A self-assembled nasal mucosa nano-vaccine and a preparation method thereof

Abstract

The present application relates to a kind of self-assembled nasal mucosa nano vaccine and its preparation method, and nano vaccine includes glycyrrhiza saponin plant body core, the antigen loading layer formed by glutathione-antigen complex, the biological adhesion layer formed by chitosan and the hydrophilic protective layer formed by hyaluronic acid.It is formed by self-assembly of glycyrrhiza saponin and phospholipid plant body, and glutathione-antigen complex, chitosan and hyaluronic acid are sequentially loaded, and stable nano vaccine system is obtained.The nano vaccine has higher antigen encapsulation rate, good biocompatibility and mucosal delivery capacity, can significantly promote dendritic cell uptake and maturation, improve antigen-specific IgG, IgA and secretory IgA level, enhance mucosal immunity and systemic immune response, promote Th1 / Th2 immune balance, suitable for infant nasal mucosa immunity and related disease prevention.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a self-assembled nasal mucosal nanovaccine and its preparation method. Background Technology

[0002] Vaccines are currently a crucial means of preventing and controlling infectious diseases, establishing an immune protective barrier by inducing a specific immune response in the body. Traditional vaccines are primarily administered via intramuscular or subcutaneous injection, effectively inducing humoral and systemic immune responses. However, for respiratory pathogens such as influenza viruses, coronaviruses, and respiratory syncytial viruses, the initial sites of invasion are typically the mucous membranes of the nasal cavity, pharynx, and lungs. The immune response induced by traditional vaccines mainly focuses on the peripheral circulatory system, offering relatively limited local immune protection to the mucous membrane surfaces, making it difficult to effectively block pathogen invasion in its early stages.

[0003] Nasal mucosal immunization has become an important direction in vaccine research in recent years due to its advantages such as convenient administration, non-invasiveness, high compliance, and the ability to simultaneously induce systemic and mucosal immunity. Studies have shown that nasal mucosal immunization can not only induce the production of serum IgG antibodies but also stimulate the production of secretory IgA antibodies in mucosa-associated lymphoid tissue, thereby forming an immune barrier on the first line of defense against pathogen invasion. However, the nasal mucosa has a sophisticated self-defense mechanism, including a mucus barrier, a ciliary clearance system, and various protease degradation systems. Exogenous antigens entering the nasal cavity are easily embedded in mucus and rapidly cleared by cilia, and may also be inactivated by local enzymatic degradation, resulting in a short residence time, low bioavailability, and insufficient immunogenicity of antigens on the nasal mucosa surface. Therefore, how to improve the stability, residence time, and cellular uptake efficiency of antigens in the nasal mucosa has become a crucial technical problem that urgently needs to be solved in the development of nasal mucosal vaccines.

[0004] To improve antigen delivery efficiency, existing technologies typically employ delivery systems such as liposomes, polylactic-co-glycolic acid (PLGA) nanoparticles, chitosan nanoparticles, and inorganic nanomaterials to encapsulate antigens. Liposomes offer good biocompatibility and cell membrane affinity, but suffer from poor structural stability, easy aggregation during storage, and limited immunostimulatory capacity. While polymer nanoparticles can improve antigen stability, their preparation processes are complex, and degradation products from some materials may affect biosafety. Inorganic nanomaterials generally suffer from insufficient biodegradability and difficulties in long-term safety evaluation. On the other hand, immunoadjuvants, as an important component for enhancing the body's immune response, are widely used in vaccine systems. Some saponin adjuvants have attracted widespread attention due to their effects in promoting dendritic cell maturation, enhancing antigen presentation, and activating cellular and humoral immunity. Existing research indicates that while some saponin adjuvants possess strong immunostimulatory capabilities, they also suffer from high hemolytic toxicity, insufficient stability, and poor formulation adaptability. Plant granules / saponin-phospholipid complexes have been studied as nasal mucosal adjuvants, but when they are directly assembled with antigens, there are still problems that are difficult to balance between antigen load stability, particle behavior in the mucus environment, mucosal residence and continuous cellular uptake, thus limiting their application in the field of mucosal vaccines.

[0005] Furthermore, existing nanovaccine systems generally suffer from insufficient mucosal adhesion, limited antigen delivery efficiency, and unsatisfactory immune enhancement effects, making it difficult to simultaneously achieve multiple functions such as antigen protection, mucosal retention, cell uptake promotion, and immune enhancement. Therefore, developing a novel self-assembled nanovaccine system with stable structure, good biocompatibility, and excellent mucosal delivery and immune enhancement capabilities remains a crucial technical challenge that urgently needs to be addressed in this field. Summary of the Invention

[0006] This invention aims to provide a self-assembled nasal mucosal nanovaccine and its preparation method. The GSH-antigen complex is self-assembled with glycyrrhizin plant bodies and modified with chitosan / hyaluronic acid. After binding with the antigen, it is modified with reduced glutathione and / or chitosan to solve the problems of insufficient structural stability, low mucosal delivery efficiency, low antigen utilization rate and limited immune enhancement effect of existing nanovaccines.

