A mRNA delivery carrier, preparation method and application of an oral vaccine

Abstract

The application belongs to the field of immunology and relates to an mRNA delivery carrier, a preparation method and application of an oral vaccine. A calcium alginate microsphere and a cholic acid-fluorine co-modified polyethyleneimine (DFP) form a double protection system, the compact crosslinked structure of the calcium alginate microsphere resists the acid-base environment of the gastrointestinal tract and nuclease degradation, and the complex formed by the DFP and the mRNA can further prevent the mRNA from being enzymatically degraded. The application provides preparation of an oral mRNA delivery system for activating intestinal mucosal immunity and application of the oral mRNA delivery system in a diarrhea vaccine, provides effective protection against porcine diarrhea coronavirus infection, and provides a new idea for research and development of intestinal mucosal vaccines.

Description

Technical Field

[0001] This invention belongs to the field of immunology and relates to the preparation of oral vaccines. Background Technology

[0002] Porcine diarrhea viruses are highly prevalent pathogens in the swine industry, causing acute diarrhea, dehydration, and other symptoms in pigs, leading to high mortality rates in piglets and declining production performance in adult pigs, resulting in significant economic losses to the industry. Porcine diarrhea coronaviruses (such as porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus) are the core pathogenic types, exhibiting extremely high pathogenicity. Mortality rates in infected piglets can reach up to 90%, and infection rapidly damages intestinal mucosal epithelial cells, causing intestinal barrier collapse and severe dehydration and malabsorption. Transmission primarily occurs via the fecal-oral route, with the virus spreading rapidly across populations by contaminating feed, water, equipment, and the farming environment through feces. Enhancing intestinal mucosal immunity is crucial for effectively blocking viral invasion and transmission in the early stages. Traditional vaccines, administered via intramuscular or subcutaneous injection, stimulate a systemic immune response, but their effectiveness in inducing intestinal mucosal immunity is poor, making it difficult to form an effective protective barrier in the intestinal mucosa. Orally administered vaccines have the advantage of activating intestinal mucosal immunity, effectively neutralizing the diarrhea virus before it invades intestinal cells, reducing the risk of transmission. However, oral vaccines are unable to induce a highly efficient immune response due to multiple barriers such as stomach pH, ​​digestive enzymes, and intestinal mucosal barrier.

[0003] After alginate undergoes a cross-linking reaction with calcium ions, it forms a polyelectrolyte gel. In acidic environments (such as the stomach, pH 1-2), it forms a hydrophobic protonated layer of alginate. This dense structure effectively protects the internal load from destruction by gastric acid and digestive enzymes. It swells and degrades in neutral or weakly alkaline environments (such as the intestines, pH 6-7). This property can be used to achieve carrier protection in the stomach and targeted release in the intestines.

[0004] Fluorinated polymers, due to their low surface tension, hydrophobicity, and self-assembly ability, can stably load mRNA. Their hydrophobic and oleophobic properties result in good biocompatibility and low cytotoxicity, allowing them to easily penetrate mucosal barriers and be readily endocytosed by cells but not readily fused with cell membranes. They show great promise as a nucleic acid delivery and vaccine adjuvant platform. Further modification with deoxycholic acid allows them to bind to bile acid transport proteins on the surface of intestinal epithelial cells, improving transepithelial efficiency and thus enhancing vaccine utilization by antigen-presenting cells in the innate immune layer.

[0005] Within the innate immune layer, dendritic cells, acting as immune sentinels, detect and capture antigens, and further migrate to the mesenteric lymph nodes, thereby initiating innate and adaptive immune responses against viral infections. Deoxycholic acid-modified fluorinated polymers stimulate the IRF3 and NF-κB pathways by activating TLR4 in dendritic cells, promoting cytokine secretion; they also promote T / B cell differentiation, enhance intestinal mucosal immunity, and induce long-term immune responses. Summary of the Invention

[0006] This invention proposes an mRNA delivery vector, a method for preparing an oral vaccine, and its application. The aim is to provide the preparation of an oral mRNA delivery system for activating intestinal mucosal immunity and its application in diarrhea vaccines, providing effective protection against swine diarrhea coronavirus infection.

