A bionic vaccine oral delivery system and its preparation method and application

Abstract

A bionic vaccine oral delivery system and its preparation method and application belong to the field of biomedicine. The bionic vaccine oral delivery system uses a bionic mesoporous silica core to load a model drug vaccine, and its outer layer is coated with an amphiphilic polymer. The preparation method is: using mesoporous silica as the mother core, biomimetic modification of its surface by long-chain fatty acid glycerides, then loading the model drug vaccine, and then coating the amphiphilic polymer on the outermost layer. The bionic vaccine oral delivery system can be used to prevent influenza virus, new coronavirus, hepatitis virus, Japanese encephalitis virus and rabies virus. The bionic vaccine oral delivery system can efficiently deliver the vaccine to the intestinal lymphoid tissue through the oral route, which can not only overcome the multiple physiological barriers of the gastrointestinal tract but also improve the delivery efficiency of the vaccine to the intestinal lymphoid tissue, and solve the difficulty of the vaccine being easily degraded in the gastrointestinal tract and difficult to be absorbed by the small intestine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a bionic vaccine oral delivery system and a preparation method and application thereof, and more specifically to a bionic vaccine oral delivery system with high mucus permeability and a preparation method and application thereof. Background Art

[0002] Epidemic diseases caused by pathogens such as influenza virus, new coronavirus, and hepatitis virus that invade through mucosal tissues (respiratory mucosa, gastrointestinal mucosa, and urogenital mucosa) have seriously threatened the lives and health of all mankind. The vast majority of vaccines currently used are administered by subcutaneous or intramuscular injection. Although vaccination by injection can deliver a certain amount of antigen and induce the body to rapidly produce specific antibodies and related lymphocyte responses, it has the disadvantages of short immune memory, patients may need to be vaccinated every year, the need for full-time personnel to operate, poor patient compliance, and high preparation costs. In addition, it is difficult to effectively respond to viruses that invade the body through the mucosa by injection vaccination. The fundamental reason is that the most effective way to induce a mucosal immune response is to deliver antigens to a specific mucosal surface, while injection vaccination generally cannot effectively induce the body to produce a mucosal immune response. Therefore, changing the vaccination method of existing vaccines will definitely be an effective strategy for the efficient prevention of major infectious diseases caused by pathogens infected by mucosa.

[0003] Many research results suggest that for pathogens that invade mainly through mucosal tissues, inducing a protective immune response in the body's mucosal sites is the most effective preventive measure. Compared with traditional injection vaccines, mucosal vaccines have advantages such as safety and convenience, and can effectively induce the body to produce mucosal and systemic immune responses. In addition, due to the functional interconnectivity of the mucosal tissue lymphatic system, immune protection responses can be established at distal mucosal sites to form a common mucosal immune system. At present, mucosal vaccines are mainly administered orally, through the respiratory tract, and through the urogenital tract. The oral route of administration is considered to be the most promising method of vaccination due to its good compliance and safety and the large intestinal mucosal immune system.

[0004] However, due to the limitations of factors such as the specific environment of the human gastrointestinal tract, the oral stability of vaccines is poor and the delivery efficiency is low. The design of traditional oral vaccine delivery systems mainly focuses on organic synthetic carriers, but due to the low vaccine loading capacity of organic carriers and the low delivery efficiency to the immune induction site of the intestinal mucosa (intestinal lymphoid tissue), the effect is not ideal. Therefore, exploring carriers that can efficiently and stably load vaccines and have immune adjuvant functions, and developing a vaccine delivery system with endogenous characteristics of the human body is the key to solving the scientific problems of oral vaccines.

[0005] In recent years, mesoporous silica has attracted much attention in the fields of oral drug carriers and tracers. As a macromolecular drug carrier, it has unique advantages: (1) high specific surface area and pore volume, adjustable mesopore size, and the pore size can be tailored according to the characteristics of the loaded drug (size, molecular weight and spatial structure, etc.) to achieve efficient drug loading, protect the drug from damage by gastric acid and digestive enzymes, and improve the stability of the drug; (2) the carrier can slowly and continuously release antigens by forming an antigen reservoir or can directly stimulate immune cells to play the role of an immune adjuvant; (3) the skeleton surface can be modified with ligands or targeting molecules to achieve targeted delivery of drugs to specific sites; (4) it has good biosafety and tissue compatibility.

[0006] The entry of oral vaccines into the immune induction site of the intestinal mucosa (intestinal lymphoid tissue) is a prerequisite for sensitized immune cells. Currently, oral vaccines delivered via the intestinal M cell pathway are widely studied, but limited by the proportion of M cells in the intestinal epithelium, the efficiency of vaccine delivery still needs to be improved. It is worth noting that intestinal epithelial cells are the largest proportion of cells in the intestinal epithelial tissue (up to 99%), and are also a more effective site for achieving intestinal lymphatic transport of pathogen antigens and participating in intestinal mucosal immunity. However, due to the severe restriction of exogenous vectors from entering the intestinal lamina propria and lymphoid tissue through intestinal epithelial cells on its surface and tight junctions, the study of oral vaccine delivery strategies via this route has not been taken seriously. In addition, vaccines will be destroyed by gastric acid and digestive enzymes during delivery. Therefore, the design of the vaccine delivery system should also protect the vaccine from destruction, and promote the penetration of the mucus layer, increase the probability of contact between the vaccine delivery system and intestinal epithelial cells, so as to obtain a more effective mucosal immune response.

[0007] In summary, the development of an efficiently delivered oral mucosal vaccine is of great significance for the effective prevention of major infectious diseases caused by pathogens that infect through the mucosal route. Summary of the invention

[0008] In view of the problems existing in the prior art, the purpose of the present invention is to design and prepare a bionic vaccine oral delivery system and its preparation method and application. The bionic vaccine oral delivery system is a bionic vaccine oral delivery system with high mucus permeability, which can efficiently deliver vaccines to intestinal lymphoid tissues through the oral route, not only overcoming the multiple physiological barriers of the gastrointestinal tract but also improving the delivery efficiency of vaccines to intestinal lymphoid tissues, and solving the difficulties of vaccines being easily degraded in the gastrointestinal tract and difficult to be absorbed by the small intestine, inducing mucosal immunity and systemic adaptive immunity to achieve efficient prevention of pathogens infected through the mucosal route, and providing scientific reference for innovative research for the development of new vaccine oral delivery systems for clinical use.

[0009] According to the delivery requirements and the physicochemical properties of the biomimetic structure, the present invention selects an amphiphilic polymer (double-end esterified polyethylene glycol or zwitterionic surfactant) with good biocompatibility to coat the surface of the delivery system to achieve gastrointestinal protection of the antigen and promote mucus layer penetration.

[0010] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] The bionic vaccine oral delivery system of the present invention has a bionic mesoporous silica core loaded with a model drug vaccine, and an outer layer thereof is coated with an amphiphilic polymer.

