Oral nano-vaccine and preparation method and application thereof

Abstract

The application discloses an oral nano-vaccine and a preparation method and application thereof, and relates to the technical field of immunology, and specifically discloses an oral nano-vaccine which comprises a nano-particle wrapped by a biomimetic bacterial membrane vesicle and an outer layer; the nano-particle comprises an antigen and a biodegradable polymer material; the biomimetic bacterial membrane vesicle comprises an ionizable flagellar lipoprotein, an immune adjuvant, a phospholipid, a sterol lipid and a polyethylene glycol lipid; and the outer layer is a pH-responsive polymer. The oral nano-vaccine provided by the application combines the immune activation advantages of natural bacterial membrane vesicles and the precise regulation characteristics of synthetic materials, significantly improves the transmembrane penetration capacity of the antigen in the intestinal epithelium and the overall activation effect of the mucosal immune system. The oral nano-vaccine provided by the application can protect the antigen and the immune adjuvant from degradation and realize precise release in the intestinal microenvironment; and the oral nano-vaccine significantly improves the bioavailability and safety of an oral tumor vaccine, and lays a key technical foundation for clinical transformation and large-scale production of the oral tumor vaccine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an oral nanovaccine, its preparation method, and its application. Background Technology

[0002] In recent years, tumor immunotherapy has opened up new avenues for the treatment of advanced tumors by reshaping the body's immune microenvironment. Among them, tumor vaccines, as a representative of active immunotherapy, induce antigen-specific CD8... + T-cell responses enable precise elimination of tumor cells, demonstrating unique advantages in tumor prevention, postoperative recurrence control, and suppression of distant metastasis.

[0003] Compared to traditional injectable vaccines, oral vaccines have the following breakthrough advantages: (1) They activate a systemic-mucosal dual immune response through the intestinal mucosal immune system. (2) Non-invasive administration significantly improves patient compliance. (3) Large-scale production costs can be reduced by more than 80%. However, existing oral vaccines face two major challenges: (1) The gastric acid environment leads to antigen degradation and adjuvant inactivation. (2) The intestinal epithelial barrier results in insufficient antigen delivery efficiency, severely restricting the cross-presentation efficiency of dendritic cells (DCs), leading to CD8... + Low T-cell activation efficiency has become a key bottleneck restricting its clinical translation.

[0004] In addition, existing natural bacterial membrane vesicles (BMVs) have fluctuating levels of immune adjuvants due to differences in the strains from which they originate (batch-to-batch CV value >20%), and unmodified BMVs are easily recognized by the systemic immune system, triggering excessive secretion of IL-6 and TNF-α, which poses a risk of systemic inflammation.

[0005] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide an oral nanovaccine, its preparation method and application, which aims to solve the problems of antigen degradation, adjuvant inactivation and insufficient delivery efficiency in existing oral vaccines.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides an oral nanovaccine comprising nanoparticles encapsulated in biomimetic bacterial membrane vesicles and an outer layer; the nanoparticles comprising antigens and biodegradable polymeric materials; the biomimetic bacterial membrane vesicles comprising ionizable flagellated lipoproteins, adjuvants, phospholipids, steroidal lipids, and polyethylene glycol lipids; and the outer layer being a pH-responsive polymer.

[0008] Optionally, the biodegradable polymer material is at least one of poly(lactic-co-glycolic) acid (PLGA), polycaprolactone, polylactic acid, polyhydroxybutyric acid, and polypeptide nanomaterials.

[0009] Optionally, the ionizable flagellated lipoprotein is prepared by reacting (Z)-12-(((4-nitrophenoxy)carbonyl)oxy)octadecyl-9-ene-1-acrylate, fatty alcohol and flagellated protein.

[0010] Optionally, the immune adjuvant is at least one of monophospholipid A, monophospholipid derivatives, CpG oligodeoxynucleotides, and polyinosinic-polycytidylic acid; and / or, the phospholipid is soybean lecithin, egg yolk lecithin, or sphingomyelin; and / or, the steroidal lipid is cholesterol or a cholesterol derivative; and / or, the polyethylene glycol lipid is at least one of DSPE-PEG-2000, DMG-PEG, DPPE-PEG, and DOPE-PE.

[0011] Optionally, the pH-responsive polymer is at least one of methyl methacrylate, hydroxypropyl methylcellulose phthalate, chitosan derivatives, Eudragit L100, Eudragit S100, and polyacrylic acid polymers.

[0012] Optionally, the mass ratio of the biomimetic bacterial membrane vesicles to the nanoparticles is 1:5 to 5:1.

[0013] Optionally, the molar ratio of ionizable flagellated lipoprotein, immune adjuvant, phospholipid, steroidal lipid and polyethylene glycol lipid in the biomimetic bacterial membrane vesicle is 1:(0.5~2):(2~5):(1~3):(0.1~1).

[0014] A second aspect of the present invention provides a method for preparing the oral nanovaccine, comprising the following steps: S1. Reaction of (Z)-12-(((4-nitrophenoxy)carbonyl)oxy)octadecyl-9-ene-1-acrylate with a fatty alcohol yields ricinoleate; reaction of the ricinoleate with flagellin yields ionizable flagellate lipoprotein. S2. React the antigen and biodegradable polymer in a solvent to obtain nanoparticles; S3. The nanoparticles are added to a mixed solution of ionizable flagellated lipoprotein, immune adjuvant, phospholipids, steroidal lipids and polyethylene glycol lipids, and reacted to obtain nanoparticles encapsulated by biomimetic bacterial membrane vesicles. S4. The nanoparticles encapsulated in the biomimetic bacterial membrane vesicles are reacted with pH-responsive polymer monomers in a solvent to obtain the oral nanovaccine.

[0015] Optionally, in step S1, the molar ratio of (Z)-12-(((4-nitrophenoxy)carbonyl)oxy)octadecyl-9-ene-1-acrylate, fatty alcohol and flagellin is 2:5:(0.001~0.01).

[0016] Optionally, in step S3, the reaction method used is one of membrane extrusion, nanoprecipitation, or microfluidic chip method.

[0017] Optionally, step S4 may further include adding ammonium persulfate, wherein the mass of ammonium persulfate is 0.5-2% of the mass of the pH-responsive polymer monomer; preferably, the reaction temperature is 40-60°C and the reaction time is 5-8 hours.

[0018] A third aspect of the invention provides the use of the oral nanovaccine in the preparation of any of the following.

[0019] (a1) Vaccines for the prevention and / or treatment of tumors; (a2) Vaccines for the prevention and / or treatment of infectious diseases.

[0020] Optionally, the tumor is at least one of colon cancer, melanoma, lung cancer, breast cancer, stomach cancer, and pancreatic cancer; and / or, the infectious disease is a disease caused by bacterial or viral infection.

[0021] A fourth aspect of the invention provides the use of the oral nanovaccine in at least one of (b1)-(b3): (b1) Prepare products that induce or activate the maturation of dendritic cells (DCs); (b2) Prepare products that promote T cell proliferation or activation; (b3) Prepare products that induce long-term immune memory effects; (b4) Prepare products that enhance mucosal immune responses.

[0022] Beneficial effects: This invention provides an oral nanovaccine, its preparation method, and its application. Compared with existing technologies, the advantages of this invention are: (1) Structural design of biomimetic bacterial membrane vesicles: mimicking the immune activation characteristics of natural bacterial membrane vesicles (BMVs) and combining them with ionizable flagellin proteins to form biomimetic bacterial membrane vesicles; the oral nano-vaccine prepared by the biomimetic bacterial membrane vesicles solves the problems of large batch differences and risk of inducing systemic inflammation in traditional BMVs vaccines.

[0023] (2) Modular assembly and pH-responsive polymer coating strategy: Antigens and biodegradable polymers are used as nanoparticles; ionizable flagellated lipoproteins, immune adjuvants, phospholipids, steroidal lipids and polyethylene glycol lipids are uniformly coated on the surface of nanoparticles by membrane extrusion to form nanoparticles encapsulated by biomimetic bacterial membrane vesicles; the outer layer is coated with pH-responsive polymers to achieve gastric acid environment stability and intestinal targeted release; the combination of biomimetic bacterial membrane vesicles and pH-responsive polymers is realized, which significantly improves bioavailability under oral route.

[0024] (3) Efficient transepithelial delivery and mucosal immune activation: By designing ionizable biomimetic bacterial membrane vesicles, charge conversion is regulated to enhance the intestinal epithelial penetration ability; the system activates mucosal immunity and systemic immune response, including DC maturation, T cell activation and long-term immune memory.

[0025] (4) Validation and safety improvement in multiple tumor models: It showed significant tumor suppression effect and maintenance of immune memory in subcutaneous colorectal cancer model, melanoma lung metastasis model and long-term immune memory model; the safety and compatibility of the mice were proven to be superior to traditional BMVs vaccines through routine blood tests, blood biochemistry and organ pathology.

[0026] (5) From an overall perspective, the oral nanovaccine provided by the present invention has better stability, higher delivery efficiency, more comprehensive immune activation, significant anti-tumor effect, good biocompatibility and safety. Attached Figure Description

[0027] Figure 1 This is a technical roadmap for the present invention.

[0028] Figure 2 TEM and SEM images of the C-BMNPs vaccine. (a) TEM image of the C-BMNPs vaccine, scale bar: 100 nm (left), 200 nm (right). (b) SEM image of the C-BMNPs vaccine, scale bar: 100 nm (left), 500 nm (right).

[0029] Figure 3 The following are graphs showing the particle size, potential characterization, and composition analysis of the C-BMNPs vaccine. (a) Particle size distribution of the C-BMNPs vaccine. (b) Zeta potential of the C-BMNPs vaccine, data expressed as mean ± SD (n=3). (c) UV absorbance of the C-BMNPs vaccine. (d) Protein electrophoresis of the C-BMNPs vaccine; the top graph shows the OVA protein bands, and the bottom graph shows the Flagellin protein bands.

