M cell-targeting peptide and chitosan-modified dendritic mesoporous silica oral adjuvant and application thereof

Abstract

The present application relates to the technical field of biological agents, and discloses a kind of M cell targeted peptide and chitosan modified dendritic mesoporous silica oral adjuvant and application thereof.The oral adjuvant includes dendritic mesoporous silica (DFNSs), and M cell targeted peptide (CKS9) modified chitosan (CS), the dendritic mesoporous silica is mixed with M cell targeted peptide modified chitosan at mass ratio 4-6:1, and is made into CKS9-CS-DFNSs oral nano adjuvant.The CKS9-CS-DFNSs oral nano adjuvant provided in the present application has good stability and safety, can effectively induce mucosal immune response and humoral immune response by oral immunization, improve the effect of oral vaccine immunization, and can be widely used in the preparation of animal inactivated vaccine, recombinant protein vaccine and polypeptide vaccine.The oral nano adjuvant preparation has low cost, simple process and is easy to mass-produce.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to an oral adjuvant of M-cell-targeting peptide and chitosan-modified dendritic mesoporous silica and its application. Background Technology

[0002] Vaccines are biological products used for preventative inoculation. They stimulate the animal's immune system to produce a specific immune response, forming immunity against specific pathogens and thus preventing corresponding diseases. In animal disease control, vaccines are a key measure for preventing and controlling infectious diseases, and are also the most economical and effective tool. Vaccines typically contain antigenic components, such as viral or bacterial proteins or polysaccharides. The effectiveness of a vaccine is closely related to adjuvants, which are non-specific immune enhancers that bind to the vaccine. Adjuvants themselves are not antigenic, but when used with vaccine antigens, they enhance the body's immune response to the antigens, improving the vaccine's protective effect. In vaccine production, adjuvants can also reduce the amount of immunogen used, lowering vaccine costs.

[0003] In intensive animal farming, most pathogens invade the animal body primarily through mucosal surfaces (such as the oral cavity, nasal cavity, and intestines). Therefore, a sustained and effective mucosal immune response is crucial for protecting animal health. Currently, animal vaccines mainly use injectable adjuvants, such as aluminum salt adjuvants, oil emulsion adjuvants, and immunologically active substances (polysaccharides, propolis, Toll-like receptor 9 agonists, etc.). Injectable adjuvants induce a specific immune response in animals through intramuscular or subcutaneous injection. However, they suffer from drawbacks such as high animal stress, high labor costs, and the inability to generate effective mucosal protective antibodies. In particular, they are poorly effective against intestinal diseases, such as porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and enterotoxigenic Escherichia coli. Therefore, developing vaccine adjuvants that target mucosal immunity may be one of the best strategies to improve vaccine immunogenicity. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing an oral adjuvant of M-cell-targeting peptide and chitosan-modified dendritic mesoporous silica, as well as its preparation method and application.

[0005] Invention Concept: The intestine is the body's largest digestive and absorptive organ, characterized by a highly developed mucosal immune system and extensive mucosal membranes that come into contact with the internal and external environments of the body. Oral immunization offers advantages such as convenient administration, high patient compliance, and good safety. Therefore, developing oral vaccine adjuvants is one of the best ways to induce both highly effective humoral and mucosal immune responses in vaccines. However, the acidic environment of the intestine and various digestive enzymes can degrade vaccine structures, while chemical and epithelial barriers hinder the absorption of macromolecules, limiting antigen uptake and immunogenicity. Therefore, protecting antigens and enabling them to cross the intestinal barrier for targeted delivery is crucial for oral vaccine development. M cells are intestinal epithelial cells capable of absorbing macromolecules from the intestinal lumen, capturing intestinal antigens, and initiating an intestinal mucosal immune response. Chitosan (CS), a natural positively charged active polysaccharide, possesses high biocompatibility, degradability, and good chemical modification activity. It can enhance immune cell activity, promote antigen retention at the mucosal site, and increase the uptake rate of antigens by M cells. Dendritic fibrous nano-silica (DFNSs) oral nanodelivery systems offer advantages such as large specific surface area, adjustable pore and particle size, good biocompatibility, uniform distribution of target molecules in the pore space, simultaneous loading of multiple therapeutic drugs, low cost, strong controllability, and simple production, and are commonly used for oral drug delivery. Therefore, this invention combines CKS9, CS, and DFNSs into a CKS9-CS-DFNSs oral adjuvant through chemical and physical modification methods to overcome the limitations of oral vaccines and thereby enhance the protective effect of vaccines.