[0007] The technical solution of this invention is implemented as follows:

[0008] On the one hand, the present invention provides an mRNA delivery vector, the structural formula of which is shown in (I):

[0009]

[0010] (I);

[0011] Where n = any integer between 35 and 58.

[0012] Furthermore, it also includes pharmaceutically acceptable salts, stereoisomers, or solvates of the polymer as shown in formula (I).

[0013] Secondly, a method for preparing the above-mentioned mRNA delivery vector is provided, the steps of which are: grafting tridecylfluoroheptyl ethylene oxide with polyethyleneimine, and then mixing the grafted product with deoxycholic acid, EDC and NHS in an organic solvent to obtain the mRNA delivery vector.

[0014] The above-mentioned polyethyleneimine is PEI 1.8K The structural formula is shown in (II):

[0015] ;

[0016] Where n is any integer between 35 and 58.

[0017] The deoxycholic acid is as shown in formula (III):

[0018]

[0019] (III);

[0020] The molar ratio of the above-mentioned tridecafluoroheptyl ethylene oxide to polyethyleneimine is 10-50:1; the molar ratio of the grafted product to deoxycholic acid, EDC, and NHS is 0.5-2:1:1.2:1.2.

[0021] The preferred molar ratio of the reactants of tridecafluoroheptyl ethylene oxide and polyethyleneimine is 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 45:1, 46:1, 47:1, 48:1, 49:1, or 50:1.

[0022] In some specific embodiments of the present invention, the molar ratio of the deoxycholic acid derivative to the grafted product in the above preparation method is 1:1.9, 1:1.89, 1:1.88, 1:1.87, 1:1.86, 1:1.85, 1:1.84, 1:1.83, 1:1.82, 1:1.81, 1:1.8, 1:1.79, 1:1.78, 1:1.77, 1:1.76, 1:1.75, 1:1.74, 1:1.73, 1:1.72, 1:1.71, or 1:1.705.

[0023] In some specific embodiments of the present invention, the above preparation method includes:

[0024] A methanol solution of tridecafluoroheptyl ethylene oxide was added dropwise to PEI. 1.8K The grafted product was obtained by stirring and gradient dialysis in a methanol solution.

[0025] Deoxycholic acid, EDC, NHS, and DMF are mixed, stirred, and then mixed with the grafted product. The mixture is reacted and dialyzed to obtain the mucosal delivery adjuvant.

[0026] Among them, the tridecafluoroheptyl ethylene oxide and PEI 1.8K The reactant molar ratios are 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 45:1, 46:1, 47:1, 48:1, 49:1, or 50:1;

[0027] The molar ratio of the deoxycholic acid to the grafted product is 1:1.7-1.9, for example, 1:1.9, 1:1.89, 1:1.88, 1:1.87, 1:1.86, 1:1.85, 1:1.84, 1:1.83, 1:1.82, 1:1.81, 1:1.8, 1:1.79, 1:1.78, 1:1.77, 1:1.76, 1:1.75, 1:1.74, 1:1.73, 1:1.72, 1:1.71 or 1:1.70.

[0028] In some specific embodiments of the present invention, the above preparation method includes:

[0029] The tridecafluoroheptyl ethylene oxide and the PEI 1.8K The molar ratio of the reactants is 25:1;

[0030] The molar ratio of the reactants deoxycholic acid, EDC, and NHS is 1:1.2:1.2;

[0031] The molar ratio of the deoxycholic acid to the grafted product is 1:1.79.

[0032] The tridecylfluoroheptyl ethylene oxide is shown in formula (IV):

[0033] .

[0034] (IV).

[0035] Thirdly, the present invention also provides the application of the above-mentioned mRNA delivery vector in the preparation of biological products for the prevention and treatment of intestinal infections; wherein the mRNA is an mRNA encoding a target viral receptor binding domain protein.

[0036] For example, the present invention provides a method for preparing an oral vaccine, comprising the steps of: mixing and combining the above-mentioned mRNA delivery vector and mRNA encoding receptor binding domain protein, and then combining it with acceptable excipients or adjuvants to obtain an oral vaccine.