[0007] To achieve the above objectives, this invention discloses a self-assembled nasal mucosa nanovaccine with a core-shell structure. The vaccine comprises a plant body core, an antigen-loading layer, a bioadhesion layer, and a hydrophilic protective layer. The plant body core is a glycyrrhizin plant body; the antigen-loading layer is a glutathione-antigen complex formed by glutathione and antigen; the bioadhesion layer is chitosan; and the hydrophilic protective layer is hyaluronic acid. This invention constructs a multi-layered core-shell structured nanovaccine composed of a glycyrrhizin plant body core, a glutathione-loading layer, a chitosan bioadhesion layer, and a hyaluronic acid hydrophilic protective layer. The glycyrrhizin plant body promotes dendritic cell maturation and induces IL-12 secretion; glutathione promotes intracellular antigen release and cross-presentation; chitosan enhances nasal mucosa adhesion and cellular uptake; and hyaluronic acid improves system stability and mucus diffusion capacity. This invention utilizes the synergistic effect of multiple components to achieve a continuous enhancement of the "mucus penetration-mucosal residence-cellular uptake-antigen presentation-immune activation" process. It synergistically promotes Th1-related cellular immune responses, enhances IFN-γ and IgG2a levels, and maintains Th2-related humoral immune responses and sIgA secretion, thereby correcting the natural Th2 bias and resulting in a Th1 / Th2 immune response, thus improving the overall protective effect of the vaccine. Nasal mucosal immunization effectively enhances mucosal immunity and child-friendly vaccination. Combined with glycyrrhizin, which primarily induces Th1-type immune responses, it synergistically improves children's vaccine response. Glycyrrhizin plant bodies can self-assemble with antigens to form nano-vaccines, generating a Th1-biased immune response. Simultaneous delivery of adjuvants and antigens effectively enhances the immunogenicity of immune cells. Nasal mucosal immunization can improve mucosal and systemic immunity levels, promote antigen-presenting cell uptake and cross-presentation, target CD8T cells, and enhance the immune protective function of the vaccine. Furthermore, glycyrrhizin plant bodies have good biocompatibility and safety, and the nano-vaccines have a stable structure and are easy to prepare on a large scale.

[0008] Preferably, the glycyrrhizin plant body is composed of phospholipids and glycyrrhizin in a ratio of 1:0.7-1.4; the phospholipids are one or more of soybean phospholipids, lecithin, and hydrogenated phospholipids; the glycyrrhizin is one or more of glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin G2, and ural glycyrrhizin; the chitosan has a molecular weight of 50-300 kDa, and the hyaluronic acid has a molecular weight of 10-100 kDa. Phospholipids are self-assembling frameworks. Possessing hydrophilic heads and hydrophobic tails, they are amphiphilic molecules that readily form nanovesicles and micelle structures in aqueous systems. When glycyrrhizin comes into contact with phospholipids, the hydrophobic structure of the saponin inserts into the hydrophobic region of the phospholipid, thus forming stable nanoparticles. The nanostructures formed by phospholipids can provide a protective microenvironment for antigens, reducing the risk of antigen degradation and improving antigen encapsulation efficiency and stability. Phospholipid materials have good biocompatibility with cell membranes, which is beneficial for promoting the recognition and uptake of antigen-presenting cells by antigen delivery systems, improving antigen presentation efficiency. The thick mucus layer of the nasal mucosa and its rapid clearance make it difficult for antigens to reach epithelial cells. Phospholipid nanostructures can improve mucosal diffusion and epithelial penetration, thereby improving antigen utilization. Phospholipids not only serve as carrier materials but also synergistically enhance the body's immune response by improving antigen delivery and presentation efficiency. Glycyrrhizin belongs to the triterpenoid saponin class of chemicals. As a natural amphiphilic molecule, glycyrrhizin phospholipids interact with each other, with the hydrophobic region embedded in the phospholipid bilayer and the hydrophilic end extending into the aqueous phase. This amphiphilic structure facilitates hydrophobic interactions and interfacial assembly with the phospholipids, promoting nanostructure formation and enhancing system stability. Glycyrrhizin can promote the maturation of antigen-presenting cells, enhancing antigen presentation capacity and thus strengthening the body's specific immune response. It can also promote Th1-type immune responses, increasing cellular immunity and enhancing the body's ability to clear pathogen-infected cells. Furthermore, it can promote a balanced Th1 / Th2 immune response. Glycyrrhizin can enhance the recognition and uptake of antigens by mucosa-associated lymphoid tissue, thereby promoting the production of secretory IgA antibodies. It can also promote antigen transport across biological membranes, improving antigen utilization. Glycyrrhizin exhibits better biocompatibility and safety. In addition, glycyrrhizin plant bodies are stable self-assembled nanoframeworks formed by the hydrophobic structure of glycyrrhizin embedded in the phospholipid bilayer. The hydrophobic saponin portion of glycyrrhizin is embedded in the hydrophobic region of the phospholipid bilayer, while the hydrophilic sugar chain portion is exposed in the aqueous phase. The phospholipid complex nanostructure formed by glycyrrhizin and phospholipid through intermolecular interactions forms a stable complex with phospholipid through hydrophobic interactions, hydrogen bonding, and van der Waals forces, thereby self-assembling into a nanoscale plant body structure. The formed plant body can not only improve the stability and bioavailability of glycyrrhizin, but also serve as an antigen-loading backbone to promote antigen delivery and enhance the uptake and presentation capacity of dendritic cells for antigens, thereby inducing the body to produce a stronger Th1 / Th2 immune response and mucosal immune response.

[0009] Preferably, the mass ratio of glutathione to antigen in the glutathione-antigen complex is 0.5-2:1. In this invention, glutathione plays a role in antigen stabilization, loading bridging, and intracellular release regulation. Glutathione can not only form stable complexes with antigens through sulfhydryl, amino, and carboxyl groups, improving antigen stability and loading efficiency, but also act as a bridging molecule to promote the binding between antigens and glycyrrhizin plant bodies, enhancing the structural stability of the nanovaccine. Glutathione can regulate antigen protein conformation, promoting antigen recognition by immune cells. The reversible interaction mediated by glutathione facilitates rapid intracellular release of antigens, improving cross-presentation efficiency, thereby enhancing Th1-related cellular immune responses and achieving a synergistic effect of antigen delivery and immune enhancement.

