A method for preparing a transmucosal barrier and immune-inducing carrier based on amphoteric dextran.

Abstract

This invention discloses a method for preparing a transmucosal barrier and immunomodulatory carrier based on amphoteric dextran, belonging to the field of biomedical technology. Based on the novel adjuvant dextran and polyphenol-metal complexation, this invention first modifies the dextran to amphoteric properties. Then, leveraging the self-assembly characteristics of polyphenols (such as tannic acid TA), metal ions (such as Ca²⁺) are further introduced. + Increased cross-linking strength drives DEX-CB to complex with active antigens, forming nanoparticles through self-assembly via hydrophobic and coordination interactions. These nanoparticles can load various antigens, and their surface amphoteric dextran can penetrate mucus, be taken up by cells, and thus stimulate the immune system.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing nanoparticles by cross-linking amphoteric dextran with polyphenols and metal ions to encapsulate active antigens, and its advantages as a mucosal vaccine delivery system. Background Technology

[0002] Oral vaccines have become a research hotspot in the global immunization field due to their unique advantages of being non-invasive, facilitating large-scale vaccination, and inducing mucosal immunity. Compared to traditional injectable vaccines, oral vaccines do not require specialized medical personnel, reducing the risk of iatrogenic infections, making them suitable for resource-scarce areas. Furthermore, they can induce secretory IgA through the gut-associated lymphoid tissue (GALT), providing the first line of immune defense for the mucosal surface. Although historical evidence has indicated the feasibility of oral vaccination, its progress has been tortuous. The gastrointestinal tract presents a harsh physiological and immune environment, including the degradation by gastric acid and enzymes, the mucus barrier of the small intestinal epithelium, and the epithelial cell barrier.

[0003] Overcoming the epithelial mucus barrier hinges on the surface properties of particles. Neutral charges and hydrophilic surfaces have been proven to facilitate mucus penetration, and surface PEGylation is generally considered the "gold standard" for assessing mucus penetration ability. Zwitterionic polymers are polymers with a pair of oppositely charged groups in their repeating units. Equal density of positive and negative charges can promote efficient mucus transport by avoiding electrostatic adhesion to mucins; high surface charge density creates a hydrophilic surface, minimizing mucus trapping of hydrophobic particles, and zwitterionic polymers exhibit superior cellular uptake capacity compared to PEG. Nanocarriers based on natural polysaccharides have attracted significant attention due to their biocompatibility and immune-activating capabilities. Dextran, a biodegradable polysaccharide, is easily chemically modified and possesses immune-activating properties. Dextran can bind to dectin-1 (C-type lectin) and complement receptor 3 (CR3), promoting the secretion of cytokines and the activation of B and T cells, thereby enhancing humoral and cellular immune responses. However, traditional PEG, zwitterionic materials, and dextran all have significant shortcomings in terms of mucus penetration, transepithelial delivery, and immunostimulation: although PEG has good hydrophilicity and biocompatibility, its interaction with epithelial cells is weak, its transmembrane efficiency is low, and it has the problem of immune inertness; zwitterionic materials themselves have almost no immunostimulatory ability; although dextran is a natural polysaccharide with certain biocompatibility and weak immunomodulatory activity, its mucus penetration ability is limited, and its transepithelial transport efficiency is still not ideal. Summary of the Invention

[0004] To address the problems of existing technologies, this invention provides a method for preparing a transmucosal barrier carrier based on amphoteric dextran that induces immunity. This is a vaccine delivery system capable of overcoming intestinal mucus layer barriers, cellular uptake, and inducing enhanced mucosal immunity. In this invention, after amphotericizing dextran, the amphoteric dextran is cross-linked with polyphenols and metal ions, encapsulating active antigens and self-assembling to form nanoparticle vaccine carriers. The amphoteric dextran on the carrier surface exhibits excellent mucus penetration, cellular uptake, and immune activation functions. This invention focuses on using amphoteric dextran as a vaccine carrier, aiming to develop a highly efficient delivery system that promotes mucus penetration and enhances immune responses, providing a new approach for nanoparticle drug delivery systems with promising application prospects. The method of this invention includes the following steps:

[0005] A method for preparing a transmucosal barrier vector based on amphoteric dextran that induces immunity includes the following steps: 1) Synthesis of amphoteric dextran: Amphoteric modification of dextran (DEX) was performed to obtain amphoteric dextran.

