Cation-based mannose modified liposome gel microsphere oral delivery system and application thereof

Abstract

The invention discloses a mannose modified liposome gel microsphere oral delivery system based on cations and application of the mannose modified liposome gel microsphere oral delivery system. The oral delivery system comprises a core layer and a wrapping layer, the core layer is mannose modified cationic liposome loaded with a mucous membrane adjuvant retinoic acid and an antigen, and the wrapping layer is sodium alginate gel vulcanized and modified by cysteine. The mannose modified cationic liposome is prepared by coupling mannose on the surface of the cationic liposome, and the cysteine modified sodium alginate gel is used for protecting antigens in the core layer from being eroded by gastric juice, enhancing the adhesiveness of a delivery system in the intestinal tract and prolonging the retention time of the delivery system in the intestinal tract; in addition, the R-MLip can be expanded and released under the environment that the pH of the intestinal tract is increased. The inside of the gel microsphere of the oral delivery system shows a regular net-shaped structure and has abundant holes and channels, and the oral delivery system shows a good prospect in the aspect of enhancing mucous membrane and systemic immunity and possibly becomes a potential substitute of a traditional attenuated live vaccine in the future.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to an oral delivery system for cation-based mannose-modified liposome gel microspheres and its application. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is an RNA coronavirus that infects the epithelial cells of the small and large intestines of pigs of all ages, causing severe diarrhea, vomiting, and dehydration. The virus has a high transmissibility and mortality rate, exceeding 90% in suckling piglets, causing enormous economic losses to the pig industry. Currently, there are no specific drugs for treatment. Vaccination is an important means of preventing and controlling infectious diseases in young and adult animals. The most common preventative method is still intramuscular vaccination. However, although intramuscular vaccination can rapidly induce the production of specific serum IgG antibodies, it is relatively less effective in stimulating mucosal and cellular immunity. For mucosal infections, inducing a local immune response is crucial. Locally secreted IgA (sIgA) antibodies can prevent pathogen attachment and invasion and neutralize toxins. However, intramuscular injection cannot induce a strong mucosal immune response in the intestine (the main site of infection and viral shedding). One way to induce intestinal immunity is through oral vaccination. Therefore, developing safe and effective oral vaccines is of great significance.

[0003] Live attenuated vaccines are pathogens that have undergone various treatments, resulting in structural changes and reduced virulence while retaining their immunogenicity. When administered, they do not cause disease but act as antigens to induce humoral and cellular immune responses, stimulating the body to produce specific memory B cells and memory T cells, thus providing long-term or lifelong protection. Compared to inactivated vaccines, these vaccines offer stronger immunogenicity and a longer duration of action. However, direct oral administration can lead to particle defects due to the highly acidic environment of the stomach, interfering with immunization. The ideal delivery strategy is to encapsulate the antigen in a pH-responsive and biocompatible gel material to protect it from degradation by acids and enzymes in the gastrointestinal tract, maintain antigen activity during gastric transfer, and achieve rapid release in the small intestine.

[0004] Most intestinal infections require a robust mucosal immune response based on IgA. However, most antigens are poorly immunogenic when administered orally, failing to elicit sufficient mucosal immunity, thus necessitating the use of appropriate mucosal adjuvants. The most common mucosal adjuvants are bacterial material, certain inorganic components, cytokines, and related carriers that enhance antigen presentation. However, due to safety concerns such as acute toxicity and potential delayed side effects, the number of such adjuvants available for routine vaccination is relatively small. Therefore, developing novel, safe, and effective mucosal vaccine adjuvants is crucial.

[0005] Vitamin A is an important micronutrient that regulates immune responses on mucosal surfaces. Previous studies have shown that retinoic acid (RA), a metabolite of vitamin A, can enhance the mucosal homing ability of T cells and B cells. While RA itself does not directly promote the secretion of IgA antibodies, it is essential for the differentiation of IgA-secreting cells (IgA-ASCs). Studies have shown that oral administration of RA during vaccination in mice can enhance mucosal immune responses. RA is an effective mucosal adjuvant in vaccines, but it is insoluble in water and light-labile, resulting in limited bioavailability and a limited ability to induce systemic immune responses. Summary of the Invention

[0006] This invention designs a novel adjuvant system based on cationic liposomes (as shown in Figure 1) to induce mucosal and systemic immunity. Since intestinal mucus carries a negative charge, cationic liposomes with the opposite charge are used to penetrate the mucus layer and reach intestinal epithelial cells. To target intestinal macrophages, the liposome surface is modified with mannose (MLip). Retinyl ether (RA) and antigens, such as porcine epidemic diarrhea virus (PEDV), are loaded into the liposomes (PR-MLip). RA acts as an immunostimulant, promoting the differentiation of IgA plasma cells (IgA-ASC), while PEDV acts as an antigen, stimulating IgA-ASC to produce a large amount of IgA antibodies, thereby inducing a mucosal immune response. Due to the insufficient adhesiveness of pH-responsive sodium alginate, it is modified with cysteine ​​and used to encapsulate the liposomes, preparing liposome gel microspheres (PR-MLip@Gel). These microspheres protect the antigen from gastric acid erosion, enhance its adhesion in the intestine, and prolong its residence time in the intestine.

[0007] Using BALB / c mice as a model, the immunomodulatory effect of retinoic acid liposome gel microspheres as an adjuvant for porcine epidemic diarrhea virus (PEDV) live attenuated vaccine was evaluated, particularly in terms of mucosal immune response. Following oral administration, PR-MLip@Gel reached the intestine. Due to the increased pH, the gel microspheres rapidly expanded and released PR-MLip, which penetrated the mucus layer to reach the small intestinal epithelium. There, it was selectively internalized by intestinal macrophages, promoting the maturation of antigen-presenting cells (APCs), thereby activating a strong antigen-specific mucosal secretory immunoglobulin A (sIgA) and serum immunoglobulin G (IgG) response to prevent PEDV infection. This oral delivery system shows promising potential in enhancing mucosal and systemic immunity and may serve as a potential alternative to traditional live attenuated vaccines in the future.

[0008] The technical solution adopted in this invention is as follows:

[0009] An oral delivery system for cation-based mannose-modified liposome gel microspheres includes a core layer and an encapsulation layer; the core layer is a mannose-modified cationic liposome (R-MLip) loaded with the mucosal adjuvant retinoic acid (RA) and an antigen, and the encapsulation layer is a sodium alginate gel (Cys-Alg) modified with cysteine ​​sulfide.

[0010] The mannose-modified cationic liposome (R-MLip) is prepared by coupling mannose to the surface of cationic liposomes. The cationic liposomes penetrate the mucus layer and reach the intestinal epithelial cells by means of the electrostatic interaction between the positive charge and the negatively charged mucus layer of the intestine. The mannose is used to achieve selective targeting of intestinal macrophages.

[0011] The cysteine-modified sodium alginate gel is used to solve the problem of insufficient adhesion of unmodified sodium alginate. It can protect the antigen in the core layer from gastric juice erosion, enhance the adhesion of the delivery system in the intestine and prolong its residence time in the intestine, and can expand and release R-MLip in an environment with increased intestinal pH.

[0012] The mass ratio of the core layer R-MLip to the wrapping layer Cys-Alg is 1:1 to 1:4.

[0013] The oral delivery system gel microspheres exhibit a regular network structure with abundant pores and channels, ranging in diameter from 200 to 250 μm.

[0014] The antigen is selected from one or more of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), and bovine coronavirus (BCoV); or the antigen is a live attenuated vaccine, inactivated vaccine, recombinant protein antigen (such as S protein, N protein), or nucleic acid vaccine of the above-mentioned viruses.

[0015] The cysteine-modified pH-responsive sodium alginate gel not only prevents adjuvants and antigens from being eroded by gastric juice, but also enhances intestinal adhesion and prolongs the duration of action.

[0016] The raw materials for preparing the mannose-modified cationic liposomes include egg yolk lecithin, DC-cholesterol, and Man-PEG. 2000 -DSPE and retinoic acid; the mass fractions of each raw material are: lecithin 5-25 parts, DC-cholesterol 5-25 parts, Man-PEG 2000 -DSPE 0.1-0.3 parts, retinoid 0.15-0.6 parts.

[0017] More preferably, the mass ratio of lecithin to DC-cholesterol is 3:1.

[0018] In the sodium alginate gel modified with cysteine ​​sulfate in the encapsulation layer, the molar ratio of sodium alginate to anhydrous L-cysteine ​​hydrochloride is 2:1 to 1:2. The concentration of the attenuated PEDV virus is 1×10⁻⁶. 5 -1×10 6 TCID50 / mL; the concentration of the core layer R-MLip is 5-15 mg / mL, and the volume ratio of PEDV attenuated live virus to PR-MLip is 1:1-1:5.

[0019] Preferably, the molar ratio of sodium alginate to cysteine ​​is 1:0.5 to 1:1. More preferably, the molar ratio of sodium alginate to cysteine ​​is 1:1.

[0020] The method for preparing the oral delivery system includes the following steps:

[0021] (1) Preparation of retinoic acid-loaded mannose-modified liposomes (RA-MLip): Egg yolk lecithin, DC-cholesterol, and Man-PEG were added. 2000 -DSPE and retinoic acid are dissolved in anhydrous ethanol, and the organic solvent is removed by rotary evaporation to form a film. After adding PBS buffer, the film is hydrated and sonicated at 0-80℃ to obtain RA-MLip.