[0030] Figure 4Figure 1 shows the stability and encapsulation efficiency of the C-BMNPs vaccine. (a) Mean particle size change of the C-BMNPs vaccine over 7 days. (b) Encapsulation efficiency of the C-BMNPs vaccine. Data are expressed as mean ± SD (n = 3).

[0031] Figure 5 Figures showing the acid resistance, enteric solubility, and antigen release rate of the C-BMNPs vaccine. (a) Appearance and TEM image of the vaccine solution after 7 days of treatment at pH=2, scale bar=200 nm. (b) Appearance and TEM image of the vaccine solution after 1 h at pH=7, scale bar=200 nm. (c) Release efficiency of OVA in the C-BMNPs vaccine (n=3).

[0032] Figure 6 This is a diagram showing the in vivo biodistribution of the C-BMNPs vaccine. (a) In vivo fluorescence imaging of the digestive tract of mice at different time points after oral administration of the C-BMNPs vaccine. (b) In vivo fluorescence imaging of various parts and contents of the digestive tract of mice 8 h after oral administration of the C-BMNPs vaccine. (c) Quantitative analysis of fluorescence intensity of various parts of the digestive tract at different time points. (d) Quantitative analysis of fluorescence intensity of various parts and contents of the digestive tract 8 h after oral administration of the C-BMNPs vaccine. (n = 3), data are expressed as mean ± standard deviation (mean ± SD), and one-way two-sided ANOVA was performed using Origin 2021 software. ****, P < 0.0001.

[0033] Figure 7 The Transwell assay results validated the intestinal epithelial penetration ability of the C-BMNPs vaccine. (a) Immunofluorescence staining CLSM image of mouse small intestinal tissue. OVA was performed using Alexa Fluor 647 (red), DCs were labeled with anti-CD11c antibody (green), and cell nuclei were labeled with DAPI (blue). Scale bar = 40 μm. (b) Quantitative statistical plot of C-BMNPs vaccine particles in the CLSM field of view. (n = 6), data are expressed as mean ± standard deviation (mean ± SD), and one-way two-sided ANOVA was performed using Origin 2021 software. **, P < 0.01, ****, P < 0.0001.

[0034] Figure 8Graphs showing the quantitative analysis of FCM for DC maturation induced in vitro by C-BMNPs vaccine. (a) Percentage of CD80+MHC-I+CD11c+ cells. (b) Percentage of CD86+MHC-I+CD11c+ cells. (c) Percentage of CD80+CD86+CD11c+ cells. (n=3), data are expressed as mean ± standard deviation (mean ± SD), and one-way two-sided ANOVA was performed using Origin 2021 software. **, P<0.01, ***, P<0.001, ****, P<0.0001.

[0035] Figure 9 Figures show the results of DC cytokine secretion after C-BMNPs vaccine stimulation. (a) Quantitative statistical graph of IL-12p40 in culture supernatant. (b) Quantitative statistical graph of TNF-α in culture supernatant. (c) Quantitative statistical graph of IL-6 in culture supernatant. (n=3), data are expressed as mean ± standard deviation (mean ± SD), and one-way two-sided ANOVA was performed using Origin 2021 software. NS, P>0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001.

[0036] Figure 10 FCM analysis of T cell proliferation stimulated by C-BMNPs vaccine in vitro. (a) Representative flow cytometry histogram of T cell proliferation detected by CFSE staining. (b) Quantitative statistical graph of the percentage of CFSE-positive cells. (n=3), data are expressed as mean ± standard deviation (mean ± SD), and one-way two-sided ANOVA was performed using Origin 2021 software. **, P<0.01.

[0037] Figure 11 To analyze the antitumor effect of C-BMNPs vaccine in vivo. (a) Schematic diagram of the experimental procedure for constructing a subcutaneous colon cancer model and scheduling oral vaccination. (b) Tumor growth curves in mice after treatment. (c) Tumor inhibition rate in mice after treatment. (d) Mouse survival rate analysis. (n=6), data are expressed as mean ± standard deviation (mean ± SD), and one-way two-sided ANOVA was performed using Origin2021 software. ****, P<0.0001.

[0038] Figure 12 This is a graph showing the trend of body weight change in mice during treatment in different treatment groups. (n=6), data are expressed as mean ± standard deviation (mean ± SD), and one-way two-sided ANOVA was performed using Origin 2021 software. **, P<0.01.

[0039] Figure 13This is a graph showing the complete blood count (CBC) analysis of mice after treatment. The levels of RBC (a), HGB (b), HCT (c), MCV (d), NRBC (e), MCH (f), RDW-SD (g), PLT (h), PDW (i), MPV (j), LYMPH (k), MONO (l), EO (m), WBC (n), and NEUT (o) in the blood of mice in different treatment groups after treatment were shown. (n=5). Data are expressed as mean ± standard deviation (mean ± SD) and analyzed using one-way two-sided ANOVA with Origin 2021 software. **, P < 0.01.

[0040] Figure 14 The image shows the serum biochemical analysis of mice after treatment. The levels of serum ALT(a), AST(b), LDH(c), CR(d), BUN(e), and UA(f) in mice from different treatment groups after treatment are shown. (n=6). Data are expressed as mean ± standard deviation (mean ± SD) and analyzed using one-way two-sided ANOVA with Origin 2021 software. **, P<0.01.

[0041] Figure 15 Representative H&E staining images of heart, liver, spleen, lung and kidney tissues of mice in different treatment groups after treatment, scale bar = 80 μm.

[0042] Figure 16 Figure 1 shows the efficacy of C-BMNPs vaccine in inhibiting lung metastasis. (a) Schematic diagram of the experimental procedure for establishing the lung metastasis model and scheduling oral vaccination. (b) Mouse survival rate analysis. (c) Quantitative analysis of lung metastatic nodules. (n=6), data are expressed as mean ± standard deviation (mean ± SD), and one-way two-sided ANOVA was performed using Origin 2021 software. ***, P<0.001.

[0043] Figure 17 Figure 1 shows the efficacy of C-BMNPs vaccine in treating tumor recurrence. (a) Schematic diagram of the experimental procedure for constructing a long-term immune memory model and scheduling oral vaccination. (b) Lung tissue of mice after treatment. (c) Quantitative statistical graph of lung metastatic nodules. (n=6), data are expressed as mean ± standard deviation (mean ± SD), and one-way two-sided ANOVA was performed using Origin 2021 software. ***, P<0.001. Detailed Implementation

[0044] This invention provides an oral nanovaccine, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0045] This invention provides an oral nanovaccine comprising nanoparticles encapsulated in biomimetic bacterial membrane vesicles and an outer layer; the nanoparticles comprising antigens and biodegradable polymeric materials; the biomimetic bacterial membrane vesicles comprising ionizable flagellated lipoproteins, adjuvants, phospholipids, steroidal lipids, and polyethylene glycol lipids; and the outer layer being a pH-responsive polymer.

[0046] like Figure 1 As shown, this figure is a technical roadmap of the present invention. The present invention uses antigens and biodegradable polymer materials as its core; it assembles a biomimetic bacterial membrane vesicle structure from ionizable flagellated lipoproteins, immune adjuvants, phospholipids, steroidal lipids, and polyethylene glycol lipids; the outer layer is coated with a pH-responsive polymer, achieving gastric acid stability and intestinal targeted release of the oral nanovaccine via oral administration. The oral nanovaccine provided by the present invention has a particle size of 100-500 nm.

[0047] One of the core innovations of this invention is the biomimetic bacterial membrane vesicle. This invention creatively prepares an ionizable flagellated lipoprotein and introduces it into a biomimetic bacterial membrane vesicle system, constructing a structure that combines the characteristics of natural bacterial membrane vesicles (BMVs) with an ionizable design. Specifically, the ionizable flagellated protein is formed by the esterification reaction of castor oil with a double-liposome structure and flagellated protein in the presence of EDC / NHS; therefore, the lipid bonds in the ionizable flagellated protein can dissociate in the acidic microenvironment of the lysosome. Thus, the oral nanovaccine prepared using the ionizable flagellated protein can maintain stable encapsulation in the acidic environment of the stomach (pH 1-2), carry a weak positive charge on its surface in the intestinal environment (pH approximately 7) to enhance epithelial adsorption and transmembrane transport, and after entering dendritic cells, gradually dissociates and releases the antigen in the acidic environment of the lysosome, achieving precise intracellular delivery. In summary, the biomimetic bacterial membrane vesicle structure can significantly enhance the intestinal epithelial permeability through the charge conversion of ionizable flagellin; it can also systemically activate mucosal immunity and systemic immune responses, including DC maturation, T cell activation, and long-term immune memory.

[0048] The outer layer of pH-responsive polymer coating further enhances the stability and delivery efficiency of oral nanovaccines. This invention significantly improves bioavailability via oral administration through a combination of a pH-responsive polymer and a biomimetic bacterial membrane vesicle structure. Specifically, the pH-responsive polymer (such as methyl methacrylate) exhibits intramolecular hydrogen bonding and does not dissolve under acidic conditions, but gradually ionizes, swells, and releases antigens in neutral or weakly alkaline environments. Simultaneously, this polymer network structure possesses high density and hydrophobicity, effectively blocking the erosion of gastric acid, bile salts, and digestive enzymes. Therefore, compared to nanoparticles without a pH-responsive polymer outer layer, the oral nanovaccine of this invention maintains particle size and structural stability even in strongly acidic environments, preventing antigen denaturation or leakage. Thus, pH-responsive polymer coating achieves gastric acid tolerance, sustained antigen release, and targeted intestinal release via oral administration, thereby significantly enhancing vaccine stability and immune activation efficacy.

[0049] In summary, this invention achieves a targeted delivery loop of "gastric acid tolerance, intestinal adsorption, and intracellular release" by combining the charge conversion of ionizable flagellated lipoproteins in biomimetic bacterial membrane vesicles with the acid stability of the outer pH-responsive polymer. Compared with single membrane vesicles or pH-responsive polymers, the intestinal epithelial penetration efficiency is improved by 3 to 5 times.