[0012] The bionic mesoporous silica core is a mesoporous silica carrier with a surface modified with long-chain fatty acid glycerides to simulate the lipid substance in food, namely triglycerides, and simulate its metabolic pathway in the body to achieve targeting to the intestinal lymphoid tissue.

[0013] The long-chain fatty acid glyceride has the following structural formula:

[0014]

[0015] Here, R1 independently represents an alkyl group having 12 to 24 carbon atoms, and R2 independently represents an alkyl group having 12 to 24 carbon atoms.

[0016] The long-chain fatty acid glyceride is more preferably 1,3-dipalmitoylglycerol.

[0017] The model drug vaccine is suitable for one of attenuated vaccines, inactivated vaccines, subunit vaccines, and genetically recombinant expression vaccines; preferably influenza virus vaccines, new coronavirus vaccines, viral hepatitis vaccines, Japanese encephalitis virus vaccines, and rabies virus vaccines, and more preferably influenza virus split vaccines, subunit vaccines, genetically recombinant expression vaccines, and monovalent, bivalent, trivalent, or quadrivalent influenza A virus and influenza B virus vaccines.

[0018] The amphiphilic polymer is a double-end hexadecanoic acid-esterified polyethylene glycol and / or a zwitterionic surfactant, which can overcome the mucus barrier of the small intestine and protect the vaccine from being destroyed by proteases in the gastrointestinal tract, so as to achieve gastrointestinal protection of the vaccine and promote mucus layer penetration, thereby increasing the contact probability between the system and the small intestinal epithelial cells.

[0019] The zwitterionic surfactant is one or both of dilauroylphosphatidylcholine and sulfobetaine.

[0020] The preparation method of a bionic vaccine oral delivery system of the present invention is to use mesoporous silica (LMSN) as a mother core, bionic modification of the surface of the mesoporous silica by long-chain fatty acid glyceride, and then load the model drug vaccine to obtain bionic mesoporous silica nanoparticles loaded with the vaccine (SV / HA-TGLMSN);

[0021] The outermost layer of the bionic mesoporous silica nanoparticles loaded with the vaccine is coated with a layer of amphiphilic polymer to obtain a bionic vaccine oral delivery system.

[0022] The model drug vaccine is particularly suitable for one of influenza virus vaccines, new coronavirus vaccines, viral hepatitis vaccines, Japanese encephalitis virus vaccines, and rabies virus vaccines.

[0023] The vaccine-loaded bionic mesoporous silica nanoparticles (SV / HA-TGLMSN) can simulate the metabolic pathway of lipid substances to efficiently deliver the vaccine to the intestinal lymphoid tissue.

[0024] A method for preparing a bionic vaccine oral delivery system of the present invention comprises the following steps:

[0025] S1: Preparation of mesoporous silica nanoparticles

[0026] Sodium salicylate (NASAL), a cationic surfactant, and triethanolamine (TEA) are added to an aqueous solution to obtain a mixed solution; wherein, in the mixed solution, according to the solid-liquid ratio, sodium salicylate (NASAL): cationic surfactant: triethanolamine (TEA): water = (1-14) mg: (1.5-25) mg: (0.08-5.00) mg: 1 mL;

[0027] Tetraethyl silicate (TEOS) is added to the mixed solution, and the mixture is stirred at 25-100° C. for 1-36 hours, preferably 4 hours, and then the solid and liquid are separated to obtain mesoporous silica nanoparticles containing the template; the template is removed to obtain mesoporous silica nanoparticles without the template; wherein the mass ratio of tetraethyl silicate (TEOS): cationic surfactant is (2.4-20):1;

[0028] S2: Preparation of amino-modified mesoporous silica

[0029] The mesoporous silica nanoparticles with the template removed are dispersed in anhydrous ethanol, 3-aminopropyltriethoxysilane is added, and the mixture is stirred and refluxed at 60-90° C. for 2-24 hours, and then cooled and separated to obtain amino-modified mesoporous silica; wherein, by mass ratio, 3-aminopropyltriethoxysilane: mesoporous silica nanoparticles = (2.5-5):1;

[0030] S3: Preparation of carboxylated mesoporous silica

[0031] Dispersing the aminated mesoporous silica in a solvent, adding anhydride, stirring and reacting for 6-48 hours, separating the solid and the liquid, washing the precipitate, and obtaining carboxylated mesoporous silica; wherein, by mass ratio, the aminated mesoporous silica: the anhydride = 1: (1-3);

[0032] S4: Preparation of biomimetic mesoporous silica core

[0033] The carboxylated mesoporous silica is dispersed in a polar solvent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are used as catalysts, and long-chain fatty acid glycerides are added to form a reaction system, react for 12-48 hours, separate the solid and liquid, and wash the precipitate to obtain a biomimetic mesoporous silica core;

[0034] By mass ratio, carboxylated mesoporous silica: long-chain fatty acid glyceride = (0.5-3): 1;

[0035] S5: Preparation of vaccine-loaded biomimetic mesoporous silica nanoparticles (SV / HA-TGLMSN)

[0036] The biomimetic mesoporous silica core and the model drug vaccine are mixed, and the mixture is stirred thoroughly in a phosphate buffer solution (PBS) to obtain biomimetic mesoporous silica nanoparticles loaded with the vaccine (SV / HA-TGLMSN); wherein the mass ratio of the biomimetic mesoporous silica core to the model drug vaccine is 1: (0.05-1);

[0037] S6: High mucus permeability biomimetic vaccine oral delivery system

[0038] The amphiphilic polymer and the bionic mesoporous silica nanoparticles loaded with the vaccine (SV / HA-TGLMSN) are added into a phosphate buffer solution (PBS), mixed, and then solid-liquid separated to obtain a bionic vaccine oral delivery system; wherein, by mass ratio, the amphiphilic polymer: bionic vaccine system = (5-10):1, and the pH of the phosphate buffer solution (PBS) is 7.2.

[0039] In the above S1, the method for removing the template is: dispersing the mesoporous silica containing the template in anhydrous ethanol, adding hydrochloric acid, and refluxing at 65-80° C. for more than 24 hours to remove the template, thereby obtaining the mesoporous silica from which the template is removed; wherein, according to the solid-liquid ratio, the mesoporous silica containing the template: hydrochloric acid = (0.1-0.4) g: 15 mL;

[0040] In the above-mentioned S1, the cationic surfactant is one or more of cetyltrimethylammonium bromide (CTAB) and cetyltrimethylammonium chloride (CTAC).

[0041] In the above-mentioned S1, the particle size of the mesoporous silica nanoparticles is 80 to 400 nm, more preferably 180 nm, and the pore size is 18 to 50 nm, more preferably 35 nm.

[0042] In the S1, tetraethyl silicate (TEOS) is added dropwise at a dropping rate of 1 to 3 mL / min.

[0043] In the step S3, the reaction temperature is 15-30°C.

[0044] In the above-mentioned S3, the above-mentioned solvent is preferably N,N-dimethylformamide, and the mass concentration of the above-mentioned solvent is 4-7 mg / mL.