[0006] 2) Direct mixing method for synthesizing nanoparticles: Amphoteric dextran solution, aqueous solution containing active antigen, and solution containing metal ions are added sequentially to water. Tannic acid (TA) solution is rapidly added under magnetic stirring to form nanoparticles through self-assembly. After centrifugation and washing, nanoparticles are obtained by cross-linking amphoteric dextran with polyphenols and metal ions to encapsulate active antigens.

[0007] In the above technical solution, the carrier can simultaneously overcome the intestinal mucus layer, enhance cellular uptake, and induce mucosal immune enhancement.

[0008] In the above technical solution, in step 1), the modification of zwitterions includes, but is not limited to: carboxybetaine, sulfobetaine, phosphorylcholine, etc.; the molecular weight of the dextran is 35,000-650,000, preferably 35,000-45,000.

[0009] In the above technical solution, in step 1), when carboxybetaine is used for modification, the method for synthesizing amphoteric dextran (DEX-CB) through chemical reaction is as follows: dextran and epichlorohydrin are dissolved in an aqueous sodium hydroxide solution, heated and stirred to react, then sodium hydroxide and N,N-dimethylglycine are added, and the reaction is continued to be heated and stirred. After dialysis to remove impurities, the dextran (DEX-CB) modified with carboxybetaine is freeze-dried to obtain the dextran (DEX-CB) modified with carboxybetaine.

[0010] In the above technical solution, in step 1), the mass ratio of dextran to N,N-dimethylglycine is 1:3-5, preferably 1:3.3. Before adding sodium hydroxide, the concentration of the sodium hydroxide aqueous solution is 0.01-0.1 g / mL, preferably 0.053 g / mL; the concentration range of dextran in the sodium hydroxide aqueous solution is 20-120 mg / mL, preferably 60 mg / mL; after adding sodium hydroxide, the concentration range of the sodium hydroxide aqueous solution is 20-200 mg / mL, preferably 133 mg / mL. The ratio of dextran to epichlorohydrin is 600 mg: 2-3 mL, preferably 600 mg: 2.5 mL, for example, the mass of dextran is 600 mg, and the volume of epichlorohydrin is 2-3 mL, preferably 2.5 mL.

[0011] In the above technical solution, in step 1), the conditions for the first heating and stirring reaction are: temperature 30-70℃, preferably 45-55℃; rotation speed 300-700 rpm, preferably 300 rpm; reaction time 12-48 h, preferably 12 h; the conditions for the second heating and stirring reaction are: temperature 40-80℃, preferably 55-65℃; rotation speed 300-700 rpm, preferably 300 rpm; reaction time 48-60 h, preferably 48 h; during dialysis, the solvent is water, and the molecular weight cutoff of the dialysis bag is 1-30 kDa, preferably 10 kDa; the freeze-drying involves pre-freezing followed by freeze-drying; the pre-freezing time is 2-96 h, the pre-freezing temperature is -20--80℃; the freeze-drying temperature is -45--80℃, and the freeze-drying time is 2-96 h.

[0012] In the above technical solution, in step 2), the active antigen includes, but is not limited to, small molecule proteins, recombinant proteins, virus-like particles, etc.; the concentration of the active antigen aqueous solution (an aqueous solution containing the active antigen) is 1-20 mg / mL, preferably 5 mg / mL; the concentration of amphoteric dextran in the amphoteric dextran solution is 1-20 mg / mL, preferably 5 mg / mL; the metal ion is one of calcium ions, zinc ions, iron ions, or aluminum ions, preferably calcium ions; the solution containing metal ions can be calcium chloride solution, zinc sulfate solution, or aluminum chloride solution; the concentration of metal ions in the solution containing metal ions is 1-20 mg / mL, preferably 5 mg / mL; the concentration of tannic acid in the tannic acid solution is 1-20 mg / mL, preferably 5 mg / mL.

[0013] In the above technical solution, in step 2), the amphoteric dextran solution is the amphoteric dextran solution after filtration through a membrane, and the membrane is a 0.22-0.8µm membrane.

[0014] In the above technical solution, the conditions for the magnetic stirring reaction in step 2) are: temperature 18-30 ℃, rotation speed 600-1000 rpm, preferably 800 rpm; the magnetic stirrer can be replaced with a metal bath; the reaction time is 10-30 min, preferably 20 min.

