Preparation method and application of mannose modified polyaspartic acid derivative

Abstract

The invention discloses a preparation method of a mannose modified polyaspartic acid derivative, the prepared mannose modified polyaspartic acid derivative can be further used for preparing inorganic nanoparticles and nanoparticles entrapped with fluorescence labeled antigens, and meanwhile, the invention provides a vaccine adjuvant prepared from the inorganic nanoparticles. Due to the introduction of mannose groups, the nanoparticles entrapped with the fluorescently-labeled antigen have the APCs targeting capability; the antibody has pH sensitivity, can keep stable outside cells, and declines in acidic environments such as cytolysosome to release antigens and promote cross presentation of the antigens; and the released Mn < 2 + > further enhances the immune intensity by activating a cGAS-STING pathway.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterial preparation, and in particular relates to a preparation method and application of a mannose-modified polyaspartic acid derivative. Background Art

[0002] Vaccine adjuvants are non-specific immunopotentiators that can assist in vaccination or carry antigens to enhance immune efficacy. Adjuvants can prolong the retention time in the body and promote the differentiation of lymphocytes by changing the morphology of antigens or increasing the surface area of ​​antigens, thereby accelerating, expanding and prolonging immune responses, improving immune response capabilities, reducing the antigen dose required for immune responses and reducing the frequency of immunization. According to the mechanism of action of adjuvants, some are antigen delivery carrier adjuvants, such as mineral salts, microparticles, etc., and some are immunopotentiator adjuvants, such as cytokines, Toll-like receptor agonists, etc. Among them, antigen delivery carrier adjuvants can assist antigens in prolonging the time of stimulating the body, so that antigens can be effectively recognized and taken up by antigen presenting cells (APCs) and delivered to the immune system. Therefore, the process of antigen presentation by this type of adjuvant cannot be separated from the participation of APCs. Improving the efficiency of APCs in taking up antigens will increase the stimulating effect of antigens on the body and enhance the immune response. APCs include macrophages and dendritic cells. Studies have shown that APCs have highly expressed mannose receptors on their surfaces, which can specifically recognize and bind to ligands with mannose ends, mediate the phagocytosis of APCs, and initiate the body's immune response. Adjuvants, as nonspecific immunopotentiators, are used in immunotherapy to enhance the body's immune response to antigens. Therefore, it is necessary to design APCs-targeted adjuvants and adjuvants that can promote antigen cross-presentation in the development of new adjuvants.

[0003] Polyamino acids are polymer materials made by linking amino acids with peptide bonds. Amino acids are the basic substances that constitute proteins required for animal nutrition. Polyamino acids prepared by linking them with peptide bonds have good biocompatibility and can be degraded and absorbed in the body. The literature ("Poly (hydrophobic amino acid)-based self-adjuvanting nanoparticles for group Astreptococcus vaccine delivery.". Journal of Medicinal Chemistry, 2021; 64: 2648-2658) reported a class of polyamino acids that can be used as adjuvants. After studying the ability of amino acid structure and arrangement to induce humoral immune response, it was found that some polyamino acids showed higher immunity induction ability than commercial adjuvants. Although polyamino acids composed of specific sequences can induce the production of specific antibodies after subcutaneous injection and act as adjuvants, general polyamino acids are not modified with mannose targeting APCs. Safe, biodegradable, and biocompatible adjuvants can greatly improve the efficiency of immunotherapy.

[0004] In the research work that has been reported, there are many polymer carriers with mannose as a ligand. For example, the literature ("Macrophage-targeted mannose-decorated PLGA-vegetable oil hybrid nanoparticles loaded with anti-inflammatory agents", Colloids and Surfaces B: Biointerfaces, 2022; 213: 112423) reported that mannose-modified hybrid nanoparticles of polylactic-co-glycolic acid (PLGA) and vegetable oil effectively delivered two lipophilic anti-inflammatory drugs to macrophages. Compared with unmodified nanoparticles, the cell uptake fluorescence of mannose-modified nanoparticles was enhanced, and the results showed that the ability to target macrophages was improved, but the polymer was not directly modified with mannose. The literature (“Application of Mannose-Functionalized Microgel as a Novel Vaccine Delivery Platform for Subunit Vaccines”, Advanced Functional Materials, 2021; 31(45): 2105742) reported that microgels prepared with 1-vinyl-2-pyrrolidone as a monomer and N-acryloyl glucosamine or N-acryloyl mannosamine as a copolymer were used as potential carriers for subunit vaccine delivery. The results showed that the microgels effectively improved the humoral and cellular immune responses of subunit vaccines and played the role of adjuvants. However, the main chain does not use amino acids with better biocompatibility, and the side chain uses mannosamine as a ligand instead of mannose with good targeting.

[0005] In addition, calcium phosphate, as one of the natural components in human bones and teeth, has good biocompatibility, is easy to be biodegraded, has low cost, and is easy to mass produce. At the same time, calcium phosphate is pH-sensitive and is easily decomposed in low pH values ​​such as lysosomes and tumor cells in acidic environments, thereby releasing the bound drugs or antigens, which is conducive to achieving controlled release of drugs or antigens. At present, the preparation of nano calcium phosphate can generally be divided into two types: dry method and wet method, including coprecipitation method, emulsion method, mechanochemical method, hydrothermal method, sol-gel method, flame spray method, etc. The calcium phosphate obtained by different preparation methods has different morphologies, and the calcium phosphate nanoparticle morphology generally includes spherical, fibrous, lamellar, nanorods, etc. For example, the patent application with publication number CN104984354A discloses a polyacrylic acid-calcium phosphate composite nano drug carrier and its preparation method and application. The polyacrylic acid-calcium phosphate composite nano carrier is based on precipitation method, but its stabilizer polyacrylic acid is difficult to biodegrade and does not have APCs targeting. Manganese is a trace element that is vital to the human body. It is involved in a variety of physiological processes including growth and development, reproductive function, and neural signal transmission. For example, the patent application with publication number CN112691120A discloses the use of divalent manganese in the preparation of immunoenhancing drugs or anti-tumor drugs, specifically involving divalent manganese as a dendritic cell, macrophage or CD8 positive T cell activator and its use as a dendritic cell, macrophage or CD8 positive T cell activator. The patent application with publication number CN114615986A discloses an organic-inorganic hybrid nanoparticle containing metal ions, wherein the nanoparticle includes a macromolecule and a metal ion, wherein the macromolecule includes polyaspartic acid belonging to a polyamino acid, and the metal ion is a metal ion with cGAS-STING activation ability, including a manganese ion. However, its macromolecule is not modified with mannose, so it has no ability to target APCs.

