, Pseudomonas aeruginosa synthetic peptide or rubella virus synthetic peptide; the other molecular adjuvants include one or more selected from peptidoglycan, lipoteichoic acid, monophosphoryl lipid A, imiquimod, resiquimod, CpG-ODN, bacterial flagellin, Poly I:C, 3pRNA, short double-stranded RNA, muramyl dipeptide, bisphosphonate, lipophilic statin, N-acetylglucosamine, β-glucan, trehalose diborate, cGAMP, cholera toxin, Escherichia coli heat-labile enterotoxin, cholera toxin B subunit, pertussis toxin, tetanus toxin, diphtheria toxin, interferon, GM-CSF, IL-1, IL-2, IL-12, IL-6, Quil A, QS-21, and heat shock protein.

[0009] Furthermore, when the oxidation-reduction reaction occurs between potassium permanganate and polyetherimide, the mass ratio of polyetherimide to potassium permanganate is 20:1 - 100:1.

[0010] As a preference, the mass ratio of the PEI aqueous solution to the potassium permanganate aqueous solution includes 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1.

[0011] As a preference, in the novel manganese-based nanoparticle PMn, the valence state of the manganese ion includes divalent.

[0012] Furthermore, the particle size range of the novel manganese-based nanoparticles is 20 - 500 nm.

[0013] Preferably, the particle size range of the novel manganese-based nanoparticles is 100 - 300 nm.

[0014] The preparation method of the above-mentioned novel manganese-based nanoparticles includes the following steps: S01: Prepare an aqueous solution of low molecular weight polyetherimide, and then slowly drop it into an aqueous solution of potassium permanganate to undergo a redox reaction to obtain a complex solution. The complex solution contains a manganese oxide-polyetherimide complex, and the mass ratio of the polyetherimide aqueous solution to the potassium permanganate aqueous solution is 20:1 - 100:1; S02: Centrifuge, wash the reaction solution, and resuspend the precipitate with an aqueous solution by ultrasonic treatment to obtain the novel manganese-based nanoparticles.

[0015] Furthermore, the low molecular weight polyetherimide is a polyetherimide with a molecular weight lower than 10,000.

[0016] As a preference, the molecular weight of the low molecular weight polyetherimide is 1000 - 3000.

[0017] The novel manganese-based nanoparticles (PMn) prepared by the above preparation method.

[0018] An adjuvant-like function-based sustained-release vaccine delivery system (also known as an adjuvant-like function-based sustained-release vaccine delivery tool), the adjuvant-like function-based sustained-release vaccine delivery system is composed of a shell part and a content; the shell part is a hollow yeast microcapsule; the content is novel manganese-based nanoparticles; the novel manganese-based nanoparticles are obtained by the redox reaction of potassium permanganate and polyetherimide; the novel manganese-based nanoparticles are manganese oxide-polyetherimide complexes; the surface of the novel manganese-based nanoparticles is positively charged; the manganese-based nanoparticles are located in the internal hollow part of the yeast microcapsule.

[0019] Alternatively, an adjuvant-like function-based sustained-release vaccine delivery system (also known as an adjuvant-like function-based sustained-release vaccine delivery tool), the adjuvant-like function-based sustained-release vaccine delivery system is composed of a shell part and a content; the shell part is a hollow yeast microcapsule; the content is the novel manganese-based nanoparticles obtained by the above preparation method.

[0020] Furthermore, the sustained-release vaccine delivery system with adjuvant-like function can be used to load protein antigens or other molecular adjuvants.

[0021] Furthermore, the mass ratio of the yeast microcapsule to manganese is 6:1 - 600:1.

[0022] Furthermore, the encapsulation rate of the sustained-release manganese-based delivery system with adjuvant-like function for protein antigens and / or other molecular adjuvants is greater than 80%.