[0015] In the above technical solution, in step 2), the molar ratio of the active antigen in the aqueous solution containing the active antigen, the amphoteric dextran in the amphoteric dextran solution, the metal ions in the solution containing metal ions, and the tannic acid in the tannic acid solution is 1:2-10:50-100:50-100, preferably 1:4:84:74. The concentration of amphoteric dextran in the reaction solution is 0.5-3 mg / mL, preferably 1.6 mg / mL.

[0016] In the above technical solution, in step 2), the centrifugal washing speed is 10000-11000 rpm, preferably 10000 rpm; the centrifugation time is 10-20 min, preferably 15 min; and the number of washing cycles is 3-5 times, preferably 3 times.

[0017] In the above technical solution, in step 2), the size of the amphoteric dextran-loaded active antigen nanoparticles formed is 50-600 nm.

[0018] In the above technical solution, step 2) further includes: dispersing the prepared carrier in physiological saline or phosphate buffer for preservation.

[0019] In the above technical solution, step 2) further includes: preparing the obtained carrier into freeze-dried particles, that is, after centrifugation and washing, freeze-drying to obtain a carrier based on amphoteric dextran that crosses the mucosal barrier and induces mucosal immunity; wherein, the freeze-drying is: first pre-freezing, then freeze-drying; the pre-freezing time is 2-96 h, the pre-freezing temperature is -20--80 ℃; the freeze-drying temperature is -45--80 ℃, and the freeze-drying time is 2-96 h.

[0020] In the above technical solution, a freeze-drying protectant is used during the freeze-drying process. That is, the carrier prepared in step 2) is mixed with the freeze-drying protectant and freeze-dried to obtain freeze-dried particles. The freeze-drying protectant is selected from one or more of trehalose, sucrose, lactose, and inulin, preferably trehalose. The freeze-drying protectant is an aqueous solution of the freeze-drying protectant with a mass concentration of 2.5%-10%, preferably 5%. The ratio of the prepared carrier to the aqueous solution of the freeze-drying protectant is 0.5-2 mg:0.5 mL, preferably 1 mg:0.5 mL.

[0021] The above-mentioned technical solution also includes: dispersing the prepared carrier in physiological saline or phosphate buffer for preservation.

[0022] In the above technical solution, a freeze-drying protectant is used in the freeze-drying process. The freeze-drying protectant is selected from one or more of trehalose, sucrose, lactose, and inulin, preferably trehalose, with a mass concentration of 2.5%-10%, preferably 5%.

[0023] This invention also relates to a transmucosal barrier carrier based on amphoteric dextran that induces mucosal immunity, prepared by the above-described method. The prepared carrier is a nanoparticle structure formed through self-assembly via hydrophobic and coordination interactions, wherein the particle surface is amphoteric dextran and the particle interior contains active antigens. The nanoparticles can load multiple antigens, and the surface amphoteric dextran can penetrate mucus, be taken up by cells, and thus stimulate immunity. The size of the transmucosal barrier carrier based on amphoteric dextran that induces mucosal immunity is 50-600 nm.

[0024] The zwitterionic dextran used in this invention combines the natural polysaccharide backbone of dextran with zwitterionic groups, which significantly enhances its ability to penetrate adhesion and cross epithelial cells. At the same time, the dextran backbone can be recognized by cell surface receptors, promoting transepithelial transport and immune stimulation.

[0025] This invention is based on the novel adjuvant dextran and the polyphenol-metal complexation effect. First, the dextran is modified to be amphoteric. Based on the self-assembly properties of polyphenols (such as tannic acid TA), metal ions (such as Ca²⁺) are further introduced. + This invention increases cross-linking strength, driving DEX-CB to complex with active antigens, and self-assembles into nanoparticles through hydrophobic and coordination interactions. The amphoteric dextran-active antigen nanoparticles in this invention have an amphoteric dextran surface with electrically neutral and hydrophilic properties, significantly improving mucus penetration ability to overcome the intestinal mucus layer. Dextran is a novel natural polysaccharide adjuvant that can enhance cellular uptake and induce mucosal immune enhancement.