[0006] Therefore, it is of great significance to prepare a polymer with good biosafety and biodegradability, which can target antigen-presenting cells and stabilize inorganic nanoparticles. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing a mannose-modified polyaspartic acid derivative.

[0008] The second object of the present invention is to provide a use of the mannose-modified polyaspartic acid derivative prepared by the method in the preparation of inorganic nanoparticles.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present invention provides a method for preparing a mannose-modified polyaspartic acid derivative, comprising the following steps:

[0011] Disperse polysuccinimide in DMSO, and slowly drop a DMSO solution containing mannose compounds into the above solution at a temperature of 60 to 80° C. (preferably 70° C.) for reaction for 6 to 48 hours (preferably 18 hours, 24 hours, or 32 hours), wherein the molar ratio of polysuccinimide to mannose compounds is 1:1 to 100 (preferably 1:8.3, 1:20, or 1:31.9), and add NaOH to adjust the pH to 9-10, and continue the ring-opening reaction for 2 to 12 hours (preferably 4 hours, 6 hours, or 9 hours), and adjust the pH to 7 with HCl, and dialyze with water to remove DMSO, and then centrifuge to remove the precipitate, and lyophilize to obtain the mannose-modified polyaspartic acid derivative.

[0012] The molecular weight of the polysuccinimide is 8000-20000 (preferably 8000, 15000, 20000).

[0013] The mannose compound is selected from 4-aminophenyl α-D-pyranoside and D-mannosamine.

[0014] The second aspect of the present invention provides a use of the mannose-modified polyaspartic acid derivative prepared by the method in preparing inorganic nanoparticles.

[0015] The inorganic nanoparticles are prepared from mannose-modified polyaspartic acid derivatives, CaCl2 and Na3PO4, and the molar ratio of CaCl2 to Na3PO4 is 1:0.1-5 (preferably 1:0.33, 1:0.51, 1:0.67, 1:0.95, 1:1.33, 1:2.02, 1:2.67).

[0016] The preparation method of the inorganic nanoparticles comprises the following steps:

[0017] A 5-15 mg / ml (preferably 5, 10, 15 mg / ml) mannose-modified polyaspartic acid derivative solution is mixed with a 25-65 mmol / L (preferably 25, 33, 35, 50, 65 mmol / L) CaCl2 solution, stirred for 20-40 min (preferably 30 min), deionized water is added, and then a 25-100 mmol / L (preferably 25, 50, 100 mmol / L) Na 3PO4 solution, the molar ratio of CaCl2 to Na3PO4 is 1:0.1-5 (preferably 1:0.33, 1:0.51, 1:0.67, 1:0.95, 1:1.33, 1:2.02, 1:2.67), and stirring is continued for 10-40 min (preferably 15 min), the final concentration of the mannose-modified polyaspartic acid derivative is 1-5 mg / mL (preferably 1, 2, 3 mg / mL), and the inorganic nanoparticle solution is obtained.

[0018] The inorganic nanoparticles are prepared from mannose-modified polyaspartic acid derivatives and MnCl2, CaCl2, and Na3PO4, wherein the molar ratio of CaCl2 to Na3PO4 is 1:0.1 to 5 (preferably 1:0.33, 1:0.51, 1:0.67, 1:0.95, 1:1.33, 1:2.02, and 1:2.67); the molar ratio of CaCl2 to MnCl2 is 1:0.1 to 2 (preferably 1:0.51, 1:1).

[0019] The preparation method of the inorganic nanoparticles comprises the following steps:

[0020] A 5-15 mg / ml (preferably 5, 10, 15 mg / ml) mannose-modified polyaspartic acid derivative solution is mixed with a 25-65 mmol / L (preferably 25, 33, 35, 50, 65 mmol / L) CaCl2 solution and a 15-65 mmol / L (preferably 17, 25, 50 mmol / L) MnCl2 solution, stirred for 20-40 min (preferably 30 min), deionized water is added, and then a 25-100 mmol / L (preferably 25, 50, 100 mmol / L) NaCl2 solution is added. 3PO4 solution, the molar ratio of CaCl2 to Na3PO4 is 1:0.1-5 (preferably 1:0.33, 1:0.51, 1:0.67, 1:0.95, 1:1.33, 1:2.02, 1:2.67), the molar ratio of CaCl2 to MnCl2 is 1:0.1-2 (preferably 1:0.51, 1:1), and stirring is continued for 10-40 min (preferably 15 min), the final concentration of the mannose-modified polyaspartic acid derivative is 1-5 mg / mL (preferably 1, 2, 3 mg / mL), and the inorganic nanoparticle solution is obtained.

[0021] The third aspect of the present invention provides a use of a mannose-modified polyaspartic acid derivative prepared by the method in preparing nanoparticles encapsulating fluorescently labeled antigens.

