Preparation method and application of a mannose-modified polyaspartic acid derivative

Abstract

The application discloses a preparation method of a mannose-modified polyaspartic acid derivative, and the prepared mannose-modified polyaspartic acid derivative can be used for further preparation of inorganic nanoparticles and nanoparticles loaded with fluorescently labeled antigens, and a vaccine adjuvant prepared from the inorganic nanoparticles is provided. 2+ The introduction of the mannose group enables the nanoparticles loaded with fluorescently labeled antigens to have the ability of targeting APCs, has pH sensitivity, can be kept stable outside cells, is degraded and releases antigens in an acidic environment such as a cell lysosome, and promotes cross-presentation of the antigens; the released Mn 2+ The cGAS-STING pathway is activated to further enhance the immune intensity.

Description

Technical Field

[0001] This invention belongs to the field of biomaterial preparation technology, specifically, it relates to a method for preparing mannose-modified polyaspartic acid derivatives and their applications. Background Technology

[0002] Vaccine adjuvants are non-specific immune enhancers that assist in vaccination or the transport of antigens to enhance immune efficacy. Adjuvants can prolong the residence time of antigens in the body and promote lymphocyte differentiation by altering the antigen's morphology or increasing its surface area, thereby accelerating, expanding, and prolonging the immune response, improving immune responsiveness, reducing the antigen dose required for the immune response, and decreasing the frequency of immunization. Based on their mechanism of action, adjuvants can be classified as antigen delivery carriers, such as mineral salts and microparticles, or as immune enhancers, such as cytokines and Toll-like receptor agonists. Antigen delivery carrier adjuvants assist in prolonging the stimulation time of antigens on the body, enabling antigens to be effectively recognized, taken up, and delivered to the immune system by antigen-presenting cells (APCs). Therefore, the antigen presentation process of these adjuvants relies heavily on the participation of APCs. Improving the efficiency of APCs in antigen uptake will enhance the stimulatory effect of antigens on the body, thus enhancing the immune response. APCs, including macrophages and dendritic cells, have been shown to have highly expressed mannose receptors on their surface. These receptors specifically recognize and bind to ligands with mannose terminals, mediating phagocytosis and initiating the body's immune response. Adjuvants, as non-specific immune enhancers, are used in immunotherapy to enhance the body's immune response to antigens. Therefore, designing APC-targeting adjuvants and adjuvants that promote antigen cross-presentation is essential in the development of novel adjuvants.

[0003] Polyamino acids (POAs) are polymeric materials composed of amino acids linked by peptide bonds. Amino acids are the basic building blocks of proteins required for animal nutrition, and POAs prepared by linking them with peptide bonds exhibit good biocompatibility and can be degraded and absorbed in vivo. The literature (“Poly(hydrophobic amino acid)-based self-adjuvanting nanoparticles for group Astreptococcus vaccine delivery.”. Journal of Medicinal Chemistry, 2021; 64:2648-2658) reports a class of POAs that can act as adjuvants themselves. By studying the ability of amino acid structure and arrangement to induce humoral immune responses, it was found that some POAs exhibited higher immune-inducing capabilities than commercial adjuvants. While POAs composed of specific sequences can induce the production of specific antibodies after subcutaneous injection, acting as adjuvants, general POAs do not undergo mannose modification targeting APCs. Safe, biodegradable, and biocompatible adjuvants can significantly improve the efficiency of immunotherapy.

[0004] In existing research, many polymer carriers have used 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) reports that mannose-modified hybrid nanoparticles of polylactic-co-glycolic acid copolymer (PLGA) and vegetable oil effectively deliver two lipophilic anti-inflammatory drugs to macrophages. Compared with unmodified nanoparticles, the mannose-modified nanoparticles showed enhanced cellular uptake fluorescence, indicating an improved ability to target macrophages, 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 using 1-vinyl-2-pyrrolidone as a monomer and N-acrylamide or N-acrylmannosamine as comonomers are potential carriers for subunit vaccine delivery. The results showed that they effectively improved the humoral and cellular immune responses of subunit vaccines, acting as 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, which has better targeting.