[0022] (2) Preparation of mannose-modified liposomes loaded with antigen and retinoic acid (PR-MLip): The RA-MLip obtained in step (1) is mixed with the antigen and adsorbed overnight to obtain PR-MLip;

[0023] (3) Preparation of sodium alginate gel modified by cysteine ​​sulfide (Cys-Alg): Sodium alginate was dissolved in water, EDC and NHS were added and reacted, and L-cysteine ​​hydrochloride anhydrous product was added and the reaction was continued in the dark. After dialysis, Cys-Alg was obtained.

[0024] (4) Preparation of PR-MLip@Gel: After mixing the PR-MLip obtained in step (2) with the Cys-Alg obtained in step (3) evenly, inject it into the calcium chloride solution using a syringe and crosslink at room temperature to obtain cysteine-modified calcium alginate-encapsulated mannose-modified liposomes loaded with PEDV attenuated live virus and retinoic acid.

[0025] In step 1), the mass ratio of egg yolk lecithin to DC-cholesterol is 3:1, preferably hydrated at 40°C for 1 hour, followed by sonication for 90 seconds to obtain RA-MLip.

[0026] The cross-linking time in step (4) is 15-45 minutes.

[0027] The preferred method for preparing RA-MLip involves mixing 15 mg of egg yolk lecithin, 5 mg of DC-cholesterol, and 0.3 mg of Man-PEG. 2000 -DSPE and 0.6 mg RA were dissolved in 4 mL of anhydrous ethanol. The solution was evaporated under reduced pressure to form a film. Then, 4 mL of PBS solution was added, and the film was hydrated at 40°C for 1 hour, followed by sonication for 90 seconds to obtain the RA-MLip suspension. Furthermore, the RA-MLip prepared using the optimal process conditions had a particle size distribution of approximately 240 nm.

[0028] Application of the oral adjuvant delivery system in the preparation of porcine epidemic diarrhea virus vaccine.

[0029] The vaccine is an oral vaccine; it is used to induce the production of secretory immunoglobulin A (sIgA) in the intestinal mucosa and serum immunoglobulin G (IgG), thereby achieving dual protection of mucosal immunity and systemic immunity.

[0030] The antigens described in this invention are antigens for oral vaccines, preferably attenuated live attenuated enteropathogenic virus vaccine strains similar to porcine epidemic diarrhea virus (PEDV) attenuated live virus. Specific examples include: porcine transmissible gastroenteritis virus (TGEV) attenuated live vaccine strain antigen, porcine rotavirus (RV) attenuated live vaccine strain antigen, porcine deltacoronavirus (PDCoV) attenuated live vaccine strain antigen, bovine viral diarrhea virus (BVDV) attenuated live vaccine strain antigen, and Newcastle disease virus (NDV) attenuated live vaccine strain antigen. Other enteroassociated bacterial antigens (such as pathogenic Escherichia coli K88 strain antigen and attenuated Salmonella strain antigen) and parasite antigens (such as porcine coccidia antigen) may also be included.

[0031] The concentration of the antigen is 1×10⁻⁶. 5 -1×10 6 TCID50 / mL (viral antigen) or 1×10 6 -1×10 7 CFU / mL (bacterial antigen); the concentration of R-MLip is 5-15 mg / mL, and the volume ratio of antigen to R-MLip is 1:1-1:5; the loading mass ratio of retinoic acid (RA) to antigen is (1:10)-(1:50).

[0032] The oral delivery system of the present invention will be specifically described below using PEDV attenuated live virus as an example.

[0033] An oral drug delivery system based on cationic mannose-modified liposome gel microspheres is disclosed. This system utilizes sulfide-modified sodium alginate gel to encapsulate mannose-modified cationic liposomes loaded with the mucosal adjuvant retinoic acid and attenuated PEDV live virus. Retinoic acid promotes the differentiation of IgA antibody-secreting cells, leading to rapid secretion of large amounts of IgA antibodies upon antigen stimulation, effectively regulating the intestinal mucosal immune response. The cationic liposomes effectively load retinoic acid and adsorb antigens through electrostatic interactions, improving the stability of both the adjuvant and antigen, and facilitating penetration into the intestinal mucus layer. The mannose modification further enhances bioavailability by facilitating uptake by intestinal macrophages. Encapsulating the liposomes with cysteine-modified sodium alginate not only prevents the adjuvant and antigen from being eroded by gastric juices but also enhances intestinal adhesion and prolongs the duration of action.

[0034] The specific preparation method is as follows:

[0035] (1) Egg yolk lecithin, DC-cholesterol, and Man-PEG 2000 -DSPE and retinoic acid were dissolved in anhydrous ethanol, and the organic solvent was removed by rotary evaporation to form a film. After hydration with PBS buffer, the film was sonicated for 5 hours to obtain retinoic acid-loaded mannose-modified liposomes (RA-MLip).

[0036] Preferably, the egg yolk lecithin, DC-cholesterol, and Man-PEG are... 2000 - The mass fractions of DSPE and retinoic acid are 5-25, 5-25, 0.1-0.3, and 0.15-0.6, respectively;

[0037] Preferably, the hydration temperature is 0-80℃;

[0038] (2) The RA-MLip obtained in (1) is mixed with PEDV attenuated live virus and adsorbed overnight to obtain mannose-modified liposomes (PR-MLip) loaded with PEDV attenuated live virus and retinoic acid.

[0039] Preferably, the PEDV attenuated live virus is 1×10⁻⁶. 5 -1×10 6 TCID50 / mL;

[0040] Preferably, the concentration of PR-MLip is 5-15 mg / mL;

[0041] Preferably, the volume ratio of the attenuated PEDV virus to PR-MLip is 1:1 to 1:5;

[0042] (3) Sodium alginate was dissolved in water, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added and reacted for 30 minutes. Then, anhydrous L-cysteine ​​hydrochloride was added and the reaction was continued in the dark for 12 hours. After dialysis, sulfide-modified sodium alginate (Cys-Alg) was obtained.

[0043] Preferably, the mass-to-volume ratio of sodium alginate to water is 0.5:100-2.5:100;

[0044] Preferably, the molar ratio of sodium alginate to anhydrous L-cysteine ​​hydrochloride is 2:1 to 1:2;

[0045] Preferably, the molar ratio of EDC to NHS is 1:1;

[0046] (4) After mixing the PR-MLip obtained in (2) with the Cys-Alg in (3) evenly, inject it into the calcium chloride solution using a syringe and crosslink it at room temperature to obtain cysteine-modified calcium alginate-encapsulated mannose-modified liposomes loaded with PEDV attenuated live virus and retinoic acid (PR-MLip@Gel).

[0047] Preferably, the mass ratio of PR-MLip to Cys-Alg is 1:1 to 1:4;

[0048] Preferably, the mass-to-volume ratio of calcium chloride to water is 1:100-3:100;

[0049] Preferably, the crosslinking time is 15-45 minutes.

[0050] Beneficial effects:

[0051] This invention discloses a novel adjuvant system based on cationic liposomes to induce mucosal and systemic immunity. Cationic liposomes with opposite charges penetrate the mucus layer and reach intestinal epithelial cells. The liposome surface is modified with mannose (MLip) to target intestinal macrophages. Reticulocyte rashes (RA) and porcine epidemic diarrhea virus (PEDV) are loaded into the liposomes (PR-MLip). Retinoic acid, as an immune enhancer, promotes the differentiation of IgA plasma cells (IgA-ASC), while PEDV, as an antigen, stimulates IgA-ASC to produce a large amount of IgA antibodies, thereby inducing a mucosal immune response. Modification of sodium alginate with cysteine ​​for encapsulating the liposomes overcomes the insufficient adhesiveness of sodium alginate, resulting in liposome gel microspheres (PR-MLip@Gel). These microspheres protect the antigen from gastric acid erosion, enhance its adhesion in the intestine, and prolong its residence time in the intestine.

[0052] Using BALB / c mice as a model, PR-MLip@Gel reached the intestine after oral administration. Due to the increased pH, the gel beads rapidly expanded and released PR-MLip, which penetrated the mucus layer to reach the small intestinal epithelium. There, it was selectively internalized by intestinal macrophages, promoting the maturation of antigen-presenting cells (APCs), thereby activating a strong antigen-specific mucosal secretory immunoglobulin A (sIgA) and serum immunoglobulin G (IgG) response to prevent porcine epidemic diarrhea virus (PEDV) infection. This oral delivery system shows promising potential in enhancing mucosal and systemic immunity and may become a potential alternative to traditional live attenuated vaccines in the future. Attached Figure Description

[0053] Figure 1 Preparation and characterization of PR-MLip@Gel; A. Schematic diagram of PR-MLip preparation; B. MLip particle size distribution; C. PR-MLip particle size distribution; D. Zeta potential of MLip, RA-MLip, and PR-MLip; E. Scanning electron microscopy analysis (MLip, RA-MLip, and PR-MLip from left to right, scale bar: 1 μm); F. Stability of PR-MLip under different storage conditions; G. 1H NMR spectra of alginate (Alg) and cysteine-modified alginate (Cys-Alg); H. Infrared spectra of alginate, cysteine, and cysteine-modified alginate; I. Schematic diagram of PR-MLip@Gel preparation; J. Inverted fluorescence microscopy image of MLip@Gel (scale bar: 200 μm); K. Scanning electron microscopy analysis of gel microspheres; Data are expressed as mean ± standard deviation (n=3).