[0022] The nanoparticles encapsulating fluorescently labeled antigens are prepared from antigens, mannose-modified polyaspartic acid derivatives, CaCl2 and Na3PO4, the molar ratio of CaCl2 to Na3PO4 is 1:0.1-5 (preferably 1:0.33, 1:0.51, 1:0.67, 1:0.95, 1:1.33, 1:1.5, 1:2.02, 1:2.67), the mass ratio of antigens to mannose-modified polyaspartic acid derivatives is 1:1-5 (preferably 1:4), and the antigen is selected from OVA-FITC.

[0023] The method for preparing the nanoparticles carrying fluorescently labeled antigens comprises the following steps:

[0024] The antigen, a solution of a mannose-modified polyaspartic acid derivative at a concentration of 5 to 15 mg / ml (preferably 5, 10, 15 mg / ml) and a CaCl2 solution at a concentration of 25 to 65 mmol / L (preferably 25, 33, 35, 50, 65 mmol / L) are mixed, the pH is adjusted to 9, and the mixture is incubated at a temperature of 1 to 5°C (preferably 4°C) for 1 to 3 h (preferably 2 h), deionized water is added, and then a solution of 25 to 100 mmol / L (preferably 25, 33, 35, 50, 65 mmol / L) is added. The mixture is stirred for 10 to 40 minutes (preferably 30 minutes) to obtain the nanoparticle solution containing the fluorescently labeled antigen, wherein the antigen is selected from OVA-FITC.

[0025] The nanoparticles encapsulating fluorescently labeled antigens are prepared from antigens, mannose-modified polyaspartic acid derivatives, MnCl2, CaCl2, and Na3PO4, wherein the molar ratio of CaCl2 to Na3PO4 is 1:0.1 to 5 (preferably 1:0.33, 1:0.51, 1:0.67, 1:0.95, 1:1.33, 1:2, and 1:2.67); the molar ratio of CaCl2 to MnCl2 is 1:0.1 to 2 (preferably 1:0.5), the mass ratio of antigens to mannose-modified polyaspartic acid derivatives is 1:1 to 5 (preferably 1:4), and the antigen is selected from OVA-FITC.

[0026] The method for preparing the nanoparticles carrying fluorescently labeled antigens comprises the following steps:

[0027] The antigen, a solution of a mannose-modified polyaspartic acid derivative at a concentration of 5 to 15 mg / ml (preferably 5, 10, 15 mg / ml), a CaCl2 solution at a concentration of 25 to 65 mmol / L (preferably 25, 33, 35, 50, 65 mmol / L), and a MnCl2 solution at a concentration of 15 to 65 mmol / L (preferably 17, 25, 50 mmol / L) are mixed, the pH is adjusted to 9, and the mixture is incubated at a temperature of 1 to 5°C (preferably 4°C) for 1 to 3 h (preferably 2 h), and the deoxyribonucleic acid is added. Deionized water is added, and then a Na3PO4 solution with a concentration of 25 to 100 mmol / L (preferably 25, 50, 100 mmol / L) is added, the molar ratio of CaCl2 to Na3PO4 is 1:0.1 to 5 (preferably 1:0.33, 1:0.51, 1:0.67, 1:0.95, 1:1.33, 1:2, 1:2.67), and stirring is continued for 10 to 40 minutes (preferably 30 minutes) to obtain the nanoparticle solution encapsulating the fluorescently labeled antigen, wherein the antigen is selected from OVA-FITC.

[0028] In a fourth aspect of the present invention, a vaccine adjuvant is provided, wherein the vaccine adjuvant is prepared from an aqueous antigen solution and the inorganic nanoparticle solution in a volume ratio of 1:1, and the antigen is selected from a classical swine fever virus E2 (CSFV-E2) antigen.

[0029] The mass ratio of the inorganic nanoparticles to the antigen is 1 to 5:1 (preferably 3:1).

[0030] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0031] The mannose-modified polyaspartic acid derivative provided by the present invention can be prepared into inorganic nanoparticles, and the nanoparticle solution can be formed as a particle vaccine adjuvant, which prolongs the retention time of the antigen in the body through the reservoir effect; as a multifunctional carrier, different antigens can be loaded onto APCs to activate immunity; the introduction of mannose groups enables the nanoparticles encapsulating fluorescently labeled antigens to have the ability to target APCs; the inorganic nanoparticles are pH-sensitive, remain stable in the weak alkaline environment outside the cell, and degrade and release antigens in the acidic environment such as cell lysosomes, thereby promoting the cross-presentation of antigens; the released Mn 2+ Immune strength is further enhanced by activating the cGAS-STING pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the nuclear magnetic resonance measurement results of the aminophenylmannose-grafted polyaspartic acid derivative prepared in Example 1.

[0033] Figure 2This is a schematic diagram of the infrared measurement results of the aminophenylmannose-grafted polyaspartic acid derivative prepared in Example 1.

[0034] Figure 3 Schematic diagram of the particle size distribution results of prescriptions 2, 8, 11, and 13 in Example 4.

[0035] Figure 4 Schematic diagram of field emission scanning electron microscopy of prescriptions 2, 11, and 13 in Example 4.

[0036] Figure 5 Schematic diagram of pH sensitivity of prescriptions 2, 11, and 13 in Example 4.

[0037] Figure 6 This is a schematic diagram of the cell uptake fluorescence microscopy results of formulations 14, 15, 16, and 17 loaded with fluorescent antigens in Example 4.

[0038] Figure 7 Schematic diagram of the flow cytometry results of cellular uptake of formulations 14, 15, 16, and 17 loaded with fluorescent antigens in Example 4.

[0039] Figure 8 Schematic diagram of the flow cytometry results of cellular uptake of formulations 14, 15, 16, and 17 loaded with fluorescent antigens in Example 4.