[0006] To solve the above-mentioned technical problems, the present invention discloses the following technical solution:

[0007] In a first aspect, the present invention discloses an oral adjuvant of M-cell targeting peptide and chitosan modified dendritic mesoporous silica (CKS9-CS-DFNSs oral nanoadjuvant).

[0008] In some embodiments, the CKS9-CS-DFNSs oral nanoadjuvant comprises dendritic mesoporous silica (DFNSs) and chitosan modified with M-cell-targeting peptides (CKS9-CS). DFNSs is the core of the drug / antigen loading, soluble in 0.9% physiological saline, and negatively charged; CKS9-CS is key to targeting M cells and improving intestinal uptake efficiency, soluble in phosphate buffer with pH ≤ 6.2, and positively charged.

[0009] In some embodiments, the DFNSs and CKS9-CS are mixed at a mass ratio of 4-6:1 to prepare CKS9-CS-DFNSs oral nanoadjuvants, and in some embodiments, they are mixed at a mass ratio of 5:1.

[0010] In some embodiments, the CKS9-CS-DFNSs oral nanoadjuvant is spherical with a diameter of 190-220 nm, a zeta potential of 25-30 mV, and a PDI dispersion index of 0.2-0.3.

[0011] In some embodiments, the DFNSs are spherical, appearing as mesoporous, dendritic structures under transmission electron microscopy, with a diameter of 150-200 nm (e.g., 180-190 nm), a zeta potential of -20 to -15 mV, and a PDI dispersion index of 0.1-0.2. The drug loading capacity of dendritic mesoporous silica is higher than that of ordinary silica, and the pore volume of dendritic mesoporous silica is 1.5-1.8 cm³. 3 / g, the pore volume of ordinary mesoporous silica is ≤1 cm³. 3 / g, and it is stable.

[0012] In some embodiments, the amino acid sequence of the M cell targeting peptide (CKS9) is shown in SEQ ID NO. 1 (CKSTHPLSC), and the amino group of its tail cysteine ​​is acetylated to form an acetylated M cell targeting peptide.

[0013] In some embodiments, the chitosan (CS) has a weight-average molecular weight of 50-100 kDa, a degree of deacetylation ≥85%, and is soluble at pH ≤6.2.

[0014] In some embodiments, CKS9-CS is formed by chemically linking CKS9 and CS. In some embodiments, it is formed by linking M cell targeting peptide and chitosan via an amide bond. In some embodiments, the substitution rate of CKS9 is 6-7 mol%, and in some embodiments, it is 6.49 mol%. In this invention, the substitution rate of CKS9 refers to the molar ratio of CKS9 to CS.

[0015] Secondly, the present invention discloses a method for preparing the CKS9-CS-DFNSs oral nanoadjuvant described in the first aspect above.

[0016] In some embodiments, the preparation method includes:

[0017] (1) Disperse dendritic mesoporous silica in physiological saline or citrate buffer solution, autoclave, centrifuge, and resuspend the resulting precipitate in physiological saline to obtain DFNSs dispersion;

[0018] (2) Dissolve the chitosan modified with M cell targeting peptide in phosphate buffer, filter to remove bacteria, and prepare CKS9-CS solution;

[0019] (3) The DFNSs dispersion and CKS9-CS solution are thoroughly mixed under sterile conditions to prepare CKS9-CS-DFNSs oral nanoadjuvant.

[0020] In step (1), the mass-to-volume ratio of the dendritic mesoporous silica to physiological saline or citrate buffer solution is 4-6 mg / mL, such as 5 mg / mL; in some embodiments, the volume ratio of the physiological saline used for resuspension to the volume ratio of the initially added physiological saline or citrate buffer solution is 1:0.5-1.5, and in some embodiments it is 1:1.

[0021] In step (2), the pH of the phosphate buffer solution is below 6.2, such as 6.0.

[0022] In step (2), the concentration of the CKS9-CS solution is 0.5-1.5 mg / mL, and in some embodiments it is 1 mg / mL.

[0023] In step (3), the volume ratio of the DFNSs material dispersion to the CKS9-CS solution is 1:0.5-1.5, and in some embodiments it is 1:1.

[0024] Thirdly, this invention discloses a vaccine.

[0025] In some embodiments, the vaccine is obtained by combining the M-cell targeting peptide and chitosan-modified dendritic mesoporous silica oral adjuvant prepared by the method described in the first aspect with the antigen.