[0054] Figure 2. Tolerance and intestinal retention capacity of MLip@Gel in the gastrointestinal environment; A. Representative images of the degradation process of MLip@Gel in simulated gastric juice (SGF) and simulated intestinal juice (SIF) (scale bar: 500 μm); B. Particle size variation curves of MLip@Gel in simulated gastric juice (SGF) and simulated intestinal juice (SIF); C. Swelling rate of MLip@Gel in water, simulated gastric juice (SGF), and simulated intestinal juice (SIF); D. Release curves of simulated retinoic acid (RA) and porcine epidemic diarrhea virus (PEDV) of PR-MLip and PR-MLip@Gel under gastrointestinal conditions; E. Statistical count of microspheres of MLip@Gel (sodium alginate-based, Alg) and MLip@Gel (cysteine-modified sodium alginate-based, Cys-Alg) after washing; F. Comparison of adhesion ability between MLip@Gel (Alg) and MLip@Gel (Cys-Alg); G Images of residual amounts of MLip@Gel (Alg) and MLip@Gel (Cys-Alg) on ​​the surface of the small intestine before and after three washes; H. Curves showing changes in thiol content of cysteine-modified sodium alginate (Cys-Alg) in simulated gastric juice (SGF) and simulated intestinal juice (SIF); I. In vitro imaging of the distribution of fluorescein isothiocyanate (FITC), FITC-labeled MLip, and FITC-labeled MLip@Gel in the gastrointestinal tract after oral administration; J. Quantitative analysis of the content of fluorescein isothiocyanate (FITC), FITC-labeled MLip, and FITC-labeled MLip@Gel; Data are expressed as mean ± standard deviation (n=3).

[0055] Figure 3 Drug absorption and targeted delivery; A. FITC, FITC-MLIP, and FITC-MLip@Gel across the small intestine; B. FITC, FITC-MLIP, and FITC-MLip@Gel in the small intestine; C. Cell viability of HEK 239T cells (C), RAW264.7 cells (D), and DCs (E) under different MLips, MLip@Gel, and RA-MLip@Gel; Confocal images of RAW264.7 cell uptake (magnification: 40x); G. Quantitative fluorescence analysis of RAW264.7 cell uptake. Data are expressed as mean ± standard deviation (n=3).

[0056] Figure 4In vitro immune activation of the vaccine; A. Representative flow cytometry images of MHC-II, CD86, and CD80 on DCs. B. Expression of CD86 (B), CD80 (C), and MHC II (D) on DCs; C. Expression of CD86 (E), CD80 (F), and MHC II (G) on RAW264.7 cells; D. Expression of IL-4, IFN-γ, and TNF-α cytokines in DCs after in vitro immune stimulation; E. Expression of IL-4, IFN-γ, and TNF-α cytokines in RAW264.7 cells after in vitro immune stimulation.

[0057] Figure 5 A. Vaccine-induced immune activation and induction of mucosal immune responses; B. Vaccine immunization schedule, expression of CD86 (B), CD80 (C), and MCH II (D) on DCs isolated 96 hours after the first immunization (n=3), post-immunization; C. Representative flow cytometry images of α4β7+CCR9+ on intestinal epithelial lymphocytes in each group; D. Analysis of the proportion of α4β7+CCR9+ cells on intestinal epithelial lymphocytes; E. GP / N value of IgA antibody in mouse intestinal lavage fluid; H / N value of IgA antibody in mouse serum (from top to bottom, 0, 14, 28, and 42 days). Unless otherwise stated, data are expressed as mean ± standard deviation, n=6.

[0058] Figure 6 Vaccine-induced systemic immune response; A. P / N ratio of IgG antibodies in mouse serum (from left to right: 0, 14, 28, and 42 days); B. Neutralization titer in mouse serum (from left to right: 14, 28, and 42 days); C. Splenic lymphocyte proliferation index; D. Cytokine levels secreted by mouse spleen cells 48 hours after stimulation (from left to right: IL-4, IFN-γ, and TNF-α); E-flux analysis of mouse spleen lymphocytes; F. Proportion analysis of CD3e+CD4+ T lymphocytes; G. Proportion analysis of CD3e+CD8+ T lymphocytes; H. Proportion analysis of B220+CD19+ B lymphocytes; I. Proportion analysis of CD3e+CD49b+ NK lymphocytes; Data are expressed as mean ± standard deviation (n=6).

[0059] Figure 7 Positive / negative (P / N) ratios of immunoglobulin A (IgA) antibodies in mouse fecal samples (from left to right: 0 days, 14 days, 28 days, and 42 days post-immunization).

[0060] Figure 8 Positive / negative (P / N) ratios of immunoglobulin G (IgG) antibodies in mouse fecal samples (from left to right: 0 days, 14 days, 28 days, and 42 days).

[0061] Figure 9 The curve showing the change in body weight of mice during immunization.

[0062] Figure 10 Following immunization, the heart, liver, spleen, lungs, and kidney tissues were stained with hematoxylin and eosin (H&E) (scale bar: 200 μm).

[0063] Figure 11 After immunization, the small intestinal tissue was stained with hematoxylin and eosin (H&E) (scale bar: 200 μm). Detailed Implementation

[0064] The materials used in the following embodiments are:

[0065] Penicillin / streptomycin solution, fetal bovine serum (FBS), DMEM medium, RPMI 1640 medium, and phosphate-buffered saline (PBS) were purchased from Nanjing Cyper Biotechnology Co., Ltd. (Nanjing, China). IFN-γ, TNF-α, and IL-4 ELISA kits were purchased from Nanjing Liangwei Biotechnology Co., Ltd. (Nanjing, China). Unless otherwise specified, all reagents were of analytical grade and purchased from the supplier.

[0066] Example 1: Preparation and characterization of cysteine-modified sodium alginate (Cys-Alg)

[0067] Reference for the preparation method of Cys-Alg: Haishan Wu, Jian Nan, Liu Yang, Hyun JinPark, Jinglei Li. Insulin-loaded liposomes packaged in alginate hydrogels promote the oral bioavailability of insulin. Journal of Controlled Release, 2022, 353:51-62.

[0068] 177.39 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 106.50 mg of N-hydroxysuccinimide (NHS) were added to 20 mL of sodium alginate solution (2%, by volume). After incubation at room temperature for 45 minutes, 145.86 mg of anhydrous L-cysteine ​​hydrochloride was added, and the pH was adjusted to 5–6 with 0.1 mol / L sodium hydroxide (NaOH) solution. The reaction was continued for 12 hours at room temperature in the dark. Two dialyses were performed with 5 mmol / L hydrochloric acid (8,000–14,000 Daltons) to remove unreacted cysteine ​​and small molecule impurities, in the dark. After two dialyses with 1 mmol / L hydrochloric acid, the pH was adjusted to 4. The sample was lyophilized and stored at 4°C in a sealed, light-protected environment.

[0069] One milligram of dried Cys, Alg, or Cys-Alg was ground and mixed uniformly with 100 milligrams of dried potassium bromide (KBr) powder, and pressed into translucent thin films. The analysis was performed using Fourier transform infrared spectroscopy (FTIR) in the wavenumber range of 4000–400 cm⁻¹. Alg and Cys-Alg were dissolved in heavy water (D₂O). Their structural characteristics were analyzed by ¹H nuclear magnetic resonance spectroscopy (Bruker, Germany). The thiol content in Cys-Alg was determined using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reagent. 4.0 milligrams of Cys-Alg were dissolved in 1 mL of water, and the pH was adjusted to 4.0 with 0.1 mol / L hydrochloric acid solution. The Cys-Alg solution was then mixed with an equal volume of DTNB solution (0.3 mg / mL), and reacted at room temperature in the dark for 15 minutes. The absorbance was measured at 412 nm using a UV spectrophotometer.

[0070] The cross-linking rate (mol%) of cysteine ​​and sodium alginate was calculated based on Equation 1.

[0071] Crosslinking rate (%) = Thiol (mol) / Sodium alginate (mol) × 100 (1).

[0072] Example 2: Preparation and Characterization of PR-MLip

[0073] Based on the results of the single-factor study, the effects of three levels of four factors—the ratio of egg yolk lecithin to DC-cholesterol, the amount of RA, hydration temperature, and sonication time—on the encapsulation efficiency of liposomes were determined. An orthogonal experimental design was used (Table 1), and the results were analyzed using SPSS software.

[0074] Table 1. Factor Level Table for Orthogonal Experiment

[0075] level Egg yolk lecithin: DC-cholesterol RA dosage Hydration temperature (°C) Ultrasound time (s) 1 2:1 2% 20 90 2 3:1 3% 40 120 3 4:1 4% 60 150

[0076] To further optimize the liposome preparation process, based on the results of single-factor investigation, an orthogonal experiment was conducted at three levels with egg yolk lecithin:DC-cholesterol (A), RA dosage (B), hydration temperature (C), and sonication time (D) as influencing factors, in order to obtain RA-MLip with high encapsulation efficiency, good stability, and good reproducibility.

[0077] The results of the orthogonal experiment and analysis are shown in Table 2 below. The order of influence of each factor and level on the results is A2>A1>A3, B2>B1>B3, C2>C3>C1, D1>D3>D2, and the optimal combination is A2B2C2D1. Three batches of RA-MLip were prepared using the optimal combination obtained from the orthogonal experiment. After testing, the encapsulation efficiencies of the three batches of RA-MLip were 78.96%, 79.24%, and 78.65%, respectively, and the loading rates were 2.37%, 2.38%, and 2.36%, respectively, all higher than those of the other groups in the orthogonal experiment. This indicates that the optimal combination is the best process condition for preparing RA-MLip.