[0005] Furthermore, calcium phosphate, as a natural component of human bones and teeth, possesses good biocompatibility, is easily biodegradable, low in cost, and easy to mass-produce. Simultaneously, calcium phosphate is pH-sensitive, readily decomposing in low-pH environments such as lysosomes and the acidic environments of tumor cells, thereby releasing bound drugs or antigens, which is beneficial for controlled drug or antigen release. Currently, the preparation of nano-calcium phosphate can generally be divided into dry and wet methods, including co-precipitation, emulsion, mechanochemical, hydrothermal, sol-gel, and flame spray methods. Calcium phosphate obtained through different preparation methods exhibits different morphologies; calcium phosphate nanoparticles typically include spherical, fibrous, sheet-like, and nanorod-like forms. For example, patent application CN104984354A discloses a polyacrylic acid-calcium phosphate composite nanocarrier, its preparation method, and its application. This polyacrylic acid-calcium phosphate composite nanocarrier is based on a precipitation method, but its stabilizer, polyacrylic acid, is difficult to biodegrade and lacks APC targeting specificity. Manganese is a vital trace element for the human body, participating in various physiological processes including growth and development, reproductive function, and nerve signal transmission. For example, patent application CN112691120A discloses the application of divalent manganese in the preparation of immunomodulatory or antitumor drugs, specifically involving divalent manganese as an activator of dendritic cells, macrophages, or CD8-positive T cells, and its use in activating dendritic cells, macrophages, or CD8-positive T cells. Patent application CN114615986A discloses an organic-inorganic hybrid nanoparticle containing metal ions. The nanoparticle comprises macromolecules and metal ions, wherein the macromolecules include polyaspartic acid (a polyamino acid), and the metal ions are those with cGAS-STING activation capabilities, including manganese ions. However, its macromolecules are not modified with mannose, therefore it lacks the ability to target APCs.

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

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

[0008] A second objective of this invention is to provide an application of the mannose-modified polyaspartic acid derivative prepared by the method described above in the preparation of inorganic nanoparticles.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

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

[0011] Polysuccinimide was dispersed in DMSO. Under conditions of 60–80°C (preferably 70°C), a DMSO solution containing mannose compounds was slowly added dropwise to the above solution and reacted for 6–48 h (preferably 18 h, 24 h, or 32 h). The molar ratio of polysuccinimide to mannose compounds was 1:1–100 (preferably 1:8.3, 1:20, or 1:31.9). NaOH was added to adjust the pH to 9–10, and the ring-opening reaction was continued for 2–12 h (preferably 4 h, 6 h, or 9 h). The pH was adjusted to 7 with HCl. DMSO was removed by dialysis with water, and then the precipitate was removed by centrifugation and lyophilized to obtain the mannose-modified polyaspartic acid derivative.

[0012] The molecular weight of the polysuccinimide is 8,000 to 20,000 (preferably 8,000, 15,000, or 20,000).

[0013] The mannose compounds are selected from 4-aminophenyl α-D-mannopyranoside and D-mannosamine.

[0014] In a second aspect, the present invention provides the application of the mannose-modified polyaspartic acid derivative prepared by the method described above in the preparation of inorganic nanoparticles.

[0015] The inorganic nanoparticles are prepared by mannose-modified polyaspartic acid derivatives with 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, 1:2.67).

[0016] The method for preparing the inorganic nanoparticles includes the following steps:

[0017] A solution of mannose-modified polyaspartic acid derivative with a concentration of 5–15 mg / ml (preferably 5, 10, or 15 mg / ml) is mixed with a CaCl2 solution with a concentration of 25–65 mmol / L (preferably 25, 33, 35, 50, or 65 mmol / L), and stirred for 20–40 min (preferably 30 min). Deionized water is then added, followed by a solution of Na+ with a concentration of 25–100 mmol / L (preferably 25, 50, or 100 mmol / L). The solution of Na3PO4, wherein 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), is stirred for 10-40 min (preferably 15 min), and the final concentration of the mannose-modified polyaspartic acid derivative is 1-5 mg / mL (preferably 1, 2, or 3 mg / mL), is obtained to produce the inorganic nanoparticle solution.

[0018] The inorganic nanoparticles are prepared by mannose-modified polyaspartic acid derivatives with MnCl2, CaCl2, and Na3PO4. 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, or 1:2.67); the molar ratio of CaCl2 to MnCl2 is 1:0.1 to 2 (preferably 1:0.51 or 1:1).