[0023] Furthermore, the protein antigen includes one or more selected from hepatitis B surface antigen HBsAg, recombinant PreS1, PreS2, recombinant core protein, hepatitis C virus antigen, hepatitis E virus antigen, anthrax toxin-binding protein, pneumococcal protein, group A M protein, streptococcal C5a peptidase, enolase-binding protein, serine carboxylesterase, outer membrane protein OMP of Neisseria meningitidis serogroup B, tumor cell lysate protein, bacterial lysate protein, chicken ovalbumin, Treponema pallidum surface lipoprotein, bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myoalbumin, legumelin, gliadin, myoglobin, collagen, fibronectin or polypeptide antigen; the polypeptide antigen includes one or more selected from group A streptococcal M peptide, TRP2, HGP100, p15E, Pseudomonas aeruginosa synthetic peptide or rubella virus synthetic peptide; the other molecular adjuvants include one or more selected from peptidoglycan, lipoteichoic acid, monophosphoryl lipid A, imiquimod, resiquimod, CpG-ODN, bacterial flagellin, Poly I:C, 3pRNA, short double-stranded RNA, muramyl dipeptide, bisphosphonate, lipophilic statin, N-acetylglucosamine, β-glucan, trehalose diborate, cGAMP, cholera toxin, Escherichia coli heat-labile enterotoxin, cholera toxin B subunit, pertussis toxin, tetanus toxin, diphtheria toxin, interferon, GM-CSF, IL-1, IL-2, IL-12, IL-6, Quil A, QS-21, and heat shock protein.

[0024] The present invention can also provide a combination of drugs for enhancing immune response, and the combination of drugs includes the above-mentioned sustained-release vaccine delivery system with adjuvant-like function and an adjuvant.

[0025] As a preference, the adjuvant includes zoledronate ZOL, oligonucleotide CpG and / or STING protein small molecule agonist cGAMP.

[0026] A preparation method of a sustained-release vaccine delivery system with adjuvant-like function includes the following steps: S01: Preparation of yeast microparticles: Weigh an appropriate amount of yeast granules, dissolve them in an aqueous sodium hydroxide solution, and then subject them to an oil bath treatment at the first temperature. Collect the reaction precipitate, resuspend it in water, adjust the pH value to weakly acidic, and then place it in an oil bath at the second temperature for treatment. Wash and dry to obtain the yeast microparticles; the first temperature is higher than the second temperature. S02: Preparation of manganese-based nanoparticles: Prepare an aqueous solution of low molecular weight polyetherimide, and then slowly drop it into an aqueous potassium permanganate solution. An oxidation-reduction reaction occurs to obtain a complex solution, and the complex solution contains manganese oxide-polyetherimide complexes. The mass ratio of the aqueous polyetherimide solution to the aqueous potassium permanganate solution is 20:1 - 100:1; centrifuge and wash to collect the manganese-based nanoparticles. S03: Prepare the slow-release vaccine delivery system with adjuvant-like function by the reverse microemulsion method: S031: Disperse the yeast microparticles prepared in S01 in a mixed solvent of an organic solvent immiscible with water and a surfactant, and slowly drop the manganese-based nanoparticle solution into it under stirring for reaction; S032: After the reaction is completed, centrifuge to remove the oil phase, and wash the precipitate successively with an organic solvent immiscible with water, absolute ethanol, and ultrapure water. Resuspend it with ultrapure water and collect the slow-release vaccine delivery system with adjuvant-like function. In some preferred embodiments, the organic solvent immiscible with water is cyclohexane.

[0027] In some preferred embodiments, the surfactant is Igepal CO-520.

[0028] Beneficial technical effects: The present invention provides two novel vaccine carriers: PMn and YC@PMn.

[0029] First, the present invention innovatively uses a low molecular weight cationic polymer polyetherimide (PEI) to reduce potassium permanganate, and an oxidation-reduction reaction occurs under mild conditions to prepare a stable manganese oxide-polyetherimide complex (PMn). During the reaction process of the present invention, PEI acts as both a reducing agent and a stabilizer, wrapping around the surface of the newly formed manganese-based nanoparticles to prevent further aggregation and precipitation of manganese oxide. Moreover, the surface of the manganese oxide-polyetherimide complex is positively charged, and subsequently, protein antigens or other molecular adjuvants can be efficiently loaded through electrostatic adsorption to achieve specific uses in the preparation of vaccines. The preparation method of the present invention overcomes the disadvantages of the cumbersome and harsh reaction conditions for the traditional preparation of manganese oxide nanoparticles, which is not conducive to large-scale production. In particular, the low molecular weight of PEI reduces cytotoxicity and can also stimulate innate immunity by triggering "danger signals" to exert an immune adjuvant-like function. Experiments show that PMn can be efficiently taken up by DC cells, promote the ability of antigens to escape from lysosomes, and can also significantly promote DC cells to secrete type I IFNs, proving that it itself also has a good adjuvant-like function.