[0078] The specific steps are as follows: Add 15 mg of egg yolk lecithin, 5 mg of DC-cholesterol, and 0.3 mg of Man-PEG. 2000 -DSPE and 0.6 mg RA were dissolved in 4 mL of anhydrous ethanol. The solution was evaporated under reduced pressure to form a film. Then, 4 mL of PBS solution was added, and the film was hydrated at 40°C for 1 hour, followed by sonication for 90 seconds to obtain the RA-MLip suspension. Furthermore, the RA-MLip prepared under optimal process conditions had a particle size distribution of approximately 240 nm, a potential of approximately 29 mV, a PDI of less than 0.2, and an RSD of less than 5%, indicating that the process is stable, reliable, and has good reproducibility. Specific parameters are shown in Table 3.

[0079] Table 2. Results and Analysis of Orthogonal Experiments

[0080]

[0081] Table 3. Specific parameters of the three batches of RA-MLip prepared based on the orthogonal experimental results

[0082]

[0083] Phosphatidylcholine, DC-cholesterol, and mannose-polyethylene glycol-distearate phosphatidylethanolamine (Man-PEG) were added. 2000DR-DSPE and retinoic acid (RA) were dissolved in anhydrous ethanol, with a final concentration of 1 mg / mL for each substance. The solution was mixed at a volume ratio of 15:5:0.3:0.6, and the anhydrous ethanol was removed by rotary evaporation under reduced pressure at 40 °C to obtain a uniform film. The film was dispersed in 3 mL of phosphate-buffered saline (PBS) and incubated in a 40 °C water bath for 1 hour to promote complete hydration of the lipid membrane. Then, it was sonicated for 90 seconds, and the solution was filtered three times through a 0.45 μm microporous membrane to obtain RA-MLip, which was then freeze-dried and stored at 4 °C protected from light. Blank liposomes (MLip) were prepared without the addition of retinoic acid. PR-MLip was prepared by dispersing 15.0 mg of RA-MLip liposomes in 1 mL of PBS, adding 250 μL of porcine epidemic diarrhea virus (PEDV) virus suspension (1×10⁻⁶). 5 (TCID50 / mL), adsorbed overnight.

[0084] After MLip, RA-MLip, and PR-MLip were prepared into 1 mg / mL solutions, their particle size, polydispersity index, and zeta potential were measured using a Malvern Zetasizer Nano ZSE. The morphology and size of the newly prepared MLip, RA-MLip, and PR-MLip were imaged using field emission scanning electron microscopy (SEM, Quanta FEG 250, FEI, USA). Encapsulation efficiency (EE) and drug loading (LC) were determined according to the literature method [Aung Myo Htet, Ei Ei Thin, Mi Mi Saw, Soe Win. Chemical analysis of hydroquinone and retinoic acid contents in facial whitening creams. Asian Journal of Pharmaceutical Sciences, 2016, 11(1):89-90. Young IlJeong, Kyu Don Chung, Da Hye Kim, Yoon Hyuk Kim, Yeon Soo Lee, Ki Choon Choi. All-trans retinoic acid-incorporated nanoparticles of deoxycholic acid-conjugated dextran for treatment of CT26 colorectal carcinoma cells. International Journal of Nanomedicine, 2013, 8(1):485-493.]. The RA-MLip solution was dialyzed in pure water (8000-12000 Kda) for 6 hours. Take 0.5 mL of RA-MLip solution before and after dialysis, and demulsify with an equal volume of methanol solution. Then, determine the RA content in the solution using high-performance liquid chromatography (HPLC). A KromaSil C18 column (5 μm, 250 mm × 4.6 mm) was used, with methanol and 2% acetic acid aqueous solution (92:8 v / v) as the mobile phase. The injection volume was 20 μL, and the flow rate was 1.0 mL / min. The detection wavelength was 350 nm. EE (%) and LC (%) were calculated using Equations 2 and 3, respectively.

[0085] EE (%) = Cr / C0 × 100 (2)

[0086] LC (%) = Cr×V / (M + C0×V)×100 (3)

[0087] Where Co is the concentration of RA in liposomes before dialysis, Cr is the concentration of RA in liposomes after dialysis, V is the volume of the liposome solution, and M is the mass of the liposomes.

[0088] Example 3: Preparation and Characterization of PR-MLip@Gel

[0089] 37.50 mg of PR-MLip was added to 5 mL of 1.5% Cys-Alg solution and mixed thoroughly. Then, using a 1 mL sterile syringe (30 g), the mixture was injected into 50 mL of 3% (w / v) CaCl2 solution at a rate of 5 mL / min. The cross-linking reaction was carried out at room temperature for 20 minutes to obtain PR-MLip@Gel.

[0090] PR-MLip-free gels and PR-MLip@Gel were freeze-dried and dehydrated, then disrupted with liquid nitrogen. After gold sputtering, their internal structure was observed using scanning electron microscopy. To investigate the distribution of liposomes within the gel microspheres, FITC was used instead of RA to prepare liposome gel microspheres, which were then observed using an inverted fluorescence microscope.

[0091] Example 4 Performance Testing

[0092] 4.1 RA-MLip Storage Stability Determination

[0093] To test the storage stability of RA-MLip, the encapsulation efficiency of RA-MLip was measured every 5 days under the following conditions: 4°C with light, 4°C without light, 25°C with light, and 25°C without light.

[0094] 4.2 In vitro degradation and release test

[0095] To investigate the degradation mechanism of this oral drug delivery system, liposome gel microspheres (MLip@Gel) were placed in simulated gastric fluid (SGF, pH 1.2, without pepsin) and simulated intestinal fluid (SIF, pH 6.8, without trypsin). Morphological changes were observed under a microscope at 0, 0.5, 1, 2, and 6 hours. Furthermore, the swelling properties of MLip@Gel were evaluated. 50 mg of lyophilized MLip@Gel was incubated in different media (water, SGF, and SIF), and removed at the sampling time points, dried, and weighed.

[0096] To investigate the release behavior of PR-MLip@Gel, RA-MLip and PR-MLip@Gel were first incubated in SGF for 2 hours, then transferred to SIF and incubated for another 24 hours. At predetermined time points, 0.5 mL of sample was taken and an equal volume of simulated solution was added. The release amount of RA was detected by high performance liquid chromatography (HPLC), and the release amount of PEDV was detected by real-time quantitative PCR (qPCR).

[0097] 4.3 Cytotoxicity test

[0098] The safety of the oral delivery vector was verified using the MTT assay. HEK 293T cells, RAW264.7 cells, or dendritic cells (DCs) were treated with different concentrations (0, 62.5, 125, 250, 500, and 1000 μg / mL) of MLip, MLip@Gel, and RA-MLip@Gel, respectively, and co-incubated at 37 °C for 24 hours. After washing three times with PBS, 20 μL of MTT solution was added to each well. After incubation for another 4 hours, 150 μL of dimethyl sulfoxide was added, and formazan crystals were dissolved by shaking. Cell viability was determined by measuring absorbance at 490 nm using a microplate reader.

[0099] 4.4 Determination of sulfhydryl content in cysteine-modified alginate (Cys-Alg)

[0100] At 37°C, 40 mg of Cys-Alg was incubated in 10 mL of simulated gastric fluid (SGF) or simulated intestinal fluid (SIF). The thiol content of 50 μL of sample (with the same volume of solvent replenished) was determined every 1 hour by the dithiodinitrobenzoic acid (DTNB) method.

[0101] The thiol groups grafted onto the material can form disulfide bonds with thiol-containing substances in the intestinal mucus layer, thereby improving intestinal adhesion. From this perspective, the thiol content is related to the final effect. However, in the preparation process, Cys-Alg prepared using different ratios of sodium alginate and cysteine ​​showed the highest crosslinking rate for Cys-Alg-3 (sodium alginate:cysteine ​​= 1:1.5, molar ratio), but it was poorly soluble in deionized water and unsuitable for subsequent preparation of gel microspheres. Cys-Alg-1 (sodium alginate:cysteine ​​= 1:0.5, molar ratio) and Cys-Alg-2 (sodium alginate:cysteine ​​= 1:1, molar ratio) showed better solubility in deionized water, with Cys-Alg-2 exhibiting a higher crosslinking rate. Therefore, Cys-Alg-2 was used for subsequent experiments. Specific data are shown in Table 4.

[0102] Table 4

[0103] Group SA (mg) Cys (mg) EDC (mg) NHS (mg) Crosslinking rate (%) Molar ratio (Alg:Cys) Cys-Alg -1 100 36.47 88.7 53.21 5.42 1:0.5 Cys-Alg-2 100 72.94 88.7 53.21 5.81 1:1 Cys-Alg -3 100 109.41 88.7 53.21 6.19 1:1.5

[0104] 4.5 In vitro intestinal adhesion experiment

[0105] Liposomes (MLip), calcium alginate liposome gel microspheres (MLip@Gel (Alg)), and cysteine-modified calcium alginate liposome gel microspheres (MLip@Gel (Cys-Alg)) were placed on the surface of upright fresh porcine small intestine tissue. Their adhesion behavior was observed by photographing at different time points. To further quantify the intestinal adhesion rate of the liposome gel microspheres, methylene blue was loaded into the liposomes. A section of fresh porcine small intestine approximately 5 cm long was used for the experiment. Fifty MLip@Gel (Alg) and MLip@Gel (Cys-Alg) microspheres were dispersed in phosphate-buffered saline (PBS) and allowed to flow over the surface of the porcine small intestine epithelium, followed by three rinses with PBS. The number of microspheres remaining on the small intestine epithelium surface after each rinse was recorded.