[0019] The method for preparing the inorganic nanoparticles includes the following steps:

[0020] A solution of mannose-modified polyaspartic acid derivative with a concentration of 5–15 mg / ml (preferably 5, 10, or 15 mg / ml) is mixed with a CaCl2 solution with a concentration of 25–65 mmol / L (preferably 25, 33, 35, 50, or 65 mmol / L) and a MnCl2 solution with a concentration of 15–65 mmol / L (preferably 17, 25, or 50 mmol / L). The mixture is stirred for 20–40 min (preferably 30 min), then deionized water is added, followed by a solution of Na+ with a concentration of 25–100 mmol / L (preferably 25, 50, or 100 mmol / L). A 3PO4 solution is prepared, wherein 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, or 1:2.67), and the molar ratio of CaCl2 to MnCl2 is 1:0.1–2 (preferably 1:0.51 or 1:1). The mixture is stirred for 10–40 min (preferably 15 min), and the final concentration of the mannose-modified polyaspartic acid derivative is 1–5 mg / mL (preferably 1, 2, or 3 mg / mL), thereby obtaining the inorganic nanoparticle solution.

[0021] A third aspect of the present invention provides the application of the mannose-modified polyaspartic acid derivative prepared by the method described above in the preparation of nanoparticles loaded with fluorescently labeled antigens.

[0022] The nanoparticles encapsulating the fluorescently labeled antigen are prepared from the antigen, a mannose-modified polyaspartic acid derivative, CaCl2, and Na3PO4. 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, 1:1.5, 1:2.02, or 1:2.67), and the mass ratio of the antigen to the mannose-modified polyaspartic acid derivative is 1:1 to 5 (preferably 1:4). The antigen is selected from OVA-FITC.

[0023] The method for preparing the nanoparticles loaded with fluorescently labeled antigens includes the following steps:

[0024] The antigen, a solution of mannose-modified polyaspartic acid derivative at a concentration of 5–15 mg / ml (preferably 5, 10, or 15 mg / ml), and a CaCl2 solution at a concentration of 25–65 mmol / L (preferably 25, 33, 35, 50, or 65 mmol / L) are mixed and the pH is adjusted to 9. The mixture is then incubated at 1–5°C (preferably 4°C) for 1–3 hours (preferably 2 hours). Deionized water is then added, followed by the addition of a solution at a concentration of 25–100 mmol / L (preferably...). A Na3PO4 solution (25, 50, or 100 mmol / L) is prepared, wherein 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, or 1:2.67), and the mixture is stirred for 10–40 min (preferably 30 min) to obtain the nanoparticle solution loaded with the fluorescently labeled antigen, wherein the antigen is selected from OVA-FITC.

[0025] The nanoparticles encapsulating the fluorescently labeled antigen are prepared from the antigen, a mannose-modified polyaspartic acid derivative, and MnCl2, 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, 1:2, or 1:2.67); the molar ratio of CaCl2 to MnCl2 is 1:0.1–2 (preferably 1:0.5); the mass ratio of the antigen to the mannose-modified polyaspartic acid derivative is 1:1–5 (preferably 1:4); and the antigen is selected from OVA-FITC.

[0026] The method for preparing the nanoparticles loaded with fluorescently labeled antigens includes the following steps:

[0027] The antigen, a solution of mannose-modified polyaspartic acid derivative at a concentration of 5–15 mg / ml (preferably 5, 10, or 15 mg / ml), a CaCl2 solution at a concentration of 25–65 mmol / L (preferably 25, 33, 35, 50, or 65 mmol / L), and a MnCl2 solution at a concentration of 15–65 mmol / L (preferably 17, 25, or 50 mmol / L) are mixed and the pH is adjusted to 9. The mixture is then incubated at 1–5°C (preferably 4°C) for 1–3 hours (preferably 2 hours). Then, a desmoplastic agent is added. Deionized water is then added to a Na3PO4 solution with a concentration of 25–100 mmol / L (preferably 25, 50, or 100 mmol / L), wherein 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, or 1:2.67), and the mixture is stirred for 10–40 min (preferably 30 min) to obtain the nanoparticle solution loaded with the fluorescently labeled antigen, wherein the antigen is selected from OVA-FITC.