[0030] Furthermore, PMn is loaded into hollow yeast microcapsules to obtain a more stable vaccine delivery system with adjuvant-like function and sustained release effect (or a vaccine delivery tool with adjuvant-like function and sustained release effect). This vaccine delivery system itself has the function of an adjuvant and can further load protein antigens and / or other molecular adjuvants, ultimately achieving multi-level loading and co-delivery of antigens and adjuvants. In addition, after being encapsulated by yeast microcapsules, this delivery system also has a sustained release function. Animal experiments prove that this novel sustained release vaccine delivery system with adjuvant-like function provided by the present invention can significantly induce high levels of antigen-specific IgG antibodies in mice, mainly including IgG1 antibodies and IgG2a antibodies, enhancing humoral and cellular immune responses, and having broad clinical application prospects. Brief Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to actual scale. Obviously, the following-described drawings are some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings based on these drawings without creative efforts.

[0032] Figure 1 Transmission electron microscopy and elemental mapping of the novel manganese-based nanoparticle PMn synthesized in one embodiment of the present invention (scale bar is 100 nm); Figure 2X-ray photoelectron spectroscopy of the novel manganese-based nanoparticles PMn synthesized in one embodiment of the present invention; Figure 3 Characterization of the particle size and potential of the novel manganese-based nanoparticles PMn and PMn / OVA in one embodiment of the present invention; Figure 4 Representative flow cytometry and statistical charts of in vitro DC2.4 cell uptake of PMn / Cy5-OVA in one embodiment of the present invention; Figure 5 Representative confocal fluorescence images of PMn / Cy5-OVA promoting antigen lysosomal escape on DC2.4 cells in one embodiment of the present invention; Figure 6 Evaluation of the activation of the STING pathway by PMn / OVA promoting the expression of type I IFN mRNA in DC cells in one embodiment of the present invention; Figure 7 OVA-specific IgG and IgG1 antibody levels in the serum of mice after immunization with PMn / OVA in one embodiment of the present invention; Figure 8 Schematic diagram of the preparation of the sustained-release vaccine delivery system YC@PMn@antigen in one embodiment of the present invention; Figure 9 Particle size and potential diagrams of YC and YC@PMn in one embodiment of the present invention; Figure 10 Transmission electron microscope images of YC and YC@PMn (scale bar is 1μm) in one embodiment of the present invention; Figure 11 Element mapping diagram of YC@PMn in one embodiment of the present invention; Figure 12 Laser confocal fluorescence image of YC@PMn@Cy5-OVA in one embodiment of the present invention; Figure 13 Laser confocal fluorescence image of YC@PMn@Cy5-OVA after being phagocytosed by DC cells in one embodiment of the present invention; Figure 14 Result diagram of OVA-specific antibody levels in the serum of mice after immunization in one embodiment of the present invention. Detailed implementation mode

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0034] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0035] As used herein, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0036] As used in this specification, the term "about" typically represents + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0037] In this specification, certain embodiments may be disclosed in a format within a certain range. It should be understood that this description of "within a certain range" is only for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0038] Glossary of terms: The "manganese oxide-polyetherimide complex" described in the present invention refers to the product obtained by the redox reaction of potassium permanganate and polyetherimide, which contains manganese oxide and the oxidation product of polyetherimide, and the valence state of manganese in this complex includes divalent.

[0039] Example 1 This example provides an example of preparing manganese oxide-polyetherimide complex (PMn) nanoparticles.

[0040] Prepare an aqueous solution of low molecular weight polyetherimide PEI (for example, PEI 1.8kAn aqueous solution), and then an aqueous solution of potassium permanganate was slowly added dropwise. The mass ratio of PEI to potassium permanganate was 40:1, and the reaction was stirred (the reaction temperature could be set at room temperature or heated at 30°C - 60°C; the more potassium permanganate was added, the higher the reaction temperature and the shorter the reaction time; the less potassium permanganate was added, the lower the reaction temperature and the longer the reaction time). Then, it was centrifuged at 10000g for 60 min, washed twice with ultrapure water, and finally resuspended in a 5% glucose aqueous solution and sonicated to obtain manganese oxide-polyetherimide composite PMn nanoparticles (which can be simply referred to as novel manganese-based nanoparticles). The prepared PMn nanoparticles were stored at -80°C.