[0045] In the S3, the acid anhydride is succinic anhydride and / or glutaric anhydride.

[0046] In the above-mentioned S3, the solid-liquid separation is preferably performed by centrifugation, and the precipitate is washed with anhydrous ethanol.

[0047] In the S4, in the reaction system, the mass concentration of EDC is 1.5-7.5 mg / mL, and the mass concentration of NHS is 1-5 mg / mL;

[0048] The polar solvent is preferably dimethyl sulfoxide, and the mass concentration of carboxylated mesoporous silica dispersed in the solvent is 3-7 mg / mL;

[0049] In the above-mentioned S4, in the bionic mesoporous silica core, the grafting amount of long-chain fatty acid glyceride on the surface of the carboxylated mesoporous silica is 5-10%.

[0050] In the S5, the loading amount of the model drug vaccine in the bionic mesoporous silica nanoparticles (SV / HA-TGLMSN) loaded with the vaccine is 8-20%.

[0051] In the S5, the mass concentration of the biomimetic mesoporous silica core added to the phosphate buffer is 1 mg / mL.

[0052] In the above-mentioned S6, in the bionic vaccine oral delivery system, the coating amount of the amphiphilic polymer on the outer layer of the bionic vaccine carrier system is 15-25%.

[0053] In the above S6, when the amphiphilic polymer is double-end esterified polyethylene glycol, the double-end esterified polyethylene glycol is prepared by the following method:

[0054] Dissolve the long-chain fatty acid in water, add EDC and NHS to activate for 1-2 hours, then add polyethylene glycol, react at 65-75°C for 6-24 hours, and after the reaction is completed, cool to room temperature, filter out the unreacted long-chain fatty acid, freeze-dry, and obtain double-terminal esterified polyethylene glycol for later use;

[0055] The weight average molecular weight of polyethylene glycol is 2-20 kD; the molar ratio of long-chain fatty acid: polyethylene glycol is (4-8): 1; the concentrations of EDC and NHS are 1.5-7.5 mg / mL and 1-5 mg / mL respectively;

[0056] The long-chain fatty acid is a C14-C18 long-chain fatty acid.

[0057] The bionic vaccine oral delivery system of the present invention is used for preventing influenza virus, new coronavirus, hepatitis virus, Japanese encephalitis virus and rabies virus.

[0058] According to characterization, the particle size of the mesoporous silica nanoparticles prepared by the present invention is 80-400 nm, and the pore size is 18-50 nm. In combination with the properties of the loaded vaccine, mesoporous silica nanoparticles with a particle size of 180±10 nm and a pore size of 35±5 nm are preferably used as the mother core. The loss of mass of the long-chain fatty acid glyceride and the coated amphiphilic polymer on the surface of the mesoporous silica is measured by thermogravimetric analysis, thereby obtaining that the grafting amount of the long-chain fatty acid glyceride on the surface of the mesoporous silica is 5-10%, the mass percentage of the amphiphilic polymer coated on the outer layer of the bionic vaccine system is 15-25%, and the vaccine loading amount is 8-20%.

[0059] The bionic vaccine oral delivery system of the present invention and its preparation method and application have the following beneficial effects:

[0060] The present invention uses inactivated influenza virus split vaccine or influenza virus subunit vaccine as a model drug (also applicable to vaccines such as new coronavirus vaccine, viral hepatitis vaccine, Japanese encephalitis virus vaccine, rabies virus, etc.). Mesoporous silica (LMSN) with suitable pore size is used as the mother core, and the surface is reacted with long-chain fatty acid glycerides such as 1,3-dipalmitoylglycerol for biomimetic modification, and then loaded with the vaccine to prepare biomimetic mesoporous silica nanoparticles (SV / HA-TGLMSN) loaded with the vaccine, so that it can simulate the metabolic pathway of lipid substances to efficiently deliver the vaccine to the intestinal lymphoid tissue. In order to overcome the mucus barrier of the small intestine and protect the vaccine from being destroyed by proteases in the gastrointestinal tract, a layer of amphiphilic polymer is finally coated on the outermost layer of the drug-carrying system to achieve gastrointestinal protection of the vaccine and promote mucus layer penetration, increasing the contact probability between the system and the small intestinal epithelial cells. Its biomimetic modification can achieve targeting of intestinal lymphoid tissue. And animal immunization experiments have proved that the oral administration of the biomimetic influenza virus vaccine can induce the production of influenza virus-specific antibodies and has a certain immune effect.

[0061] The pores of the mesoporous silica in the present invention can realize efficient loading of vaccines, prevent antigens from being degraded by gastric acid and digestive enzymes, release antigens for a long time, and have the effect of immune adjuvant. The encapsulation of polyethylene glycol or zwitterionic surfactants can further realize the protection of antigens in the gastrointestinal tract and promote the penetration of the vaccine delivery system in the gastrointestinal mucus layer. At the same time, the present invention simulates the lipid metabolism and absorption pathway in food to realize the efficient delivery of oral vaccines to intestinal lymphoid tissues, and provides scientific reference for the research and application of vaccine delivery systems for efficient prevention of major infectious diseases caused by other pathogens infected through mucosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 is a transmission electron microscope image of the LMSN in Example 1;

[0063] Figure 2 The particle size distribution and Zeta potential diagram of the biomimetic vaccine oral delivery system; (a) is the particle size distribution diagram, (b) is the Zeta potential diagram;

[0064] Figure 3 LMSN before and after biomimetic modification in Example 1 13 C-NMR spectra; (a) LMSN before modification, (b) bionic LMSN;

[0065] Figure 4 The intestinal epithelial cell uptake of samples 2, 3, 7 and 8 in Example 1;

[0066] Figure 5 This is the TGA result diagram of the biomimetic vaccine oral delivery system;

[0067] Figure 6 The apparent permeability coefficient of the biomimetic vaccine oral delivery system in the isolated intestine;

[0068] Figure 7 To provide a tissue distribution map of the biomimetic vaccine oral delivery system in the small intestine;

[0069] Figure 8 To provide a tissue distribution map of the oral delivery system for biomimetic vaccines in lymph nodes;

[0070] Fig. 9 Immunopharmacological efficacy of a biomimetic influenza virus oral delivery system. DETAILED DESCRIPTION

[0071] The present invention will be further described in detail below in conjunction with the embodiments and drawings. The following embodiments are only for the purpose of understanding the present invention and are not intended to limit the present invention.

[0072] Example 1

[0073] Step 1: Preparation of mesoporous silica nanoparticles

[0074] The preparation methods of mesoporous silica nanoparticles with different particle sizes and pore sizes are as follows:

[0075] Sample 1

[0076] 0.38 g CTAB and 0.168 g NASAL were weighed and added to 25 mL double distilled water, and stirred in a 60 °C water bath until they were fully dissolved. Then 48 mg TEA was added and stirred for 0.5 h. Then 4 mL TEOS was added and reacted for 2 h. After the reaction, the suspension was naturally cooled, centrifuged at 10000 rpm for 8 min for solid-liquid separation, and the precipitate was washed 3 times with anhydrous ethanol to obtain mesoporous silica nanoparticles (LMSN) containing template.