[0028] In a fourth aspect, the present invention provides a vaccine adjuvant, which is prepared by mixing an aqueous antigen solution and an inorganic nanoparticle solution in a volume ratio of 1:1, wherein the antigen is selected from the 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] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0031] The mannose-modified polyaspartic acid derivative provided by this invention can be prepared into inorganic nanoparticles and form a nanoparticle solution as a particulate vaccine adjuvant, prolonging the retention time of antigens in vivo 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 carrying fluorescently labeled antigens to target APCs; the inorganic nanoparticles are pH sensitive, remaining stable in a weakly alkaline extracellular environment, while degrading and releasing antigens in acidic environments such as lysosomes, promoting antigen cross-presentation; the released Mn 2+ Further enhance immune strength by activating the cGAS-STING pathway. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the NMR 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 This is a schematic diagram showing the particle size distribution results of formulations 2, 8, 11, and 13 in Example 4.

[0035] Figure 4 This is a field emission scanning electron microscope schematic diagram of prescriptions 2, 11, and 13 in Example 4.

[0036] Figure 5 This is a schematic diagram of the pH sensitivity of formulations 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 containing fluorescent antigens in Example 4.

[0038] Figure 7 This is a schematic diagram of the cell uptake flow cytometry results for formulations 14, 15, 16, and 17 containing fluorescent antigens in Example 4.

[0039] Figure 8 This is a bar chart showing the cell uptake flow cytometry results of formulations 14, 15, 16, and 17 containing fluorescent antigens in Example 4.

[0040] Figure 9 This is a schematic diagram of the cGAS-STING pathway activation results of formulations 15, 16, and 17 containing fluorescent antigens in Example 4.

[0041] Figure 10 This is a schematic diagram showing the results of specific IgG antibody titer determination after immunization with six groups of vaccines.

[0042] Figure 11 The diagram shows the in vivo imaging results for prescriptions 15, 16, and 17. Detailed Implementation

[0043] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

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

[0046] Polysuccinimide (0.03 mmol, 250 mg) with a molecular weight of 8000 was dispersed in 3 mL of DMSO. Under magnetic stirring at 70 °C, 3 mL of DMSO solution containing 0.25 mmol, 70 mg of 4-aminophenyl α-D-mannopyranoside was slowly added dropwise to the above solution, and the reaction was carried out for 18 h, yielding a clear, brownish-red solution. NaOH was added to adjust the pH to 9-10, and the ring-opening reaction was continued for 4 h. The pH was then adjusted to 7 with 5% HCl. DMSO was removed by dialysis with water, followed by centrifugation to remove the precipitate, and lyophilization to obtain 205.76 g of aminophenylmannopyranoside-grafted polyaspartic acid derivative. Figure 1 This is a schematic diagram of the NMR results of the aminophenylmannose-grafted polyaspartic acid derivative prepared in Example 1. Figure 2 This is a schematic diagram of the infrared spectroscopy results for the aminophenylmannose-grafted polyaspartic acid derivative prepared in Example 1. The NMR and infrared data 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] Where p is 95 and n is 800.

[0050] Example 2

[0051] Synthesis of mannosamine-grafted polyaspartic acid derivative (MAN-PASP-2)

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

[0053] Example 3

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

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

[0056] Example 4

[0057] Preparation of Inorganic Nanoparticles and Antigen-Loaded Nanoparticles

[0058] Table 1. Formulations of different inorganic nanoparticles

[0059]

[0060] The preparation method of mannose-modified polyaspartic acid derivative solution includes the following steps:

[0061] The mannose-modified polyaspartic acid derivatives prepared in Examples 1, 2, and 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.

[0062] The preparation method of polyaspartic acid (PASP) solution includes 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 method for preparing CaCl2 solution includes the following steps:

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

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

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

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

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

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

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

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

[0073] At room temperature, following the formulation 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, followed by 1 mL of Na3PO4 solution, and stirring was continued for 15 min to obtain a polyaspartic acid-calcium phosphate composite nanoparticle solution.