[0041] The above examples are only for illustration and can be implemented within the range of material ratios described in the specification of the present invention.

[0042] The transmission electron microscopy and elemental mapping of the synthesized PMn nanoparticles are as Figure 1 shown; the X-ray photoelectron spectroscopy results are as Figure 2 shown, and the results show that the valence states of manganese include Mn 2+ .

[0043] Example 2 This example provides the loading of antigens with the PMn nanoparticles synthesized in Example 1 and the verification of its effects.

[0044] In this example, the model antigen ovalbumin (OVA) was taken as an example. After the PMn nanoparticles were loaded with OVA, they were labeled as PMn / OVA.

[0045] Experimental method steps: S01: Preparation of the PMn core: First, an aqueous solution of PEI 1.8k was prepared, and then an aqueous solution of potassium permanganate was slowly added dropwise. The mass ratio of PEI to potassium permanganate was 50:1, and the reaction was stirred at room temperature for 1 - 4 h. Then, it was centrifuged at 10000g for 60 min, washed twice with ultrapure water, and finally resuspended in a 5% glucose aqueous solution and sonicated to obtain PMn nanoparticles.

[0046] S02: Under the condition of vigorous vortexing, an aqueous solution of OVA protein was gradually added dropwise to the aqueous solution of PMn nanoparticles, and then vortexed for at least 30 s. Subsequently, it was incubated at room temperature for 20 min to obtain PMn / OVA loaded with OVA. The encapsulation efficiency was measured by the ultrafiltration method. An aqueous solution of free protein with the same concentration and the prepared nanoparticle solution were placed in an ultrafiltration tube (the molecular weight cut-off was 300 kDa), centrifuged at 3000g for 10 min, and then the protein concentrations inside and outside the ultrafiltration tube were detected using a micro BCA protein quantification kit, and the encapsulation efficiency of the antigen protein could be calculated. The loading efficiency of the PMn nanoparticles in this example for loading OVA was high, and the encapsulation efficiency reached more than 95%.

[0047] The results are shown in Figure 3 , and the prepared PMn nanoparticles have a hydrated particle size of about 100 nm, as shown in Figure 3 A. The potential of the PMn nanoparticles is about +40 mV. After the antigen OVA is adsorbed on the surface, the hydrated particle size changes little, but the potential drops to less than +30 mV ( Figure 3 B). PEI is rich in primary amines, secondary amines, tertiary amines and other groups, has high chemical activity, and its cytotoxicity decreases significantly with the decrease of molecular weight. It can also stimulate innate immunity by triggering "danger signals" and play an immune adjuvant-like function. During the reaction process, PEI acts as both a reducing agent and a stabilizer, wrapping on the surface of the newly formed manganese-based nanoparticles to prevent further aggregation and precipitation of manganese oxide. Moreover, the strongly positively charged manganese-based nanoparticles can also efficiently load protein antigens and / or other molecular adjuvants through electrostatic interaction.

[0048] Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APCs) in the body, and they are also the only APCs that can activate naive T cells, connecting the innate immune system and the adaptive immune system, which is particularly important during the immune response process. Therefore, this example further verifies the effect of PMn / OVA on DC cells in vitro.

[0049] Experimental methods and steps: (1) Label OVA with the fluorescent dye Cy5, and then prepare the PMn / Cy5-OVA preparation with reference to Example 2. Plate the DC2.4 cells in the logarithmic growth phase, and add free Cy5-OVA and PMn / Cy5-OVA respectively under the conditions of having blood and antibodies. The final concentration is 10 μg / mL Cy5-OVA for both. After continuing to culture for 1 h, detect the uptake of the samples by the cells through a flow cytometer. The results are shown in Figure 4 .

[0050] The results show that compared with the free antigen protein OVA, PMn / OVA is more easily taken up by DC2.4 cells. After 1 h of administration, the positive rate is above 95%.