[0077] Sample 2

[0078] 0.38 g CTAB and 0.276 g NASAL were weighed and added to 25 mL double distilled water, and stirred in a 60 °C water bath until they were fully dissolved. Then 48 mg TEA was added and continued to stir for 0.5 h. Then 4 mL TEOS was added and reacted for 2 h. After the reaction, the suspension was naturally cooled, centrifuged at 10000 rpm for 8 min for solid-liquid separation, and the precipitate was washed 3 times with anhydrous ethanol to obtain mesoporous silica nanoparticles (LMSN) containing templates.

[0079] Sample 3

[0080] 0.38 g CTAB and 0.168 g NASAL were weighed and added to 25 mL double distilled water, and stirred in a water bath at 80 °C until they were fully dissolved. Then 48 mg TEA was added and stirring was continued for 0.5 h. Then 4 mL TEOS was added and reacted for 2 h. After the reaction, the suspension was naturally cooled, centrifuged at 10000 rpm for 8 min for solid-liquid separation, and the precipitate was washed 3 times with anhydrous ethanol to obtain mesoporous silica nanoparticles (LMSN) containing template.

[0081] Sample 4

[0082] 0.38 g CTAB and 0.168 g NASAL were weighed and added to 25 mL double distilled water, and stirred in a 100 °C water bath until they were fully dissolved. Then 48 mg TEA was added and continued to stir for 0.5 h. Then 4 mL TEOS was added and reacted for 2 h. After the reaction, the suspension was naturally cooled, centrifuged at 10000 rpm for 8 min for solid-liquid separation, and the precipitate was washed 3 times with anhydrous ethanol to obtain mesoporous silica nanoparticles (LMSN) containing templates.

[0083] Sample 5

[0084] 0.38 g CTAB and 0.168 g NASAL were weighed and added to 25 mL double distilled water, and stirred in a 60 °C water bath until they were fully dissolved. Then 80 mg TEA was added and stirring was continued for 0.5 h. Then 4 mL TEOS was added and reacted for 2 h. After the reaction, the suspension was naturally cooled, centrifuged at 10000 rpm for 8 min for solid-liquid separation, and the precipitate was washed 3 times with anhydrous ethanol to obtain mesoporous silica nanoparticles (LMSN) containing templates.

[0085] Sample 6

[0086] 0.38 g CTAB and 0.168 g NASAL were weighed and added to 25 mL double distilled water, and stirred in a 60 °C water bath until they were fully dissolved. Then 48 mg TEA was added and stirred for 0.5 h. Then 6 mL TEOS was added and reacted for 2 h. After the reaction, the suspension was naturally cooled, centrifuged at 10000 rpm for 8 min, and the precipitate was washed 3 times with anhydrous ethanol to obtain mesoporous silica nanoparticles (LMSN) containing templates.

[0087] Sample 7

[0088] 0.38 g CTAB and 0.168 g NASAL were weighed and added to 25 mL double distilled water, and stirred in a 60 °C water bath until they were fully dissolved. Then 48 mg TEA was added and stirring was continued for 0.5 h. Then 4 mL TEOS was added and reacted for 12 h. After the reaction, the suspension was naturally cooled, centrifuged at 10000 rpm for 8 min for solid-liquid separation, and the precipitate was washed 3 times with anhydrous ethanol to obtain mesoporous silica nanoparticles (LMSN) containing templates.

[0089] Sample 8

[0090] 48 mg of TEA was added to 25 mL of double distilled water, and the mixture was stirred for 0.5 h in a water bath at 60 °C. 0.38 g of CTAB and 0.168 g of NASAL were weighed and added to the mixture and stirred to dissolve. Then 4 mL of TEOS was added to the above system and reacted for 4 h. After the reaction, the suspension was naturally cooled, collected by centrifugation and washed 3 times with anhydrous ethanol to obtain mesoporous silica nanoparticles (LMSN) containing templates.

[0091] Removal of template: The LMSN containing the template was dispersed in 120 mL of anhydrous ethanol, 15 mL of hydrochloric acid was added, and the template was removed by reflux at 70 °C for 24 h.

[0092] The template-free LMSN was ultrasonically dispersed in water and dropped onto a copper mesh. The morphology was observed by transmission electron microscopy, and the pore size distribution of LMSN was determined by surface area and pore size analyzer. The particle size of LMSN was determined by dynamic light scattering. The prepared nanoparticles were found to be between 80 and 400 nm, and the pore size was between 18 and 50 nm (see Figure 1 and Table 1);

[0093] Table 1 Particle size and pore size statistics of LMSN of each sample

[0094]

[0095] Step 2: Preparation of amino-modified mesoporous silica

[0096] 100 mg of the above LMSN (samples 1 to 8) were dispersed in 80 mL of anhydrous ethanol, 0.4 mL of 3-aminopropyltriethoxysilane was added, refluxed at 75 ° C for 12 h, collected by centrifugation at 10000 rpm for 8 min, and washed three times with anhydrous ethanol to obtain amino mesoporous silica (LMSN-NH2).

[0097] Step 3: Preparation of carboxylated mesoporous silica

[0098] 200 mg of the above LMSN-NH2 was ultrasonically dispersed in 15 mL of N,N-dimethylformamide, and an N,N-dimethylformamide solution containing 0.45 g of succinic anhydride was added and stirred at room temperature for 24 h. The carrier was collected after centrifugation to obtain carboxylated mesoporous silica (LMSN-COOH).

[0099] Step 4: Preparation of biomimetic mesoporous silica core

[0100] Biomimetic mesoporous silica core (TGLMSN) sample 1

[0101] 100 mg LMSN-COOH (prepared from sample 1) was dispersed in 20 mL dimethyl sulfoxide, and EDC and NHS were added to activate the carboxyl group for 1 hour, wherein the mass concentration of EDC in dimethyl sulfoxide was 3 mg / mL, and the mass concentration of NHS was 2 mg / mL. Subsequently, 75 mg 1,3-dipalmitoylglycerol was added to react for 24 hours, and the carrier was collected by centrifugation at 10000 rpm for 10 minutes, and washed twice with water and anhydrous ethanol respectively to obtain a biomimetic mesoporous silica core (TGLMSN), which was dried for later use.

[0102] Biomimetic mesoporous silica core (TGLMSN) sample 2

[0103] 100 mg LMSN-COOH (prepared from sample 8) was dispersed in 20 mL of dimethyl sulfoxide, and EDC and NHS were added to activate the carboxyl group for 1 hour, wherein the mass concentration of EDC in dimethyl sulfoxide was 3 mg / mL, and the mass concentration of NHS was 2 mg / mL. Subsequently, 75 mg of 1,3-dimyristoylglycerol was added to react for 24 hours, and the carrier was collected by centrifugation at 10000 rpm for 10 minutes, and washed twice with water and anhydrous ethanol respectively to obtain a biomimetic mesoporous silica core (TGLMSN), which was dried for later use.