[0040] Fig. 9 Schematic diagram of the cGAS-STING pathway activation results of formulations 15, 16, and 17 loaded with fluorescent antigens in Example 4.

[0041] Fig.10 Schematic diagram of the results of specific IgG antibody titer determination after immunization with six groups of vaccines.

[0042] Fig.11 Schematic diagram of the in vivo imaging results of prescriptions 15, 16, and 17. DETAILED DESCRIPTION

[0043] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0044] Example 1

[0045] Synthesis of aminophenylmannose-grafted polyaspartic acid derivative (MAN-PASP-1)

[0046] Polysuccinimide (0.03mmol, 250mg) with a molecular weight of 8000 was dispersed in 3mL of DMSO. Under the condition of 70℃, 3mL of DMSO solution containing 4-aminophenyl α-D-pyranoside (0.25mmol, 70mg) was slowly added dropwise to the above solution under magnetic stirring for 18h to obtain a brown-red clear solution. NaOH was added to adjust the pH to 9-10, and the ring-opening reaction was continued for 4h. The pH was adjusted to 7 with 5% HCl. DMSO was removed by dialysis with water, and then the precipitate was removed by centrifugation and freeze-dried to obtain 205.76g of aminophenylmannose-grafted polyaspartic acid derivative. Figure 1 This is a schematic diagram of the nuclear magnetic resonance measurement results of the aminophenylmannose-grafted polyaspartic acid derivative prepared in Example 1. Figure 2 This is a schematic diagram of the infrared measurement results of the aminophenylmannose-grafted polyaspartic acid derivative prepared in Example 1. The NMR and infrared data results show that mannose has been successfully grafted onto polyaspartic acid.

[0047] The structure of the aminophenylmannose-grafted polyaspartic acid derivative is shown below:

[0048]

[0049] Among them, p is 95 and n is 800.

[0050] Example 2

[0051] Synthesis of Mannosamine-grafted Polyaspartic Acid Derivative (MAN-PASP-2)

[0052] Polysuccinimide (0.016mmol, 250mg) with a molecular weight of 15000 was dispersed in 3mL of DMSO. Under the condition of 70℃, 3mL of DMSO solution containing D-mannosamine (0.51mmol, 92mg) was slowly added dropwise to the above solution under magnetic stirring for 24h to obtain a brown-red clear solution. NaOH was added to adjust the pH to 9-10, and the ring-opening reaction was continued for 6h. The pH was adjusted to 7 with 5% HCl. DMSO was removed by dialysis with water, and then the precipitate was removed by centrifugation and freeze-dried to obtain 218.88g of polyaspartic acid derivative grafted with mannosamine.

[0053] Example 3

[0054] Synthesis of aminophenylmannose-grafted polyaspartic acid derivative (MAN-PASP-3)

[0055] Polysuccinimide (0.0125mmol, 250mg) with a molecular weight of 20,000 was dispersed in 3mL of DMSO. Under the condition of 70°C, 3mL of DMSO solution containing 4-aminophenyl α-D-pyranoside (0.25mmol, 70mg) was slowly added dropwise to the above solution under magnetic stirring for 32h to obtain a brown-red clear solution. NaOH was added to adjust the pH to 9-10, and the ring-opening reaction was continued for 9h. The pH was adjusted to 7 with 5% HCl. DMSO was removed by dialysis with water, and then the precipitate was removed by centrifugation and freeze-dried to obtain 202.56g of aminophenylmannose-grafted polyaspartic acid derivative.

[0056] Example 4

[0057] Preparation of inorganic nanoparticles and antigen-loaded nanoparticles

[0058] Table 1 Prescriptions of different inorganic nanoparticles

[0059]

[0060] The method for preparing the mannose-modified polyaspartic acid derivative solution comprises the following steps:

[0061] The mannose-modified polyaspartic acid derivatives prepared in Example 1, Example 2, and Example 3 were dissolved in water to obtain mannose-modified polyaspartic acid derivative solutions with concentrations of 5 mg / ml, 10 mg / ml, and 15 mg / ml, respectively, which were referred to as MAN-PASP-1 solution, MAN-PASP-2 solution, and MAN-PASP-3 solution, respectively.

[0062] The preparation method of polyaspartic acid (PASP) solution comprises the following steps:

[0063] Polyaspartic acid was dissolved in water to obtain a polyaspartic acid (PASP) solution with a concentration of 10 mg / ml.

[0064] The preparation method of CaCl2 solution comprises the following steps:

[0065] CaCl2 was dissolved in water to obtain CaCl2 solutions with concentrations of 25mmol / L, 33mmol / L, 35mmol / L, 50mmol / L, and 65mmol / L, respectively.

[0066] The preparation method of MnCl2 solution comprises the following steps:

[0067] MnCl2 was dissolved in water to obtain MnCl2 solutions with concentrations of 17mmol / L, 25mmol / L, and 50mmol / L, respectively.

[0068] The preparation method of Na3PO4 solution comprises the following steps:

[0069] Na3PO4 was dissolved in water to obtain Na3PO4 solutions with concentrations of 25mmol / L, 50mmol / L, and 100mmol / L, respectively.

[0070] The preparation methods of Prescriptions 1 to 7 include the following steps:

[0071] At room temperature, according to the formula in Table 1, 1 mL of the MAN-PASP-1 solution prepared in Example 1 was mixed with 1.5 mL of the CaCl2 solution and magnetically stirred for 30 min. 1.5 mL of deionized water was added, and then 1 mL of the Na3PO4 solution was added, and stirring was continued for 15 min to obtain a polyaspartic acid-calcium phosphate composite nanoparticle solution.