[0026] In some embodiments, the antigen is an immunogenic antigen, in some embodiments it is a water-soluble protein, and in some embodiments it is BSA and / or PEDV.

[0027] In some embodiments, when the antigen is BSA, the mass ratio of the antigen to DFNSs is 1:4-6. In some embodiments, when the antigen is PEDV, the volume-to-mass ratio of the antigen to DFNSs is 1 mL:95-105 mg.

[0028] In some embodiments, the oral adjuvant in the vaccine has a drug loading rate of 10%-20% and an encapsulation rate of 70%-80%.

[0029] Fourthly, the present invention discloses a method for preparing the vaccine described in the third aspect.

[0030] The preparation method includes mixing a DFNSs solution with an antigen, centrifuging, and resuspending the resulting precipitate; mixing the resuspended solution with a solution containing CKS9-CS, stirring, and obtaining an oral vaccine with CKS9-CS-DFNSs oral nanoadjuvant.

[0031] In some embodiments, the antigen is an immunogenic antigen, in some embodiments it is a water-soluble protein, and in some embodiments it is BSA and / or PEDV.

[0032] In some embodiments, when the antigen is BSA, the mass ratio of the antigen to DFNSs is 1:4-6. In some embodiments, when the antigen is PEDV, the volume-to-mass ratio of the antigen to DFNSs is 1 mL:95-105 mg.

[0033] In some embodiments, the concentration of the DFNSs solution is 4-6 mg / mL, such as 5 mg / mL; the DFNSs solution is prepared by dissolving DFNSs in physiological saline or citrate buffer solution and then autoclaving.

[0034] In some embodiments, the centrifugation is performed at 10,000-15,000 rpm for 8-12 minutes.

[0035] In some embodiments, the resuspension is a resuspension with physiological saline, wherein the volume ratio of the physiological saline used for resuspension to the volume of the initially added physiological saline or citrate buffer solution is 1:0.5-1.5, and in some embodiments it is 1:1.

[0036] In some embodiments, the solvent of the CKS9-CS solution is a phosphate buffer with a pH below 6.2, such as a phosphate buffer with pH=6; in some embodiments, the concentration of the CKS9-CS solution is 0.5-1.5 mg / mL, and in some embodiments it is 1 mg / mL.

[0037] In some embodiments, the volume ratio of the supernatant to the CKS9-CS solution is 1:0.5-1.5, and in some embodiments it is 1:1.

[0038] Fifthly, the present invention discloses the use of the CKS9-CS-DFNSs oral nanoadjuvant described in the first aspect, or the CKS9-CS-DFNSs oral nanoadjuvant prepared by the preparation method described in the second aspect, or the vaccine described in the third aspect, or the vaccine prepared by the preparation method described in the fourth aspect, in the preparation of oral immunomodulators containing protein antigens.

[0039] In some embodiments, the formulation includes a vaccine, and in some embodiments, an animal vaccine.

[0040] In some embodiments, the protein antigen is bovine serum albumin (BSA); in some embodiments, the protein antigen is derived from virions, and in some embodiments, it is porcine epidemic diarrhea virus (PEDV).

[0041] In this invention, the method for preparing dendritic mesoporous silica is as follows: Dendritic mesoporous silica is prepared using a template method with triethanolamine, hexadecyltrimethylammonium bromide, sodium salicylate, and tetraethyl orthosilicate. In some embodiments, the mass ratio of triethanolamine, hexadecyltrimethylammonium bromide, sodium salicylate, and tetraethyl orthosilicate is 850-950 mg: 5-6 g: 1-2 g: 50-60 mL. In some embodiments, a first reaction is carried out with an aqueous solution of triethanolamine, a second reaction is carried out with the addition of hexadecyltrimethylammonium bromide, a third reaction is carried out with the addition of sodium salicylate, and a fourth reaction is carried out with the addition of tetraethyl orthosilicate. The precipitate is collected and calcined to obtain DFNSs. In some embodiments, the concentration of the triethanolamine aqueous solution is 2-3 mg / mL; in some embodiments, the temperature of the first reaction is 75-85°C, and the reaction time is 0.5-1.5 h; in some embodiments, the reaction time is 0.5-1.5 h; in some embodiments, the reaction time is 2.5-3.5 h; in some embodiments, the reaction time is 2.5-3.5 h; in some embodiments, the calcination is performed at 500-650°C for 5-7 h.