[0051] (2) Further, plate the DC2.4 cells in the logarithmic growth phase and inoculate them into confocal dishes. Add fluorescently labeled free Cy5-OVA and PMn / Cy5-OVA with a final concentration of 10 μg / mL Cy5-OVA. After continuing to culture for 6 h, label the lysosomes with the fluorescent dye LysoTracker, and evaluate the lysosomal escape ability of the antigen by laser confocal microscopy. The results are shown in Figure 5 .

[0052] The results showed that after co-incubating PMn / Cy5-OVA with DC2.4 cells for 6 h, the co-localization of intracellular OVA and lysosomes was significantly weakened, indicating that the manganese-based nanoparticles could promote the escape of intracellular antigen from lysosomes.

[0053] (3)Furthermore, RT-qPCR was used to examine the relative expression levels of IFN-α1, IFN-α2, and IFN-β1 mRNAs in DC2.4 cells 24 h after administration of PMn / OVA to evaluate the activation of the cGAS-STING signaling pathway.

[0054] DC2.4 cells in the logarithmic growth phase were plated and treated with PBS, OVA, and PMn / OVA, respectively, at a final concentration of 10 μg / mL OVA. After 24 h of administration, the cells were collected, total RNA was extracted, and the expression of IFN-α1, IFN-α2, and IFN-β1 mRNAs in the cells was detected by RT-qPCR to evaluate the activation of the STING pathway. See Figure 6 。

[0055] The results showed that PMn could significantly promote the secretion of type I IFNs by DC2.4 cells, demonstrating its good adjuvant-like function.

[0056] (4)OVA free of endotoxin was used to prepare the PMn / OVA preparation of Example 2. C57 mice were randomly divided into three groups, which were PBS, free antigen OVA, and PMn / OVA groups in sequence. They were immunized subcutaneously in the footpads on days 0, 14, and 21, with 10 μg OVA per mouse each time. On days 21 and 28, blood was collected from the mouse orbits using a capillary tube. After standing at low temperature for 2 - 6 h, the serum samples were collected by centrifugation, and the levels of OVA antigen-specific IgG and IgG1 antibodies in the serum were determined by indirect ELISA. See Figure 7 。

[0057] The results showed that the manganese-based nanoparticles induced higher levels of antigen-specific IgG and IgG1 antibodies in mice, indicating an enhanced humoral immune response.

[0058] Example 3 Preparation and characterization of a sustained-release vaccine delivery system YC@PMn with adjuvant-like function.

[0059] Yeast capsules (YC) of natural origin are obtained by treating Saccharomyces cerevisiae with acid-base solutions and organic reagents to form hollow capsule shells with a particle size of 2-4 µm. The surface of the capsule shells has pores ranging in size from a few nanometers to hundreds of nanometers, making them ideal loading tools. PMn nanoparticles, antigens, and / or other molecular adjuvants are co-loaded into these yeast microcapsule particles. As the core with adjuvant-like functions, PMn adsorbs antigens through electrostatic interactions, thereby achieving multi-level loading and co-delivery of antigens and adjuvants. The vaccine delivery system prepared in this way can not only stably protect antigens but also slowly release antigens and adjuvants, providing long-lasting stimulation to achieve a slow-release effect and prolong the action time of the vaccine and the immune system. The schematic diagram is shown in Figure 8 , and the specific preparation method is as follows.

[0060] Preparation of YC: Weigh 10 g of yeast particles, first treat them with 100 mL of 1 M sodium hydroxide aqueous solution in an 80 °C constant-temperature oil bath for 1 h, and collect the white precipitate by centrifugation. Then, resuspend the precipitate in 100 mL of water, adjust the pH to 4-5, and treat it in a 55 °C constant-temperature oil bath for 1 h. Next, wash the precipitate four times with isopropanol and twice with acetone, and evaporate the organic reagents to dryness. The resulting white powder is the hollow YC microparticles.

[0061] Preparation and optimization of YC@PMn: Prepared by the reverse microemulsion method. Disperse 3 mg of YC in 1 mL of cyclohexane / Igepal CO-520 (85 / 15, v / v) solvent. Under stirring at 37 °C, slowly drop 100 μg of the pre-prepared PMn nanoparticle solution into it. After reacting for 1 h, centrifuge to remove the oil phase. Wash the precipitate twice with cyclohexane, once with absolute ethanol, and once with ultrapure water. Finally, resuspend it in 600 μL of ultrapure water to obtain the optimized slow-release vaccine delivery system YC@PMn@OVA+cGAMP.