[0026] The beneficial effects of this invention are as follows: This invention utilizes a novel natural polysaccharide adjuvant, dextran, and biocompatible tannic acid and calcium ions to prepare nanoparticles through continuous chemical reactions and self-assembly, thus creating a delivery carrier capable of encapsulating different antigens. The particle surface is composed of amphoteric dextran, which significantly enhances mucus penetration, thereby overcoming the intestinal mucus layer, promoting cellular uptake, and inducing enhanced mucosal immunity. Attached Figure Description

[0027] Figure 1 The NMR spectra of DEX and DEX-CB are 1H NMR spectra.

[0028] Figure 2 Infrared spectra of DEX and DEX-CB.

[0029] Figure 3The image shown is a transmission electron microscope image of the nanoparticles from Example 1; where the scale bar is 2 µm.

[0030] Figure 4 To study the mean square displacement of the penetrating motion behavior of three types of nanoparticles in mucus using multi-particle tracking technology. <msd>The quantitative evaluation results were as follows. Among them, dextran-coated BSA nanoparticles (DEX+BSA NP) and the recognized mucus-penetrating particles, namely PEG-coated BSA nanoparticles (PEG+BSA NP), were used as control materials.

[0031] Figure 5 The cytotoxicity of three types of nanoparticles loaded with the model antigen BSA in Example 1 and Comparative Examples 1-2 on antigen-presenting cells, namely primary mouse bone marrow-derived dendritic cells (BMDCs), was evaluated.

[0032] Figure 6 Flow cytometry quantitative evaluation of the uptake capacity of three nanoparticles loaded with model antigen BSA in intestinal epithelial cells (Caco-2) in Example 1 and Comparative Examples 1-2.

[0033] Figure 7 This study evaluated the cell activation and cytokine release of primary mouse bone marrow-derived dendritic cells (BMDCs) loaded with three types of nanoparticles containing the model antigen BSA, as described in Examples 1 and 1-2. DEX-CB+BSA NP promoted the expression of (a) CD80+, (b) CD86+, and (c) MHCII+ on the surface of BMDC cells; and further promoted the secretion of (d) IL by BMDC cells. 6. (e)IL 12. (f)TNF Secretion of α-cytokines. Detailed Implementation

[0034] The following non-limiting embodiments are not intended to limit the invention in any way. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; and the reagents and materials involved are conventional reagents unless otherwise specified.

[0035] Example 1 A method for preparing nanoparticles (DEX-CB+BSA NP) by cross-linking amphoteric dextran loaded with model antigen BSA with tannic acid and calcium ions includes the following steps: 1) Synthesis of amphoteric dextran (DEX-CB): 600 mg of dextran (DEX, purchased from Sigma, molecular weight 35-45 kDa) was weighed and dissolved in 10 mL of 0.053 g / mL sodium hydroxide aqueous solution. Then, 2.5 mL of epichlorohydrin was added, and the mixture was reacted in a water bath at 45 °C and 300 rpm for 12 h. After that, 800 mg of solid sodium hydroxide and 2 g of N,N-dimethylglycine were added, and the temperature was increased to 55 °C and the reaction was continued for 48 h. The mixture was dialyzed with ultrapure water for 48 h using a 10 kDa dialysis bag to remove impurities. Then, it was pre-frozen at -80 °C for 48 h and freeze-dried at -80 °C for 48 h to obtain amphoteric dextran (DEX-CB).

[0036] 2) Preparation of BSA nanoparticles encapsulated with amphoteric dextran (DEX-CB+BSA NP): The DEX-CB obtained in step 1) was dissolved in ultrapure water at a concentration of 5 mg / mL and filtered through a 0.22 µm aqueous phase filter membrane three times to obtain a DEX-CB solution. BSA, TA, and CaCl2 solutions, each with a concentration of 5 mg / mL, were prepared separately using ultrapure water. 200 µL of BSA solution, 800 µL of DEX-CB solution, and 28 µL of CaCl2 solution were added sequentially to a 5 mL vial containing 1120 µL of ultrapure water. The magnetic stirrer was turned on and the speed adjusted to 800 rpm. At room temperature, 380 µL of TA solution was quickly added using a pipette to initiate the reaction. Stirring was continued for 20 min. The sample was collected, centrifuged three times at 10000 rpm and 4 °C for 15 min each time. 1 mg of the solid was resuspended in 0.5 mL of 5% (w / w) trehalose aqueous solution, pre-frozen at -80 °C for 48 h, and then freeze-dried at -80 °C for 48 h to obtain amphoteric dextran-encapsulated BSA nanoparticles (DEX-CB+BSA). NP).