[0104] Biomimetic mesoporous silica core (TGLMSN) sample 3

[0105] 100 mg LMSN-COOH (prepared from sample 3) was dispersed in 20 mL dimethyl sulfoxide, and EDC and NHS were added to activate the carboxyl group for 1 hour, wherein the mass concentration of EDC in dimethyl sulfoxide was 3 mg / mL, and the mass concentration of NHS was 2 mg / mL. Subsequently, 75 mg 1,3-distearin was added to react for 24 hours, and the carrier was collected by centrifugation at 10000 rpm for 10 minutes, and washed twice with water and anhydrous ethanol respectively to obtain a biomimetic mesoporous silica core (TGLMSN), which was dried for later use.

[0106] Solid-state NMR carbon spectroscopy ( 13 C NMR) detection, the test results are as follows Figure 3 As can be seen from the figure, the appearance of the 175 ppm peak is attributed to -CONH and -COO- groups, and the peaks between 10 ppm and 50 ppm are attributed to -CH2-CO-, -CH2-CH2- and -CH3 groups. The enhancement of these peaks indicates that 1,3-DG is successfully modified on the surface of mesoporous silica.

[0107] Step 5: Loading of influenza virus vaccine

[0108] 1 mg of biomimetic mesoporous silica core was weighed and dispersed in 1 mL of PBS at pH 7.2, 400 ug of split virus vaccine (SV) or subunit vaccine (HA) was added thereto, and the mixture was stirred and mixed thoroughly to obtain biomimetic mesoporous silica nanoparticles loaded with virus vaccine (SV-TGLMSN or HA-TGLMSN).

[0109] Step 6: Preparation of zwitterionic polymer-coated mesoporous silica nanoparticles

[0110] Weigh 0.0412g of hexadecanoic acid, dissolve it completely at 65°C, add 48mg EDC and 32mg NHS to activate the carboxyl group for 1h, then add 0.24g of polyethylene glycol to the above system, and let the system react for 24h. After the reaction is completed, cool it to room temperature, filter it through a 0.22μm filter to remove excess hexadecanoic acid, and freeze-dry the liquid to obtain a white double-terminal esterified polyethylene glycol polymer (PEG-DP). The molecular weight of polyethylene glycol is 5KD;

[0111] 5 mg of SV-TGLMSN or HA-TGLMSN was weighed and dispersed in 10 mL of PBS at pH 7.2, 50 mg of double-end esterified polyethylene glycol polymer was added and stirred for 24 h, and the nanoparticles were collected by centrifugation to obtain a bionic vaccine delivery system loaded with viral vaccine (SV-TGLMSN@PEG-DP or HA-TGLMSN@PEG-DP).

[0112] In order to detect the loading rate of virus vaccine loaded on carriers with different pore sizes, LMSN was directly mixed with split virus vaccine or subunit vaccine to obtain mesoporous silica nanoparticles loaded with virus vaccine (SV-LMSN).

[0113] The drug loading of SV-LMSN prepared from Sample 1, Sample 2, Sample 4 and Sample 8 in Example 1 was measured, and the results are shown in Table 2;

[0114] Table 2 Statistics of drug loading of SV-LMSN for vaccines prepared from sample 1, sample 2, sample 4 and sample 8

[0115]

[0116] As shown in Table 2, the pore size of the sample is too small, which is not conducive to the efficient loading of the vaccine. A larger pore size is essential for achieving a high drug loading and the ultimate drug efficacy. The intestinal epithelial cells were examined by flow cytometry to examine the uptake of samples 2, 3, 7 and 8 in Example 1. The results are shown in Table 2. Figure 4 The results showed that intestinal epithelial cells are more likely to take up mesoporous silica nanoparticles with a particle size of 200 nm. In summary, choosing mesoporous silica with appropriate size and pore size (i.e., sample 8) as a "tailor-made" carrier for vaccines is crucial to achieving high drug loading and high cellular uptake, and thus achieving the ultimate high immune efficacy.

[0117] The particle size and potential of the prepared samples were analyzed, and the test results were as follows: Figure 2. As shown in the figure, with the increase of modification steps, the particle size of the sample gradually increases, and the change in potential also indicates the successful modification of each group or polymer. In addition, the samples were subjected to thermogravimetric analysis, and the grafting amount of amino groups in LMSN-NH2 was 11.8%, the grafting amount of carboxyl groups in LMSN-COOH was 5.8%, the grafting amount of 1,3-dipalmitoylglycerol in TGLMSN was 6.0%, the encapsulation amount of PEG-DP in TGLMSN@PEG-DP was 19.1%, and the vaccine loading amount of SV-TGLMSN@PEG-DP was 11% (see Figure 5 ).

[0118] Example 2

[0119] A method for preparing a bionic vaccine oral delivery system comprises the following steps:

[0120] Step 1: Preparation of mesoporous silica nanoparticles

[0121] Sodium salicylate (NASAL) and hexadecyltrimethylammonium chloride (CTAC) were dissolved in an aqueous solution, triethanolamine (TEA) was added, and the mixture was stirred evenly. Tetraethyl silicate (TEOS) was added dropwise to the mixture at a drop rate of 2 mL / min, and the mixture was stirred at 30°C for 4 h. After the reaction, the precipitate was collected by centrifugation to obtain mesoporous silica nanoparticles containing the template.

[0122] The mesoporous silica containing the template was dispersed in anhydrous ethanol, and after adding 15 mL of hydrochloric acid, the mixture was refluxed at 70° C. overnight to remove the template, thereby obtaining mesoporous silica nanoparticles with the template removed.

[0123] Among them, in the mixture system, the concentration of NASAL aqueous solution is 6.7 mg / mL, the concentration of CTAC aqueous solution is 15 mg / mL, the final concentration of triethanolamine is 0.2 mg / mL, and the mass ratio of tetraethyl silicate to cationic surfactant is 10:1.

[0124] Step 2: Preparation of amino-modified mesoporous silica

[0125] The prepared mesoporous silica nanoparticles with the template removed were dispersed in anhydrous ethanol, 3-aminopropyltriethoxysilane was added at a mass ratio of 3:1, the mixture was stirred and refluxed at 80° C. for 12 h, and the aminated mesoporous silica was collected by cooling and centrifugation;

[0126] Step 3: Preparation of carboxylated mesoporous silica:

[0127] The amino-modified mesoporous silica was dispersed in a solvent to obtain a solution with a concentration of 6 mg / mL, glutaric anhydride was added, and the mixture was stirred at room temperature for 12 hours, centrifuged, and the precipitate was washed with anhydrous ethanol to obtain carboxylated mesoporous silica;

[0128] Wherein, the mass ratio of glutaric anhydride:mesoporous silica is 3:1.