[0010] Preferably, the mass ratio of glycyrrhizin plant bodies, glutathione-antigen complex, chitosan, and hyaluronic acid is 12-20:6-10:2-8:2-4. In this invention, chitosan can form a bioadhesive layer, enhancing the interaction between the nanovaccine and the nasal mucosa through its cationic properties, prolonging the residence time of the nanovaccine in the nasal cavity, and promoting the uptake of the nanovaccine by dendritic cells. Hyaluronic acid can form a hydrophilic protective layer, improving system stability by constructing a highly hydration interface (a highly hydration interface refers to a stable, dynamic water molecule protective layer formed by the adsorption and binding of a large number of water molecules on the surface of nanoparticles due to the presence of numerous hydrophilic groups), while reducing non-specific interactions between nanoparticles and mucus components, and enhancing immune cell recognition through CD44 receptor-mediated communication. Chitosan and hyaluronic acid synergistically construct a delivery system with both mucosal retention and mucus diffusion capabilities, achieving efficient delivery of the nanovaccine to the nasal mucosa and sustained immune activation.

[0011] Preferably, the glycyrrhizin plant bodies have a particle size of 20-30 nm. This not only improves the antigen loading capacity but also works synergistically with the subsequent chitosan and hyaluronic acid modification layers to ensure that the final nanovaccine is within the particle size range that dendritic cells can efficiently take up, thereby promoting Th1-related cellular immune responses and maintaining Th1 / Th2 immune balance.

[0012] The method for preparing the nano-vaccine of this invention includes: S1. Prepare glycyrrhizin plant bodies, then dissolve them in ultrapure water to obtain a glycyrrhizin plant body solution; S2. Dissolve the antigen in ultrapure water to obtain an antigen solution; S3. Add glutathione to the antigen solution and mix well to obtain a glutathione-antigen complex solution; S4. Add the glutathione-antigen complex solution to the glycyrrhizin plant body solution to form the glycyrrhizin plant body-antigen complex through self-assembly. S5. Add the chitosan solution to the glycyrrhizin plant body-antigen complex, then adjust the pH, add the hyaluronic acid solution, and stir to obtain the nano-vaccine.

[0013] Preferably, the method for preparing glycyrrhizin plant bodies in step S1 includes the following steps: S11. Dissolve phospholipids in tetrahydrofuran, then add glycyrrhizin, and stir in a water bath to obtain a mixture; S12. Filter the stirred mixture and heat it by rotary evaporation to obtain the complex.

[0014] Preferably, the mixing time of S3 is 10-30 min and the stirring speed is 200-500 rpm.

[0015] Preferably, the glutathione-antigen complex solution of S4 is added to the glycyrrhizin plant body solution at a dropping rate of 0.5-1.5 ml / min and stirred at room temperature for 1-2 hours.

[0016] Preferably, after adding the chitosan solution in S5, the mixture is stirred at room temperature for 300-400 rpm for 1-2 hours; the pH is adjusted to 5.5-6.5; and the hyaluronic acid is added with a stirring speed of 300-500 rpm for 1-2 hours.

[0017] The beneficial effects of this invention are: (1) This invention utilizes the synergistic effect of glycyrrhizin plant bodies, glutathione, chitosan and hyaluronic acid to form a plant body core through the self-assembly of glycyrrhizin and phospholipids. Glutathione forms a complex with the antigen and loads it onto the surface of the plant body. This achieves multiple technical effects, including efficient antigen loading, long-term delivery to the nasal mucosa, efficient uptake by dendritic cells, simultaneous enhancement of mucosal and systemic immunity, regulation of Th1 / Th2 immune balance, and reduction of saponin toxicity. It is particularly suitable for the construction of nasal mucosal vaccines. (2) The glycyrrhizin and phospholipids of the present invention self-assemble to form a plant microstructure nanoframework with small particle size and good stability, and load antigens to form a nanovaccine; and the nanovaccine can improve antigen stability and delivery efficiency, promote dendritic cell uptake and presentation of antigens, significantly enhance humoral immunity, cellular immunity and mucosal immune response, increase the levels of IgG, IgA, sIgA and Th1 / Th2 related cytokines, and promote the formation of memory T cells, thereby obtaining excellent immune enhancement effect and long-term immune protection effect; (3) The glycyrrhizin plant bodies of the present invention have good nasal mucosal permeability and absorption, significantly reduce the hemolytic activity of glycyrrhizin, have good safety, and can effectively induce dendritic cell maturation, making them excellent adjuvants for nasal mucosal delivery of vaccines. (4) The glycyrrhizin plant bodies of the present invention can self-assemble with antigens in water to form nano-vaccines without the need for harsh conditions such as organic solvents and high temperatures. They can be effectively taken up by antigen-presenting cells and induced to mature. After nasal mucosal inoculation, the vaccine can effectively induce a Th1-biased immune response and has higher levels of mucosal, humoral and cellular immune responses, suggesting its potential as a vaccine delivery platform for the population. It can not only improve vaccine compliance but also meet the immune characteristics of this immune-biased population. Attached Figure Description