[0037] Comparative Example 1 Preparation of dextran-loaded BSA nanoparticles (DEX+BSA NP): Dextran (DEX, purchased from Sigma, molecular weight 35000-45000) solution, BSA solution, TA solution, and CaCl2 solution, each with a concentration of 5 mg / mL, were prepared using ultrapure water. 200 µL of BSA solution, 800 µL of DEX solution, and 13 µL of CaCl2 solution were sequentially added to a 5 mL vial containing 1100 µL of ultrapure water. The magnetic stirrer was turned on and the speed was adjusted to 800 rpm. 400 µL of TA solution was quickly added using a pipette to initiate the reaction. Stirring was continued for 20 min. The sample was collected, centrifuged three times at 10000 rpm at 4 °C for 15 min each time. 1 mg of the solid was then resuspended in 0.5 mL of 5% (w / w) trehalose aqueous solution, pre-frozen at -80 °C for 48 h, and then freeze-dried at -80 °C for 48 h to obtain dextran-encapsulated BSA nanoparticles (DEX+BSA NP).

[0038] Comparative Example 2 Preparation of PEG-coated BSA nanoparticles (PEG+BSA NP): Solutions of polyethylene glycol (PEG, purchased from Maclean's, molecular weight 5000), BSA, TA, and CaCl2 were prepared with ultrapure water, each with a concentration of 5 mg / mL. 200 µL of BSA solution, 800 µL of PEG solution, and 130 µL of CaCl2 solution were sequentially added to a 5 mL vial containing 970 µL of ultrapure water. The magnetic stirrer was turned on and the speed adjusted to 800 rpm. 400 µL of TA solution was quickly added using a pipette to initiate the reaction. Stirring was continued for 20 min. The sample was collected, centrifuged three times at 10000 rpm at 4 °C for 15 min each time. 1 mg of the solid was resuspended in 0.5 mL of 5% trehalose aqueous solution, pre-frozen at -80 °C for 48 h, and then freeze-dried at -80 °C for 48 h to obtain PEG-coated BSA nanoparticles (PEG+BSA NP).

[0039] Example 1, Comparative Examples 1-2: The relevant structural characterizations are as follows Figure 1-2 .

[0040] Figure 1 The 1H NMR spectra of DEX and DEX-CB are shown: (j) is the quaternary ammonium group hydrogen signal, indicating that DEX-CB has been successfully modified with quaternary ammonium groups, and the degree of substitution is I. j / 6I a =1.40 / (6 1.00) = 0.233, which meets the expected degree of substitution.

[0041] Figure 2 Infrared spectra of DEX and DEX-CB: 1635 cm⁻¹ -1 The significant increase in the intensity of the C=O peak at 3410 cm⁻¹ indicates the presence of a carboxyl group in the DEX-CB structure; -1 The point is the stretching vibration of OH on the DEX structure, 2925 cm. -1 It is the stretching vibration of CH, 1635 cm. -1 It is a stretching vibration of C=O, 1030 cm. -1 It is a C–O stretching vibration.

[0042] Example 2 Physicochemical properties of nanoparticles prepared in Example 1 and Comparative Examples 1-2 were tested. The morphology of the nanoparticles (DEX-CB+BSA NP) prepared in Example 1 was observed using transmission electron microscopy. The results are shown in the figure. Figure 3 Electron microscopy analysis showed that DEX-CB+BSA NPs were uniform particles with a size of approximately 300 nm.

[0043] The hydrodynamic size and Zata potential of the nanoparticles (DEX-CB+BSA NP, DEX+BSA NP, PEG+BSA NP) prepared in Example 1 and Comparative Examples 1-2 were measured, and the results are shown in Table 1.

[0044] Table 1. Hydrodynamic dimensions, PDI, and Zata potential of nanoparticles prepared in Example 1 and Comparative Examples 1-2

[0045] Example 3 Test of the mucus-penetrating ability of nanoparticles prepared in Example 1 and Comparative Examples 1-2: The BSA in the nanoparticles prepared in Example 1 and Comparative Examples 1-2 was replaced with RB (Rhodamine B) fluorescently labeled BSA. The mucus-penetrating ability of the three nanoparticles (DEX-CB+BSA NP, DEX+BSA NP, and PEG+BSA NP) was evaluated using multi-particle tracking technology. The results are shown in [Figure number missing]. Figure 4 Compared with the control groups DEX+BSA NP and PEG+BSA NP, quantitative analysis showed that DEX-CB+BSA NP had a higher mean square displacement in mucus ( <msd>).