[0042] In this invention, the preparation method of the M-cell-targeting peptide-modified chitosan is as follows: using N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as catalysts, CKS9 and CS are linked via an amide reaction. In some embodiments, the mass ratio of CKS9 to CS is 1:0.5-1.5, and in some embodiments it is 1:1; in some embodiments, CKS9 is activated at room temperature under the action of EDC and NHS, mixed with a solution containing CS, reacted, the resulting reactant is dialyzed, and the product is freeze-dried to obtain the CKS9-CS complex; in some embodiments, the concentration of CS in the solution containing CS is 30-36 mg / mL; in some embodiments, the reaction is carried out at room temperature for 20-28 h; in some embodiments, the molecular weight cutoff of the dialysis bag during dialysis is 2500-3500 Da, and in some embodiments it is 3000 Da.

[0043] The technical solution provided by this invention effectively solves the problems of low delivery efficiency and poor immune protection of oral vaccines. The oral vaccine based on chitosan-encapsulated dendritic mesoporous silica oral nanoadjuvant preparation modified with M-cell targeting peptide provided by this invention, is a composite preparation of dendritic mesoporous silica material (DFNSs), M-cell targeting peptide CKS9 and chitosan CS. It can be used as an oral adjuvant to cooperate with multiple antigen components to obtain a complete vaccine. The vaccine has good stability and safety, good sustained-release properties, and can simultaneously induce mucosal immunity and humoral immunity, thereby improving the long-term immune effect of the vaccine and enhancing the protective effect against mucosal and intestinal infectious diseases.

[0044] Beneficial effects:

[0045] (1) The oral nano-adjuvant CKS9-CS-DFNSs prepared by the present invention can solve the problems of time-consuming and laborious immunization, poor mucosal protection effect and poor effect of preventing intestinal pathogen infection of existing injectable adjuvants. Moreover, the adjuvant has low preparation cost, high delivery efficiency and strong immunization ability, which can reduce vaccine cost.

[0046] (2) In the existing adjuvants, chitosan can only be water-soluble low-molecular-weight chitosan (≤10 kDa). The preparation process of low-molecular-weight water-soluble chitosan is complex and costly. The preparation of CKS9-CS in the adjuvant provided by this invention has lower material requirements and a wider range. It can use high-molecular-weight chitosan with a molecular weight of 50-100 kDa. The preparation cost of the chitosan used in this invention is significantly lower than that of low-molecular-weight water-soluble chitosan, which is conducive to industrial production.

[0047] (3) The adjuvant of the present invention is convenient for adsorbing antigens, requiring only simple shaking and mixing, and has strong stability. Attached Figure Description

[0048] Figure 1 A flowchart for preparing dendritic mesoporous silica.

[0049] Figure 2 This is a diagram showing the properties of dendritic mesoporous silica.

[0050] Figure 3 This is a scanning electron microscope image of dendritic mesoporous silica.

[0051] Figure 4 This is a transmission electron microscope (TEM) image of dendritic mesoporous silica.

[0052] Figure 5 This is a diagram showing nitrogen adsorption in dendritic mesoporous silica.

[0053] Figure 6 This is a diagram showing dendritic mesoporous silica dissolved in physiological saline.

[0054] Figure 7The chemical structure of acetylated CKS9 is given.

[0055] Figure 8 Here is the chemical structural formula of CS.

[0056] Figure 9 This is the NMR spectrum of the CKS9-CS complex.

[0057] Figure 10 The image shows the infrared spectrum of the CKS9-CS complex.

[0058] Figure 11 The chemical structural formula of the CKS9-CS complex is shown below.

[0059] Figure 12 This is a phenotypic diagram of CKS9-CS-DFNSs / BSA.

[0060] Figure 13 Scanning electron microscope image of CKS9-CS-DFNSs / BSA.

[0061] Figure 14 Transmission electron microscopy image of CKS9-CS-DFNSs / BSA.

[0062] Figure 15 The image shows the results of intestinal tissue analysis in mice, including the blank control group and the CKS9-CS-DFNSs / BSA vaccine group.

[0063] Figure 16 The image shows the results of intestinal flushing fluid analysis in mice from the blank control group, the CKS9-CS-DFNSs / BSA vaccine group, etc.

[0064] Figure 17 The graph shows the serum test results of mice in the blank group, CKS9-CS-DFNSs / BSA vaccine group, etc.