[0072] The preparation method of prescription 8 comprises the following steps:

[0073] At room temperature, according to the formula in Table 1, 1 mL of polyaspartic acid (PASP) solution was mixed with 1.5 mL of CaCl2 solution and magnetically stirred for 30 min. 1.5 mL of deionized water was added, and then 1 mL of Na3PO4 solution was added, and stirring was continued for 15 min to obtain a polyaspartic acid-calcium phosphate composite nanoparticle solution.

[0074] The preparation method of prescription 9 comprises the following steps:

[0075] At room temperature, according to the formula in Table 1, 1 mL of the MAN-PASP-2 solution prepared in Example 2 was mixed with 1.5 mL of the CaCl2 solution and magnetically stirred for 30 min. 1.5 mL of deionized water was added, and then 1 mL of the Na3PO4 solution was added, and stirring was continued for 15 min to obtain a polyaspartic acid-calcium phosphate composite nanoparticle solution.

[0076] The preparation method of prescription 10 comprises the following steps:

[0077] At room temperature, according to the formula in Table 1, 1 mL of the MAN-PASP-3 solution prepared in Example 3 was mixed with 1.5 mL of the CaCl2 solution and magnetically stirred for 30 min. 1.5 mL of deionized water was added, and then 1 mL of the Na3PO4 solution was added, and stirring was continued for 15 min to obtain a polyaspartic acid-calcium phosphate composite nanoparticle solution.

[0078] The preparation method of prescription 11 to prescription 12 comprises the following steps:

[0079] At room temperature, according to the formula in Table 1, 1 mL of the MAN-PASP-1 solution prepared in Example 1, 0.75 mL of the CaCl2 solution, and 0.75 mL of the MnCl2 solution were mixed and magnetically stirred for 30 min. 1.5 mL of deionized water was added, and then 1 mL of the Na3PO4 solution was added, and stirring was continued for 15 min to obtain a polyaspartic acid-calcium manganese phosphate composite nanoparticle solution.

[0080] The preparation method of prescription 13 comprises the following steps:

[0081] At room temperature, according to the formula in Table 1, 1 mL of the MAN-PASP-1 solution prepared in Example 1 and 1.5 mL of the MnCl2 solution were mixed and magnetically stirred for 30 min. 1.5 mL of deionized water was added, and then 1 mL of the Na3PO4 solution was added, and stirring was continued for 15 min to obtain a polyaspartic acid-manganese phosphate composite nanoparticle solution.

[0082] Table 2 shows the results of nanoparticle size and polymer dispersibility index (PDI) under different prescriptions. The detection method is as follows: take 1 ml of the solution prepared from prescriptions 1 to 13, add it to the cuvette of the laser particle size analyzer, remove bubbles, and measure the particle size and PDI at 25°C. Each sample is measured three times in parallel.

[0083] Table 2

[0084] Particle size (nm) PDI Prescription 1 282.2±5.5 0.429±0.053 Prescription 2 125.8±2.1 0.237±0.057 Prescription 3 120.2±2.7 0.305±0.036 Prescription 4 236.9±2.7 0.258±0.084 Prescription 5 492.8±8.5 0.805±0.106 Prescription 6 778.7±7.3 0.616±0.136 Prescription 7 \ \ Prescription 8 115.8±2.1 0.261±0.014 Prescription 9 143.2±3.4 0.316±0.059 Prescription 10 157.3±7.1 0.365±0.086 Prescription 11 136.2±2.8 0.246±0.008 Prescription 12 153.4±3.5 0.315±0.061 Prescription 13 169.8±6.1 0.304±0.046

[0085] As can be seen from Table 2, inorganic nanoparticles with different particle sizes can be prepared by using MAN-PASP as a stabilizer and controlling the concentration of each component. Comparison of prescriptions 1, 2, and 3: When the final concentration of MAN-PASP in prescription 1 is 1 mg / mL, the amount of chelated calcium ions is small, the formed calcium phosphate nanoparticles are about 282.2 nm, and the particle dispersion is poor, and the particle size distribution shows a bimodal distribution. When the final concentration of MAN-PASP is 2 mg / mL or above as in prescription 2, the particle size of the calcium phosphate nanoparticles is small and relatively uniform. In prescription 3, as the concentration of MAN-PASP continues to increase, the particle size of the calcium phosphate nanoparticles decreases slightly, with no significant changes.

[0086] Comparison of prescriptions 2, 4, and 5: In prescription 4, when the calcium ion concentration was 35mmol / L, the nanoparticles quickly aggregated to form flocculent precipitates. This may be due to the change in the Ca / P molar ratio in the system, which led to the formation of different types of calcium phosphate precipitates, and Man-PASP had poor stability against these precipitates. In prescription 2, when the calcium ion concentration was 50mmol / L, the calcium phosphate nanoparticles had the smallest particle size and the best dispersibility. In prescription 5, as the calcium ion concentration increased, the particle size and PDI of the nanoparticles also increased accordingly, indicating that the combination of excess calcium ions and phosphate ions promoted the growth of the nanoparticle nucleus, causing the particle size of some nanoparticles to increase significantly, while the particle size of other nanoparticles increased less or not significantly, resulting in a wider particle size distribution and an increase in PDI.

[0087] Comparison of prescriptions 3, 6, and 7: In prescription 7, when the concentration of phosphate ions added was 100mmol / L, flocculent precipitates were directly formed in the system. This may be because a large number of negatively charged phosphate ions are easy to agglomerate after combining with calcium ions, forming unstable calcium phosphate precipitates, which destroy the stability of the nanoparticle system. On the contrary, in prescription 6, when the phosphate ion concentration is as low as 25mmol / L, due to the increase in the Ca / P ratio, the calcium ions combined with the limited phosphate ions increased, and the crystal nuclei continued to grow, resulting in larger particle size and poorer dispersibility. Therefore, the ratio of calcium ions to phosphate ions needs to be within an appropriate range to ensure that the formed nanoparticle system is stable and uniform.