[0046] Example 4 Cytotoxicity detection of nanoparticles prepared in Example 1 and Comparative Examples 1-2: The cell viability of primary mouse bone marrow-derived dendritic cells (BMDCs) treated with 100 ug / mL BSA, DEX, DEX-CB, DEX-CB+BSA NP, DEX+BSA NP, PEG+BSA NP, and lipopolysaccharide (LPS) was determined using the MTS method. The results are shown in [Figure 1]. Figure 5 Compared with the control group of normally growing cells without any treatment, DEX-CB+BSA NP did not show cytotoxicity in cells, demonstrating good biosafety properties.

[0047] Example 5 An example 1, assay of the cellular uptake capacity of the nanoparticles prepared in Comparative Examples 1-2: The BSA in the nanoparticles prepared in Example 1 and Comparative Examples 1-2 was replaced with FITC (fluorescein isothiocyanate)-labeled BSA. Nanoparticles formed by loading FITC-labeled BSA onto PEG, DEX, and DEX-CB were prepared respectively. Specifically, the nanoparticles formed by loading FITC-labeled BSA onto DEX-CB in Example 1 were designated DEX+FITC-BSA NP, the nanoparticles formed by loading FITC-labeled BSA onto DEX in Comparative Example 1 were designated DEX+FITC-BSA NP, and the nanoparticles formed by loading FITC-labeled BSA onto PEG in Comparative Example 2 were designated PEG+FITC-BSA NP. The cellular uptake capacity of the nanoparticles formed by loading FITC-labeled BSA onto PEG, DEX, and DEX-CB was quantitatively analyzed using flow cytometry. FITC-labeled BSA (FITC-BSA) was added as a control. The results are shown in [Figure number missing]. Figure 6 Compared with the control groups PEG+FITC-BSA NP and DEX+FITC-BSA NP, DEX-CB+FITC-BSA NP significantly promoted the uptake of Caco-2 cells.

[0048] Example 6 Example 1, assay of the cell activation and cytokine secretion capabilities of the nanoparticles prepared in Comparative Examples 1-2: The cell activation and cytokine secretion capabilities of three nanoparticles were investigated using mouse primary bone marrow-derived dendritic cells (BMDC) (enzyme-linked immunosorbent assay). Dextran (DEX) and carboxy-betained alkalized dextran (DEX-CB) were added as controls. Results are shown in [Figure number missing]. Figure 7 DEX-CB+BSA NP significantly promoted the activity of CD80, CD86, and major histocompatibility complex II (MHC II) in CD11c. + Upregulation of its expression in cells demonstrates that it can promote the activation of BMDCs, as shown in the following figures. Figure 7 (a) c). Furthermore, DEX-CB+BSA NP can also significantly promote the secretion of Th1 and Th2 cytokines by BMDCs, namely: IL-1... 6. IL 12. TNF α, results are shown Figure 7 (d) f), further verifying its ability to induce a robust immune response.< / msd> < / msd>

Claims

1. A method for preparing a carrier based on amphoteric dextran that crosses the mucosal barrier and induces mucosal immunity, characterized in that, Includes the following steps: 1) Synthesis of amphoteric dextran: Amphoteric modification of dextran yields amphoteric dextran; 2) Add the amphoteric dextran solution, the aqueous solution containing the active antigen, and the solution containing metal ions to water, and add the tannic acid solution under magnetic stirring to obtain the reaction solution. The reaction solution self-assembles to form nanoparticles, which are then centrifuged and washed to obtain a carrier based on amphoteric dextran that crosses the mucosal barrier and induces mucosal immunity.

2. The preparation method according to claim 1, characterized in that, In step 1), the zwitterionic modification includes carboxybetaine, sulfobetaine, or phosphorylcholine; the molecular weight of the dextran is 35,000-650,000.