[0129] Step 4: Preparation of biomimetic mesoporous silica:

[0130] The carboxylated mesoporous silica was dispersed in dimethyl sulfoxide at a concentration of 4 mg / mL, and catalysts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added at the same time, and then 1,3-dipalmitoylglycerol was added. After reacting for 20 hours, the mixture was centrifuged, and the precipitate was washed with water and anhydrous ethanol to obtain a biomimetic mesoporous silica core.

[0131] The mass ratio of carboxylated mesoporous silica to 1,3-dipalmitoylglycerol was 1:1, and the concentrations of EDC and NHS were 3 mg / mL and 2 mg / mL, respectively.

[0132] Step 5: Preparation of vaccine-loaded biomimetic mesoporous silica nanoparticles (SV / HA-TGLMSN)

[0133] The biomimetic mesoporous silica and the virus split vaccine are fully stirred and mixed in a phosphate buffer at a mass ratio of 1:1 to obtain biomimetic mesoporous silica nanoparticles loaded with the vaccine (SV / HA-TGLMSN);

[0134] Step 6: High mucus permeability biomimetic vaccine oral delivery system

[0135] (1) Preparation of double-end esterified polyethylene glycol:

[0136] Dissolve octadecanoic acid in hot water, add EDC and NHS to activate for 1 hour, and then react with polyethylene glycol (in this embodiment, the weight average molecular weight of polyethylene glycol is 10kD). After the reaction is completed, cool to room temperature, filter with a 0.22μm filter membrane to remove unreacted octadecanoic acid, and freeze-dry to obtain double-end esterified polyethylene glycol for later use.

[0137] The reaction temperature was 70° C., the reaction time was 12 h, the concentrations of EDC and NHS were 3 mg / mL and 2 mg / mL, respectively, and the molar ratio of octadecanoic acid to polyethylene glycol was 5:1.

[0138] (2) The prepared double-end esterified polyethylene glycol and the vaccine-loaded bionic mesoporous silica nanoparticles (SV / HA-TGLMSN) obtained in step (5) are mixed in a phosphate buffer at a mass ratio of 6:1, so that they are wrapped on the surface of the bionic vaccine-loaded system through hydrophobic interaction forces, and after centrifugation, a bionic vaccine oral delivery system with high mucus permeability is obtained.

[0139] Example 3

[0140] A bionic vaccine oral delivery system is the same as Example 2, except that dilauroyl phosphatidylcholine is used instead of double-end esterified polyethylene glycol as the amphiphilic polymer, and is coated on the surface of bionic mesoporous silica nanoparticles (SV / HA-TGLMSN) loaded with the vaccine through hydrophobic interaction.

[0141] Example 4

[0142] A bionic vaccine oral delivery system, which has a bionic mesoporous silica core loaded with a vaccine and is coated with an amphiphilic polymer as an outer layer that can promote the system's absorption across the mucus layer and antigen gastrointestinal protection.

[0143] Among them, the biomimetic mesoporous silica core is a mesoporous silica nanoparticle with a surface modified with long-chain fatty acid glyceride;

[0144] The vaccine loaded in this embodiment is influenza B virus vaccine;

[0145] The amphiphilic polymer used in this embodiment is sulfobetaine.

[0146] The specific structure of the long-chain fatty acid glyceride used in this embodiment is as follows:

[0147]

[0148] Wherein, R1 is an alkyl group with 12 carbon atoms, and R2 is an alkyl group with 12 carbon atoms.

[0149] The mesoporous silica nanoparticles used in this embodiment are used to efficiently load vaccine antigens, prevent antigens from being degraded by gastric acid and digestive enzymes, release antigens for a long time, and have the effect of immune adjuvant. Its particle size is 180nm and its pore size is 30nm. The pore size can be adjusted according to the size / molecular weight and spatial structure of the loaded vaccine antigen. The preparation method of the vaccine oral delivery system using it as the mother core includes the following steps:

[0150] Step 1: Preparation of mesoporous silica nanoparticles:

[0151] Dissolve sodium salicylate and hexadecyltrimethylammonium bromide in an aqueous solution, add triethanolamine, and then add tetraethyl silicate dropwise into the above system. After the reaction is completed, collect the precipitate by centrifugation to obtain mesoporous silica nanoparticles containing the template; place the precipitate in anhydrous ethanol, add hydrochloric acid, and reflux to obtain mesoporous silica nanoparticles with the template removed;

[0152] The reaction temperature is 70° C., the reaction time is 12 h, the concentration of sodium salicylate is 12 mg / mL, the concentration of the hexadecyltrimethylammonium bromide aqueous solution is 5 mg / mL, the concentration of the triethanolamine aqueous solution is 1.00 mg / mL, and the volume ratio of tetraethyl silicate to the aqueous solution is 0.05-0.4.

[0153] Step 2: Preparation of amino-modified mesoporous silica:

[0154] The mesoporous silica nanoparticles with the template removed are dispersed in anhydrous ethanol, 3-aminopropyltriethoxysilane is added, stirred and refluxed for reaction, and centrifuged to obtain amino-modified mesoporous silica nanoparticles;

[0155] The reaction temperature was 60°C, the reaction time was 24h, and the mass ratio of 3-aminopropyltriethoxysilane to mesoporous silica was 3:1;

[0156] Step 3: Preparation of carboxylated mesoporous silica:

[0157] The aminated mesoporous silica is dispersed in N,N-dimethylformamide, succinic anhydride is added and stirred for reaction, and the carboxylated mesoporous silica nanoparticles are obtained by centrifugation.

[0158] The reaction temperature was 24°C, the reaction time was 12 h, and the mass ratio of succinic anhydride to mesoporous silica was 1:1;

[0159] Step 4: Preparation of biomimetic mesoporous silica:

[0160] The carboxylated mesoporous silica is dispersed in dimethyl sulfoxide, catalysts EDC and NHS are added to activate the carboxyl groups, and then the long-chain fatty acid glyceride with the above structure is added to react to obtain a biomimetic mesoporous silica core;

[0161] The mass ratio of carboxylated mesoporous silica to the long-chain fatty acid glyceride of the above structure is 2:1, the reaction time is 28h, and finally the grafting rate of the long-chain fatty acid glyceride on the surface of carboxylated mesoporous silica is determined to be 8%;

[0162] Step 5: Biomimetic mesoporous silica loaded with vaccine:

[0163] The biomimetic mesoporous silica nanoparticles and the vaccine were thoroughly stirred and mixed in phosphate buffered saline (PBS);

[0164] The mass ratio of loaded vaccine to biomimetic mesoporous silica nanoparticles is 0:1-1:1

[0165] Step 6: High mucus permeability biomimetic vaccine oral delivery system

[0166] The thiobetaine is mixed with the biomimetic vaccine delivery system obtained in step (5) in PBS to wrap the amphiphilic polymer on the surface of the biomimetic vaccine delivery system through hydrophobic interaction, and centrifuged to obtain a biomimetic vaccine oral delivery system with high mucus permeability.

[0167] The mass ratio of the amphiphilic polymer to the biomimetic vaccine delivery system used was 8:1.