[0088] Comparison of prescriptions 11, 12, and 13: As the proportion of manganese ions increases, the particle size of the nanoparticles gradually increases, and the PDI also shows an overall increasing trend. When the system contains only manganese ions, manganese phosphate nanoparticles are prepared with a particle size of about 169.8nm, while nanoparticles with a smaller particle size can be prepared by co-doping manganese and calcium.

[0089] Figure 3 The figure is a schematic diagram of the particle size distribution results of formulations 2, 8, 11, and 13 in Example 4. By adjusting the appropriate formulation, nanoparticles with a particle size between 100 and 200 nm can be obtained.

[0090] Figure 4 The figure is a schematic diagram of field emission scanning electron microscopy of prescriptions 2, 11 and 13 in Example 4. It is found that the particle sizes of the three nanoparticles are relatively uniform and the appearance is approximately spherical.

[0091] Figure 5 Schematic diagram of pH sensitivity of formulations 2, 11, and 13 in Example 4. It was found that the three nanoparticles remained stable in a weakly alkaline environment simulating extracellular environment, but degraded into ionic form in an acidic environment such as a simulated lysosome.

[0092] Preparation of nanoparticles loaded with fluorescently labeled antigens:

[0093] The preparation method of prescription 14 comprises the following steps:

[0094] OVA-FITC (fluorescein isothiocyanate labeled ovalbumin) was used as a model antigen. According to the ratio in Table 3, 2.5 mg of OVA-FITC was added to 1.5 mL of CaCl2 solution and 1 mL of PASP solution, the pH was adjusted to 9, incubated at 4°C for 2 h, 1.5 mL of deionized water was added, and then 1 mL of Na3PO4 solution was added, and stirring was continued for 30 min to prepare 5 mL of antigen-encapsulated polyaspartic acid-inorganic salt composite nanoparticle solution.

[0095] The preparation method of prescription 15 comprises the following steps:

[0096] OVA-FITC was used as a model antigen. According to the ratio in Table 3, 2.5 mg of OVA-FITC was added to 1.5 mL of CaCl2 solution and 1 mL of Man-PASP-1 solution, the pH was adjusted to 9, incubated at 4°C for 2 h, 1.5 mL of deionized water was added, and then 1 mL of Na3PO4 solution was added, and stirring was continued for 30 min to prepare 5 mL of antigen-encapsulated polyaspartic acid-inorganic salt composite nanoparticle solution.

[0097] The preparation method of prescription 16 comprises the following steps:

[0098] OVA-FITC was used as a model antigen. According to the ratio in Table 3, 2.5 mg of OVA-FITC was added to 1 mL of CaCl2 solution, 0.5 mL of MnCl2 solution and 1 mL of Man-PASP-1 solution, the pH was adjusted to 9, incubated at 4°C for 2 h, 1.5 mL of deionized water was added, and then 1 mL of Na3PO4 solution was added, and stirring was continued for 30 min to prepare 5 mL of antigen-encapsulated polyaspartic acid-inorganic salt composite nanoparticle solution.

[0099] The preparation method of prescription 17 comprises the following steps:

[0100] OVA-FITC was used as a model antigen. According to the ratio in Table 3, 2.5 mg of OVA-FITC was added to 1.5 mL of MnCl2 solution and 1 mL of Man-PASP-1 solution, the pH was adjusted to 9, incubated at 4°C for 2 h, 1.5 mL of deionized water was added, and then 1 mL of Na3PO4 solution was added, and stirring was continued for 30 min to prepare 5 mL of antigen-encapsulated polyaspartic acid-inorganic salt composite nanoparticle solution.

[0101] Table 3

[0102]

[0103] Test method:

[0104] Cell uptake experiment: 2×10 5 RAW264.7 (mouse mononuclear macrophage leukemia cells) were inoculated into 24-well plates at a density of 100 μL / mL, and 500 μL of cell suspension was added to each well. The plates were cultured in an incubator at 37°C and 5% CO2 for 24 hours. The old culture medium of each well was removed, and free OVA-FITC and antigen-encapsulated polyaspartic acid-inorganic salt composite nanoparticle solutions prepared by formulas 14, 15, 16, and 17 containing OVA-FITC were diluted with fresh DMEM culture medium. Two replicate wells were set up for each group. The culture was continued for 4 hours at 37°C and 5% CO2. After the culture was completed, the cells were washed 3 times with PBS buffer to remove unbound fluorescent substances on the cell surface. 200 μL of 4% paraformaldehyde was added to each well to fix the cells for 20 minutes, the paraformaldehyde was removed, and the cells were washed 3 times with PBS buffer. 500 μL of DAPI dye was added to each well, incubated for 10 minutes, and the cell nuclei were stained. The cells were washed 3 times with PBS buffer to remove excess DAPI dye. The samples were observed and analyzed using a fluorescence microscope. In addition, 500 μL of fresh DMEM medium was added to another duplicate well, and the cells were collected and transferred to a centrifuge tube by gently blowing and aspirating with a pipette or using a cell scraper. Centrifuge at 1200 rpm for 5 min. The supernatant was removed, and the cells were resuspended in PBS buffer containing 10% FBS, and the fluorescence intensity of cell uptake was measured by flow cytometry.