[0106] 4.6 In vivo biodistribution experiment

[0107] Fluorescein isothiocyanate (FITC) was used to mimic rheumatoid arthritis (RA) drugs. Mice were administered FITC, FITC-labeled MLip, and FITC-labeled MLip@Gel (n = 3 per group) by gavage at a dose of 80 mg / kg (FITC). The small intestine of the mice was harvested at 0, 2, 4, 12, and 24 hours. At 24 hours, the major organs (heart, liver, spleen, lung, and kidney) of the mice were also harvested. The samples were imaged and analyzed using the VISQUE InVivo Smart-LF in vivo imaging system.

[0108] 4.7 Small Intestinal Permeability Test

[0109] FITC was used to simulate RA drugs. The effect of this formulation on the permeability of RA drugs was studied by detecting the FITC content permeating the small intestinal loop using a fluorescent microplate reader. 0.5 mL of fluorescently labeled samples (FITC, FITC-MLip, and FITC-MLip@Gel) were injected into small intestinal loops prepared from 3 cm of fresh porcine small intestine. The small intestinal loops were incubated at 37°C with 40 mL of Krebs-Ringer buffer in the dark using a low-speed shaker. At 0, 30, 60, 90, 120, and 240 minutes, 200 μL of sample was collected for analysis, and an equal volume of Krebs-Ringer buffer was added. The apparent permeability coefficient (Papp) was calculated using Formula 4 as follows:

[0110] Papp = dQ / dt1 / S1 / C (4)

[0111] Where dQ / dt is the FITC permeation rate, S is the surface area of ​​the small intestinal segment, and C is the initial concentration of FITC.

[0112] 4.8 Cell uptake experiment

[0113] Cells were loaded at a rate of 1×10 5 RAW264.7 cells were seeded at a density of 10 cells / mL in 24-well plates with cell spreaders and cultured for 24 hours until adherence. FITC was used instead of RA drug in sample preparation. RAW264.7 cells were co-incubated for 4 hours with 1 mL of FITC, FITC-labeled unmodified liposomes (FITC-Lip), FITC-labeled mannose-modified liposomes (FITC-MLip), and mannose-added FITC-labeled mannose-modified liposomes (Man + FITC-MLip). The concentration of FITC in each sample was 50 μg / mL. After incubation, cells were washed three times with pre-chilled PBS and then fixed with 4% paraformaldehyde for 20 minutes at room temperature. The fixative was discarded, and cells were washed again. Cell nuclei were labeled with 4',6-diamidindo-2-phenylindole (DAPI). Cells were observed and photographed using a laser confocal microscope (LSM 880) (Zeiss, Germany). The treatment of dendritic cells was the same as that of RAW264.7 cells, except that the Man + FITC-MLip group was not set up.

[0114] 4.9 In vitro immune stimulation experiment

[0115] In cell experiments, ovalbumin (OVA) was used instead of porcine epidemic diarrhea virus (PEDV) as the antigen. Two mL of phosphate-buffered saline (PBS), OVA, rheumatoid arthritis drug (RA), MLip@Gel, and liposome gel microspheres loaded with RA and OVA (OR-MLip@Gel) were added to 6-well plates seeded with RAW264.7 cells or dendritic cells, with OVA at a concentration of 10 μg / mL and RA at a concentration of 10 μg / mL. After 24 hours of incubation, cells were collected and labeled in the dark for 30 minutes with MHC II-APC, CD80-PE, and CD86-FITC antibodies (Biolegend, USA). Flow cytometry was used to analyze the expression of MHC II, CD80, and CD86 on RAW264.7 cells and dendritic cells. After 48 hours of incubation, 0.5 ml of supernatant was collected from each group, and the levels of cytokines (interferon-γ (IFN-γ), interleukin-4 (IL-4), and tumor necrosis factor-α (TNF-α)) were detected by enzyme-linked immunosorbent assay (ELISA).

[0116] 4.10 Animal Immunization Experiment

[0117] 4.10.1 Mouse immunization and sample collection

[0118] Female BALB / c mice (approximately 6 weeks old, weighing 18–22 g, specific pathogen-free (SPF) grade) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Six-week-old BALB / c mice were randomly divided into 6 groups (n=9 per group). Mice were orally immunized with 200 μL of PBS, PEDV, RA, MLip@Gel, or PR-Lip@Gel. One group of mice was intramuscularly injected with 200 μL of PEDV (RA: 100 μg per mouse, PEDV: TCID50 of 10⁵ / 200 μL). Immunization was repeated every two weeks with the same dose, for a total of three immunizations. Fecal samples were collected before the first immunization and 48 hours after each immunization. Blood was collected on days 0, 14, 28, and 42 for serum separation. On day 14 after the last immunization, all mice were euthanized, and heart, liver, spleen, lung, kidney, and small intestine tissues were collected.

[0119] The experimental protocol had previously been approved by the Animal Ethics Committee of Nanjing University of Technology and was conducted in accordance with relevant guidelines and regulations.

[0120] 4.10.2 In vivo immune stimulation experiment

[0121] Ninety-six hours after the initial immunization, bone marrow dendritic cells were isolated from the femur and tibia of mice and labeled with MHCII-APC, CD80-PE, and CD86-FITC antibodies, and then analyzed by flow cytometry.

[0122] 4.10.3 Enzyme-linked immunosorbent assay (ELISA)

[0123] Using PEDV spike protein as an antigen, PEDV-specific IgG and IgA antibodies in mouse serum, feces, and intestinal lavage fluid were detected by ELISA.

[0124] 4.10.4 Serum neutralization test

[0125] Mouse serum was inactivated in a 56°C water bath for 30 minutes, and then inactivated with DMEM medium at a 2-fold gradient (2... -1 Up to 2 -9 Dilute 50 μL of the diluted serum with 50 μL of PEDV (100 TCID50 / 50 μL), neutralize at 37°C for 1 hour, and then add the mixture to 96-well plates containing HEK 293T cells. After incubation at 37°C for 2 hours, discard the serum-virus mixture and add 100 μL of DMEM medium containing 5 μg / mL trypsin for maintenance culture. Observe and record the lesion condition daily, and calculate the results according to the Karber method.

[0126] 4.10.5 Splenic lymphocyte proliferation test

[0127] On day 42 post-immunization, splenic lymphocytes were isolated from the spleen of mice and seeded into 96-well plates. After cell adhesion, 100 μL of concanavalin A (ConA) at 10 μg / mL was added as a stimulant, and an equal volume of RPMI-1640 medium was added to the negative control group. After 72 hours of culture, 50 μL of cell culture supernatant was collected for subsequent cytokine detection. Cell proliferation was then assessed using the MTT assay, and the stimulation index (SI) was calculated according to Formula 5.

[0128] SI = (Optical density (OD) value of the immunized group - OD value of the blank control group) / (OD value of the negative control group - OD value of the blank control group) (5)

[0129] 4.10.6 Cytokine Level Measurement

[0130] The collected cell culture supernatants were quantitatively analyzed using mouse TNF-α, mouse IL-4, and mouse IFN-γ ELISA kits. The specific procedures were performed according to the kit manufacturer's instructions.

[0131] 4.10.7 Flow cytometry immunophenotyping analysis

[0132] Splenic lymphocytes were isolated from the spleen of mice, and intraepithelial lymphocytes were isolated from the small intestine of mice for flow cytometry analysis. Splenic cells (103) were labeled with fluorescein-labeled cell surface-specific anti-mouse antibodies CD3e-FITC / CD4-PE / CD8a-APC, B220-PerCPCy5.5 / CD19-FITC, and CD3e-FITC / CD49b-APC (Biolegend, USA). 6 Cells were labeled with fluorescein-labeled cell surface-specific anti-mouse antibody α4β7-APC / CCR9-PE (Biolegend, USA) to label small intestinal intraepithelial lymphocytes (10 cells). 6 (Number of samples) Detection was performed using flow cytometry, and data analysis was conducted using NovoExpress software.

[0133] 4.10.8 Security Assessment

[0134] The safety of the oral vaccine was assessed by weight monitoring and histopathological examination. Collected mouse hearts, livers, spleens, lungs, kidneys, and small intestines were fixed in 10% formalin solution. After paraffin embedding and sectioning, hematoxylin and eosin (H&E) staining was performed for observation.

[0135] Statistical analysis

[0136] Graphing and statistical analysis were performed using Prism GraphPad 8.0 or Origin software. Unless otherwise specified, all experiments were repeated three times (n = 3), and data are presented as mean ± standard deviation. One-way ANOVA was used to compare whether significant differences existed between groups. P < 0.05 was considered significant, P < 0.01 was considered highly significant, and P < 0.001 was considered extremely significant.