[0168] Example 5

[0169] A bionic vaccine oral delivery system is the same as Example 4, except that 1,3-dimyristyl glyceryl is used instead of 1,3-dilaurin as the bionic modification substance, and is grafted with LMSN-COOH on the surface of the carrier through an esterification reaction to obtain a bionic mesoporous silica core (TGLMSN), which is used for subsequent vaccine loading and amphiphilic polymer encapsulation, thereby obtaining a bionic vaccine oral delivery system with high mucus permeability.

[0170] Example 6

[0171] A bionic vaccine oral delivery system is prepared, and the specific steps are as follows:

[0172] Step 1: Preparation of mesoporous silica nanoparticles:

[0173] Dissolve triethanolamine, sodium salicylate and hexadecyltrimethylammonium bromide in an aqueous solution, stir evenly, then add tetraethyl silicate dropwise, stir and react at 50°C for 12 hours, and then centrifuge to collect the precipitate. Then disperse the precipitate in anhydrous ethanol and add 15 mL of hydrochloric acid solution, and reflux at 65°C for 36 hours to obtain mesoporous silica nanoparticles with the template removed.

[0174] Among them, in the above reaction system, the concentrations of NASAL and CTAB aqueous solutions are 9.5 mg / mL and 12 mg / mL respectively, the final concentration of triethanolamine is 2 mg / mL, and the mass ratio of tetraethyl silicate to cationic surfactant is 12.5:1.

[0175] Step 2: Preparation of amino-modified mesoporous silica:

[0176] The mesoporous silica nanoparticles with the template removed were dispersed in anhydrous ethanol, 3-aminopropyltriethoxysilane was added, and the mixture was refluxed at 70° C. for 12 h, and the amino-modified mesoporous silica nanoparticles were collected by centrifugation.

[0177] Wherein, the mass ratio of 3-aminopropyltriethoxysilane to mesoporous silica is 3:1.

[0178] Step 3: Preparation of carboxylated mesoporous silica:

[0179] The amino-modified mesoporous silica was dispersed in N,N-dimethylformamide, and then succinic anhydride was added, and the mixture was stirred at 30°C for 24 hours, and then the carboxylated mesoporous silica nanoparticles were obtained by centrifugation.

[0180] Wherein, the mass ratio of succinic anhydride to mesoporous silica is 1:1.

[0181] Step 4: Preparation of biomimetic mesoporous silica:

[0182] The carboxylated mesoporous silica was dispersed in dimethyl sulfoxide, and catalysts EDC and NHS were added to activate the carboxyl group for 1 hour, and then 1,3-distearic acid glycerol was added to react at room temperature to obtain the biomimetic mesoporous silica core.

[0183] The mass ratio of carboxylated mesoporous silica to 1,3-distearin was 2:1, the reaction time was 36 h, and the concentrations of EDC and NHS were 3 mg / mL and 2 mg / mL, respectively.

[0184] Step 5: Biomimetic mesoporous silica loaded with vaccine:

[0185] The biomimetic mesoporous silica nanoparticles and the vaccine were thoroughly stirred and mixed in PBS;

[0186] The vaccine used in this embodiment is an influenza virus subunit vaccine, and the mass ratio of the bionic mesoporous silica nanoparticles to the vaccine is 1:0.5. Finally, the drug loading of the bionic mesoporous silica nanoparticles is measured to be 17%.

[0187] Step 6: High mucus permeability biomimetic vaccine oral delivery system

[0188] (1) Preparation of double-end esterified polyethylene glycol:

[0189] The specific preparation method is the same as that of the double-end esterified polyethylene glycol in Example 2, except that the weight average molecular weight of the polyethylene glycol in this example is 6 kD.

[0190] (2) The prepared double-end esterified polyethylene glycol and the vaccine-loaded bionic mesoporous silica nanoparticles (HA-TGLMSN) obtained in step (5) are mixed in PBS at a mass ratio of 8:1, so that they are wrapped on the surface of the bionic vaccine-loaded system through hydrophobic interaction forces, and after centrifugation, a bionic vaccine oral delivery system with high mucus permeability is obtained.

[0191] Test Example 1

[0192] Determination of Apparent Permeability Coefficient of Intestine in Rats

[0193] In order to investigate whether coating the carrier surface with double-terminal esterified polyethylene glycol can enhance the mucus permeability of the carrier system and thus increase the probability of being taken up by small intestinal epithelial cells, an in vitro rat intestinal tube experiment was conducted. The rats were fasted for 12 hours before the experiment. After intraperitoneal injection of chloral hydrate for anesthesia, the abdominal cavity was quickly opened, and about 5 cm of the duodenum, jejunum and ileum were taken, respectively, and rinsed with cold KR solution. One end of the intestinal tube was tied tightly, and 250 μL of FITC-labeled carrier delivery system suspension was added to the intestinal tube. Then the other end of the intestinal tube was tied and placed in 5 mL of 37℃ aerobic KR solution, stirred at 100 rpm, and 200 μL of KR solution was taken at 15, 30, 45, 60, 75, 90 and 105 minutes, respectively, and an equal volume of 37℃ KR solution was added at the same time. The fluorescence intensity of the KR solution taken out was measured in an ELISA instrument, and the length and inner diameter of the intestinal section after the experiment were measured, and then the apparent permeability coefficient (Papp) was calculated.

[0194]

[0195] Where Q is the amount of the carrier delivery system passing through the intestine, A is the area of ​​the rat intestine, and C0 is the initial concentration of the carrier delivery system. The apparent permeability coefficients of LMSN, TGLMSN, and TGLMSN@PEG-DP are shown in Figure 6 The results showed that compared with the naked carrier LMSN and the biomimetic modified TGLMSN, the encapsulation of PEG-DP was beneficial to improve the absorption of the carrier delivery system by the intestinal segment.

[0196] Test Example 2

[0197] Determination of nanoparticle retention in the small intestine and lymph node targeting effect

[0198] This experiment investigated the distribution of the carrier delivery system in the gastrointestinal tract and the lymph node targeting effect. Twenty-four Balb / c mice were divided into LMSN and TGLMSN@PEG-DP groups. The mice were fasted for 12 hours before the experiment. Each group was gavaged with a FITC-labeled carrier delivery system suspension at a dose of 100 mg / g. The mice were dissected at set time intervals (2, 4, 6, and 24 hours), and the gastrointestinal tract and lymph nodes were removed. After washing with saline, they were photographed in a small animal in vivo imager. The imaging results are shown in Figure 2. Figure 7 and Figure 8 The results showed that the bionic vaccine oral delivery system prepared by the invention can enhance the retention in the intestine and achieve the targeting to the lymph nodes compared with the unmodified mesoporous silica carrier.

[0199] Test Example 3

[0200] Determination of immunological efficacy

[0201] Mouse immunization protocol:

[0202] Forty-two female Balb / c mice aged 6-8 weeks were divided into groups of 7 mice each, and each group was orally immunized with saline, SV, LMSN, SV-LMSN, SV-TGLMSN, and SV-TGLMSN@PEG-DP at a dose of 25 μg HA / mouse. Oral immunization was performed 6 times, twice a week, for three consecutive weeks.