[0018] Figure 1 Hemolytic levels of glycyrrhizin saponins and glycyrrhizin plant bodies; Figure 2 Changes in absorbance during the interaction of nano-vaccines with mucus; Figure 3 Particle size changes during the interaction of nanovaccines with mucus; Figure 4 Potential changes during the interaction of nano-vaccines with mucus; Figure 5 Flow cytometry images of OVA, P-OVA and BMDCs incubated for 2 h and 6 h; Figure 6 Flow cytometry images of GSH-P-OVA, CS-P-OVA and BMDCs incubated for 2 h and 6 h; Figure 7 Cell uptake rates of OVA, P-OVA, GSH-P-OVA, CS-P-OVA and BMDCs after incubation for 2 h and 6 h; Figure 8 Cell uptake of OVA, P-OVA, GSH-P-OVA, CS-P-OVA and BMDCs after 2 h and 6 h of incubation; Figure 9 IgG levels of OVA, GSH-P-OVA, CS-P-OVA, and GEL in nasal lavage fluid; Figure 10 The IgG1 levels of OVA, GSH-P-OVA, CS-P-OVA, and GEL in nasal lavage fluid; Figure 11 The IgG2a levels of OVA, GSH-P-OVA, CS-P-OVA, and GEL in nasal lavage fluid; Figure 12 The IgG1 / IgG2a levels of OVA, GSH-P-OVA, CS-P-OVA, and GEL in nasal lavage fluid; Figure 13 IgA levels of OVA, GSH-P-OVA, CS-P-OVA, and GEL in nasal lavage fluid; Figure 14 The sIGA levels of OVA, GSH-P-OVA, CS-P-OVA, and GEL in nasal irrigation fluid; Figure 15 The IFN-γ levels of OVA, GSH-P-OVA, CS-P-OVA, and GEL in nasal lavage fluid; Figure 16 IL-2 levels of OVA, GSH-P-OVA, CS-P-OVA, and GEL in nasal lavage fluid; Figure 17 IL-4 levels of OVA, GSH-P-OVA, CS-P-OVA, and GEL in nasal lavage fluid; Figure 18 IL-10 levels of OVA, GSH-P-OVA, CS-P-OVA, and GEL in nasal lavage fluid; Figure 19 Results of dendritic cell maturation in lymph nodes after intranasal immunization with OVA, GSH-P-OVA, CS-P-OVS, GEL, and Poly(I:C) in mice; Figure 20 The proportion of memory lymphocytes among CD8 lymphocytes in the spleen; Detailed Implementation

[0019] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, specific embodiments are described below. The present invention will be further described in detail below. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0021] Example 1: Safety experiment of glycyrrhizin plant bodies; (1) Preparation of glycyrrhizin plant bodies: S1. Take 0.5g of soybean lecithin and add it to 100ml of tetrahydrofuran. After stirring for 5 minutes, a light yellow clear liquid is obtained. Add 0.35g of glycyrrhizin and react for 3-5 hours under water bath conditions of 40-60℃ and stirring speed of 400-800rpm. After filtering with a 0.22μm microporous membrane, place it in a rotary evaporator and evaporate it at 30℃ to obtain glycyrrhizin plant bodies. (2) Safety test: 1 ml of red blood cell suspension was mixed with 1 ml of different concentrations (0.05 / 0.1 / 0.5 / 0.75 / 1 mg·mL) respectively. -1 A solution of glycyrrhizin and glycyrrhizin plant bodies was mixed. Distilled water and physiological saline were used as control groups.

[0022] Characterization of glycyrrhizin plant bodies: Based on the experimental results Figure 1 Within the concentration range of 0.05-0.1 mg / mL, there was no significant difference between the glycyrrhizin group and the glycyrrhizin plant body group. In the concentration range of 0.5-0.75 mg / mL, differences began to appear. At 0.5 mg / mL, the hemolysis rate of both the glycyrrhizin group and the glycyrrhizin plant body group was 1%. However, at 0.75 mg / mL, the hemolysis rate of the glycyrrhizin group was 1.5%, while the hemolysis rate of the glycyrrhizin plant body group was 2%. Within the concentration range of 0.75-1 mg / mL, significant differences emerged. At 1 mg / mL, the hemolysis rate of glycyrrhizin was 82%, while the hemolysis rate of glycyrrhizin plant bodies was 2.1%. Glycyrrhizin has good immune-enhancing activity, but when it exists alone in its free state, it easily interacts with cholesterol in the cell membrane, leading to erythrocyte membrane damage and hemolysis. After self-assembling with phospholipids to form plant bodies, the hydrophobic triterpenoid structure of glycyrrhizin is embedded inside the phospholipid bilayer, thereby reducing the chance of direct contact with the cell membrane and significantly reducing hemolytic activity. According to the hemolysis experiment results, the plant body structure of the present invention can significantly improve the biocompatibility and safety of glycyrrhizin.