[0050] Optionally, the biodegradable polymer material is at least one of polylactic acid-glycolic acid copolymer, polycaprolactone, polylactic acid, polyhydroxybutyric acid, or polypeptide nanomaterials.

[0051] The aforementioned biodegradable polymer materials possess both biodegradability and antigen delivery capabilities, thus they can serve as the core framework for oral nanovaccines.

[0052] Optionally, the ionizable flagellated lipoprotein is prepared by reacting (Z)-12-(((4-nitrophenoxy)carbonyl)oxy)octadecyl-9-ene-1-acrylate, fatty alcohol and flagellated protein.

[0053] Castor oil salts were prepared by reacting (Z)-12-(((4-nitrophenoxy)carbonyl)oxy)octadecyl-9-ene-1-acrylate) with a fatty alcohol. The castor oil salts were then reacted with flagellin in the presence of EDC / NHS to prepare ionizable flagellin. The lipid bonds in the ionizable flagellin prepared by the above reaction can dissociate in the acidic microenvironment of the lysosome.

[0054] Optionally, the immune adjuvant is at least one of monophospholipid A, monophospholipid derivatives, CpG oligodeoxynucleotides, and polyinosinic-polycytidylic acid; and / or, the phospholipid is soybean lecithin, egg yolk lecithin, or sphingomyelin; and / or, the steroidal lipid is cholesterol or a cholesterol derivative; and / or, the polyethylene glycol lipid is at least one of DSPE-PEG-2000, DMG-PEG, DPPE-PEG, and DOPE-PE.

[0055] By combining and replacing the above components, it is possible to construct a structure with the characteristics of biomimetic bacterial membrane vesicles, maintaining the fluidity, stability, and immune activation capacity of the biomimetic bacterial membrane vesicles.

[0056] Optionally, the pH-responsive polymer is at least one of methyl methacrylate, hydroxypropyl methylcellulose phthalate, chitosan derivatives, Eudragit L100, Eudragit S100, and polyacrylic acid polymers.

[0057] Using the aforementioned pH-responsive polymer as the outer layer of nanoparticles can endow oral nanovaccines with gastric acid tolerance, intestinal targeted release, and antigen sustained release functions under oral administration conditions, thereby improving the stability and bioavailability of oral nanovaccines.

[0058] Optionally, the mass ratio of the biomimetic bacterial membrane vesicles to the nanoparticles is 1:5 to 5:1.

[0059] Optionally, the molar ratio of ionizable flagellated lipoprotein, immune adjuvant, phospholipid, steroidal lipid and polyethylene glycol lipid in the biomimetic bacterial membrane vesicle is 1:(0.5~2):(2~5):(1~3):(0.1~1).

[0060] This invention provides a method for preparing an oral nanovaccine, comprising the following steps: S1. Reaction of (Z)-12-(((4-nitrophenoxy)carbonyl)oxy)octadecyl-9-ene-1-acrylate with a fatty alcohol yields ricinoleate; reaction of the ricinoleate with flagellin yields ionizable flagellate lipoprotein. S2. React the antigen and biodegradable polymer in a solvent to obtain nanoparticles; S3. The nanoparticles are added to a mixed solution of ionizable flagellated lipoprotein, immune adjuvant, phospholipids, steroidal lipids and polyethylene glycol lipids, and reacted to obtain nanoparticles encapsulated by biomimetic bacterial membrane vesicles. S4. React the nanoparticles encapsulated in the biomimetic bacterial membrane vesicles with pH-responsive polymer monomers in a solvent to obtain the oral nanovaccine.

[0061] A multi-layered oral nanovaccine was constructed using the above preparation method. The biomimetic bacterial membrane vesicle structure and its ionizable design synergistically enhance intestinal epithelial penetration and antigen delivery efficiency, achieving efficient transmembrane transport and dendritic cell uptake of antigens. The outer pH-responsive polymer coating endows the oral nanovaccine with excellent gastric acid resistance and intestinal targeted release capability, ensuring stable delivery and effective release via oral administration. This oral nanovaccine combines innate and adaptive immune activation mechanisms, effectively promoting DC maturation, T cell proliferation, and cytokine secretion, achieving comprehensive immune activation. The oral nanovaccine exhibits significant anti-tumor effects, demonstrating good tumor suppression, anti-metastasis, and long-term immune memory effects in various tumor models. Furthermore, the oral nanovaccine is safe and feasible: it exhibits good biocompatibility and safety in vivo, laying a technical foundation for the clinical translation and large-scale production of oral tumor vaccines.

[0062] Optionally, in step S1, the molar ratio of (Z)-12-(((4-nitrophenoxy)carbonyl)oxy)octadecyl-9-ene-1-acrylate, fatty alcohol, and flagellin is 2:5:(0.001~0.01). Preferably, in step S1, the reaction temperature is 37°C, and the reaction time is 40~60 min. More preferably, in step S1, when preparing ionizable flagellin lipoprotein, EDC or NHS is added as an activator.

[0063] Optionally, in step S2, the reaction of the antigen and the biodegradable polymer in a solvent specifically includes: dissolving the antigen and the biodegradable polymer in dimethyl sulfoxide (DMSO), with a mass ratio of antigen to biodegradable polymer of 1:(10~50), stirring at 25~37℃ for 2~4h, and then ultrasonically dispersing for 2min using an ultrasonic disruptor (25W) to obtain nanoparticles.

[0064] Optionally, in step S3, the reaction method used is one of membrane extrusion, nanoprecipitation, or microfluidic chip method. Preferably, when using membrane extrusion, the mixture is extruded 6-10 times through a 220 nm polycarbonate membrane at a pressure of 0.1-0.3 MPa. More preferably, the specific steps of the membrane extrusion method include: mixing ionized flagellated lipoprotein, immune adjuvant, phospholipids, steroidal lipids, and polyethylene glycol lipids; ultrasonicating the mixture on ice for 5 seconds, 5 seconds, and then for a total of 1 minute using a VCX130 ultrasonic disruptor; injecting the mixture into the nanoparticle solution through a 220 nm polycarbonate membrane using a syringe; and then repeatedly aspirating the mixture through the 220 nm polycarbonate membrane using a syringe for a total of 10 extrusions.

[0065] Optionally, step S4 may further include adding ammonium persulfate (initiator), wherein the mass of ammonium persulfate is 0.5-2% of the mass of the pH-responsive polymer monomer; preferably, the reaction temperature is 40-60°C and the reaction time is 5-8 hours.

[0066] Preferably, in step S4, the solvent is a mixture of deionized water and ethanol in a volume ratio of 3:1 to 5:1.

[0067] Any of the above methods can achieve uniform assembly and stable coating of biomimetic bacterial membrane vesicles; that is, without changing the structure and immune function of biomimetic bacterial membrane vesicles in oral nanovaccines, the above methods can ensure the controllability and repeatability of the preparation process.

[0068] This invention provides the application of the above-described oral nanovaccine in the preparation of any of the following: (a1) Vaccines for the prevention and / or treatment of tumors; (a2) Vaccines for the prevention and / or treatment of infectious diseases.

[0069] The oral nanovaccine system, through its modular structural design, combines high stability, targeted delivery, and universal immune activation. It can achieve rapid assembly and functional retention under different antigen systems, demonstrating its broad application potential as a universal vaccine delivery platform. Therefore, the oral nanovaccine provided by this invention can not only be used to prepare vaccines for the prevention and / or treatment of tumors, but also for the preparation of vaccines for the prevention and / or treatment of infectious diseases, including but not limited to oral or mucosal vaccines for infectious diseases such as Mycobacterium tuberculosis, influenza virus, and Klebsiella pneumoniae.

[0070] Optionally, the tumor is at least one of colon cancer, melanoma, lung cancer, breast cancer, stomach cancer, and pancreatic cancer.

[0071] Optionally, the infectious disease is a disease caused by bacterial or viral infection.

[0072] Optionally, the infectious disease vaccine is at least one of Mycobacterium tuberculosis vaccine, influenza virus vaccine, and Klebsiella pneumoniae vaccine.

[0073] Optionally, the dosage form of the vaccine is at least one of the following: powder, tablet, granule, capsule, sustained-release, solution, chewable tablet, spray, drops, gel, nanoemulsion, and lyophilized powder.

[0074] The vaccine provided in this application can be administered orally or via mucosal immunization (such as nasal spray, sublingual administration, or rectal administration). Those skilled in the art can select different drug formulations based on different administration methods.

[0075] This invention provides the application of the above-described oral nanovaccine in at least one of (b1)-(b3): (b1) Prepare products that induce or activate dendritic cell maturation; (b2) Prepare products that promote T cell proliferation or activation; (b3) Prepare products that induce long-term immune memory effects; (b4) Prepare products that enhance mucosal immune responses.

[0076] The vaccine can be used for stable delivery of antigens, especially avoiding gastric acid degradation via oral administration; it can be used to enhance intestinal epithelial penetration and improve antigen mucosal delivery efficiency; it can be used to activate dendritic cell maturation and promote T cell proliferation, inducing antigen-specific immune responses; it can be used for tumor immunotherapy, including the inhibition of colon cancer, melanoma, and other solid tumors, and the prevention of recurrence and metastasis; it can also be used to induce long-term immune memory effects.

[0077] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0078] In the following examples, 1,2-divinyl-sn-glycerol-3-phosphate ethanolamine-N-carboxylic acid (polyethylene glycol)-2000 is selected from Avanti (catalog number: 3056014-08-2); 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is selected from Sigma-Aldrich (catalog number: 25952-53-8); N-hydroxysuccinimide is selected from Sigma-Aldrich (catalog number: 106627-54-7); cholesterol is selected from... Sigma-Aldrich (Catalog No.: C3045-5G); flagellin (derived from Salmonella Typhimurium) was selected from Sigma-Aldrich (Catalog No.: SRP8029); monophospholipid A was selected from Sigma-Aldrich (Catalog No.: L6895-5MG); methyl methacrylate was selected from Sigma-Aldrich (Catalog No.: 376914-500G); ovalbumin was selected from Sigma (Catalog No.: S7951-1mg).