[0062] Preparation and optimization of YC@PMn@OVA microparticle vaccine: Taking the model antigen OVA as an example, prepare the microparticle vaccine by the reverse microemulsion method. Disperse 3 mg of YC in 1 mL of cyclohexane / Igepal CO-520 (85 / 15, v / v) solvent. Under stirring at 37 °C, slowly drop 75 μg of the pre-prepared PMn nanoparticle solution into it. After reacting for 1 h, then slowly drop 75 μg of the OVA antigen solution into it and continue to react for 1 h. Subsequently, centrifuge to remove the oil phase. Wash the precipitate twice with cyclohexane, once with absolute ethanol, and once with ultrapure water. Finally, resuspend it in 375 μL of ultrapure water to obtain the optimized slow-release vaccine delivery system YC@PMn@OVA.

[0063] Preparation and optimization of YC@PMn@OVA+ZOL: Disperse 3 mg of YC in 1 mL of cyclohexane / Igepal CO-520 (85 / 15, v / v) solvent. Under the condition of stirring at 37 °C, slowly drop 75 μg of the pre-prepared PMn nano-solution into it. After reacting for 1 h, then slowly drop 75 μg of OVA antigen and 187.5 μg of zoledronic acid (ZOL) adjuvant solution into it, and continue to react for 1 h. Subsequently, centrifuge to remove the oil phase, wash the precipitate with cyclohexane twice, anhydrous ethanol once, and ultrapure water once, and finally resuspend it with 375 μL of ultrapure water to obtain the optimized sustained-release vaccine delivery system YC@PMn@OVA+ZOL.

[0064] Preparation and optimization of YC@PMn@OVA+CpG: Disperse 4.8 mg of YC in 2 mL of cyclohexane / Igepal CO-520 (85 / 15, v / v) solvent. Under the condition of stirring at 37 °C, slowly drop 120 μg of the pre-prepared PMn nano-solution into it. After reacting for 1 h, then slowly drop 120 μg of OVA antigen and 9 μg of TLR9 agonist (CpG adjuvant) solution into it, and continue to react for 1 h. Subsequently, centrifuge to remove the oil phase, wash the precipitate with cyclohexane twice, anhydrous ethanol once, and ultrapure water once, and finally resuspend it with 600 μL of ultrapure water to obtain the optimized sustained-release vaccine delivery system YC@PMn@OVA+CpG.

[0065] Preparation and optimization of YC@PMn@OVA+cGAMP: Disperse 4.8 mg of YC in 2 mL of cyclohexane / Igepal CO-520 (85 / 15, v / v) solvent. Under the condition of stirring at 37 °C, slowly drop 120 μg of the pre-prepared PMn nano-solution into it. After reacting for 1 h, then slowly drop 120 μg of OVA antigen and 30 μg of STING agonist (cGAMP adjuvant) into it, and continue to react for 1 h. Subsequently, centrifuge to remove the oil phase, wash the precipitate with cyclohexane twice, anhydrous ethanol once, and ultrapure water once, and finally resuspend it with 600 μL of ultrapure water to obtain the optimized sustained-release vaccine delivery system YC@PMn@OVA+cGAMP.

[0066] The above embodiments are only examples and can be implemented within the range of material ratios described in the specification of the present invention.

[0067] Characterization: The hydrated particle size and Zeta potential of the carrier were characterized by a particle size analyzer; the appearance morphology of the microparticles was observed by high-resolution transmission electron microscopy and elemental mapping analysis was performed; the content of manganese in the solution was determined by inductively coupled plasma optical emission spectrometer (ICP-OES); the encapsulation efficiency of the carrier against antigen protein was detected by a micro BCA protein quantification kit; the encapsulation efficiency of adjuvant CpG was determined by PAGE method; the encapsulation efficiencies of adjuvants ZOL and cGAMP were determined by HPLC method.

[0068] In the sustained-release vaccine delivery system prepared in this example, the encapsulation efficiency of the antigen protein was 80% - 100%; the encapsulation efficiencies of ZOL and cGAMP were greater than 70%, and the encapsulation efficiency of CpG was greater than 75%.