[0023] Example 2: Encapsulation efficiency and drug loading of nano-vaccines; Preparation of nano-vaccines: (1) Preparation of P-OVA vaccine: Dissolve 2.5 mg OVA (antigen) in 5 ml of ultrapure water to a concentration of 0.5 mg·mL. -1 Dissolve 5 mg or 10 mg of DG-P (glycyrrhizin plant body) in 5 ml of water to obtain a concentration of 1 mg / mL. -1 Or 2 mg·mL -1Add the OVA solution to DG-P at a rate of 1 ml / min and stir continuously at room temperature for 60 min to obtain P-OVA, which is a pale blue clear liquid. (2) Preparation of GSH-P-OVA vaccine: Dissolve 2.5 mg OVA in 5 ml of ultrapure water to a concentration of 0.5 mg·mL. -1 1.25 mg, 2.5 mg, or 5 mg of GSH (glutathione) were dissolved in OVA solution to achieve concentrations of 0.25, 0.5, and 1 mg / mL, respectively. -1 10 mg DG-P was dissolved in an equal volume of water to obtain a concentration of 2 mg / mL. -1 GSH-OVA solution was added to DG-P at a rate of 1 ml / min and stirred continuously at room temperature for 60 min to obtain GSH-P-OVA; (3) Preparation of CS-P-OVA vaccine: 200 mg chitosan was dissolved in 100 ml of 1% acetic acid solution to prepare a 0.2% chitosan solution. 1 ml or 0.5 ml of the chitosan solution was added to 2 ml of ultrapure water. Based on the ratio of chitosan to DG-P (CS:DG-P = 1:2, 1:4, w / w), 2 ml of GSH-P-OVA (GSH:OVA:DG-P = 1:1:4, DG-P concentration 2 mg·mL⁻¹) was added to each solution. -1 Add the chitosan to the CS (chitosan) solution and stir continuously at room temperature for 60 minutes to obtain CS-P-OVA; (4) Preparation of HA-P-OVA vaccine: 200 mg of chitosan was dissolved in 100 ml of 1% acetic acid solution to prepare a 0.2% chitosan solution. 15 mg of hyaluronic acid (HA) was dissolved in 10 ml of water to obtain 1.5 mg / mL. -1 HA solution. Take 0.5 ml of chitosan solution and add ultrapure water to 1.5 ml. According to the ratio of chitosan to DG-P (CS:DG-P=1:2, 1:4, w / w), add 2 ml of GSH-P-OVA (GSH:OVA:DG-P=1:1:4, DG-P concentration is 2 mg·mL⁻¹). -1 Add 0.5 ml of HA solution to CS solution and stir continuously at room temperature for 60 min to obtain CS-P-OVA. Then add 0.5 ml of HA solution to CS-P-OVA and stir continuously at room temperature for 60 min to obtain HA-P-OVA.

[0024] Add 1 mL of the nano-vaccine suspension to a 1.5 mL conical centrifuge tube, centrifuge at 16000 rpm for 15 min at 4°C, take the supernatant, and use the BCA protein concentration assay kit to determine the protein concentration in the sample. Encapsulation efficiency (EE) = (Amount of OVA added - Amount of free OVA in the supernatant) / Amount of OVA added × 100%; Drug loading capacity (LC) = (Amount of OVA added - Amount of free OVA in the supernatant) / Total amount of nanovaccine × 100%.

[0025] Example 3: Stability test of nano-vaccines in PBS 7.4; Using PBS to simulate the stability of the nasal cavity pH environment, high concentrations of P-OVA were obtained by using 8 mg·mL⁻¹ of DG-P and 2 mg·mL⁻¹ of OVA. Similarly, high concentrations of GSH-P-OVA and CS-P-OVA were prepared according to the optimized ratio. The above high-concentration nanovaccines were diluted 4-fold with PBS 7.4, and the particle size of the nanovaccines was determined.

[0026] Example 4: Particle size and potential testing of nano-vaccines; At room temperature, 1 mL of a nano-vaccine suspension of appropriate concentration was placed in a clean sample cell, and the particle size, zeta potential, and PDI value of the nano-vaccine were measured using a particle size-potential analyzer.

[0027] Table 1. Composition of Nanoparticle Vaccine Formulation (w / w)

[0028] Table 2 Physicochemical Properties of Nano-Vaccines

[0029] The results show that glycyrrhizin plant bodies can form stable nanoparticle systems with antigens through self-assembly, with an average particle size of approximately 100-200 nm, good dispersibility, and a Zeta potential of approximately -20 mV, forming a stable basic delivery structure. After introducing GSH, the encapsulation efficiency and drug loading of the nanovaccine are significantly improved, reaching approximately 70%–86% and 22%–25% respectively. Simultaneously, the system particle size changes (approximately 125–155 nm). The GSH-P-OVA particle size of 348.07 is due to GSH excess leading to aggregation and the formation of larger particles, while simultaneously increasing both encapsulation efficiency and drug loading. In this invention, GSH binds to antigens at multiple points in the system through thiol and hydrogen bonding, and also acts as a "molecular bridging agent" to promote tight assembly between antigens and DG-P, thereby improving antigen encapsulation efficiency. However, higher GSH levels can cause slight aggregation between nanoparticles, leading to increased particle size but not reducing the overall antigen loading capacity of the system. After modification with chitosan (CS), the zeta potential of the nanovaccine surface changed from negative to positive (approximately +40 mV to +55 mV), indicating that chitosan was successfully coated onto the nanoparticle surface. Simultaneously, the particle size of the system showed a certain increasing trend in the PBS environment (approximately 180–270 nm), suggesting that the CS layer has a certain hydration swelling and weak interparticle bridging effect in the physiological environment. The drug loading of the CS-modified system decreased slightly (approximately 12%–16%) because chitosan is a non-antigen polymer material; its introduction increases the overall mass of the nanoparticles, thus leading to a decrease in apparent drug loading under mass ratio calculations, but it does not affect the effective encapsulation ability of the antigen. After the introduction of hyaluronic acid (HA), the zeta potential of the nanoparticles recovered to a near-neutral level (approximately -2 mV), and the particle size significantly decreased and tended to stabilize (approximately 150–160 nm), indicating that HA can effectively neutralize the strong positive charge caused by CS and form a stable hydration layer structure on the particle surface. This hydration layer not only inhibits non-specific aggregation between nanoparticles but also improves the stability of the system in PBS 7.4, thereby enhancing overall dispersibility and structural uniformity. Simultaneously, the introduction of HA improves the interfacial hydration state to some extent, contributing to enhanced stable delivery in a mucus environment. All nanovaccine systems exhibited good structural stability in PBS 7.4, with minimal overall particle size changes, indicating no significant disintegration. The CS-modified system showed a slight increase in particle size, while the GSH and HA-modified systems demonstrated better stability maintenance, further proving that multilayer structure modification can effectively regulate the behavior of nanoparticles in a physiological environment.