[0105] Figure 6 This is a schematic diagram of the cell uptake fluorescence microscopy results of formulations 14, 15, 16, and 17 loaded with fluorescent antigens in Example 4. Figure 7 Schematic diagram of the flow cytometry results of cellular uptake of formulations 14, 15, 16, and 17 loaded with fluorescent antigens in Example 4. Figure 8 The figure is a bar graph showing the results of cell uptake flow cytometry of formulas 14, 15, 16, and 17 loaded with fluorescent antigens in Example 4. Formulas 15, 16, and 17 all showed better uptake effects than formula 14, indicating that MAN-PASP can improve the targeting of nanoparticles to macrophages due to the grafting of mannose. Figure 6It can be seen that only a small amount of the antigen OVA-FITC alone enters the cell, and the observed fluorescence signal is very weak. However, when nanoparticles are used as adjuvants and antigen carriers, the efficiency of antigen uptake is greatly improved. When OVA-FITC is adsorbed by nanoparticles, its intracellular uptake efficiency is significantly increased. Compared with prescription 14, the fluorescence intensity of prescription 15 is significantly enhanced, indicating that grafting mannose to polyaspartic acid significantly improves the efficiency of macrophage antigen uptake. Since there are a large number of mannose receptors expressed on the surface of macrophages, this enables nanoparticles containing mannose to actively target macrophages and be taken up more into the cell. In addition, the fluorescence intensity of prescription 17 is slightly lower, while prescription 16 after complexing with calcium shows obvious fluorescence improvement. In prescription 16, calcium-manganese phosphate composite nanoparticles grafted with mannose show the ability to effectively present antigens to antigen presenting cells (APCs) and have good application potential.

[0106] from Figure 7 , Figure 8 It can be seen that the adsorption of OVA-FITC in nanoparticles can significantly increase the fluorescence intensity of its entry into cells, indicating that mannose-modified polyaspartic acid derivatives as antigen carriers are easier to be taken up by cells than single antigens, and the fluorescence intensity is significantly different (P < 0.001). The nanoparticles grafted with mannose, prescription 15, showed a better uptake effect than prescription 14, with a significant difference (P < 0.001), which further verified the targeting effect of mannose in improving the uptake of antigens by macrophages and enhanced the uptake efficiency of OVA-FITC by Raw264.7 cells. The fluorescence intensity of prescription 16 was higher than that of prescription 15 and prescription 17. There was no significant difference compared with prescription 15, but there was a significant difference compared with prescription 17 (P < 0.001), indicating that calcium phosphate nanoparticles have better cell uptake ability than simple manganese phosphate nanoparticles, and the combination of calcium and manganese improves the uptake of manganese phosphate nanoparticles. Enhancing the uptake of antigens and adjuvants by APCs plays a key role in stimulating stronger immune responses and improving vaccine effectiveness.

[0107] Fig. 9 The following is a schematic diagram of the cGAS-STING pathway activation results of formulas 15, 16, and 17 loaded with fluorescent antigens in Example 4. The results show that formula 15 can hardly activate cGAS-STING related proteins, but formula 16 with manganese added can significantly increase the expression of cGAS, STING, and IRF3, because manganese is an effective cGAS activator that can enhance the affinity of cGAMP binding to STING, thereby producing more IRF3 and cytokines and generating an effective immune response. At the same time, formula 16 showed better protein expression than formula 15 because STING is activated in Ca 2+ Oligomerization is more likely to occur in the presence of

[0108] A 2 mg / ml aqueous solution of classical swine fever virus E2 (CSFV-E2) antigen was mixed with the polyaspartic acid-calcium phosphate composite nanoparticle solution (6 mg / ml) prepared in Prescription 2 at a volume ratio of 1:1, and incubated at 4°C for 4 h to prepare a CSFV-E2 vaccine (abbreviated as CSFV-E2+Prescription 2).

[0109] A 2 mg / ml aqueous solution of classical swine fever virus E2 (CSFV-E2) antigen was mixed with the polyaspartic acid-calcium phosphate composite nanoparticle solution (6 mg / ml) prepared in Prescription 8 at a volume ratio of 1:1, and incubated at 4°C for 4 h to prepare a CSFV-E2 vaccine (abbreviated as CSFV-E2+Prescription 8).

[0110] A 2 mg / ml aqueous solution of classical swine fever virus E2 (CSFV-E2) antigen was mixed with the polyaspartic acid-calcium phosphate composite nanoparticle solution (6 mg / ml) prepared by prescription 11 in a volume ratio of 1:1, and incubated at 4°C for 4 h to prepare a CSFV-E2 vaccine (abbreviated as CSFV-E2+prescription 11).

[0111] A 2 mg / ml aqueous solution of classical swine fever virus E2 (CSFV-E2) antigen was mixed with a polyaspartic acid-calcium phosphate composite nanoparticle solution (6 mg / ml) prepared by prescription 13 in a volume ratio of 1:1, and incubated at 4°C for 4 h to prepare a CSFV-E2 vaccine (abbreviated as CSFV-E2+prescription 13).

[0112] The groups were as follows: the first group was the blank control, which was only vaccinated with PBS; the second group was vaccinated with CSFV-E2 antigen; the third group was vaccinated with CSFV-E2 + prescription 8; the fourth group was vaccinated with CSFV-E2 + prescription 2; the fifth group was vaccinated with CSFV-E2 + prescription 11; and the sixth group was vaccinated with CSFV-E2 + prescription 13.

[0113] Animal in vivo test: 30 female ICR mice were randomly divided into 6 groups, 5 mice in each group. Immunization was performed by subcutaneous injection. The dose of the second group was 100 μg per mouse, 100 μL PBS was injected per mouse in the first group, and 100 μL of the corresponding vaccine was injected per mouse in the third to sixth groups. On the 10th day after the initial immunization, the corresponding drug was injected again according to the above dose for booster immunization. On the 15th, 30th and 45th days after the initial immunization, the mice were blooded, the blood was collected in a centrifuge tube containing an anticoagulant, and the serum was collected to determine the specific antibody titer of CSFV-E2 by ELISA.