[0069] The results of YC loaded with PMn nanoparticles are shown in Figure 9 . The particle size analyzer showed that there was no obvious change in the particle size before and after loading, the Zeta potential increased, but it was still negatively charged overall, suggesting that the positively charged PMn nanoparticles were located in the hollow inner core of YC.

[0070] The transmission electron microscopy results showed that the morphology of YC was ellipsoidal, with a major axis of about 3 - 5 μm, having a hollow shell structure, low internal electron density. After loading PMn nanoparticles, the electron density increased significantly and the color deepened, as shown in Figure 10 . The elemental mapping diagram is shown in Figure 11 .

[0071] The antigen protein was labeled with the fluorescent dye Cy5 to obtain Cy5-OVA. Subsequently, the YC@PMn@Cy5-OVA preparation was prepared as described above, and the uptake was observed using a laser confocal fluorescence microscope. The results are shown in Figure 12 . The results showed that the antigen protein was loaded inside the cavity of YC.

[0072] Furthermore, cell uptake was investigated: The antigen protein was labeled with the fluorescent dye Cy5 to obtain Cy5-OVA. Subsequently, it was prepared into the YC@PMn@Cy5-OVA preparation. DC2.4 cells in the logarithmic growth phase were plated and inoculated in a confocal dish. Under the condition of having blood and antibodies, the prepared YC@PMn@Cy5-OVA was added, with a final concentration of 10 μg / mL Cy5-OVA. After continued culture for 1 h, in order to qualitatively analyze the intracellular distribution of the microparticle vaccine, the uptake was observed by laser confocal fluorescence microscopy imaging, as shown in Figure 13 . The results proved that YC@PMn@OVA could be efficiently taken up by DC2.4 cells at the in vitro cell level.

[0073] Example 4 This example provides an efficacy verification of the sustained-release microparticle vaccine delivery system YC@PMn@OVA prepared in Example 3.

[0074] The effector cells of the adaptive immune system are mainly two types of lymphocytes, T and B. Naive B cells differentiate into effector B cells after being activated by antigens and secrete antibodies to participate in the humoral immune response. Mouse IgG antibodies are divided into many subclasses. The isotype switch between different IgG subclasses is mainly affected by cytokines. IL-4 mainly induces the production of IgG1, while IFN-γ mainly induces the isotype switch of IgG2a. Therefore, IgG1 and IgG2a are widely used as markers of Th2- and Th1-type biased adaptive immune responses, respectively.

[0075] In this example, the in vivo antibody levels induced by the sustained-release vaccine delivery system YC@PMn@OVA with adjuvant-like function were initially investigated in mice.

[0076] Immunization protocol: The optimal formulation was prepared using endotoxin-free OVA. C57 mice were randomly divided into seven groups, and the groups were as follows in sequence: PBS group, OVA + cGAMP group, PMn@OVA + cGAMP group, YC@PMn@OVA group, YC@PMn@OVA + ZOL group, YC@PMn@OVA + CpG group, and YC@PMn@OVA + cGAMP group. Immunization was performed subcutaneously in the inguinal region on day 0 and day 14. Each mouse received 100 μL of the formulation, 20 μg of OVA / mouse and / or 50 μg of ZOL / mouse, 1.5 μg of CpG / mouse, and 5 μg of cGAMP / mouse each time. On days 14 and 21, mice were bled from the orbital sinus using a capillary tube. After standing at low temperature for 2 - 6 h, serum samples were collected by centrifugation, and the levels of OVA antigen-specific IgG, IgG1, and IgG2a antibodies in the serum were measured by indirect ELISA. The results are shown in Figure 14 , Figure 14 A shows the immunization results at 14 days; Figure 14 B shows the immunization results at 21 days.

[0077] The experiment found that after administration, high levels of antigen-specific IgG antibodies, including IgG1 and IgG2a antibodies, were induced in mice. Compared with the PMn@OVA + cGAMP group, the antibody levels induced by the YC@PMn@OVA-related experimental groups were higher. The above results indicate that the sustained-release vaccine delivery system can more effectively promote immune responses, including humoral immunity and cellular immunity, and the combined use of other adjuvants can further enhance the level of immune response.

[0078] It should be noted that in this article, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent to such a process, method, article or device. Without further limitations, an element defined by the statement "including one..." does not exclude the existence of additional identical elements in the process, method, article or device including such an element.