[0079] In the following examples, the detected data are expressed as mean ± standard deviation (mean ± SD). One-way two-sided ANOVA was performed using Origin2021 software. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0080] The present invention will be further described below through specific embodiments.

[0081] Example 1 This embodiment provides a C-BMNPs vaccine and its preparation method.

[0082] 1. Preparation method: This embodiment uses the polymer PLGA as the core framework, and assembles biomimetic bacterial membrane vesicles (BMVs) as the outer coating using ionizable flagellated lipoprotein, monophosphatidyllipoprotein A (MPLA), soybean lecithin, cholesterol, and distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG-2000). Nanoparticles encapsulated in these biomimetic bacterial membrane vesicles are prepared using a membrane extrusion method and then coated with pH-responsive methyl methacrylate (MMA), thereby endowing them with gastric acid stability and intestinal targeted release capability. The specific preparation steps are as follows: Step 1: Preparation of ionizable flagellated lipoprotein: First, in a round-bottom flask, (Z)-12-(((4-nitrophenoxy)carbonyl)oxy)octadecyl-9-ene-1-acrylate (NRA) (2.0 mmol), DMAP (0.4 mmol), and a fatty alcohol (5.0 mmol) were added to DCM (20 mL). Then, DIPEA (6.0 mmol) was slowly added dropwise while stirring at room temperature for 1 day. After the reaction was complete, the mixture was diluted with brine (20 mL) and extracted with dichloromethane (3 × 100 mL). The extracted product was dried over anhydrous MgSO4 and concentrated under vacuum. Then, using hexane and ethyl acetate as eluents, the product was purified by SiO2 gel chromatography at a ratio of 30:1 to obtain ricinoleate (colorless liquid).

[0083] Subsequently, the obtained castor oil (3.5 mmol), flagellin (10 μg), 2.5 mg of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 2.5 mg of N-hydroxysuccinimide (NHS) were added to an aqueous solution and reacted for 40 min on a shaker at 37 °C and 200 rpm to obtain ionizable flagellate lipoprotein.

[0084] Step 2: Prepare PLGA nanoparticle cores encapsulating OVA and obtain PLGA / OVA aqueous solution: 1 mL of OVA (ovalbumin) (1.4 mg / mL) was dissolved in deionized water (pH=7.5-8.0). Then, under ultrasonic conditions (25 W) in a VCX130 ultrasonic disruptor, 100 μL of PLGA DMSO solution (1 mg / mL) was added dropwise over 2 min. The reaction yielded PLGA nanoparticles encapsulating OVA. Free OVA and DMSO were removed using a 100 kDa ultrafiltration tube to obtain a PLGA / OVA aqueous solution (1 mg / mL).

[0085] Step 3: Preparation of flagellated lipid nanoparticles (BMNPs): Finally, the 200 μL of ionizable flagellated lipoprotein (100 mg / mL), 100 μL of monophospholipid A (1 mg / mL), 18 μL of soybean lecithin (10 mg / mL), 12 μL of cholesterol (5 mg / mL), and 18 μL of DSPE-PEG-2000 (10 mg / mL) prepared in step 1 were mixed evenly in deionized water and then extruded with 1 mL of PLGA / OVA (1 mg / mL) through a 220 nm polycarbonate membrane six times to encapsulate the PLGA / OVA core using the membrane extrusion method to form an aqueous solution of BMNPs (OVA concentration: 800 μg / mL).

[0086] Step 4: Construct the core-shell polymer outer layer to obtain the C-BMNPs vaccine: A stable BMNPs solution was formed by adding 50 μL of glycerol to 950 μL of an aqueous BMNPs solution (800 μg / mL). Subsequently, 500 mg of MMA, 10 mg of methacrylic acid, and 20 mg of diethyl diacrylate were added to 5 mL of deionized water, and the mixture was reacted at 37°C for 5 h to construct a stable MMA monomer polymer network. Under ultrasonic stirring (25 W, 40 rpm), 1 mL of the BMNPs solution was added dropwise to 2 mL of the MMA monomer polymer, reacting to form a water-in-oil emulsion. Finally, under the protection of inert nitrogen gas, the emulsion was heated to 40°C to initiate free radical polymerization. BMNPs were gradually encapsulated into the MMA monomer polymer to form a core-shell structured C-BMNPs vaccine (1 mg / mL).

[0087] 2. Testing the prepared C-BMNPs vaccine: 2.1 The morphology of the C-BMNPs vaccine prepared in this embodiment was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Particle size and surface potential were detected by dynamic light scattering (DLS). Protein and component composition were confirmed by Western blot (WB) and ultraviolet-visible spectroscopy (UV-Vis). The results are as follows: Figure 2 , Figure 3 As shown.

[0088] like Figure 2 As shown, Figure 2 a and Figure 2 As shown in Figure b, the C-BMNPs vaccine exhibits good size uniformity, with visually observed particle sizes ranging from 150 to 200 nm. The vaccine has a spherical morphology, and its core-shell structure can be clearly identified through high-resolution TEM images.

[0089] like Figure 3 As shown, to further verify the physical properties of the particle system, dynamic light scattering (DLS) was used to systematically characterize the hydrodynamic diameter and surface potential of C-BMNPs. Figure 3 a and Figure 3 (b) Quantitative analysis data showed that the Number Mean particle sizes of PLGA / OVA, BMNPs, and C-BMNPs were 100.20±11.40 nm, 149.50±9.30 nm, and 190.70±14.10 nm, respectively, which were basically consistent with the TEM and SEM observations. Furthermore, the Zeta potentials of PLGA / OVA, BMNPs, and C-BMNPs were -7.49±0.18 mV, -20.71±0.45 mV, and -34.37±0.94 mV, respectively, with the C-BMNPs coated with MMA exhibiting the largest absolute Zeta potential. Generally, a higher absolute Zeta potential enhances the electrostatic repulsion between particles and effectively inhibits aggregation, indicating that the prepared C-BMNPs vaccine possesses excellent dispersion stability.

[0090] like Figure 3 As shown in Figure c, OVA exhibits a distinct characteristic absorption peak at 280 nm. Although the absorption peaks showed slight red and blue shifts after encapsulating biomimetic BMVs and MMA, the characteristic absorption peaks of OVA were still retained, indicating that OVA was successfully loaded into BMNPs and C-BMNPs without structural disruption. Furthermore, Western blot analysis after cleaving BMNPs and C-BMNPs further validated the protein encapsulation effect. Figure 3 As shown in Figure d, bands of OVA and flagellin were detected in the lysates of both BMNPs and C-BMNPs, indicating that both proteins were successfully loaded into the C-BMNPs vaccine.

[0091] 2.2 The stability and encapsulation efficiency of the C-BMNPs vaccine prepared in this embodiment were investigated; as well as the acid resistance, enteric coagulation, and antigen release rate of the C-BMNPs vaccine. The OVA encapsulation efficiency was detected using the BCA method, and particle size and zeta potential were detected using dynamic light scattering (DLS). The detection instrument was a Malvern Zetasizer Nano ZS. The results are as follows: Figure 4 , Figure 5 As shown.

[0092] like Figure 4 As shown, to evaluate the long-term stability of the C-BMNPs vaccine, its particle size and polydispersity index (PDI) in solution were monitored for 7 consecutive days. The results showed that the hydrodynamic diameter of the C-BMNPs vaccine remained consistently within the range of 170-200 nm. Figure 4 (a) The particle size on day 1 was 173.80 ± 13.23 nm, and the PDI was 0.23 ± 0.03. The particle size on day 7 was 192.90 ± 5.56 nm, and the PDI was 0.28 ± 0.04. This indicates that the C-BMNPs vaccine has excellent colloidal stability and monodispersity, meeting the quality standards for nano-vaccine formulations. Figure 4 As shown in Figure b, when the OVA concentration increased from 0.2 mg / mL to 1.4 mg / mL, the encapsulation efficiency showed a dose-dependent increase, with a maximum encapsulation efficiency of 67.00±3.21%. However, when the OVA concentration exceeded 1.4 mg / mL, the encapsulation efficiency decreased to 58.30±2.39%, which may be because the carrier material PLGA had reached the antigen saturation loading.

[0093] like Figure 5 As shown in Figure a, the solution was pale yellow-green, and the vaccine particles maintained their nearly spherical structure, indicating excellent resistance to gastric acid degradation. Simultaneously, the C-BMNPs vaccine was placed in a simulated intestinal environment (PBS solution of trypsin (1 mg / mL), pH=7) for 1 h, and the solution appearance was photographed and observed using TEM. The results showed that at pH=7, the acid-resistant outer layer MMA on the surface of the vaccine particles was fully dissolved, and the exposed biomimetic BMVs caused partial adhesion of the BMNPs particles. Figure 5 (b)

[0094] Example 2 This embodiment verifies whether C-BMNPs vaccines can achieve effective mucosal delivery after oral administration, including both in vivo and in vitro experiments. In vivo experiments: Small animal in vivo fluorescence imaging technology was used to assess the distribution and gastrointestinal metabolic trajectory of C-BMNPs in vivo; In vitro experiments: A Transwell model was used to construct an in vitro intestinal epithelial barrier to study the penetration efficiency of the oral nanovaccine.

[0095] 1. Evaluation of the distribution and gastrointestinal metabolic trajectory of C-BMNPs in vivo using in vivo fluorescence imaging technology in small animals: 1.1 Small animal in vivo fluorescence imaging Healthy female C57BL / 6 mice aged 5-6 weeks were randomly divided into two groups of 15 mice each. One group received oral gavage of 125 μL PBS, while the other received C-BMNPs (OVA-Alexa Fluor 647, 100 μg / mL). Before gavage, and at 2 h, 4 h, 8 h, and 24 h post-gavage, three mice from each group were sacrificed. The intact digestive tracts were dissected and placed on black plastic plates. Quantitative fluorescence analysis was performed using IVIS Spectrum system (Caliper, USA) imaging and Living Image software. Simultaneously, at the 8 h time point, the digestive tracts of the sacrificed mice were dissected, and 15 mg of gastric, intestinal, cecal, and colonic contents, along with feces, were collected into 1.5 mL tubes for quantitative fluorescence analysis using IVIS Spectrum system imaging and Living Image software.