[0065] Figure 18 The graph shows the results of the examination of the spleen, kidney, liver, duodenum, jejunum and ileum of mice in the blank group, CKS9-CS-DFNSs / BSA vaccine group and other groups.

[0066] Figure 19 The graph shows the effects of the control group, the CKS9-CS-DFNSs / PEDV vaccine group, etc., on piglet feces.

[0067] Figure 20 Dissection and mesenteric lymph node diagrams of piglets in the blank group, CKS9-CS-DFNSs / PEDV vaccine group, etc. Detailed Implementation

[0068] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0069] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0070] The substitution rate of CKS9 mentioned in this invention refers to the molar amount of chitosan CS that is replaced by CKS9. The percentage of substitution is calculated by comparing it with the NMR peak intensity of chitosan.

[0071] Example 1: Preparation of drug-loaded nanoparticles—dendritic mesoporous silica (DFNSs)

[0072] Dendritic mesoporous silica is spherical mesoporous silica. Transmission electron microscopy reveals that the mesopores resemble a dendritic structure, exhibiting a larger specific surface area and higher drug loading capacity than ordinary mesoporous silica. For example... Figure 1 As shown, the preparation steps are as follows:

[0073] (1) Weigh 900 mg of triethanolamine (TEA) and place it in a round-bottom flask. Add 360 mL of pure water and heat and stir in a constant temperature heater for 1 hour at a reaction temperature of 80℃ and a rotation speed of 700 rpm.

[0074] (2) Weigh 5.7 g of hexadecyltrimethylammonium bromide (CTAB) and add it to the reaction solution in step (1) and react for 1 hour;

[0075] (3) Weigh 1.5 g of sodium salicylate and add it to the reaction liquid in step (2), and react for 3 hours;

[0076] (4) Add 54 mL of tetraethyl orthosilicate (TEOS) to the reaction solution in step (3) and react for 3 hours;

[0077] (5) Place the reaction product from step (4) into a 50 mL centrifuge tube (EP), centrifuge at 8000 rpm for 15 minutes, discard the supernatant, and collect the precipitate;

[0078] (6) Wash the precipitate from step (5) three times with anhydrous ethanol, and then wash it three times with pure water;

[0079] (7) The product from step (6) is dried and calcined at 600°C for 6 hours to remove the template, thus obtaining white powdered DFNSs, such as Figure 2 As shown.

[0080] The product in step (7) was analyzed by scanning electron microscopy, transmission electron microscopy, and nitrogen adsorption. The results are as follows: Figure 3 , Figure 4 and Figure 5 As shown, it is proven to be mesoporous silica with a dendritic structure.

[0081] Dissolve DFNSs in physiological saline, such as Figure 6 As shown in Table 1, the particle size, potential and PDI were measured using a particle size analyzer.

[0082] Table 1. Particle size, potential, and PDI of dendritic mesoporous silica

[0083]

[0084] Example 2: Preparation of chitosan complex modified with M-cell-targeting peptides

[0085] This positively charged complex can adsorb onto the negatively charged particle surface. It consists of an acetylated M-cell targeting peptide (AC-CKSTHPLSC, CKS9) and high molecular weight (50-100 kDa) CS. The preparation steps are as follows:

[0086] (1) Weigh 100 mg of acetylated CKS9 and dissolve it in 2 mL of water (pH=6) containing EDC (100 mg) and NHS (40 mg), and activate it at room temperature for 6 hours. The chemical structure of acetylated CKS9 is as follows. Figure 7 As shown;

[0087] (2) Mix the product from step (1) with 3 mL of CS solution (containing 100 mg of CS, pH=6) (the structural formula of CS is shown in Figure 1). Figure 8 As shown), the reaction was carried out at 220 rpm for 24 hours at room temperature;

[0088] (3) Place the reaction product from step (2) in a 3000 Da dialysis bag, dialyze for 3 days, and freeze-dry the product.

[0089] The product was analyzed by NMR and IR, and the results are as follows: Figure 9 and Figure 10 As shown in the infrared spectrum, a peak generated by the C=O stretching vibration of the amide bond is observed at 1630.76, proving that CKS9 has been grafted onto CS, resulting in the CKS9-CS complex, the chemical formula of which is shown below. Figure 11 As shown (a indicates that a molar amount of a group is replaced by CKS9), the substitution rate of CKS9 in this embodiment is 6.49%.

[0090] Example 3: Preparation of oral nanoadjuvants consisting of chitosan-encapsulated dendritic mesoporous silica modified with M-cell-targeting peptides.