[0079] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the claims of the present invention. All of these fall within the protection scope of the present invention.

Claims

1. Use of a novel manganese-based nanoparticle in the preparation of a vaccine, characterized in that, The novel manganese-based nanoparticles are obtained by the redox reaction of potassium permanganate and polyetherimide; the novel manganese-based nanoparticles are manganese oxide-polyetherimide complexes; the surface of the novel manganese-based nanoparticles is positively charged; the novel manganese-based nanoparticles are used for loading protein antigens, loading adjuvants or acting as adjuvants.

2. The application according to claim 1, wherein When the redox reaction occurs between potassium permanganate and polyetherimide, the mass ratio of the polyetherimide to the potassium permanganate is 20:1 - 100:

1.

3. The application according to claim 1, wherein The particle size range of the novel manganese-based nanoparticles is 20 - 500 nm.

4. The preparation method of the novel manganese-based nanoparticles according to claim 1, characterized in that It includes the following steps: S01: Prepare an aqueous solution of low molecular weight polyetherimide, and then slowly drop it into an aqueous solution of potassium permanganate to undergo a redox reaction to obtain a complex solution. The complex solution contains manganese oxide-polyetherimide complexes, and the mass ratio of the aqueous solution of polyetherimide to the aqueous solution of potassium permanganate is 20:1 - 100:1; S02: Centrifuge, wash the reaction solution, and resuspend the precipitate with an aqueous solution by ultrasonic treatment to obtain the novel manganese-based nanoparticles.

5. The preparation method according to claim 4, characterized in that, The low molecular weight polyetherimide is a polyetherimide with a molecular weight less than 10,000.

6. The novel manganese-based nanoparticles prepared by the preparation method according to claim 4 or 5.

7. A sustained-release vaccine delivery system with adjuvant-like function, characterized in that, The sustained-release vaccine delivery is composed of a shell part and a content; the shell part is a hollow yeast microcapsule; the content is novel manganese-based nanoparticles; the novel manganese-based nanoparticles are obtained by the redox reaction of potassium permanganate and polyetherimide; the novel manganese-based nanoparticles are manganese oxide-polyetherimide complexes; the surface of the novel manganese-based nanoparticles is positively charged; the manganese-based nanoparticles are located in the internal hollow of the yeast microcapsule.

8. The sustained-release vaccine delivery system with adjuvant-like function according to claim 7, characterized in that, The sustained-release vaccine delivery system with adjuvant-like function can be used for loading protein antigens or loading molecular adjuvants.

9. The sustained-release vaccine delivery system with adjuvant-like function according to claim 7, characterized in that, The mass ratio of the yeast microcapsule to manganese is 6:1 - 600:

1.

10. The sustained-release vaccine delivery system with adjuvant-like function according to claim 8, wherein The protein antigens include one or more selected from hepatitis B surface antigen HBsAg, recombinant PreS1, PreS2, recombinant core protein, hepatitis C virus antigen, hepatitis E virus antigen, anthrax toxin binding protein, pneumococcal protein, group A M protein, streptococcal C5a peptidase, enolase binding protein, serine carboxylesterase, outer membrane protein OMP of Neisseria meningitidis serogroup B, tumor cell lysate protein, bacterial lysate protein, chicken ovalbumin, Treponema pallidum surface lipoprotein, bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myoalbumin, legumin, gliadin, myoglobin, collagen, fibronectin or polypeptide antigen; the polypeptide antigens include one or more selected from group A streptococcal M peptide, TRP2, HGP100, p15E, Pseudomonas aeruginosa synthetic peptide or rubella virus synthetic peptide; the other molecular adjuvants include one or more selected from peptidoglycan, lipoteichoic acid, monophosphoryl lipid A, imiquimod, resiquimod, CpG-ODN, bacterial flagellin, Poly I:C, 3pRNA, short double-stranded RNA, muramyl dipeptide, bisphosphonate, lipophilic statin, N-acetylglucosamine, β-glucan, trehalose diborate, cGAMP, cholera toxin, Escherichia coli heat-labile enterotoxin, cholera toxin B subunit, pertussis toxin, tetanus toxin, diphtheria toxin, interferon, GM-CSF, IL-1, IL-2, IL-12, IL-6, Quil A, QS-21, and heat shock protein.

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