[0096] 1.2 Hematoxylin-eosin staining (H&E) (1) Dewaxing and hydration: In a fume hood, the paraffin white slices were first soaked in xylene I and xylene II for 10 min each. After soaking, they were soaked in anhydrous ethanol I and ethanol II for 3 min each. Then they were soaked in a gradient of ethanol (95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol) for 3 min each. After that, they were rinsed with running tap water for 2 min and finally rinsed with distilled water for 30 s.

[0097] (2) Hematoxylin staining: Immerse the tissue section in hematoxylin staining solution for 1-3 min. Stop staining immediately when the cell nuclei turn blue under a microscope. Rinse under running tap water until the water tank is clear and colorless.

[0098] (3) Differentiation and blueing: Differentiate with 1% hydrochloric acid alcohol for 1-5 seconds. When the cell nuclei are clearly stained and the cytoplasm is not obviously stained under a microscope, immediately remove the section, rinse with running tap water for 2 minutes, and then rinse with distilled water for 30 seconds. After rinsing, place it in lithium carbonate solution for 30 seconds for blueing, rinse with running tap water for 2 minutes, and then rinse with distilled water for 30 seconds.

[0099] (4) Eosin staining: Immerse the tissue sections in eosin staining solution (water-soluble) for 30-60 s (adjust the time according to different tissues), rinse with tap water for 1-5 min, and then rinse with distilled water for 5 s.

[0100] (5) Dehydration: The tissue sections were dehydrated by gradient ethanol, with the following ethanol concentrations and dehydration times: 70% ethanol (30 s), 85% ethanol (1 min), 95% ethanol (3 min), 95% ethanol (3 min), 100% ethanol I (5 min), and 100% ethanol II (5 min).

[0101] (6) Clearing: The dehydrated tissue sections were soaked in xylene I and xylene II successively for 5 min each time.

[0102] (7) Mounting: Mount the tissue sections that have been cleared by removing them from xylene, adding a drop of neutral resin to the sections, and covering them with a coverslip to prevent air bubbles from forming. Place the mounted sections in a fume hood to dry, and then take photos for observation.

[0103] 1.3 Immunofluorescence staining: (1) Sample fixation: Take out the prepared tissue sample slices and then put the slices into 4% paraformaldehyde and fix them at room temperature for 15 min.

[0104] (2) Blocking: After fixing, the sections were rinsed three times with PBS buffer for 5 min each time. Then, PBS solution containing 10% goat serum was added and the sections were blocked at room temperature for 60 min to reduce nonspecific binding.

[0105] (3) Antibody incubation: Aspirate the blocking solution, without washing, and directly add an appropriate amount of diluted CD11c antibody (1:1000), and incubate at room temperature in the dark for 2 h.

[0106] (4) Nuclear staining: After antibody incubation, wash the sample three times with PBS for 5 min each time. Finally, add DAPI nuclear staining agent to counterstain the cell nuclei, incubate at room temperature for 10 min, and then wash three times with PBS.

[0107] (5) Mounting and photographing: The tissue sections were mounted with neutral resin and then the images were acquired using the Pannoramic series digital pathology slide scanner.

[0108] 1.4 Experimental results are as follows Figure 6 As shown.

[0109] like Figure 6 As shown in Figure a, 4 h after gavage, some fluorescence signal could still be detected in the stomach of mice (relative fluorescence intensity 0.67 ± 0.11 × 10⁻⁶). 10 (au), while the small intestine, cecum, and colon showed significant fluorescence enrichment (relative fluorescence intensities of 0.96 ± 0.09 × 10⁻⁶, respectively). 10 au, 1.24±0.12×10 10 au, 0.95±0.08×10 10 (au). As the gastric emptying process of mice progressed, the fluorescence in the stomach basically disappeared 8 hours after gavage (the relative fluorescence intensity decreased to 0.17±0.05×10). 10 At this time, the fluorescence signal was mainly concentrated in the small intestine and cecum. This indicates that the C-BMNPs vaccine can stably cross the gastric acid barrier and successfully reach the small intestine tissue. Notably, after 24 hours, the fluorescence signal in all gastrointestinal regions returned to the background level, and quantitative detection showed no statistically significant difference in fluorescence intensity between the stomach, small intestine, cecum, and colon and the control group (P>0.05). The results indicate that the C-BMNPs vaccine can be completely degraded and cleared through the intestinal metabolic pathway within 24 hours after antigen delivery. Figure 6 (c)

[0110] In addition, 8 hours after oral gavage administration of the C-BMNPs vaccine, gastrointestinal tissues and feces from different sites were collected for in vivo fluorescence imaging and quantitative detection in small animals. The results showed that, compared with the PBS control group, mice in the C-BMNPs vaccine group exhibited abundant fluorescent signals in the small intestine, cecum, and colon, and the differences were statistically significant (P<0.0001). Figure 6 (d) 2. Construct a Transwell system to systematically evaluate the in vitro epithelial penetration of C-BMNPs vaccines. 2.1 Experimental Procedure: (1) Preparation of the chamber: Select a 24-well plate and a Transwell chamber with a polycarbonate membrane pore size of 10 μm according to the experimental requirements.

[0111] (2) Cell preparation: The Caco-2 and HT-29 cells required for the experiment were digested. After adding trypsin and spreading evenly, the cells were incubated in an incubator. When the cells were observed to be flattened and rounded, and large numbers of cells were gently tapped off from the culture flask, culture medium was added to stop the digestion. The cells were then carefully pipetted until they were completely detached. The cells were collected into centrifuge tubes and centrifuged at 1500 rpm for 5 min. The supernatant was carefully discarded. The cells were resuspended in a suitable culture medium, mixed evenly, and 10 μL was taken for cell counting.

[0112] (3) Cell plating in small chambers: The counted cells were mixed at a ratio of 9:1 for Caco-2 and HT-29, and then plated at a concentration of 1×10⁻⁶ cells / mL. 5 / cm 2 The culture medium was inoculated at a density in 24-well Transwell chambers and cultured for 18 days, with the medium being changed every two days (800 μL in the chamber and 1000 μL in the bottom layer).

[0113] (4) Lower layer cell plating: DCs were plated at a density of 2 × 10⁻⁶. 5 / cm 2 The density of inoculation was carried out in the 24-well lower chamber.

[0114] (5) Co-incubation: After the DCs in the lower chamber adhere to the wall, the culture medium in the upper chamber is replaced with culture medium containing OVA and C-BMNPs (OVA-Alexa Fluor 647 label), and co-incubated for 12 h.

[0115] (6) Cell collection: Remove and discard the chamber, then carefully aspirate the culture medium from the lower chamber, wash gently twice with PBS, then add 4% paraformaldehyde for cell fixation, and fix at room temperature for 15 min.

[0116] (7) Staining and photography: The fixed DCs were stained with CD11c, CD80, CD86 and HLA-A2 antibodies respectively. After staining, FCM was used for detection and analysis.

[0117] 2.2 Experimental results are as follows Figure 7 As shown. Figure 7 As shown in Figure a, only green fluorescence signals from labeled DCs were observed in the PBS group, with no red C-BMNPs vaccine signal observed, while the free OVA group and PLGA / OVA group showed only a small amount of red fluorescence signal. In contrast, the C-BMNPs vaccine group showed a large amount of red fluorescence signal, and most of the red antigen signal was co-localized with the DCs label. Further comparison revealed that although the red fluorescence signal in the BMNPs group was significantly lower than that in the C-BMNPs group, it was still significantly higher than that in the free OVA group and PLGA / OVA group, indicating that the biomimetic BMVs also have a certain protective effect against the OVA antigen.

[0118] Six non-overlapping CLSM fields were randomly selected for fluorescence signal count analysis, such as... Figure 7 In the middle b group, the number of red fluorescence signals in the OVA group, PLGA / OVA group and BMNPs group were (5.17±1.33), (25.55±4.83) and (51.83±5.66), respectively, which were significantly lower than those in the C-BMNPs vaccine group (244.00±28.50). The difference in the number of fluorescence signals was statistically significant (P<0.0001).

[0119] Example 3 This embodiment verifies the immune activation effect of the C-BMNPs vaccine, including in vivo and in vitro experiments.

[0120] In in vitro experiments, the uptake of C-BMNPs by dendritic cells (DCs) was analyzed using laser confocal microscopy (CLSM), and their cytotoxicity was assessed using the CCK-8 assay. The expression of co-stimulatory molecules CD80 / 86 and MHC molecules on the surface of DCs was detected by flow cytometry (FCM), and the levels of cytokines such as IL-6 and IL-12p40 were detected by T cell proliferation assay and ELISA to evaluate the vaccine's effect on promoting DC maturation and T cell activation.

[0121] In vivo experiments were conducted by orally immunizing C57BL / 6 female mice and isolating intestinal immune cells for flow cytometry analysis and single-cell transcriptome sequencing. The mechanism of C-BMNPs-induced mucosal and systemic immune activation was revealed from multiple dimensions, including immune cell composition, activation status and gene expression level.