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

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

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

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

[0078] The preparation methods of prescriptions 11 and 12 include the following steps:

[0079] At room temperature, following the formulation in Table 1, 1 mL of the MAN-PASP-1 solution prepared in Example 1, 0.75 mL of CaCl2 solution, and 0.75 mL of MnCl2 solution were mixed and magnetically stirred for 30 min. 1.5 mL of deionized water was added, followed by 1 mL of Na3PO4 solution, 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 includes the following steps:

[0081] At room temperature, following the formulation in Table 1, 1 mL of the MAN-PASP-1 solution prepared in Example 1 and 1.5 mL of MnCl2 solution were mixed and magnetically stirred for 30 min. 1.5 mL of deionized water was added, followed by 1 mL of Na3PO4 solution, 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 formulations. The detection method is as follows: Take 1 ml of the solution prepared by formulation 1 to formulation 13, add it to the cuvette of the laser particle size analyzer, remove the air bubbles, and determine the particle size and PDI at 25℃. Each sample is measured in three parallel measurements.

[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] Table 2 shows that by using MAN-PASP as a stabilizer and controlling the concentration of each component, inorganic nanoparticles with different particle sizes can be prepared. Comparing formulations 1, 2, and 3: In formulation 1, when the final concentration of MAN-PASP is 1 mg / mL, the amount of chelated calcium ions is relatively small, resulting in calcium phosphate nanoparticles with a size of approximately 282.2 nm, poor particle dispersibility, and a bimodal particle size distribution. When the final concentration of MAN-PASP is 2 mg / mL or higher, as in formulation 2, the calcium phosphate nanoparticles have small and relatively uniform particle sizes. In formulation 3, as the concentration of MAN-PASP continues to increase, the decrease in particle size of calcium phosphate nanoparticles is small and shows no significant change.

[0086] Comparing formulations 2, 4, and 5: In formulation 4, when the calcium ion concentration was 35 mmol / L, the nanoparticles rapidly aggregated to form flocculent precipitates. This may be due to the change in the Ca / P molar ratio in the system leading to the formation of different types of calcium phosphate salt precipitates, and Man-PASP exhibited poor stability towards these precipitates. In formulation 2, when the calcium ion concentration was 50 mmol / L, the calcium phosphate nanoparticles obtained had the smallest particle size and the best dispersibility. In formulation 5, with increasing calcium ion concentration, the nanoparticle size and PDI also increased accordingly, indicating that the combination of excess calcium ions and phosphate ions promoted the growth of nanoparticle nuclei, resulting in a significant increase in the particle size of some nanoparticles, while the increase in the particle size of other nanoparticles was less or not obvious, thus leading to a wider particle size distribution and an increased PDI.

[0087] Comparing formulations 3, 6, and 7: In formulation 7, when the added phosphate ion concentration was 100 mmol / L, flocculent precipitate formed directly in the system. This is likely because a large number of negatively charged phosphate ions easily aggregate after combining with calcium ions, forming unstable calcium phosphate precipitates and compromising the stability of the nanoparticle system. Conversely, in formulation 6, when the phosphate ion concentration was as low as 25 mmol / L, the increased Ca / P ratio led to increased binding of calcium ions with the limited number of phosphate ions, resulting in continuous crystal nucleation and larger particle size with poorer dispersibility. Therefore, the ratio of calcium ions to phosphate ions needs to be within an appropriate range to ensure a stable and uniform nanoparticle system.

[0088] Comparing formulations 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 approximately 169.8 nm, while co-doping with manganese and calcium can prepare nanoparticles with even smaller particle sizes.

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

[0090] Figure 4 The image shows a field emission scanning electron microscope (FEM) diagram of formulations 2, 11, and 13 in Example 4. It was found that the particle size of the three nanoparticles was relatively uniform, and their appearance was approximately spherical.

[0091] Figure 5 This is a schematic diagram illustrating the 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 conditions, but degraded into ionic forms in acidic environments such as simulating lysosomes.

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

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

[0094] OVA-FITC (fluorescein isothiocyanate-labeled ovalbumin) was used as the model antigen. Following the proportions 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, and the mixture was incubated at 4 °C for 2 h. Then, 1.5 mL of deionized water and 1 mL of Na3PO4 solution were added, and the mixture was stirred for another 30 min to prepare 5 mL of a polyaspartic acid-inorganic salt composite nanoparticle solution encapsulating the antigen.