[0137] Results and discussion

[0138] 1. Preparation and characterization of PR-MLip

[0139] The oral delivery process of this invention faces several challenges: the strong acidity of gastric juice causes antigen denaturation, and the intestinal mucus layer and tightly connected intestinal epithelium are insufficient, resulting in inadequate mucosal immune strength. To address these challenges, this invention uses pH-responsive alginate to encapsulate antigen-loaded liposomes, protecting the antigen from gastric juice erosion and enabling rapid release upon arrival in the intestine. The positively charged cationic liposomes easily penetrate the intestinal mucus layer, and their cell-membrane-like structure facilitates the uptake of adjuvants and antigens by antigen-presenting cells. The sodium alginate undergoes sulfidation modification, enhancing intestinal adhesion and prolonging the antigen's duration of action in the intestine. Retinoic acid promotes the differentiation of IgA antibody-secreting cells, recruits immune cells, and enhances the mucosal immune response.

[0140] Therefore, we first constructed a positively charged mannose-modified liposome gel microsphere for targeted drug delivery to the intestine. Figure 1 A). Retinoic acid-loaded liposomes (RA-MLip) were prepared using a thin-film hydration method, and negatively charged attenuated PEDV (PR-Lip) was adsorbed via electrostatic interactions. The liposomes were evaluated using various methods. The size distribution and surface charge of MLip, RA MLip, and PR MLip were measured by dynamic light scattering (DLS). DLS results showed that the particle sizes of MLip, RA MLip, and PR MLip were 215.70±5.86 nm, 236.43±8.07 nm, and 318.23±2.61 nm, respectively. Figure 1 B, C). The polydispersity index (PDI) was 0.167±0.016, 0.170±0.014, and 0.269±0.039, respectively. This indicates that the prepared liposomes have good dispersibility. After loading RA and PEDV, the zeta potential of the cationic liposomes decreased significantly, indicating that RA and PEDV were successfully loaded onto the liposomes. Figure 1 D). SEM images showed an increase in the size of MLip, RAMLip, and PR MLip compared to DLS results, possibly due to the flattening of the liposomes during drying. Figure 1E). The encapsulation efficiency and loading rate of retinoic acid in RAMLip were 78.18±1.52% and 2.35±0.05%, respectively. These results fully demonstrate the successful preparation of RAMLip. RA is insoluble in water. Although RA is relatively stable in air, it is easily degraded under light and high temperature. Liposome encapsulation can improve the solubility and stability of RA. The stability of RA-MLip was studied under different storage conditions, with the encapsulation efficiency of RA as the indicator. The encapsulation efficiency of RA-MLip decreased significantly when stored under light. The encapsulation efficiency of RA decreased by 18.67% and 31.40% at 4°C and 25°C, respectively. After 30 days of storage in the dark at 4°C, the encapsulation efficiency of retinoic acid in liposomes decreased slightly, but remained stable in the range of 70-75%. Figure 1 F). Furthermore, the particle size, potential, and PDI of RA-MLip did not change significantly, indicating that it has good stability.

[0141] 2. Preparation and characterization of PR-MLip@Gel

[0142] To facilitate delivery into the intestines, the formulation initially needs to remain stable in the gastric environment. Therefore, we used calcium alginate (Ca-Alg) as the outer shell, which is stable under acidic conditions, decomposes in weakly alkaline environments, and possesses some adhesiveness. However, Ca-Alg hydrogels exhibit relatively weak adhesion to the intestinal mucosa and a relatively short residence time on the small intestinal surface. We used cysteine-modified alginate (Cys-Alg) instead of alginate to form the hydrogel. First, cysteine ​​was grafted onto the surface of sodium alginate (Na-Alg) using an amidation reaction mediated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS). To confirm successful grafting of cysteine ​​onto Na-Alg, Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (¹H NMR) were performed. In the ¹H NMR spectrum (… Figure 1 The new peak appearing at approximately 2.9-3.0 ppm in Cys-Alg represents the methylene proton in Cys, confirming the successful modification of cysteine. In the FTIR spectrum ( Figure 1 The peaks at 1248, 1412, and 1604 cm⁻¹ in Cys-Alg represent the stretching vibrations of the CN bond in the primary amine, the bending vibrations of the CN bond in the primary amide, and the bending vibrations of the NH bond in the amide, respectively. Furthermore, a weak thiol characteristic peak appeared at 2526 cm⁻¹, indicating successful synthesis of Cys-Alg. The thiol content in Cys-Alg was determined using the dithionitrobenzene (DTNB) method, and the crosslinking rate (mol%) was calculated to be 5.77 ± 0.24%. Figure 1As shown in Figure 1, PR MLip was added to the gel solution and mixed, then rapidly injected into a calcium chloride solution to obtain liposome gel microspheres (PR-MLip@Gel). These PR-MLip@Gels exhibited regular spherical shapes with diameters ranging from 200 to 250 μm. Next, the internal microstructure of the gel microspheres was observed using scanning electron microscopy (SEM). After gelation, the gel microspheres displayed a regular network structure with abundant pores and channels, such as... Figure 1 As shown in K. To further explore the distribution of liposomes in the gel microspheres, the fluorescence distribution was observed using an inverted fluorescence microscope. Figure 1 In J, the gel microspheres are uniformly distributed in a spherical shape, indicating that the liposomes are uniformly distributed in the calcium alginate gel microspheres.

[0143] 3 Resistance PR-MLip@Gel Gastrointestinal Environment

[0144] In most cases, substance transport through the stomach takes approximately 1–2 hours, and through the small intestine approximately 1–6 hours [Yunkai Tang, Yawei Du, Junna Ye, Lianfu Deng, Wenguo Cui. Intestine-Targeted Explosive Hydrogel Microsphere Promotes Uric Acid Excretion for Gout Therapy. Advanced Materials, 2023, 36(3):2310492]. To verify the intestinal targeting of MLip@Gel, we determined an appropriate retention time and evaluated the detection of MLip@Gel in simulated gastrointestinal fluids by comparing its appearance and diameter in different simulated fluids. After incubation in simulated gastric fluid (SGF, pH=1.2) for 6 hours, the morphology of MLip@Gel remained largely unchanged (Fig. 2A); however, fragmentation occurred shortly after contact with simulated intestinal fluid (SIF, pH=6.8), and the gel structure collapsed significantly within 1 hour. Figure 2 Data in B indicate that MLip@Gel is relatively stable in SGF. In SIF, MLip@Gel initially increases significantly, then the gel structure collapses and becomes non-spherical. Furthermore, when measuring the swelling rate, MLip@Gel ( Figure 2(C) It was found that the swelling rates of the expanded MLip@Gel microspheres in deionized water and SGF were similar, at 785.02±4.77% and 1065.85±6.55%, respectively. The swelling rate of MLip@Gel in SIF reached 2939.28±60.96% within 1 hour, eventually reaching 4181.89±95.56%. Macroscopically, MLip@Gel exhibited ideal tolerance to SGF. Next, we evaluated the release characteristics of RA and PEDV in SGF and SIF. Figure 2 As shown in Figure D, the release rates of RA and PEDV in the PR-MLip@GelSGF group were lower than those in the RA-MLIP group. The cumulative release within 2 hours did not exceed 15%, significantly lower than the 40.45±0.36% in the RA-Lip group, indicating that the gel shell significantly inhibited drug release in the stomach. Upon transfer to simulated intestinal fluid, the release of RA and PEDV in vivo accelerated in the PR-MLip@Gel group. The cumulative release within 2 hours was 51.28±0.58% and 54.08±2.58%, respectively, close to that of RA MLip (54.83±1.16%), followed by a slower release. The final cumulative release of RA and PEDV reached 72.17±0.30% and 66.60±0.95%, respectively, lower than the cumulative release of RA in the RA MLip group (87.96±0.44%), which may be due to the hindering effect of the gel network structure on drug release. Overall, after oral administration, PR-MLip@Gel remains intact in the stomach with only a small amount of drug leakage, and rapidly expands and degrades in the small intestine, releasing PR MLip.

[0145] 4. Enhanced retention of liposome gel microspheres in the intestine

[0146] Intestinal retention time is crucial for improving and maintaining local drug concentrations in the intestine. To evaluate the effect of Cys-Alg on enhanced intestinal adhesion, the intestinal adhesion of Cys-Alg was first elucidated in vitro by affinity assays on the surface of fresh porcine small intestinal epithelium. MLip, MLip@Gel (Alg), and MLip@Gel (Cys-Alg) were placed on the mucosa of upright porcine small intestine. MLip slid off immediately. However, liposome gel microspheres could adhere to the mucosal surface for a longer period, with MLip@Gel (Cys-Alg) showing more microspheres adhering to the small intestinal surface and a smaller surface area after 120 minutes. Subsequently, MLip@Gel and MLip@Gel (Cys-Alg) were added to the small intestinal epithelial surface in a PBS solution, retaining a certain number of microspheres on the small intestinal surface. PBS was used to simulate intraperitoneal fluid. The small intestinal surface was rinsed, and the adhesion rate of the microspheres was analyzed by calculating the number of microspheres remaining after each rinse. Figure 2E, F, G). The adhesion rates of MLip@Gel and MLip@Gel-modified Arg (cysteine-modified) were 50.67±5.03% and 73.33±3.05%, respectively, indicating that cysteine-modified Arg significantly enhances its adhesion to intestinal epithelial cells. This may be because after thiol reduction, disulfide bonds are formed within the thiol polymer itself, which increases viscosity and thus enhances adhesion to the intestine. The formation of disulfide bonds was investigated by measuring the remaining thiol groups. Figure 2 H). The remaining thiol content in SGF remained essentially unchanged, while in SIF it decreased to 67.9% after 1 hour. Next, the in vitro biodistribution of liposome gel microspheres in the mouse gastrointestinal tract was investigated using FITC mimicry. After oral administration, the cumulative fluorescence intensity in the gastrointestinal tract of the FITC and FITC-MLIP groups was significantly lower than that of the FITC-MLip@Gel group ( Figure 2 I, J). The fluorescence signal of the FITC group gradually weakened over time, almost disappearing at 12 hours, while the fluorescence signal of the FITC-MLip@Gel group could still be detected at 24 hours, indicating that FITC-Lip@Gel can effectively prolong the residence time in the intestine and promote intestinal absorption.