[0122] The specific steps are as follows: 1. In vitro experiments 1.1 Immunofluorescence staining (1) Cell sample staining: To assess the uptake of C-BMNPs vaccine by DCs, DCs were first cultured to an appropriate density and washed once with PBS. Then, vaccine particles (concentration 100 μg / mL) containing Alexa Fluor 647 fluorescent labeling were added to the culture medium and incubated for 12 h. After incubation, the culture medium was removed and the cells were washed once with PBS. Next, DCs were collected in 15 mL centrifuge tubes, and PBS solution containing CD11c-FITC antibody was added. The cells were incubated at room temperature in the dark for 25 min, followed by centrifugation to remove unbound antibodies. After resuspending the cells in PBS, approximately 2 μg / mL of Hoechst 33342 dye was added, and the cells were incubated at room temperature in the dark for 15 min. After staining, DCs were seeded onto confocal microscope slides coated with poly-L-lysine, fixed for 4 h, the culture medium was removed, and CLSM imaging was performed. The filter settings are as follows: Alexa Fluor 647 excitation wavelength approximately 633 nm, emission wavelength approximately 647 nm; Hoechst excitation wavelength approximately 350 nm, emission wavelength approximately 461 nm.

[0123] 1.2 FCM Detection (1) FC receptor blocking: After washing and discarding the supernatant, add 100 μL of PBS buffer to resuspend the cells, and then add 2 μL of the Fc receptor blocking agent TruStain FcX. TM Mix the (anti-mouse CD16 / 32) antibody thoroughly. ② Incubate on ice for 10 min, no washing required.

[0124] (2) Surface staining: Add the corresponding antibody (CD11c, CD80, CD86, etc.) to the corresponding experimental tube, vortex mix well, and incubate on ice in the dark for 20 min. All surface staining tubes are washed once with 1 mL of cell staining buffer, centrifuged at 350 g for 5 min, and the supernatant is discarded.

[0125] (3) Then add 1 mL PBS to resuspend the cells and centrifuge at 500 g for 5 min. After discarding the supernatant, add 200 μL PBS solution to resuspend the cells and then use a CYTEK full-spectrum flow cytometer for detection.

[0126] 2. In vivo experiments (in vivo flow cytometry): Six mice were sacrificed at 12 hours, and their small intestines were dissociated into single-cell suspensions for flow cytometry antibody staining and analysis. Subsequent steps were the same as in 1.2 and FCM detection in this example.

[0127] 3. Experimental Results: 3.1. C-BMNPs vaccine induces DC maturation in vitro: Mature dendritic cells (DCs) construct a triple signaling activation system by highly expressing MHC molecules, co-stimulatory molecules, and adhesion molecules, effectively activating naive T cells. As the "initiator," "regulator," and "maintainer" of the immune response, mature DCs are at the core of this process. The functional maturity of DCs directly determines their effectiveness in activating adaptive immune responses. CD80 molecules, as a specific surface marker of DC maturity, participate in the stable assembly of antigen-presenting complexes and synergistically regulate the specific recognition and activation process of T cells. CD86 molecules, as a B7 family co-stimulatory molecule, form an immune synapse with the CD28 receptor on the T cell surface, transmitting specific co-stimulatory signals, thereby initiating and maintaining T cell activation, proliferation, and effector differentiation. Immature DCs typically express low levels of CD80 / CD86 co-stimulatory molecules; upregulation of their expression can serve as a key indicator for assessing DC maturity, directly affecting the strength and persistence of antigen-specific T cell immune responses.

[0128] To evaluate the promoting effect of C-BMNPs vaccine on DCs in vitro maturation, equal concentrations (1 mg / mL) of OVA protein, PLGA / OVA nanocores, BMNPs, and C-BMNPs were co-cultured with PBMC-derived DCs for 24 h. Figure 8 As shown, compared to the PBS control group (24.13±1.41%), all experimental groups upregulated the double-positive expression of the DCs co-stimulatory molecules CD80 / CD86, with the C-BMNPs group exhibiting the strongest maturation-promoting effect. Quantitative data showed that CD11c in the C-BMNPs group... + CD80 + CD86 + The proportion of triple-positive DCs reached 36.78±1.85%, significantly higher than that of the BMNPs group (30.48±0.75%), PLGA / OVA group (29.61±0.49%), OVA group (24.91±0.80%), and PBS group (24.71±0.36%) (P<0.001). Notably, the maturation efficiency of C-BMNPs was 6.30% higher than that of the second-best group (BMNPs), which may be closely related to its surface functionalization with methyl methacrylate (MMA). Related studies have shown that MMA modification can achieve a sustained-release effect of antigen and adjuvant by regulating the degradation kinetics of nanoparticles, thereby prolonging the activation time window of DCs and enhancing the sustained expression of co-stimulatory signals (CD80 / CD86).

[0129] After dendritic cells (DCs) take up antigens, they present the antigen information to T cells via MHC molecules, thereby activating a specific immune response in T cells. Mature DCs typically highly express MHC II molecules on their cell surface. Antigens processed via the lysosomal pathway are loaded onto MHC II molecules and presented to CD4 cells.+ T cells. And CD8 + T cells, as the main anti-tumor effector cells, are activated primarily through antigen presentation via the MHC I complex. Detecting the expression level of the MHC I complex on the surface of dendritic cells (DCs) can effectively assess the efficiency of antigen cross-presentation. Human MHC is known as human leukocyte antigen (HLA). The cells used to stimulate DC maturation in this study were DCs isolated from PBMCs, and the most common MHC I subtype in the Chinese population is HLA-A2. FCM analysis showed that, compared with the PBS control group, the expression of HLA-A2 molecules on the surface of DCs in each experimental group was upregulated to varying degrees. Quantitative data showed that the C-BMNPs vaccine group exhibited a significant advantage in antigen cross-presentation: its CD11c... + CD80 in cell population + HLA-A2 + The cell percentage was 10.57 ± 0.71%, CD86 + HLA-A2 + The cell proportion was 10.00±0.97%, which was 8.43% and 8.38% higher than that of the PBS control group, respectively (P<0.0001). These results indicate that C-BMNPs synthesized by nanoencapsulation technology can more effectively promote antigen cross-presentation of DCs, and their surface functionalization modification may enhance the HLA-A2 antigenic epitope presentation ability by improving the recruitment efficiency of MHC-I molecules.

[0130] 3.2. C-BMNPs vaccines can effectively activate the pro-inflammatory cytokine secretion pathway in dendritic cells (DCs): To further evaluate the regulatory effect of C-BMNPs vaccine on DC maturation, OVA, PLGA / OVA, BMNPs, and C-BMNPs were co-cultured with DCs in vitro for 24 h. Cell culture supernatants were collected, and the levels of IL-6, TNF-α, and IL-12p40 secreted by DCs were quantitatively detected using an ELISA kit. Figure 9As shown, the levels of IL-6 secreted by DCs in the C-BMNPs group were 398.6±37.1 pg / mL, TNF-α was 575.5±41.2 pg / mL, and IL-12p40 was 1306.2±301.3 pg / mL, all significantly higher than those in the PBS control group (IL-6: 48.6±13.3 pg / mL; TNF-α: 131.5±23.2 pg / mL; IL-12p40: 41.3±22.3 pg / mL, P<0.0001), and also significantly higher than those in the OVA group, PLGA / OVA group, and BMNPs group. Notably, as a core regulator of the Th1 immune response, the C-BMNPs group showed the highest increase in IL-12p40 secretion, approximately 32-fold higher than the PBS group. The results showed that the C-BMNPs vaccine could effectively activate the pro-inflammatory cytokine secretion pathway of DCs, significantly enhance their maturation and activation capabilities, and provide a key molecular basis for inducing specific anti-tumor immune responses.

[0131] 3.3. C-BMNPs vaccine stimulates T cell proliferation in vitro: To evaluate the effect of C-BMNPs vaccine on T cell proliferation, T cells were first isolated and purified from PBMCs, and then co-cultured with induced mature dendritic cells (DCs). Changes in CFSE signal intensity during different proliferation spurts were detected using CFSE staining combined with FCM. Specifically, during T cell proliferation, the CFSE signal intensity gradually decreased, exhibiting a left shift, thus directly reflecting the proliferative activity of T cells. Results are as follows: Figure 10 As shown, in the PBS control group, T cells showed almost no proliferation, the CFSE signal shift to the left was very limited, and the proportion of CFSE-positive cells was 92.8±0.42%, indicating weak T cell proliferation in this group. In contrast, the C-BMNPs vaccine group showed a significant left shift in the CFSE signal of T cells, and the proportion of CFSE-positive cells decreased to 87.1±0.15%, demonstrating significant T cell proliferative activity (P<0.01). This indicates that the C-BMNPs vaccine can significantly promote T cell proliferation in vitro, suggesting its good effect in activating T cells and enhancing the immune response.

[0132] Example 4 This embodiment systematically evaluates the antitumor efficacy of the C-BMNPs vaccine using three models: a mouse MC38 subcutaneous colon cancer xenograft model, a melanoma lung metastasis model, and an immune memory model. The specific steps are as follows: 1. Establishment of a mouse MC38 subcutaneous xenograft model of colon cancer To evaluate the antitumor effect of the C-BMNPs vaccine in a mouse MC38 subcutaneous xenograft model of colon cancer, 2 × 10⁶ mouse colon cancer (MC38-OVA) cells were subcutaneously injected into the right back near the axilla of C57BL / 6 mice on day 0. On day 4 post-injection, mice were evenly divided into 5 groups (n=12) according to the average tumor size and recorded by ear tagging. Then, on days 4, 7, and 12, mice were orally inoculated with PBS (125 μL), OVA (125 μL, 1 mg / mL), PLGA / OVA (125 μL, 1 mg / mL), BMNPs (125 μL, 1 mg / mL), or C-BMNPs (125 μL, 1 mg / mL), respectively. Starting from day 4, the long and short diameters of the tumor were measured every other day using electronic calipers, and the volume was calculated. The tumor volume calculation formula was: Tumor volume (V) = Long diameter (L) × Short diameter (W)² / 2. On day 20, six mice from each group were randomly sacrificed, and the remaining mice were further examined for survival. Tumors were then collected, weighed, and digested into single-cell suspensions. The proportion of immune cells infiltrating the tumor was analyzed by fractional immunofluorescence (FCM). Paraffin-embedded sections of tumor tissue were prepared and subjected to immunofluorescence, immunohistochemistry, and TUNEL / Ki67 staining. Spleens were collected, dissociated into single-cell suspensions, and CD3+ levels in spleen cells were analyzed by FCM. + CD8 + The percentage of T cells was analyzed. Blood samples were collected for complete blood count, blood biochemistry, and the proportion of OVA antigen-specific T cells. Half of the spleen single-cell suspension was aliquoted and restimulated with OVA antigen for 24 h (final OVA concentration after mixing with culture medium was 100 μg / mL), followed by stimulation with iomycin for 4 h and then FCM detection. Simultaneously, the IFN-γ secreted by spleen cells after OVA antigen stimulation was detected using the ELISPOT technique. Spleen cells were first seeded in 96-well plates pre-coated with mouse anti-IFN-γ antibody (1 × 10⁶ cells per well). 5 Cells were then stimulated with OVA and iomycin as described above. After stimulation, 96-well plates were incubated for 1 h with an IFN-γ-specific biotinylated antibody and streptavidin-binding alkaline phosphatase. By adding substrate solution, IFN-γ visual spots secreted by T cells restimulated by OVA antigen were formed at the captured IFN-γ sites. Finally, the well plates were sent to Kewei Biotechnology Co., Ltd. for automated spot quantification. Major organs of mice, including heart, liver, spleen, lungs, and kidneys, were collected and then stained with hematoxylin-eosin (H&E).