[0091] (1) Weigh 100 mg of the DFNSs prepared in Example 1 and dissolve it in 20 mL of physiological saline and autoclave it; centrifuge at 12000 rmp for 10 minutes, wash 3 times, and collect the supernatant and precipitate; resuspend the precipitate in 20 mL of sterile physiological saline for subsequent oral nanoadjuvant preparation;

[0092] (2) Weigh 20 mg of the CKS9-CS complex prepared in Example 2 and dissolve it in 20 mL of phosphate buffer at pH=6 and filter it for sterilization;

[0093] (3) Mix the liquid resuspended in physiological saline in step (1) with the liquid in step (2) at a volume ratio of 1:1, stir well, and prepare the oral nano-adjuvant CKS9-CS-DFNSs.

[0094] The particle size, potential, and PDI of the oral nanovaccine CKS9-CS-DFNSs were measured using a particle size analyzer, and the results are shown in Table 2.

[0095] Table 2. Particle size, potential, and PDI of oral nanovaccine CKS9-CS-DFNSs

[0096]

[0097] Example 4: Preparation of an oral nanovaccine consisting of a chitosan-encapsulated dendritic mesoporous silica nanoparticle modified with a BSA-based M-cell-targeting peptide.

[0098] The vaccine's drug delivery core consists of DFNSs, and the outer layer is formed by the encapsulation of a CKS9-CS complex. The preparation steps are as follows:

[0099] (1) Weigh 100 mg of the DFNSs prepared in Example 1 and dissolve it in 20 mL of physiological saline and autoclave it; then, add 20 mg of BSA, mix and stir; centrifuge at 12000 rmp for 10 minutes, wash 3 times, and collect the supernatant and precipitate; the precipitate is the oral nanovaccine DFNSs / BSA, and the drug loading rate of DFNSs is 20% and the encapsulation rate is 80%; the precipitate is resuspended in 20 mL of sterile physiological saline for subsequent vaccine preparation;

[0100] (2) Weigh 20 mg of the CKS9-CS complex prepared in Example 2 and dissolve it in 20 mL of phosphate buffer at pH=6 and filter it for sterilization;

[0101] (3) Mix the liquid resuspended in physiological saline in step (1) with the liquid in step (2) at a volume ratio of 1:1, stir well, and prepare the oral nanovaccine CKS9-CS-DFNSs / BSA, as shown. Figure 12 As shown.

[0102] The particle size, potential, and PDI of the oral nanovaccine CKS9-CS-DFNSs / BSA were measured using a particle size analyzer, and the results are shown in Table 3.

[0103] Table 3. Particle size, potential, and PDI of oral nanovaccine CKS9-CS-DFNSs / BSA

[0104]

[0105] The oral nanovaccine CKS9-CS-DFNSs / BSA was examined by scanning electron microscopy and transmission electron microscopy, and the results are as follows: Figure 13 and Figure 14 As shown.

[0106] Example 5: Application of CKS9-CS-DFNSs as an oral adjuvant in enhancing the oral immunogenicity of the model antigen BSA

[0107] Six-week-old SPF mice were divided into four groups: a control group, a BSA group, a DFNSs / BSA group, and a CKS9-CS-DFNSs / BSA group. Oral vaccine immunization experiments were conducted. Mice in the control group were orally administered saline via gavage; mice in the BSA group were orally administered BSA solution; mice in the DFNSs / BSA group were orally administered DFNSs / BSA vaccine; and mice in the CKS9-CS-DFNSs / BSA group were orally administered CKS9-CS-DFNSs / BSA vaccine. Oral immunization was performed on days 1-3, 14, and 21, for a total of 42 days. Serum and intestinal flushing fluid were collected at days 14, 21, 28, 35, and 42. Spleen, kidney, liver, duodenum, jejunum, ileum, and intestinal tissue were collected from mice at day 42.

[0108] The results are as follows:

[0109] (1) Detect the intestinal tissue of mice, such as Figure 15 As shown, the CKS9-CS-DFNSs / BSA vaccine significantly increased IL-4, IL-6, IL-17 and TNF-α, indicating that the vaccine has a good immunization effect.

[0110] (2) Detect mouse intestinal flushing fluid, such as Figure 16 As shown, the levels of sIgA and specific BSA-IgA antibodies were significantly increased in the CKS9-CS-DFNSs / BSA vaccine group, and this increase persisted over time. In the later stages, the increase in the CKS9-CS-DFNSs / BSA group was higher than that in the DFNSs / BSA group and the BSA group.