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

[0096] OVA-FITC was used as the 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, and the mixture was incubated at 4 °C for 2 h. Then, 1.5 mL of deionized water and 1 mL of Na3PO4 solution were added, and the mixture was stirred for another 30 min to prepare 5 mL of polyaspartic acid-inorganic salt composite nanoparticle solution loaded with the antigen.

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

[0098] OVA-FITC was used as the 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, and the mixture was incubated at 4 °C for 2 h. Then, 1.5 mL of deionized water and 1 mL of Na3PO4 solution were added, and the mixture was stirred for another 30 min to prepare 5 mL of polyaspartic acid-inorganic salt composite nanoparticle solution loaded with antigen.

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

[0100] OVA-FITC was used as the 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, and the mixture was incubated at 4 °C for 2 h. Then, 1.5 mL of deionized water and 1 mL of Na3PO4 solution were added, and the mixture was stirred for another 30 min to prepare 5 mL of polyaspartic acid-inorganic salt composite nanoparticle solution loaded with the antigen.

[0101] Table 3

[0102]

[0103] Test method:

[0104] Cellular uptake experiment: with 2×10 5 RAW264.7 (mouse mononuclear macrophage leukemia cells) were seeded into 24-well plates at a density of 500 μL of cell suspension per well and incubated at 37°C with 5% CO2 for 24 h. The old culture medium was removed from each well, and solutions of free OVA-FITC diluted with fresh DMEM and antigen-encapsulated polyaspartic acid-inorganic salt composite nanoparticles prepared according to formulations 14, 15, 16, and 17 (containing OVA-FITC) were added, with two replicates per group. Cells were incubated at 37°C with 5% CO2 for another 4 h. After incubation, cells were washed three times with PBS buffer to remove unbound fluorescent substances from the cell surface. Cells were fixed in 200 μL of 4% paraformaldehyde per well for 20 min, the paraformaldehyde was removed, and cells were washed three times with PBS buffer. 500 μL of DAPI dye was added to each well and incubated for 10 min to stain the cell nuclei. Cells were then washed three times with PBS buffer to remove excess DAPI dye. Samples were observed and analyzed using a fluorescence microscope. Additionally, 500 μL of fresh DMEM culture medium was added to another replicate well, and cells were collected by gently pipetting or scraping with a cell scraper and transferred to centrifuge tubes. The tubes were centrifuged at 1200 rpm for 5 min. The supernatant was removed, and the cells were resuspended in PBS buffer containing 10% FBS. 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 containing fluorescent antigens in Example 4. Figure 7 This is a schematic diagram of the cell uptake flow cytometry results for formulations 14, 15, 16, and 17 containing fluorescent antigens in Example 4. Figure 8 This is a bar graph illustrating the flow cytometry results of cellular uptake of formulations 14, 15, 16, and 17, which encapsulate fluorescent antigens, in Example 4. Formulations 15, 16, and 17 all exhibited better uptake compared to formulation 14, indicating that MAN-PASP, due to mannose grafting, can enhance the targeting of nanoparticles to macrophages. Figure 6As can be seen, only a small amount of the single antigen OVA-FITC 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 increases significantly. Compared with formulation 14, formulation 15 shows a significantly enhanced fluorescence intensity, indicating that grafting mannose onto polyaspartic acid significantly improves the uptake efficiency of antigens by macrophages. Due to the large number of mannose receptors expressed on the surface of macrophages, mannose-containing nanoparticles can actively target macrophages and be more fully uptaken into cells. In addition, the fluorescence intensity of formulation 17 is slightly lower, while formulation 16, after being combined with calcium, shows a significant improvement in fluorescence. In formulation 16, the mannose-grafted calcium manganese phosphate composite nanoparticles show the ability to effectively present antigens to antigen-presenting cells (APCs), and have good application potential.

[0106] from Figure 7 , Figure 8 As can be seen, adsorbing OVA-FITC into nanoparticles significantly enhances its fluorescence intensity upon cellular entry, indicating that the mannose-modified polyaspartic acid derivative, as an antigen carrier, is more easily taken up by cells compared to the antigen alone, and the fluorescence intensity shows a significant difference (P < 0.001). The mannose-grafted nanoparticle formulation 15 exhibits a significantly better uptake effect compared to formulation 14 (P < 0.001), further validating the targeting effect of mannose in enhancing macrophage antigen uptake and improving the uptake efficiency of OVA-FITC in Raw264.7 cells. The fluorescence intensity of formulation 16 is higher than that of formulations 15 and 17, with no significant difference compared to formulation 15, but a significant difference compared to formulation 17 (P < 0.001), indicating that calcium phosphate nanoparticles have better cellular uptake capacity than manganese phosphate nanoparticles alone, and the combination of calcium and manganese enhances the uptake of manganese phosphate nanoparticles. Enhancing the uptake of antigens and adjuvants by APCs plays a crucial role in stimulating a stronger immune response and improving vaccine efficacy.