3. The preparation method according to claim 1, characterized in that, In step 1), when carboxybetaine is used for modification, the method for synthesizing amphoteric dextran through chemical reaction is as follows: dextran and epichlorohydrin are dissolved in sodium hydroxide aqueous solution, heated and stirred to react, sodium hydroxide and N,N-dimethylglycine are added, heated and stirred to react, dialyzed to remove impurities, and then freeze-dried to obtain carboxybetaine-modified dextran.

4. The preparation method according to claim 3, characterized in that, In step 1), the mass ratio of dextran to N,N-dimethylglycine is 1:3-5; before adding sodium hydroxide, the concentration of the sodium hydroxide aqueous solution is 0.01-0.1 g / mL, and the concentration of dextran in the sodium hydroxide aqueous solution is 20-120 mg / mL; after adding sodium hydroxide, the concentration of the sodium hydroxide aqueous solution is 20-200 mg / mL, and the ratio of dextran to epichlorohydrin is 600 mg: 2-3 mL. The conditions for the first heating and stirring reaction were: temperature 30-70 ℃, rotation speed 300-700 rpm, and reaction time 12-48 h; the conditions for the second heating and stirring reaction were: temperature 40-80 ℃, rotation speed 300-700 rpm, and reaction time 48-60 h. During dialysis, water is used as the solvent, and the molecular weight cutoff of the dialysis bag used is 1-30 kDa; The freeze-drying process involves pre-freezing followed by freeze-drying; the pre-freezing time is 2-96 hours, and the pre-freezing temperature is -20 to -80 °C; the freeze-drying temperature is -45 to -80 °C, and the freeze-drying time is 2-96 hours.

5. The preparation method according to claim 1, characterized in that, In step 2), the active antigen includes one or more of small molecule proteins, recombinant proteins, and virus-like particles; the concentration of the aqueous solution containing the active antigen is 1-20 mg / mL; the concentration of amphoteric dextran in the amphoteric dextran solution is 1-20 mg / mL; the metal ion is one of calcium ions, zinc ions, iron ions, or aluminum ions; the concentration of the metal ion in the solution containing the metal ion is 1-20 mg / mL; and the concentration of tannic acid in the tannic acid solution is 1-20 mg / mL. The molar ratio of active antigen in the aqueous solution containing active antigen, amphoteric dextran in the amphoteric dextran solution, metal ions in the solution containing metal ions, and tannic acid in the tannic acid solution is 1:2-10:50-100:50-100, and the concentration of amphoteric dextran in the reaction solution is 0.5-3 mg / mL. The conditions for the magnetic stirring reaction are: temperature 18-30 ℃, rotation speed 600-1000 rpm, and reaction time 10-30 min; The centrifugation speed is 10000-11000 rpm, the centrifugation time is 10-20 min, and the number of washes is 3-5.

6. The preparation method according to claim 1, characterized in that, In step 2), the amphoteric dextran solution is the amphoteric dextran solution after filtration through a 0.22-0.8µm filter membrane. Alternatively, the magnetic stirring can be replaced with a metal bath.

7. The preparation method according to claim 1, characterized in that, In step 2), after centrifugation and washing, the product is lyophilized to obtain a vector based on amphoteric dextran that crosses the mucosal barrier and induces mucosal immunity. The lyophilization process involves pre-freezing followed by freeze-drying. The pre-freezing time is 2-96 h, and the pre-freezing temperature is -20 to -80 °C. The freeze-drying temperature is -45 to -80 °C, and the freeze-drying time is 2-96 h.

8. The preparation method according to claim 1 or 7, characterized in that, Also includes: The prepared vaccine vector was dispersed in physiological saline or phosphate buffer for storage. Alternatively, a freeze-drying protectant may be used during the freeze-drying process. The freeze-drying protectant may be selected from one or more of trehalose, sucrose, lactose, and inulin.

9. The preparation method according to any one of claims 1-8 yields a carrier based on amphoteric dextran that crosses the mucosal barrier and induces mucosal immunity, characterized in that... The prepared carrier is a nanoparticle structure, in which the surface of the particles is amphoteric dextran and the interior of the particles is an active antigen.

10. The carrier based on amphoteric dextran that crosses the mucosal barrier and induces mucosal immunity according to claim 9, characterized in that, The size of the vector based on amphoteric dextran that crosses the mucosal barrier and induces mucosal immunity is 50-600 nm.

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