[0111] (3) Detect mouse serum, such as Figure 17As shown, the levels of specific BSA-IgG, BSA-IgG1, BSA-IgG2a, and BSA-IgG2b antibodies were significantly increased in the CKS9-CS-DFNSs / BSA vaccine group, and this increase persisted over time. Furthermore, in the later stages, the levels of these antibodies in the CKS9-CS-DFNSs / BSA group were higher than those in the DFNSs / BSA group and the BSA group. Figure 16 and Figure 17 The comparison revealed that both CKS9-CS-DFNSs / BSA and DFNSs / BSA could simultaneously enhance intestinal mucosal immunity and humoral immunity. However, over time, the CKS9-CS-DFNSs / BSA group was more effective in increasing serum antibody levels.

[0112] (4) The spleen, kidneys, liver, duodenum, jejunum, and ileum of the mice were examined, and the results are as follows: Figure 18 As shown, no pathological damage was found, indicating that CKS9-CS-DFNSs / BSA is safe for mice.

[0113] By implementing the above scheme, a safe and efficient oral adjuvant CKS9-CS-DFNSs can be prepared, which can significantly enhance the intestinal mucosal immune response and humoral immune response in mice, increase the level of specific antibodies, and protect the body's health.

[0114] Example 6: Application of CKS9-CS-DFNSs as an oral adjuvant in enhancing the oral immunization efficacy of PEDV vaccine

[0115] Twenty-day-old pigs that were negative for PRRS, African swine fever, and PEDV antibodies were divided into four groups: a control group, a challenge group, a PEDV group, a CKS9-CS / PEDV group, and a CKS9-CS-DFNSs / PEDV group, with 10 pigs in each group, for oral vaccine immunization experiments. Specifically, piglets in the control and challenge groups were administered saline orally via gavage, the PEDV group was administered PEDV inactivated vaccine via gavage, the CKS9-CS / PEDV group was administered CKS9-CS / PEDV vaccine via gavage, and the CKS9-CS-DFNSs / PEDV group was administered CKS9-CS-DFNSs / PEDV vaccine via gavage. The vaccine dosage for all groups was 5 mL × 10⁻¹⁰. 8 PFU / mL PEDV inactivated vaccine.

[0116] The preparation methods for CKS9-CS / PEDV vaccine and CKS9-CS-DFNSs / PEDV are described in Example 4, except that the antigen is replaced with PEDV. In step (1), 100 mg of the DFNSs prepared in Example 1 is weighed and dissolved in 20 mL of physiological saline and then autoclaved. Then, 1 mL of 1×10 8PFU / mL PEDV inactivated vaccine, mix well and stir; centrifuge at 12000 rmp for 10 minutes, wash 3 times, collect the precipitate, which is the CKS9-CS / PEDV vaccine; the obtained precipitate is resuspended in 20 mL of sterile physiological saline for subsequent vaccine preparation. Referring to the calculation method of drug loading rate and encapsulation rate in Example 4, the drug loading rate is 18% and the encapsulation rate is 76%.

[0117] Oral immunization was administered on days 1-3, 7, and 14 of the experiment. Following the initial immunization, on day 21, a PEDV challenge experiment was conducted. Piglets in the challenge group, PEDV group, CKS9-CS / PEDV group, and CKS9-CS-DFNSs / PEDV group were given 10 mL / head (1×10⁻⁶) of the vaccine. 8 PEDV live virus (PFU / mL) was used to record diarrhea and mortality in piglets. Ten days after challenge, the mesenteric lymph nodes of the piglets were dissected and collected.

[0118] The results are as follows:

[0119] (1) Statistics on diarrhea rate and mortality rate of piglets after challenge with the virus are shown in Table 4.

[0120] Table 4. Mortality rate, diarrhea rate, and mental status of piglets

[0121]

[0122] (2) After the virus challenge, the condition of the piglets' feces is shown in the figure. Figure 19 Piglets exhibited diarrhea after viral challenge, but the CKS9-CS-DFNSs / PEDV group did not show obvious diarrhea symptoms, similar to the control group, indicating a significant protective effect.

[0123] (3) Post-infection necropsy of piglets and findings of mesenteric lymph nodes, see [see details]. Figure 20 After the virus challenge, piglets showed hemorrhage and swelling of the mesenteric lymph nodes. The CKS9-CS-DFNSs / PEDV group did not show obvious hemorrhage and swelling of the mesenteric lymph nodes, similar to the control group, indicating that its protective effect was obvious.