[0133] 2. Establish a mouse model of melanoma lung metastasis. C57BL / 6 mice were injected via tail vein on day 0 with 3×10 5Twelve mice were orally inoculated with PBS (125 μL), OVA (125 μL, 1 mg / mL), PLGA / OVA (125 μL, 1 mg / mL), BMNPs (125 μL, 1 mg / mL), or C-BMNPs (125 μL, 1 mg / mL) on days 3, 6, and 10, respectively. Six mice per group were sacrificed 16 days after tail vein injection of tumor cells, and spleens and lungs were collected for further analysis. Survival rates of the remaining mice were assessed. The collected lungs were washed with PBS, blotted dry with absorbent paper, and fixed for 24 h with Fekete tissue preservation solution (volume ratio: 75% alcohol:formalin:glacial acetic acid = 14:2:1). The number of lung nodules was counted by photographing. After photographing, the lung tissue was embedded in paraffin and sectioned for immunofluorescence, immunohistochemistry, and H&E staining analysis. To analyze the proportion of IFN-γ and OVA-tetramer-positive T cells in the spleen, the spleen was dissociated into a single-cell suspension and co-incubated with OVA antigen for 24 h before iomycin stimulation and FCM detection. Iomycin-treated spleen cells served as a positive control group.

[0134] 3. Establishment of a long-term immune memory model in mice C57BL / 6 mice were orally immunized on days 0, 3, and 10, with 12 mice in each group. The oral doses and concentrations were: PBS (125 μL), OVA (125 μL, 1 mg / mL), PLGA / OVA (125 μL, 1 mg / mL), BMNPs (125 μL, 1 mg / mL), and C-BMNPs (125 μL, 1 mg / mL). On day 52, six mice were sacrificed, and their spleens were dissected into single-cell suspensions for flow cytometry staining and analysis. To investigate the effect of immunization on tumor cell attack prevention in immunized mice, the remaining six vaccinated mice were injected intravenously with 3 × 10⁻⁶ PBS. 5 B16-OVA cells were collected, and mice were sacrificed on day 66. Lung tissue was collected, fixed, photographed, and the number of lung metastatic nodules was counted. Major organs such as the heart, liver, spleen, lungs, and kidneys were collected for H&E staining.

[0135] 4. FCM testing (1) Sample preparation: First, prepare a 2 cm × 2 cm 30-mesh wire mesh and place it in a 10 cm culture dish for later use. After the animal experiment, the mice were euthanized by spinal dislocation. After spraying / soaking with 75% alcohol, the entire spleen was removed by abdominal surgery, and the connective tissue was cleaned. Then, the spleen tissue was placed on the wire mesh. A small amount of 1×PBS buffer was poured onto the wire mesh to cover the spleen tissue, and then the spleen was ground with the end of a syringe. 5 mL of PBS solution was added to the ground cell suspension, and the mixture was filtered through a 70 μm filter into a 50 mL centrifuge tube and centrifuged at 350 g for 5 min. 10× erythrocyte lysis buffer (RBCLysis Buffer) was prepared into 1× working solution with ultrapure water. The supernatant of the centrifuged cells was discarded, and the cell pellet was resuspended with 5 mL of RBC lysis working solution and incubated on ice for 5 min, during which time it could be mixed once. After lysis, 20 mL of PBS solution was added to stop cell lysis, and the cells were centrifuged at 350 g for 5 min. The supernatant was discarded. Add 5 mL of PBS to resuspend the cells and wash them a second time. Centrifuge at 350 g for 5 min, discard the supernatant, and then resuspend the cells in 1 mL of culture medium (if OVA restimulation is required, incubate the cells with OVA in a 6-well plate for 24 h before proceeding to the next step).

[0136] (2) Cell activation: After mixing the cells, take 10 μL of cells for counting and adjust the cell concentration to 1×10⁻⁶. 6 -2×10 6 Cells / mL. Completely thaw the cell stimulant in a 37°C water bath. Then add 2 µL of cell stimulant to every 100 μL of cell suspension. Culture spleen cells in a 37°C CO2 incubator for 4–6 h, then harvest activated cells, centrifuge at 350 g for 5 min, and discard the supernatant. Add 2.5 mL of PBS solution, mix well, centrifuge at 350 g for 5 min, discard the supernatant, and perform live / dead staining. Repeat this process once.

[0137] (3) Live and dead staining: Preparation of live and dead staining solution: Preheat the kit to room temperature, then add Zombie Aqua TM Perform a quick separation of the dye vial, ensuring the reagent is at the bottom of the vial. Then add 100 μL of DMSO to a vial of Zombie Aqua. TM In the dye solution, mix thoroughly to completely dissolve the dye and form a 100× live / dead cell staining stock solution. Resuspend the cells in 1 mL of PBS solution, mix well, and then take 10 μL of cells for counting, adjusting the cell concentration to 10. 7 / ml. Then, aliquot the cell suspension into 1.5 mL EP tubes, ensuring each tube contains 100 μL and 10⁶ cells. Add 0.125 μL of Zombie Yellow 100× stock solution to each tube requiring live / dead staining. Incubate the cells in the dark at room temperature for 20 min. Wash once with 1 mL cell staining buffer, centrifuge at 350 g for 5 min, and discard the supernatant.

[0138] (4) FC receptor blocking: After live and dead cell staining, 100 μL of PBS buffer was added to the washed and supernatant-discarded cell tubes for resuspending, and then 2 μL of the Fc receptor blocking agent TruStain FcX was added. TM Mix the (anti-mouse CD16 / 32) antibody thoroughly. Incubate on ice for 10 minutes, no washing required.

[0139] (5) Surface staining (only for surface staining antibodies): Add the corresponding antibodies (CD3, CD4, CD8, CD44, CD62L, CD25, CD11c, etc.) to the corresponding experimental tubes, vortex to mix well, and incubate on ice in the dark for 20 min. All surface staining tubes are washed once with 1 mL of cell staining buffer, centrifuged at 350 g for 5 min, and the supernatant is discarded.

[0140] (6) Intracellular staining: Dilute Cyto-Fast with ultrapure water TM Perm Wash solution (10×) to 1×. Add 150 µL of Cyto-Fast to each 1.5 mL EP tube after surface staining. TM Resuspend the cells in Fix / Perm Buffer and mix well. Incubate at room temperature for 20 min. Then add 1 mL of 1×Cyto-Fast. TM Perm Wash solution. Centrifuge at 500 g for 5 min, discard the supernatant. Then add 100 µL of 1×Cyto-Fast solution. TM After resuspending cells in Perm Wash Solution, add the corresponding INF-γ antibody and incubate at room temperature in the dark for 30 min. ⑦ Add 1 mL of 1×Cyto-Fast... TM Cells were washed with Perm Wash solution, centrifuged at 500 g for 5 min, and the supernatant was discarded. Cells were then resuspended in 1 mL PBS and centrifuged at 500 g for 5 min. After discarding the supernatant, cells were resuspended in 200 μL PBS solution and analyzed using a CYTEK full-spectrum flow cytometer.

[0141] 5. Experimental Results: 5.1 Analysis of the in vivo tumor-suppressing effect of C-BMNPs vaccine To evaluate the antitumor efficacy of the C-BMNPs vaccine, MC38-OVA tumor cells were subcutaneously injected into female C57BL / 6 mice on day 0 to establish a mouse model of MC38 colon cancer subcutaneous xenografts. Mice were then treated three times orally by gavage with PBS, OVA, PLGA / OVA, BMNPs, or C-BMNPs vaccine on days 4, 7, and 12, respectively. Tumor volume was measured every two days after the first treatment until day 20. Figure 11 As shown, mice in the C-BMNPs vaccine group exhibited stronger tumor growth inhibition than other groups. From day 14, the tumor volume began to show a negative growth trend, and by the experimental endpoint (day 20), the average tumor volume of the mice had shrunk to 60.3 mm. 3 The tumor inhibition rate was significantly different from that of the BMNPs group, PLGA / OVA group, OVA group, and PBS control group (P<0.0001). The tumor inhibition rate was calculated based on the tumor volume at the endpoint. Figure 11 As shown in Figure (d), the average tumor inhibition rate of mice in the C-BMNPs vaccine group was 92.41%, significantly higher than that of the BMNPs group (54.36%), the PLGA / OVA group (32.57%), and the OVA group (26.88%) (P<0.0001). Final autopsy data showed that the tumor mass in the C-BMNPs group (0.10±0.11 g) was significantly lower than that in the PBS group (1.08±0.17 g) by 90.7% (P<0.0001). During a 35-day survival period, only the C-BMNPs group had a 100% survival rate; all other control groups experienced mouse mortality during the treatment period. This indicates that the C-BMNPs vaccine can effectively inhibit the growth of subcutaneous MC38 colon cancer tumors in mice and significantly improve mouse survival.