[0107] Figure 9 This diagram illustrates the cGAS-STING pathway activation results of formulations 15, 16, and 17, which encapsulate fluorescent antigens in Example 4. The results show that formulation 15 barely activates cGAS-STING-related proteins, but formulation 16, with the addition of manganese, significantly increases the expression of cGAS, STING, and IRF3. This is because manganese is an effective cGAS activator, enhancing the affinity of cGAMP for STING, thereby generating more IRF3 and cytokines, resulting in an effective immune response. Simultaneously, formulation 16 shows better protein expression than formulation 15 because STING is more readily expressed in Ca2+. 2+ Oligoggregation is more likely to occur when it is present.

[0108] A 2 mg / ml aqueous solution of classical swine fever virus E2 (CSFV-E2) antigen was mixed with a 6 mg / ml solution of polyaspartic acid-calcium phosphate composite nanoparticles prepared by Formula 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+Formula 2).

[0109] A 2 mg / ml aqueous solution of classical swine fever virus E2 (CSFV-E2) antigen was mixed with a 6 mg / ml solution of polyaspartic acid-calcium phosphate composite nanoparticles prepared by Formula 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+Formula 8).

[0110] A 2 mg / ml aqueous solution of classical swine fever virus E2 (CSFV-E2) antigen was mixed with a 6 mg / ml solution of polyaspartic acid-calcium phosphate composite nanoparticles prepared by Formula 11 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+Formula 11).

[0111] A 2 mg / ml aqueous solution of classical swine fever virus E2 (CSFV-E2) antigen was mixed with a 6 mg / ml solution of polyaspartic acid-calcium phosphate composite nanoparticles prepared by Formula 13 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+Formula 13).

[0112] The groupings are as follows: Group 1 is the blank control, and is only injected with PBS; Group 2 is injected with CSFV-E2 antigen; Group 3 is injected with CSFV-E2+ prescription 8; Group 4 is injected with CSFV-E2+ prescription 2; Group 5 is injected with CSFV-E2+ prescription 11; and Group 6 is injected with CSFV-E2+ prescription 13.

[0113] In vivo animal experiments: Thirty female ICR mice were randomly divided into 6 groups of 5 mice each. Immunization was administered subcutaneously. Group 2 received 100 μg per mouse, Group 1 received 100 μL of PBS per mouse, and Groups 3 through 6 received 100 μL of the corresponding vaccine per mouse. A booster immunization was administered on day 10 following the initial immunization, following the same dosage. Blood samples were collected from mice on days 15, 30, and 45 post-primary immunization. Blood was collected in centrifuge tubes containing anticoagulant, and serum was collected for determination of the CSFV-E2 specific antibody titer using ELISA.

[0114] Figure 10This diagram illustrates the results of specific IgG antibody titer assays after immunization with six vaccine groups. The results show that at all time points, the antibody levels in groups four, five, and six were significantly higher than those in group three, indicating that MAN-PASP, as a stabilizer, can enhance the antibody-inducing effect of various inorganic nanoparticle adjuvants. The antibody level in group five was significantly higher than that in groups four and six, and the antibody level in group six was significantly higher than that in group four, indicating that group five showed the best effect.

[0115] Adjuvant-induced reservoir effect of nanoparticles: In vivo imaging was used to investigate the antigen reservoir induction effect of antigen-loaded nanoparticles in ICR mice.