[0124] The implementation of this program showed that CKS9-CS-DFNSs / PEDV can effectively protect piglets from infection by live PEDV, enhance intestinal mucosal immunity and humoral immune response in piglets, and significantly reduce piglet mortality and diarrhea rate. The effect was better than that of the PEDV inactivated vaccine group and the CKS9-CS / PEDV group.

[0125] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An oral adjuvant of M-cell-targeting peptide and chitosan-modified dendritic mesoporous silica, characterized in that, The mixture comprises dendritic mesoporous silica and chitosan modified with M-cell targeting peptides; the amino acid sequence of the M-cell targeting peptides is shown in SEQ ID NO. 1; the dendritic mesoporous silica and the chitosan modified with M-cell targeting peptides are mixed at a mass ratio of 4-6:1 to prepare an oral adjuvant of M-cell targeting peptides and chitosan modified with dendritic mesoporous silica.

2. The oral adjuvant of M-cell targeting peptide and chitosan-modified dendritic mesoporous silica according to claim 1, characterized in that, The oral adjuvant containing M-cell-targeting peptides and chitosan-modified dendritic mesoporous silica is spherical with a diameter of 190-220 nm, a zeta potential of 25-30 mV, and a PDI dispersion index of 0.2-0.

3.

3. The oral adjuvant of M-cell targeting peptide and chitosan-modified dendritic mesoporous silica according to claim 1, characterized in that, The dendritic mesoporous silica is spherical, and under transmission electron microscopy, the mesopores appear to open in a dendritic pattern with a diameter of 150-200 nm, a zeta potential of -20 to -15 mV, and a PDI dispersion index of 0.1-0.

2.

4. The oral adjuvant of M-cell targeting peptide and chitosan-modified dendritic mesoporous silica according to claim 1, characterized in that, The amino group of the cysteine ​​residue at the tail of the M-cell targeting peptide is acetylated; the weight-average molecular weight of the chitosan is 50-100 kDa, and the degree of deacetylation is ≥85%; the chitosan modified with the M-cell targeting peptide is linked to the M-cell targeting peptide and the chitosan by an amide bond.

5. A method for preparing the oral adjuvant of M-cell targeting peptide and chitosan-modified dendritic mesoporous silica according to any one of claims 1-4, characterized in that, Dendritic mesoporous silica was dispersed in physiological saline or citrate buffer, autoclaved, centrifuged, and the resulting precipitate was resuspended to obtain a dendritic mesoporous silica dispersion. M-cell-targeting peptide-modified chitosan was dissolved in phosphate buffer, filtered to remove bacteria, and a chitosan solution modified with M-cell-targeting peptide was prepared. The dendritic mesoporous silica dispersion and the chitosan solution modified with M-cell-targeting peptide were thoroughly mixed under sterile conditions to obtain an oral adjuvant of M-cell-targeting peptide and chitosan-modified dendritic mesoporous silica.

6. A vaccine, characterized in that, The oral adjuvant of M-cell targeting peptide and chitosan-modified dendritic mesoporous silica prepared by any one of claims 1-4 or by the preparation method described in claim 5 is obtained by compounding it with an antigen.

7. The vaccine according to claim 6, characterized in that, The antigen mentioned is an immunogenic antigen.

8. The vaccine according to claim 6, characterized in that, The antigens mentioned are bovine serum albumin (BSA) and / or porcine epidemic diarrhea virus (PEDV).

9. A method for preparing a vaccine according to any one of claims 6-8, characterized in that, Dendritic mesoporous silica was mixed with the antigen, centrifuged, and the resulting precipitate was resuspended to obtain a resuspension. The resuspension was then mixed with a solution containing chitosan modified with M-cell targeting peptides to obtain an oral vaccine with oral nano-adjuvants of M-cell targeting peptides and chitosan modified dendritic mesoporous silica.

10. The use of the oral adjuvant of the M-cell targeting peptide and chitosan-modified dendritic mesoporous silica according to any one of claims 1-4, or the oral adjuvant of the M-cell targeting peptide and chitosan-modified dendritic mesoporous silica prepared by the preparation method according to claim 5, or the vaccine according to any one of claims 6-8, or the vaccine prepared by the preparation method according to claim 9, in the preparation of an oral immunomodulator containing a protein antigen.

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