[0147] 5. Drug absorption and targeted delivery

[0148] Better mucus penetration and intestinal absorption are beneficial for drug action. To investigate the contribution of liposomes, FITC was used to mimic RA, and the protein content in the small intestine was studied by in vitro assays of FITC, FITC-MLip, and FITC-MLip@Gel. All groups showed significant time dependence. Figure 3 A, B). Cumulative level of FITC in the small intestine: FITC-Lip@Gel group (Papp = 9.90 × 10⁻⁶) -7 (cm / s) compared to the FITC group (Papp=6.08×10) -7 The concentration of FITC in the FITC-MLip@Gel group was approximately 1.79 times higher than that in the FITC-MLip group. The accumulation of FITC in vivo was initially lower in the FITC-MLip group, but gradually leveled off and even reversed. This may be due to the time difference required for the Cys-Alg hydrogel to undergo a swelling process before releasing FITC-MLip. The high transport level of FITC-MLip@Gel indicates that positively charged liposomes are more likely to penetrate the negatively charged intestinal mucus layer, promoting intestinal drug absorption.

[0149] Subsequently, we investigated the delivery of the formulation at the cellular level. APC internalization of the vaccine is crucial for immune activation. Intestinal antigen-presenting cells are primarily composed of macrophages and dendritic cells (DCs). Macrophages engulf pathogens and, together with dendritic cells, present antigens to elicit an immune response. Type C lectin receptors are expressed in phagocytes and play an important role in host recognition of pathogens and activation of the innate immune response. Mannose receptors are one such receptor. Targeting of intestinal macrophages was achieved by modifying the surface of liposomes with mannose. First, the cytotoxicity of the formulation was investigated. HEK 293T cells, RAW264.7 cells, and DCs showed cell viability exceeding 90% for MLip@gel and RAMLip@gel at all tested concentrations (0–1 mg / mL). Figure 3 CE indicates that the cytotoxicity of the delivery system is negligible. To investigate the internalization ability of APC formulations and their targeting of intestinal macrophages, an uptake assay was performed using FITC-mimicking RA. The green fluorescence intensity was quantified to assess FITC survival; FITC-Lip, FITC-MLip, and Man+FITC-MLips were effectively internalized in macrophages. Confocal images show ( Figure 3 Compared to the FITC group (F, G), the liposome group showed higher fluorescence intensity, indicating that liposomes enhanced drug internalization. The fluorescence intensity of the FITC-MLip group was significantly stronger than that of the FITC-Lip group and the Man+FITC-MLips group, with the fluorescence brightness of the Man+FITC MLip group and the FITC-Lips group being similar. These results confirm that mannose modification on the surface of liposomes can achieve targeted delivery to intestinal macrophages. Dendritic cells (DCs) are the most capable antigen-presenting APCs, so drug uptake by DCs was also investigated. The results were similar to the trend observed in macrophages, possibly because dendritic cells also have mannose receptors on their surface.

[0150] 6. Immunization of vaccines

[0151] After vaccine internalization, promoting the activation of antigen-presenting cells (APCs) and the recruitment of immune cells is crucial for activating antigen-specific immune responses and enhancing vaccine efficacy. To confirm whether the designed vaccine can serve as an effective adjuvant to promote APC maturation and antigen presentation, OVA was used as a model antigen, and RA, MLip@Gel, or OR-MLip@Gel were co-cultured with DCs and RAW264.7 cells for 24 hours. The expression levels of CD80, CD86, and MHC-II were measured. Cells treated with PBS and OVA were used as negative and positive controls, respectively. Figure 4As shown in the AG, compared with the PBS group, the expression of CD80, CD86, and MHC-II on DCs and macrophages in both the RA and MLip@Gel groups was upregulated, indicating their potential adjuvant properties. The strongest CD80 (22.79%) and CD86 (25.83%) signaling was significantly higher in the OR-MLip@Gel group than in the OVA group (CD80: 18.87%, CD86: 18.56%), indicating its superior effect in promoting DC activation. The MHC-II positivity rate in dendritic cells in the OR-MLip@Gel OVA group was 49.46%, 16.34% higher than in the OVA group. In macrophages, MHC-II expression was 36.24%, 18.25% higher than in the OVA group, indicating its effective promotion of antigen presentation. This may be due to the liposomes effectively enhancing cellular antigen uptake.

[0152] Immune cells typically exert their immune function by secreting immune-related cytokines. To further assess immune cell activation, representative cytokines such as IL-4, IFN-γ, and TNF-α were selected for detection. Cytokine secretion levels in vaccine-stimulated dendritic cells (DCs) and macrophages were measured in vitro using ELISA. Figure 4 As shown in H and I, both MLip@Gel and OR-MLip@Gel stimulated immune cells to secrete IL-4, IFN-γ, and TNF-α compared to the PBS group. The OR-MLip@Gel group, compared to the OVA group, stimulated dendritic cells (DCs) to secrete higher levels of IL-4, IFN-γ, and TNF-α, with a more pronounced effect on IFN-γ than the other two cytokines. Similar effects were observed in macrophages. This indicates that RA-MLip@Gel can upregulate the secretion levels of immune-related cytokines by OVA-stimulated immune cells in vitro. These results suggest that the proposed liposome-based antigen delivery system can increase vaccine uptake, promote macrophage activation, and stimulate dendritic cell (DC) maturation, potentially enhancing their immunostimulatory activity in vivo.

[0153] To investigate the immune activation capacity of the oral vaccine in vivo, mice were immunized according to the immunization protocol shown in Figure 5A. Bone marrow dendritic cells (BMDCs) were isolated 96 hours after the first immunization, and the expression levels of CD80, CD86, and MHC-II on the surface of BMDCs were detected. As shown in Figures 5B-D, the expression levels of CD80, CD86, and MHC-II in the PR-MLip@Gel group were significantly higher than those in the porcine epidemic diarrhea virus (PEDV) gavage injection (IG) group and the PEDV intramuscular injection (IM) group. Compared with the PBS group, the expression levels of CD80, CD86, and MHC-II on BMDCs in the RA group and the MLip@Gel group were higher, but significantly lower than those in other groups. This is consistent with the results of in vitro experiments. The results showed that the PEDV-loaded delivery system significantly promoted the expression of MHC-II, CD80, and CD86 on dendritic cells 96 hours after immunization, further demonstrating that it can accelerate the maturation of dendritic cells and promote antigen presentation.

[0154] 7. Oral vaccines induce mucosal immune responses

[0155] PEDV primarily infects small intestinal epithelial cells and causes lesions, thus mucosal protection is crucial. Secretory IgA antibodies located on the mucosal surface can effectively neutralize invading viruses

[10] . Therefore, inducing an appropriate mucosal immune response and producing sufficient secretory IgA antibodies is key to resisting PEDV infection. Studies have shown that RA can promote the expression of intestinal homing molecules such as chemokine receptor 9 (CCR9) and α4β7 in immune cells, induce the differentiation of IgA ASCs, and ultimately regulate the mucosal immune response of the intestine. Therefore, we isolated intestinal epithelial lymphocytes from mice 42 days after the first immunization and used flow cytometry to detect the expression of CCR9 and α4β7 on their surface. Figure 5 As shown in E and F, PR-MLip@Gel levels were 19.08% higher than in the PEDV (IG) group and 27.87% higher than in the RA group. These results indicate that PR-MLip@Gel can effectively upregulate the expression of CCR9 and α4β7, thus modulating mucosal immune responses. Furthermore, PEDV-specific IgA antibodies were detected by ELISA in mouse feces, intestinal lavage fluid, and mouse serum. Figure 5 As shown in Figure G, the PR-MLip@Gel PEDV (IG) group had significantly higher levels of IgA than other groups in the intestinal lavage fluid samples. The PEDV (IM) group was compared with the PBS group, RA group, and MLip@Gel group. Notably, the PR-MLip@Gel group exhibited higher IgA levels compared to the PEDV (IG) group, attributed to its superior mucus permeability and intestinal macrophage targeting ability. (Mouse fecal samples...) Figure 7) and serum samples ( Figure 5 The trend of specific IgA levels in H) was similar to that in intestinal lavage fluid, demonstrating the feasibility of this oral vaccine delivery system in generating mucosal immunity and preventing PEDV virus infection. Furthermore, the extremely low mucosal IgA titers in the PEDV(IM) group also indicate the independence of the mucosal immune system.

[0156] 8. Oral vaccines induce a systemic immune response.