[0030] Example 5: Evaluation of the interaction between nanovaccines and mucus The adhesion performance of nanovaccines in the nasal mucus environment was evaluated by using porcine gastric mucin (Mucin Type III) to simulate the nasal mucus environment. (1) Weigh Mucin Type III and add it to PBS buffer (pH 7.4) to prepare a 1% (w / v) mucin solution. Stir magnetically for 12 h at room temperature to fully dissolve and form a homogeneous system. Then centrifuge at 4000 rpm and 34 °C for 40 min and collect the supernatant. (2) Take equal volumes of P-OVA, GSH-P-OVA and CS-P-OVA nanovaccine suspensions respectively, measure the particle size of each nanovaccine, and then mix them with simulated mucus solution at a volume ratio of 1:1. Take samples and measure the particle size of each nanovaccine after mixing with the mucus. Then, after mixing, place them in a constant temperature incubator at 37℃ and take samples at 1h and 2h respectively. Measure the absorbance of each group of samples at a wavelength of 650nm. (3) Resample and mix with the mucus solution at a volume ratio of 1:1. Use a dynamic light scattering instrument to measure the changes in Zeta potential before and after mixing P-OVA, GSH-P-OVA and CS-P-OVA with the mucus.

[0031] Depend on Figure 2 It can be seen that in the absorbance change experiment after the nano-vaccine interacted with mucus, the absorbance of the Mucin group (blank control group) was 0.17, the absorbance of the OVA group was 0.18, the absorbance of the P-OVA group was 0.19, and the absorbance of the GSH-P-OVA group was 0.90. This indicates that the chitosan-modified nanoantigen can improve its binding with mucus, forming a larger complex, thus improving the adsorption and retention rate of the nano-vaccine. Figure 3 It can be seen that after the nano-vaccines bind to the mucus, the nanoparticle size of the P-OVA group increased from 80 nm to 521 nm, the GSH-P-OVA group increased from 112 nm to 263 nm, and the CS-P-OVA group increased from 132 nm to 1123 nm. This indicates that chitosan can significantly enhance the adsorption of the nano-vaccines onto the mucus. Figure 4It can be seen from the potential changes after the nanovaccine interacts with mucus. In the Mucin group (blank control group), the mucus itself carries a negative charge. After interaction with mucus, the potential of the P-OVA group changed from -30mV to -10mV, GSH-P-OVA from -25mV to -10mV, and CS-P-OVA from +35mV to -5mV. These experimental results show that the chitosan-modified CS-P-OVA interacts significantly with mucus, exhibiting a significant increase in absorbance and particle size from approximately 120 nm to over 1100 nm. Simultaneously, the Zeta potential changed from positive to near the negative potential of mucus. This indicates that chitosan can interact strongly with mucin, thereby enhancing the retention ability of the nanovaccine on the nasal mucosa surface. This demonstrates that chitosan endows the nanovaccine with excellent bioadhesion properties, providing sufficient antigen exposure time for subsequent nasal mucosal immunization.

[0032] Example 6: Induction of mouse nanovaccine uptake and maturation in antigen-presenting cells; Laboratory animals: 3-week-old healthy male C57BL / 6 mice, weighing 16-18g, obtained from the Experimental Animal Center of Hangzhou Medical College; and adapted to the environment at 18-25℃, 60-70% humidity, and a 12-hour light-dark cycle. All animal experiments were conducted in accordance with the Animal Care and Use Guidelines of the Chinese Physiological Society and with the permission of the Animal Breeding and Use Committee of Zhejiang Provincial Animal Medical Center. Adaptive feeding of laboratory animals: Forty-eight healthy male C57BL / 6 mice aged 3 weeks were used in this experiment. After being transferred to a standard animal facility, the mice underwent 3-5 days of acclimatization feeding to reduce the impact of environmental stress on the experiment.

[0033] Three-week-old male mice were selected and euthanized by cervical dislocation. The femurs and tibias were separated under sterile conditions. After the bone ends were removed, the bone marrow cavity was rinsed with pre-cooled PBS buffer, and the bone marrow cell suspension was collected. The suspension was filtered through a 70 μm cell sieve and then centrifuged at 1000 rpm for 5 min to remove the supernatant. Add erythrocyte lysis buffer and incubate at room temperature for 5 min. After lysis is terminated, centrifuge again to collect the cell pellet. Resuspend the cells in RPMI-1640 complete medium and add recombinant mouse GM-CSF (20 ng / mL) and IL-4 (10 ng / mL) to induce dendritic cell differentiation. Culture the dendritic cells in a 37°C, 5% CO2 incubator. Replace half of the medium on the third and fifth days, and collect the cells on the seventh day for later use. Bone marrow-derived dendritic cells (BMDCs) were collected at a rate of 1×10⁻⁶. 6Cells were inoculated into bacterial culture dishes at a density of 100 cells / dish and cultured overnight. Cy7 (Cyanine7) fluorescently labeled OVA, P-OVA, GSH-P-OVA, and CS-P-OVA were added, with consistent final OVA concentrations across all groups. After incubation at 37°C for 2 h and 6 h, cells were collected and washed three times with PBS to remove untaken free samples. Subsequently, CD11c-APC antibody (CD11c (Cluster of Differentiation 11c) is added for staining, and the cells were incubated on ice in the dark for 30 min. After staining, the cells were centrifuged, washed, and resuspended in PBS. Flow cytometry was used to detect the Cy7 fluorescence intensity in CD11c-positive cells, and the mean fluorescence intensity (MFI) was used to evaluate the cell uptake efficiency of different nanovaccines. BMDCs are arranged according to 1×10 6 Cells were inoculated into bacterial culture dishes at a density of 100 cells / dish and cultured overnight. OVA, P-OVA, GSH-P-OVA, CS-P-OVA, and Poly(I)-OVA (Poly(I) – Polyinosinic-polycytidylic acid, a classic dendritic cell activator) were added to each dish. After 24 h of culture, cells were collected, washed with PBS, resuspended, and stained with CD11c-APC antibody, CD80-FITC antibody, and CD86-PE antibody, respectively. CD80 (Cluster of Differentiation 80) and CD86 (Cluster of Differentiation 86) are co-stimulatory molecules upregulated during dendritic cell maturation; FITC (Fluorescein Isothiocyanate); and PE (Phycoerythrin) is a phycoerythrin fluorescent dye. After staining, the expression levels of CD80 and CD86 in the CD11c⁺ cell population were detected by flow cytometry.