[0114] Fig.10The figure is a schematic diagram of the specific IgG antibody titer determination results after six groups of vaccine immunization. The results show that at each time point, the antibody levels of the fourth, fifth and sixth groups were significantly higher than those of the third group, indicating that MAN-PASP, as a stabilizer, can improve the antibody induction effect of various inorganic nanoparticle adjuvants. The antibody level of the fifth group was significantly higher than that of the fourth and sixth groups, and the antibody level of the sixth group was significantly higher than that of the fourth group. The fifth group had the best effect.

[0115] Nanoparticle adjuvant-induced depot effect: In vivo imaging technology was used to investigate the antigen depot-inducing effect of antigen-loaded nanoparticles in ICR mice.

[0116] The concentration of OVA-FITC was controlled at 500 μg / mL, and the antigen-loaded polyaspartic acid-inorganic salt composite nanoparticle solution prepared by prescriptions 15, 16, and 17 was injected into ICR mice subcutaneously in the neck at a dose of 200 μL. At 0h, 2h, 6h, 12h, and 24h after injection, the mice were subjected to in vivo fluorescence imaging to monitor the attenuation of the injected antigen fluorescence and the localization effect. The results are shown in Figure 2. Fig.11 As shown, Fig.11 The following is a schematic diagram of the in vivo imaging results of prescriptions 15, 16, and 17. After free OVA-FITC was injected into the mouse subcutaneously, the fluorescence intensity dropped significantly at 2 hours, and almost no fluorescence was detected at the injection site after 12 hours. However, the nanoparticles loaded with fluorescently labeled antigens prepared by prescriptions 15, 16, and 17 still had some fluorescence visible 24 hours after injection, indicating that the nanoparticles loaded with fluorescently labeled antigens prepared by prescriptions 15, 16, and 17 can form an antigen reservoir at the injection site, allowing the antigen to be slowly released.

[0117] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A method for preparing a mannose-modified polyaspartic acid derivative, characterized in that: The following steps are involved: The polysuccinimide is dispersed in DMSO, and at a temperature of 60 to 80° C., a DMSO solution containing mannose compounds is slowly added dropwise to the above solution for reaction for 6 to 48 hours, the molar ratio of the polysuccinimide to the mannose compound is 1:1 to 100, NaOH is added to adjust the pH to 9-10, the ring-opening reaction is continued for 2 to 12 hours, and the pH is adjusted to 7 with HCl. The DMSO is removed by dialysis with water, and then the precipitate is removed by centrifugation and freeze-dried to obtain the mannose-modified polyaspartic acid derivative.

2. The method for preparing the mannose-modified polyaspartic acid derivative according to claim 1, characterized in that: The mannose compound is selected from 4-aminophenyl α-D-pyranoside and D-mannosamine.

3. Use of a mannose-modified polyaspartic acid derivative prepared by the method of claim 1 or 2 in the preparation of inorganic nanoparticles.

4. The use according to claim 3, characterized in that: The inorganic nanoparticles are prepared from mannose-modified polyaspartic acid derivatives, CaCl2 and Na3PO4, and the molar ratio of CaCl2 to Na3PO4 is 1:0.1-5.

5. The use according to claim 3, characterized in that: The inorganic nanoparticles are prepared from mannose-modified polyaspartic acid derivatives, MnCl2, CaCl2 and Na3PO4, wherein the molar ratio of CaCl2 to Na3PO4 is 1:0.1-5; and the molar ratio of CaCl2 to MnCl2 is 1:0.1-2.

6. Use of the mannose-modified polyaspartic acid derivative prepared by the method in preparing nanoparticles encapsulating fluorescently labeled antigens.

7. The use according to claim 6, characterized in that: The nanoparticles encapsulating fluorescently labeled antigens are prepared from antigens, mannose-modified polyaspartic acid derivatives, CaCl2 and Na3PO4, the molar ratio of CaCl2 to Na3PO4 is 1:0.1-5, the mass ratio of antigens to mannose-modified polyaspartic acid derivatives is 1:1-5, and the antigen is selected from OVA-FITC.

8. The use according to claim 6, characterized in that: The nanoparticles encapsulating fluorescently labeled antigens are prepared from antigens, mannose-modified polyaspartic acid derivatives, MnCl2, CaCl2, and Na3PO4, wherein the molar ratio of CaCl2 to Na3PO4 is 1:0.1-5; the molar ratio of CaCl2 to MnCl2 is 1:0.1-2, the mass ratio of antigens to mannose-modified polyaspartic acid derivatives is 1:1-5, and the antigen is selected from OVA-FITC.

9. The use according to claim 8, characterized in that: The method for preparing the nanoparticles carrying fluorescently labeled antigens comprises the following steps: The antigen, a mannose-modified polyaspartic acid derivative solution with a concentration of 5 to 15 mg / ml, a CaCl2 solution with a concentration of 25 to 65 mmol / L, and a MnCl2 solution with a concentration of 15 to 65 mmol / L are mixed, the pH is adjusted to 9, and the mixture is incubated at a temperature of 1 to 5°C for 1 to 3 hours. Deionized water is added, and then a Na3PO4 solution with a concentration of 25 to 100 mmol / L is added, wherein the molar ratio of CaCl2 to Na3PO4 is 1:0.1 to 5, and stirring is continued for 10 to 40 minutes to obtain the nanoparticle solution encapsulating the fluorescently labeled antigen, wherein the antigen is selected from OVA-FITC.

10. A vaccine adjuvant, which is prepared from an aqueous antigen solution in a volume ratio of 1:1 and an inorganic nanoparticle solution prepared by the application of claims 3 to 5, wherein the antigen is selected from classical swine fever virus E2 antigen.

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