[0116] The concentration of OVA-FITC was controlled at 500 μg / mL. The antigen-encapsulated polyaspartic acid-inorganic salt composite nanoparticle solutions prepared according to formulations 15, 16, and 17 were injected subcutaneously into the neck of ICR mice at a dose of 200 μL. In vivo fluorescence imaging was performed on the mice at 0h, 2h, 6h, 12h, and 24h post-injection to monitor the attenuation of antigen fluorescence and localization effect. Results are as follows: Figure 11 As shown, Figure 11 The diagram shows the in vivo imaging results for formulations 15, 16, and 17. After subcutaneous injection into mice, the fluorescence intensity of free OVA-FITC significantly decreased after 2 hours, and fluorescence was almost undetectable at the injection site after 12 hours. However, the nanoparticles encapsulating fluorescently labeled antigens prepared with formulations 15, 16, and 17 still showed some fluorescence 24 hours after injection. This indicates that the nanoparticles encapsulating fluorescently labeled antigens prepared with formulations 15, 16, and 17 can form an antigen reservoir at the injection site, allowing for the slow release of the antigen.

[0117] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of mannose-modified polyaspartic acid in the preparation of inorganic nanoparticles, characterized in that, The inorganic nanoparticles were prepared by mannose-modified polyaspartic acid derivatives and CaCl2 and Na3PO4, wherein the molar ratio of CaCl2 to Na3PO4 was 1:0.1~5. Alternatively, the inorganic nanoparticles are prepared from mannose-modified polyaspartic acid and 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. The preparation method of the mannose-modified polyaspartic acid includes the following steps: Polysuccinimide was dispersed in DMSO. Under conditions of 60-80 °C, a DMSO solution containing mannose compounds was slowly added dropwise to the above solution and reacted for 6-48 h. The molar ratio of polysuccinimide to mannose compounds was 1:1-100. NaOH was added to adjust the pH to 9-10, and the ring-opening reaction was continued for 2-12 h. The pH was adjusted to 7 with HCl. DMSO was removed by dialysis with water, and then the precipitate was removed by centrifugation and lyophilized to obtain the mannose-modified polyaspartic acid.

2. The application according to claim 1, characterized in that, The mannose compounds are selected from 4-aminophenyl α-D-mannopyranoside and D-mannosamine.

3. The application of mannose-modified polyaspartic acid in the preparation of nanoparticles loaded with fluorescently labeled antigens, characterized in that, The preparation method of the mannose-modified polyaspartic acid includes the following steps: Polysuccinimide was dispersed in DMSO. Under the condition of 60-80 °C, a DMSO solution containing mannose compounds was slowly added dropwise to the above solution and reacted for 6-48 h. The molar ratio of polysuccinimide to mannose compounds was 1:1-100. NaOH was added to adjust the pH to 9-10, and the ring-opening reaction was continued for 2-12 h. The pH was adjusted to 7 with HCl. DMSO was removed by dialysis with water, and then the precipitate was removed by centrifugation and lyophilized to obtain the mannose-modified polyaspartic acid. The nanoparticles carrying fluorescently labeled antigens were prepared from antigens, mannose-modified polyaspartic acid, CaCl2, and Na3PO4. The molar ratio of CaCl2 to Na3PO4 was 1:0.1~5, and the mass ratio of antigens to mannose-modified polyaspartic acid was 1:1~5. The antigens were selected from OVA-FITC. Alternatively, the nanoparticles carrying the fluorescently labeled antigen are prepared from the antigen, mannose-modified polyaspartic acid, 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 antigen to mannose-modified polyaspartic acid is 1:1~5; and the antigen is selected from OVA-FITC.

4. The application according to claim 3, characterized in that, The method for preparing the nanoparticles loaded with fluorescently labeled antigens includes the following steps: The antigen, a mannose-modified polyaspartic acid solution (5-15 mg / ml), a CaCl2 solution (25-65 mmol / L), and a MnCl2 solution (15-65 mmol / L) were mixed and the pH was adjusted to 9. The mixture was incubated at 1-5°C for 1-3 hours. Deionized water was then added, followed by a Na3PO4 solution (25-100 mmol / L). The molar ratio of CaCl2 to Na3PO4 was 1:0.1-5. The mixture was stirred for 10-40 minutes to obtain the nanoparticle solution containing the fluorescently labeled antigen. The antigen was selected from OVA-FITC.

5. A vaccine adjuvant, said vaccine adjuvant is prepared by mixing an aqueous solution of antigen at a volume ratio of 1:1 with an inorganic nanoparticle solution prepared according to claim 1, wherein the antigen is selected from the classical swine fever virus E2 antigen.

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