[0034] Depend on Figure 5-8It can be seen that at 2 hours, the upper right quadrant of the CS-P-OVA group had the lowest uptake, while that of the GSH-P-OVA group had the highest. After 6 hours, the order of upper right quadrant area from largest to smallest was GSH-P-OVA group, P-OVA group, CS-P-OVA group, and OVA group. At 2 hours, the uptake rate of OVA group was 60%, P-OVA group was 75%, GSH-P-OVA group was 70%, and CS-P-OVA group was 10%. At 6 hours, the uptake rate of OVA group remained at 60%, while the uptake rate of P-OVA group increased to 95%, GSH-P-OVA group to 98%, and CS-P-OVA group to 90%. At 2 hours, the uptake per cell in OVA group was 170,000, and in P-OVA group it was 280,000. The uptake per cell was 250,000 in the GSH-P-OVA group and 100,000 in the CS-P-OVA group. At 6 hours, the uptake per cell was 290,000 in the OVA group, 580,000 in the P-OVA group, 650,000 in the GSH-P-OVA group, and 290,000 in the CS-P-OVA group. Flow cytometry results showed that the nano-vaccines all enhanced the antigen uptake capacity of BMDCs. Compared with free OVA, P-OVA and GSH-P-OVA showed higher cell uptake rates and average uptakes after 2 and 6 hours of incubation, with the GSH-P-OVA group showing the highest uptake. This indicates that the introduction of glutathione is beneficial in improving the binding stability of antigens to plant bodies and promoting antigen uptake by dendritic cells. At 2 hours, the CS-P-OVA group showed the highest uptake rate. The uptake rate was low at 1 h, but significantly increased after 6 h, indicating that chitosan can promote sustained endocytosis by enhancing the interaction between nanoparticles and the cell membrane. These results suggest that glycyrrhizin plant bodies, glutathione, and chitosan can synergistically improve the uptake efficiency of antigens by dendritic cells.

[0035] Example 7: Semi-quantitative fluorescence intensity of CY7-OVA in the nasal cavity of mice In this embodiment, the gel assembly uses carbomer (CP) as a bioadhesive material and poloxamer (P407) as a thermosensitive gel material. Specifically, a certain amount of P407 and CP are added to cold water and left overnight to fully dissolve or swell. Then, a nano-vaccine suspension is added to obtain the nano-vaccine thermosensitive gel.

[0036] (1) Nasal mucosal immunization was performed using 3-week-old mice. The humoral immune effect of the vaccine was assessed by measuring OVA-specific antibodies; the mucosal immune response to nasal mucosal delivery of the nanovaccine was evaluated by measuring IgA levels in nasal irrigation fluid; serum cytokine levels were measured to assess cellular immune effects; and the maturity of dendritic cells (DCs) in cervical lymph nodes was analyzed by flow cytometry to evaluate the in vivo immunostimulatory effect of the vaccine. Figure 9-12 It can be seen that, compared with the OVA solution, the IgG concentrations in all other administration groups were significantly increased, suggesting that nasal delivery of the nano-vaccine has a better systemic immune effect than the solution. The IgG level in GSH-P-OVA was 1.8 times that of the OVA solution group. The IgG level in the Poly(I:C) positive group was also significantly higher than that in the OVA solution group. The IgG concentrations in the GSH-P-OVA and gel groups were higher than those in the positive group, and the differences were statistically significant. These results indicate that nasal mucosal delivery of the nano-vaccine can effectively generate an antigen-specific humoral immune response, and the antibody level is superior to that of the positive control Poly(I:C), thus forming a better humoral immune barrier.

[0037] (2) The immune response of the nanovaccine on the nasal mucosa was investigated by measuring the levels of OVA-specific IgA and sIgA in the nasal lavage fluid. Figure 13-14 The results showed that GSH-P-OVA, thermosensitive gel, and positive control Poly (I:C) could significantly increase the concentration of IgA in the nasal mucosa. GSH-P-OVA and CS-P-OVA could significantly increase the level of sIgA, indicating that nanovaccines can further enhance the mucosal immune barrier function.

[0038] (3) The expression levels of Th1-related cytokines IFN-γ and IL-2 and Th2-related cytokines IL-4 and IL-10 in the serum of mice after intranasal immunization with OVA, GSH-P-OVA, CS-P-OVS, GEL, and Poly(I:C) were determined by ELISA. Figure 15-18 It was found that after inoculation with GSH-P-OVA, CS-P-OVA, and thermosensitive gel, serum Th1 cytokine IFN-γ and Th2 cytokine IL-4 were significantly increased compared to OVA solution and also significantly higher than the positive control group, with statistically significant differences. Based on the changes in cytokine expression levels, it is suggested that nasal mucosal delivery of nano-vaccines can enhance cellular immune responses, with better effects than Poly(I:C).