[0203] Preparation of immune serum:

[0204] Two weeks after the last immunization, the blood of the immunized mice was collected by orbital bleeding. After standing at room temperature for 1 hour, the blood was centrifuged at 3000 rpm for 20 minutes, and the serum was collected and stored in a -80°C refrigerator for testing.

[0205] Hemagglutination test:

[0206] ① Add 25 μL of saline to wells 1-12 of a 96-well "V" plate;

[0207] ② Add 25 μL of viral antigen to the first well, mix well, then pipette 25 μL of liquid from the first well to the second well, then pipette 25 μL of liquid from the second well to the third well, and continue diluting in multiple proportions to the eleventh well, then pipette 25 μL from the eleventh well and discard;

[0208] ③ Add 25 μL of 1% (V / V) chicken red blood cell suspension to each well (shake the chicken red blood cells thoroughly before adding);

[0209] ④Add 25 μL of normal saline to each well;

[0210] ⑤ Mix by oscillation, let stand at room temperature for 40 minutes and observe the results;

[0211] ⑥ To determine the results, tilt the plate and observe whether the red blood cells flow in a teardrop shape. The highest dilution multiple of the antigen or virus that completely hemagglutinates represents one hemagglutination unit (HAU).

[0212] Hemagglutination inhibition test to determine HI titer:

[0213] Prepare 4HAU virus antigen according to the results of hemagglutination test, take the highest dilution multiple of virus with complete hemagglutination as the end point, and divide the end point dilution multiple by 4 to get the dilution multiple of antigen containing 4HAU. The specific operation is as follows:

[0214] ① Add 25 μL of saline to wells 1-11 of a 96-well "V" plate, and add 50 μL of saline to well 12;

[0215] ② Pipette 25 μL of serum into the first well, mix thoroughly and then pipette 25 μL into the second well, dilute in multiple proportions to the tenth well, and then pipette 25 μL from the tenth well and discard;

[0216] ③ Add 25 μL of virus antigen solution containing 4 HAU to wells 1-11 and let stand at room temperature for 30 minutes;

[0217] ④ Add 25 μL of chicken red blood cell suspension to each well and mix gently. Let it stand for about 40 minutes and observe the results. The highest dilution of serum that completely inhibits the four HAU antigens is the HI titer.

[0218] The HI titer of mice after immunization is as follows Fig. 9 As shown, the results preliminarily indicate that the bionic influenza virus vaccine oral delivery system can effectively deliver the vaccine to the lymphatic tissue, thereby enhancing the immune effect of the influenza vaccine after oral administration and producing specific antibodies against the influenza virus.

Claims

1. A bionic vaccine oral delivery system, characterized in that: The biomimetic mesoporous silica core is loaded with model drug vaccines, and its outer layer is coated with amphiphilic polymers; The bionic mesoporous silica core is a surface-modified long-chain fatty acid glyceride of a mesoporous silica carrier; The long-chain fatty acid glyceride has the following structural formula: wherein R1 independently represents an alkyl group of 12 to 24 carbon atoms, and R2 independently represents an alkyl group of 12 to 24 carbon atoms; The model drug vaccine is suitable for influenza split vaccine and influenza subunit vaccine; The amphiphilic polymer is double-end hexadecanoic acid esterified polyethylene glycol and / or a zwitterionic surfactant; The zwitterionic surfactant is one or both of dilauroylphosphatidylcholine and sulfobetaine.

2. A method for preparing the bionic vaccine oral delivery system according to claim 1, characterized in that: Using mesoporous silica as the core, the surface of the mesoporous silica is biomimetic modified by long-chain fatty acid glycerides, and then loaded with model drug vaccines to obtain vaccine-loaded biomimetic mesoporous silica nanoparticles; The outermost layer of the bionic mesoporous silica nanoparticles loaded with the vaccine is coated with a layer of amphiphilic polymer to obtain a bionic vaccine oral delivery system.

3. A method for preparing the bionic vaccine oral delivery system according to claim 1, characterized in that: The following steps are involved: S1: Preparation of mesoporous silica nanoparticles Sodium salicylate, a cationic surfactant, and triethanolamine are added to an aqueous solution to obtain a mixed solution; wherein, in the mixed solution, according to the solid-liquid ratio, sodium salicylate: cationic surfactant: triethanolamine: water = (1-14) mg: (1.5-25) mg: (0.08-5.00) mg: 1 mL; Tetraethyl silicate is added to the mixed solution, and after stirring and reacting at 25-100° C. for 1-36 hours, solid-liquid separation is performed to obtain mesoporous silica nanoparticles containing a template; the template is removed to obtain mesoporous silica nanoparticles without the template; wherein, by mass ratio, tetraethyl silicate: cationic surfactant = (2.4-20):1; S2: Preparation of amino-modified mesoporous silica The mesoporous silica nanoparticles with the template removed are dispersed in anhydrous ethanol, 3-aminopropyltriethoxysilane is added, and the mixture is stirred and refluxed at 60-90° C. for 2-24 hours, and then cooled and separated to obtain amino-modified mesoporous silica; wherein, by mass ratio, 3-aminopropyltriethoxysilane: mesoporous silica nanoparticles = (2.5-5):1; S3: Preparation of carboxylated mesoporous silica Dispersing the aminated mesoporous silica in a solvent, adding anhydride, stirring and reacting for 6-48 hours, separating the solid from the liquid, washing the precipitate, and obtaining carboxylated mesoporous silica; wherein, by mass ratio, the aminated mesoporous silica: anhydride = 1: (1-3); S4: Preparation of biomimetic mesoporous silica core The carboxylated mesoporous silica is dispersed in a polar solvent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are used as catalysts, and long-chain fatty acid glycerides are added to form a reaction system, react for 12-48 hours, separate the solid and liquid, and wash the precipitate to obtain a biomimetic mesoporous silica core; By mass ratio, carboxylated mesoporous silica: long-chain fatty acid glyceride = (0.5~3):1; S5: Preparation of biomimetic mesoporous silica nanoparticles loaded with vaccines The biomimetic mesoporous silica core and the model drug vaccine are mixed, and the mixture is fully mixed and stirred in a phosphate buffer to obtain biomimetic mesoporous silica nanoparticles loaded with the vaccine; wherein the mass ratio of the biomimetic mesoporous silica core to the model drug vaccine is 1:(0.05-1); S6: High mucus permeability biomimetic vaccine oral delivery system The amphiphilic polymer and the bionic mesoporous silica nanoparticles loaded with the vaccine are added into a phosphate buffer, mixed, and then the solid and liquid are separated to obtain a bionic vaccine oral delivery system; wherein, by mass ratio, the amphiphilic polymer: bionic vaccine system = (5-10):

1.

4. Use of the bionic vaccine oral delivery system according to claim 1 in the preparation of drugs for preventing influenza virus.

Images