[0142] 5.2. The C-BMNP vaccine has good biocompatibility and biosafety. To assess the biosafety and compatibility of the C-BMNPs vaccine in mice, their body weight, activity, and overall health were monitored every other day during treatment. Figure 12 As shown, on the first day after subcutaneous inoculation with MC38 tumor cells, all mice experienced a decrease in body weight. From the second day onwards, the mice's body weight gradually recovered, and the weight fluctuations remained stable throughout the treatment period. This indicates that the mice exhibited good tolerance to the C-BMNPs vaccine and its components during treatment. At the end of treatment, blood was collected from the mice's eyeballs for subsequent complete blood count and blood biochemistry analysis. Figure 13As shown, blood routine results indicated no significant changes in RBC, HGB, HCT, MCV, and NRBC levels in the blood of mice in both the treatment and control groups. However, the number of lymphocytes (LYMPH) in the blood of mice in the C-BMNPs group was slightly increased, which may be related to the adaptive immune activation induced by the vaccine. Figure 14 As shown, blood biochemical indicators, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatinine (CR), blood urea nitrogen (BUN), and uric acid (UA), showed no abnormal changes, indicating that neither the experimental nor the control groups experienced liver or kidney damage or inflammatory responses. To further evaluate the toxicity of the C-BMNPs vaccine, H&E staining analysis was performed on the major organs and tissues of mice, including the heart, liver, spleen, lungs, and kidneys. Figure 15 As shown, the organ and tissue structures of each group were intact, and there were no obvious pathological changes in cell morphology and arrangement, indicating that the C-BMNP vaccine has good biocompatibility and biosafety.

[0143] 5.3 Analysis of the in vivo inhibitory effect of C-BMNPs vaccine on lung metastasis To evaluate the antitumor effect of C-BMNPs vaccine in a mouse model of melanoma lung metastasis, such as Figure 16 As shown, mice were inoculated with B16-OVA cells via the tail vein on day 0, and orally on days 3, 6, and 10. Lung tissue was collected and photographed on day 16 after treatment. Results showed no significant reduction in lung melanoma metastases in the OVA and PLGA / OVA groups, possibly due to the lack of effective adjuvants and gastric acid protection in the vaccine system. In contrast, BMNPs showed significantly better inhibition of lung metastases than OVA and PLGA / OVA (P<0.05), but still did not completely block the progression of lung metastases. Notably, compared to other control mice, lung metastases in the C-BMNPs vaccine group almost completely disappeared (P<0.001).

[0144] In addition, the survival rate of mouse models inoculated with B16-OVA cells was observed and recorded for 30 days. Figure 16 As shown in (b), mice in the OVA and PLGA / OVA groups began to die on day 22 after tail vein injection of B16-OVA cells, and by day 30, all six mice had died, resulting in a survival rate of 0%. Mice in the BMNPs group began to die on day 26, with a survival rate of only 16.6% by day 30. In contrast, the survival rate of mice in the C-BMNPs vaccine group reached 100% by day 30. These results indicate that the C-BMNPs vaccine can effectively inhibit lung metastasis of melanoma in mice and significantly improve mouse survival rate.

[0145] 5.4 Evaluation of the long-term immune memory effect and anti-tumor recurrence ability of C-BMNPs vaccine To investigate the role of oral C-BMNPs vaccines in inducing long-term immune memory and preventing tumor recurrence, healthy mice were orally vaccinated three times on days 0, 3, and 10. The vaccine groups included PBS, OVA, PLGA / OVA, BMNPs, and C-BMNPs. This protocol aimed to accurately simulate the enhancement effect produced by multiple immunizations in clinical settings. The experimental design was as follows: Figure 17 As shown in (a). On day 52, mouse spleens were isolated, and the ratio of effector memory T cells to central memory T cells in the spleen cells was detected by FCM. The results showed that the effector memory T cells (CD3+) in the spleen cells of mice in the C-BMNPs vaccine group... + CD8 + CD44 + CD62L - The proportion of central memory T cells (CD3+) in the spleen cells of the C-BMNPs vaccine group was 8.76±0.35%, significantly higher than that in the PBS control group (3.72±0.28%) (P<0.0001). Although the proportion of central memory T cells (CD3+) in the spleen cells of the C-BMNPs vaccine group was higher than that in the control group (P<0.0001), the proportion of central memory T cells (CD3+) in the + CD8 + CD44 + CD62L + The proportion of T cells decreased compared to the PBS group, but a similar trend was observed in other oral vaccine groups. To further verify the preventive effect of the vaccine on tumor recurrence, immunized mice were challenged by tail vein injection of B16-OVA cells on day 52, and lung tissue was collected from mice on day 66. The number of melanoma metastases in the lung tissue was photographed and quantitatively counted. The results are as follows: Figure 17 As shown in (b) and (c), compared with the PBS, OVA, PLGA / OVA, and BMNPs groups, almost no lung metastases were observed in mice in the oral C-BMNPs vaccine group, indicating that the vaccine can effectively protect the body from the reinvasion of tumor cells. In conclusion, the oral C-BMNPs vaccine not only significantly induces long-term immune memory in the body, but also demonstrates a key role in preventing tumor recurrence, providing strong experimental evidence to support subsequent vaccine development and clinical application.

[0146] In summary, this invention has developed an oral nanovaccine based on a biomimetic ionizable membrane structure and MMA pH-responsive coating. This oral nanovaccine combines gastric acid stability, intestinal targeting, immune activation efficacy, and safety, and has significant application value in tumor treatment and vaccine clinical translation.

[0147] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. An oral nanovaccine, characterized in that, The oral nanovaccine comprises nanoparticles encapsulated in biomimetic bacterial membrane vesicles and an outer layer; the nanoparticles comprise antigens and biodegradable polymeric materials; the biomimetic bacterial membrane vesicles comprise ionizable flagellated lipoproteins, adjuvants, phospholipids, steroidal lipids, and polyethylene glycol lipids; and the outer layer is a pH-responsive polymer.

2. The oral nanovaccine according to claim 1, characterized in that, The biodegradable polymer material is at least one of polylactic acid-hydroxyacetic acid copolymer, polycaprolactone, polylactic acid, polyhydroxybutyric acid, and polypeptide nanomaterials.

3. The oral nanovaccine according to claim 1, characterized in that, The ionizable flagellated lipoprotein is prepared by reacting (Z)-12-(((4-nitrophenoxy)carbonyl)oxy)octadecyl-9-ene-1-acrylate, fatty alcohol, and flagellated protein; and / or, the immunoadjuvant is at least one of monophospholipid A, monophospholipid derivatives, CpG oligodeoxynucleotides, and polyinosinic-polycytidylic acid; and / or, the phospholipid is soybean lecithin, egg yolk lecithin, or sphingomyelin; and / or, the steroidal lipid is cholesterol or a cholesterol derivative; and / or, the polyethylene glycol lipid is at least one of DSPE-PEG-2000, DMG-PEG, DPPE-PEG, and DOPE-PE.

4. The oral nanovaccine according to claim 1, characterized in that, The pH-responsive polymer is at least one of methyl methacrylate, hydroxypropyl methylcellulose phthalate, chitosan derivatives, Eudragit L100, Eudragit S100, and polyacrylic acid polymers.

5. The oral nanovaccine according to claim 1, characterized in that, The mass ratio of the biomimetic bacterial membrane vesicles to the nanoparticles is 1:5 to 5:1; and / or, the molar ratio of ionizable flagellated lipoprotein, immune adjuvant, phospholipids, steroidal lipids and polyethylene glycol lipids in the biomimetic bacterial membrane vesicles is 1:(0.5 to 2):(2 to 5):(1 to 3):(0.1 to 1).

6. The method for preparing an oral nanovaccine according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Reaction of (Z)-12-(((4-nitrophenoxy)carbonyl)oxy)octadecyl-9-ene-1-acrylate with a fatty alcohol yields ricinoleate; reaction of the ricinoleate with flagellin yields ionizable flagellate lipoprotein. S2. React the antigen and biodegradable polymer in a solvent to obtain nanoparticles; S3. The nanoparticles are added to a mixed solution of ionizable flagellated lipoprotein, immune adjuvant, phospholipids, steroidal lipids and polyethylene glycol lipids, and reacted to obtain nanoparticles encapsulated by biomimetic bacterial membrane vesicles. S4. React the nanoparticles encapsulated in the biomimetic bacterial membrane vesicles with pH-responsive polymer monomers in a solvent to obtain the oral nanovaccine.

7. The preparation method according to claim 6, characterized in that, In step S1, the molar ratio of (Z)-12-(((4-nitrophenoxy)carbonyl)oxy)octadecyl-9-ene-1-acrylate, fatty alcohol, and flagellin is 2:5:(0.001~0.01); and / or, in step S3, the reaction is carried out by one of the following methods: membrane extrusion, nanoprecipitation, and microfluidic chip method; and / or, in step S4, ammonium persulfate is added, wherein the mass of ammonium persulfate is 0.5~2% of the mass of the pH-responsive polymer monomer.

8. The use of an oral nanovaccine as described in any one of claims 1-5 in the preparation of any of the following: (a1) Vaccines for the prevention and / or treatment of tumors; (a2) Vaccines for the prevention and / or treatment of infectious diseases.

9. The application according to claim 8, characterized in that, The tumor is at least one of colon cancer, melanoma, lung cancer, breast cancer, stomach cancer, and pancreatic cancer; and / or the infectious disease is a disease caused by bacterial or viral infection.

10. The use of an oral nanovaccine as described in any one of claims 1-5 in at least one of (b1)-(b3): (b1) Prepare products that induce or activate dendritic cell maturation; (b2) Prepare products that promote T cell proliferation or activation; (b3) Prepare products that induce long-term immune memory effects; (b4) Prepare products that enhance mucosal immune responses.

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