[0157] Previous research has shown that oral vaccines can induce a strong mucosal immune response. To investigate the efficacy of oral vaccines in evoking a systemic immune response, PEDV-specific IgG antibodies in mouse feces and serum were detected by ELISA. Figure 6 A and Figure 8 As shown. At all test time points, the antibody titer in the PR-MLip@Gel group was higher than that in the PEDV (IG) and PEDV (IM) groups. The highest antibody titer was observed on day 42 after the initial immunization, indicating that the vaccine delivery system significantly enhances humoral immunity and provides a good booster effect. Serum neutralization of live virus is considered to be associated with protection. Therefore, in this study, a sham virus neutralization assay was also used to investigate the effect of the vaccine on PEDV-induced neutralizing antibody (NAb) production in mice. Figure 6 B). Notably, the PR-MLip@Gel group showed the highest levels at all testing time points. Specifically, the neutralizing antibody level was 1:106.67 on day 42 post-PR-MLip@Gel immunization, compared to 1:48 in the PEDV (IG) group and 1:96 in the PEDV (IM) group, indicating that the vaccine effectively induces neutralizing antibodies against PEDV virus infection.

[0158] Splenic lymphocytes were isolated from mice 42 days after the first immunization, and then their proliferation was examined after 48 hours of ConA stimulation. Figure 6 As shown in Figure C, compared with the PBS group, the PEDV (IG) group, PEDV (IM) group, and PR-MLip@Gel group all showed proliferation. The PR-MLip@Gel group demonstrated superior ability to stimulate splenic lymphocyte proliferation. Further ELISA was used to investigate the expression levels of immune cytokines after ConA stimulation of splenic lymphocytes in vitro. IFN-γ and TNF-α are Th1 cytokines, and IL-4 is a Th2 cytokine. Figure 6As shown in Figure D, compared with the PEDV (IG) and PEDV (IM) groups, PR-MLip@Gel secreted more Th1 and Th2 cytokines after stimulation. These results indicate that PR-MLip@Gel can elicit a strong Th1 / Th2 cellular immune response and increase the secretion of cytokines involved in immune regulation. T lymphocytes and B lymphocytes are crucial in regulating immune responses and fighting pathogens, while natural killer (NK) cells are particularly important in antiviral infection and immune regulation. Therefore, the percentages of specific T lymphocytes, B lymphocytes, and NK cells in spleen lymphocytes isolated from immunized mice were detected by flow cytometry. The PBS group served as a control. Figure 6 EG analysis showed that the proportions of CD3e+CD4+ (31.08%) and CD3e+CD8+ (16.59%) T cells in the spleen lymphocytes of immunized mice were significantly higher with PR-MLip@Gel than in the PEDV (IG) group (CD3e+CD4+: 25.28%, CD3e+CD8+: 11.12%), and to some extent higher than in the PEDV group (CD34+CD4+: 27.88%, CD3e+CD8+: 12.88%). These results indicate that PR-MLip@Gel can upregulate the expression of specific T cell populations. Furthermore, the proportions of CD19+B220+ B cells and CD3e+CD49b+ NK cells in the spleen were also significantly increased in the PR-MLip@Gel group compared to other immunization groups. Figure 6 In summary, these results demonstrate that oral vaccines using this mannose-modified cationic liposome gel microsphere can induce strong mucosal and systemic immune responses.

[0159] 9. Safety Assessment

[0160] Safety assessment of the vaccine is crucial for its future clinical application. No deaths or other abnormal behaviors were observed throughout the experiment. Throughout the study, mice in each treatment group experienced a weight gain following administration, similar to the PBS group, providing preliminary evidence of the vaccine's safety. Figure 9 On day 42 post-immunization, small intestine and major organs, including heart, liver, spleen, lung, and kidney, were collected from each group of mice for histological analysis. H&E staining microscopic images showed that PR-MLip@Gel demonstrated good biocompatibility compared to the PBS group. No significant pathological changes were found in the small intestine tissue of any of the immunized groups. Figure 10 Compared with the PBS group () Figure 11There was no significant difference in the ratio of intestinal villus length to crypt depth between the PEDV (IM) and PEDV (IG) groups. However, the PR-MLip@Gel group showed a significant increase, indicating an increase in the number of intestinal epithelial cells after vaccination, which is beneficial for maintaining intestinal homeostasis. In conclusion, oral vaccines have good biosafety and promising clinical application prospects.

[0161] We have developed a cationic mannose-modified liposome gel microsphere oral delivery system (PR-MLip@Gel) that may elicit strong mucosal and systemic immune responses to combat PEDV infection. The cationic liposomes exhibit good biocompatibility, effectively load PEDV, and readily penetrate the mucus layer to reach intestinal epithelial cells. Mannose modification endows the liposomes with the ability to target intestinal macrophages, promoting drug absorption and immune cell activation, enhancing the antigen-presenting capacity of immune cells and systemic immune responses. Thiol modification of the gel material improves intestinal adhesion, prolongs antigen retention time, and increases the interaction time with the immune system. RA, as an immune enhancer, promotes the differentiation of plasma cells that secrete IgA antibodies, enhancing mucosal immune efficacy.

[0162] We further explored the immune response induced by this oral delivery system. We found that MLip@Gel reduced antigen leakage in the highly acidic gastric environment, increased its duration of action in the intestine, and promoted antigen uptake by macrophages and dendritic cells. Compared with the OVA group, the OR-MLip@Gel group significantly upregulated the expression of CD80, CD86, and MHC II on the surface of APCs, promoted their activation and antigen presentation, and increased the secretion of IL-4, IFN-γ, and TNF-α. In the PR-MLip@Gel immunization group, the activation of α4β7+CCR9+ cells in small intestinal epithelial lymphocytes was increased, and the secretion level of specific IgA antibodies was elevated, indicating that it induced a strong mucosal immune response. The increased secretion levels of specific IgG antibodies, neutralizing antibodies, IL-4, IFN-γ, and TNF-α, as well as the increased proliferation of splenic lymphocytes and the activation of CD4+ T cells, CD8+ T cells, B cells, and NK cells, all indicate that it induced a strong humoral and cellular immune response. Overall, this oral delivery system shows great potential as a vaccine delivery carrier and an effective mucosal adjuvant.

Claims

1. A cationic based mannose modified liposomal gel microsphere oral delivery system characterized in that, The core layer is a mannose-modified cationic liposome loaded with mucosal adjuvant retinoic acid and antigen, and the wrapping layer is a cysteine-sulfurized modified sodium alginate gel.

2. The oral delivery system according to claim 1, wherein, The antigen is selected from one or more of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine delta coronavirus, severe acute respiratory syndrome coronavirus 2, and bovine coronavirus; or the antigen is a live attenuated vaccine, an inactivated vaccine, a recombinant protein antigen (such as S protein and N protein), or a nucleic acid vaccine of the above viruses.

3. The oral delivery system according to claim 1, wherein, The raw materials for preparing the mannose-modified cationic liposome include lecithin, DC-cholesterol, Man-PEG 2000 -DSPE and retinoic acid; the mass fraction of each raw material is: lecithin 5-25 parts, DC-cholesterol 5-25 parts, Man-PEG 2000 -DSPE 0.1-0.3 parts, retinoic acid 0.15-0.6 parts.

4. The oral delivery system according to claim 1, wherein, In the wrapping layer of the cysteine-sulfurized modified sodium alginate gel, the molar ratio of sodium alginate to cysteine hydrochloride anhydrous is 2:1-1:

2.

5. The oral delivery system according to claim 1, wherein, The mass ratio of the core layer to the wrapping layer is 1:1-1:

4.

6. The oral delivery system according to claim 1, wherein, at a concentration of 1 x 10 5 at a concentration of 1 x 10 6 TCID50 / mL; and the retinoic acid loaded mannose-modified liposomes are at a concentration of 5-15 mg / mL, and the volume ratio of antigen to retinoic acid loaded mannose-modified liposomes is 1:1-1:

5.

7. A process for the preparation of the oral delivery system according to any one of claims 1 to 6, characterized in that, The method comprises the following steps: (1) Preparation of retinoic acid loaded mannose modified liposome (RA-MLip): Egg phosphatidylcholine, DC-cholesterol, Man-PEG 2000 -DSPE and retinoic acid were dissolved in anhydrous ethanol, and after rotary evaporation to remove the organic solvent to form a thin film, PBS buffer was added to hydrate and ultrasonic at 0-80°C to obtain RA-MLip; (2) Preparation of mannose-modified liposome loaded with antigen and retinoic acid (PR-MLip): mixing the RA-MLip obtained in step (1) with antigen, and adsorbing overnight to obtain PR-MLip; (3) Preparation of cysteine-sulfurized modified sodium alginate gel (Cys-Alg): dissolving sodium alginate in water, adding EDC and NHS for reaction, then adding L-cysteine hydrochloride anhydrous for further reaction in the dark, and then dialyzing to obtain Cys-Alg; (4) Preparation of PR-MLip@Gel: mixing the PR-MLip obtained in step (2) with the Cys-Alg obtained in step (3) uniformly, then injecting into a calcium chloride solution using a syringe, and crosslinking at room temperature to obtain cysteine-modified calcium alginate-wrapped mannose-modified liposome loaded with antigen and retinoic acid.

8. The method of claim 7, wherein the oral delivery system is prepared by, The crosslinking time in step (4) is 15-45 minutes.

9. Use of the oral adjuvant delivery system of any one of claims 1-6 in the preparation of an oral vaccine.

10. Use of the oral adjuvant delivery system of any one of claims 1-6 in the preparation of a porcine epidemic diarrhea virus vaccine.

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