[0039] (4) Maturity of dendritic cells in lymph nodes after intranasal immunization with OVA, GSH-P-OVA, CS-P-OVS, GEL, and Poly(I:C) in mice. Figure 19It can be seen that GSH-P-OVA, CS-P-OVS, and GEL significantly increased the expression of CD86 on the surface of dendritic cells, which were 2.08, 2.02, and 1.85 times higher than those in the OVA solution group, respectively. This suggests that the nasal delivery of nano-vaccines can effectively stimulate the maturation of dendritic cells in lymph nodes, thereby promoting the presentation of antigens by dendritic cells to T cells and B cells, and triggering corresponding cellular and humoral immunity.

[0040] (5) Nasal immunization in mice: Three-week-old mice were immunized via the nasal mucosa. The proportion of memory lymphocytes in CD8 lymphocytes of the spleen was measured. Figure 20 It can be seen that the hyaluronic acid (HA) modified group was significantly higher than the P-OVA group, and HA modification can significantly increase CD44. + CD62L + The proportion of CD8 lymphocytes indicates that memory CD8 T cells play a central role in resisting viral infection and providing long-term protection.

[0041] The above embodiments demonstrate that the immune platform constructed by the vaccine of the present invention is a self-assembled nasal mucosal nanovaccine that combines high-efficiency antigen loading, long-term residence in the nasal mucosa, mucus penetration, and immune enhancement. Glycyrrhizin plant bodies serve as the core carrier for antigen delivery, glutathione enhances antigen loading capacity, chitosan enhances nasal mucosal adhesion, and hyaluronic acid improves particle stability and mucus penetration. Through the synergistic effect of the above multi-layered structure, the nanovaccine exhibits high encapsulation efficiency, good physicochemical stability, and excellent mucosal delivery performance. It can significantly promote the uptake and maturation of antigens by BMDCs, increase the levels of antigen-specific IgG, IgA, and sIgA, thereby simultaneously inducing systemic and mucosal immune responses, establishing an effective immune barrier, and significantly improving the humoral and mucosal immune levels induced by the nasal mucosal vaccine.

[0042] It should be noted that the above embodiments and comparative examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the present invention.

Claims

1. A self-assembled nasal mucosal nanovaccine, characterized in that, The nanovaccine has a core-shell structure; The vaccine comprises a plant body core, an antigen-loaded layer, a bioadhesion layer, and a hydrophilic protective layer; The core of the plant body is a glycyrrhizin plant body; The antigen-loaded layer is a glutathione-antigen complex formed by glutathione and antigen; The bio-adhesive layer is chitosan; The hydrophilic protective layer is formed of hyaluronic acid.

2. The self-assembled nasal mucosal nanovaccine according to claim 1, characterized in that, The licorice saponin plant body is composed of phospholipids and licorice saponins in a ratio of 1:0.7-1.4; the phospholipids are one or more of soybean phospholipids, lecithin, and hydrogenated phospholipids; the licorice saponins are one or more of glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin G2, and ural glycyrrhizin; the chitosan has a molecular weight of 50-300 kDa, and the hyaluronic acid has a molecular weight of 10-100 kDa.

3. The self-assembled nasal mucosal nanovaccine according to claim 1, characterized in that, The mass ratio of glutathione to antigen in the glutathione-antigen complex is 0.5-2:

1.

4. The self-assembled nasal mucosal nanovaccine according to claim 1, characterized in that, The mass ratio of the glycyrrhizin plant body, glutathione-antigen complex, chitosan, and hyaluronic acid is 12-20:6-10:2-8:2-4.

5. A self-assembled nasal mucosal nanovaccine according to claim 1 or 2, characterized in that, The particle size of the licorice saponin plant bodies is 20-30 nm.

6. A method for preparing a nano-vaccine, characterized in that, A self-assembled nasal mucosal nanovaccine according to claims 1-5 comprises the following steps: S1. Prepare glycyrrhizin plant bodies, then dissolve them in ultrapure water to obtain a glycyrrhizin plant body solution; S2. Dissolve the antigen in ultrapure water to obtain an antigen solution; S3. Add glutathione to the antigen solution and mix well to obtain a glutathione-antigen complex solution; S4. Add the glutathione-antigen complex solution to the glycyrrhizin plant body solution to form the glycyrrhizin plant body-antigen complex through self-assembly. S5. Add chitosan solution to glycyrrhizin plant body-antigen complex, then add hyaluronic acid solution and stir to obtain nano-vaccine.

7. The method for preparing a nano-vaccine according to claim 6, characterized in that, The method for preparing glycyrrhizin plant bodies in S1 includes the following steps: S11. Dissolve phospholipids in tetrahydrofuran, then add glycyrrhizin, and stir in a water bath to obtain a mixture; S12. Filter the stirred mixture and heat it by rotary evaporation to obtain the complex.

8. The method for preparing a nano-vaccine according to claim 6, characterized in that, The mixing time for S3 is 10-30 min, and the stirring speed is 200-500 rpm.

9. The method for preparing a nano-vaccine according to claim 6, characterized in that, The glutathione-antigen complex solution of S4 was added to the glycyrrhizin plant body solution at a dropping rate of 0.5-1.5 ml / min and stirred at room temperature for 1-2 hours.

10. The method for preparing a nano-vaccine according to claim 6, characterized in that, After adding the chitosan solution of S5, stir at room temperature for 300-400 rpm for 1-2 hours; adjust the pH to 5.5-6.5; add the hyaluronic acid and stir at 300-500 rpm for 1-2 hours.

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