[0037] Fourthly, the present invention provides a method for preparing the above-mentioned oral vaccine, the steps of which are as follows:

[0038] (1) Add the solution containing the above-mentioned mRNA delivery vector dropwise to the mRNA solution.

[0039] Nanoparticles were obtained by self-assembly in the liquid.

[0040] (2) The nanoparticles from step (1) were added to a sodium alginate solution and microspheres were obtained by ice bath sonication and electrostatic spraying.

[0041] (3) After washing, the microspheres from step (2) are immersed in chitosan oligosaccharide solution for incubation. The microspheres are collected and freeze-dried to obtain the oral vaccine.

[0042] Furthermore, in step (1), the mass ratio of mRNA delivery vector to mRNA is 1:0.5-4; in step (2), the mass ratio of nanoparticles to sodium alginate is 8:1; and in step (3), the concentration of chitosan oligosaccharide solution is 0.1% w / v.

[0043] Fifthly, an oral vaccine prepared using the above-described method is provided.

[0044] The oral vaccines mentioned above also contain excipients or adjuvants, including at least one of the following: binders, fillers, disintegrants, lubricants, isotonic modifiers, solubilizers, cosolvents, preservatives, colorants, suspending agents, wetting agents, emulsifiers, and surfactants.

[0045] In some specific embodiments of the present invention, the intestinal infection described above includes porcine epidemic diarrhea; the administration method is oral swallowing.

[0046] In some specific embodiments of the present invention, the prevention and control described above are achieved by any of the following:

[0047] (I) It promotes cytokine secretion and activates innate immune response by activating the TLR4 pathway of conventional dendritic cells in mesenteric lymph nodes;

[0048] (II) By enhancing the uptake of antigens by conventional dendritic cells, stimulating the maturation of conventional dendritic cells, and inducing a high level of immune response;

[0049] The high level of immune response includes at least one of the following:

[0050] (i) Increased sIgA secretion levels;

[0051] (ii) Increased IgG secretion levels;

[0052] (iii) Increased RBD-specific IgG titer;

[0053] (iv) Increased RBD-specific sIgA titer in intestinal lavage fluid;

[0054] (v) Enhanced B cell activation;

[0055] (vi) Enhanced T cell activation;

[0056] (vii) Enhanced differentiation of memory B cells;

[0057] (viii) Enhanced differentiation of long-lived plasma cells;

[0058] (ix) Enhanced differentiation of memory T cells;

[0059] (x) Increased long-term IgG and sIgA titers.

[0060] The present invention has the following beneficial effects:

[0061] The calcium alginate microspheres of this invention form a dual protective system with bile acid-fluorine co-modified polyethyleneimine (DFP). The dense cross-linked structure of the calcium alginate microspheres resists the acid-base environment of the gastrointestinal tract and nuclease degradation. The complex formed by DFP and mRNA further prevents the enzymatic degradation of mRNA. In the small intestine, the calcium alginate microspheres burst, releasing DFP / mRNA nanoparticles, which activate innate immunity by activating TLR4 on the surface of dendritic cells (DCs) in the innate immune layer. After being taken up by DCs, the proton sponge effect of the PEI backbone promotes endocytosis and endosome escape, inducing a high level of adaptive immune response. Oral immunization with the C@DFP / mRNA vaccine induces high levels of mucosal immunity and IgG production, promotes the activation of B cells, T cells, and IgA plasma cells, and induces the differentiation of memory B cells and memory T cells. This invention provides effective protection against diarrhea viruses and offers a new approach for the development of intestinal mucosal vaccines. Attached Figure Description

[0062] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0063] Figure 1 This is the DFP NMR spectrum.

[0064] Figure 2 This is the Fourier transform infrared spectrum image of the DFP.

[0065] Figure 3 Here is the chemical synthesis formula for DFP.

[0066] Figure 4 This is a gel electrophoresis image showing the carrying capacity of DFP.

[0067] Figure 5 This is a particle size distribution map of DFP / mRNA NPs.

[0068] Figure 6 Transmission electron microscopy images of DFP / mRNA NPs.

[0069] Figure 7 For the cytotoxicity detection of DFP / mRNA NPs.

[0070] Figure 8 Images showing the transfection effect of DFP / mRNA NPs in DC2.4 and the results of Western blot analysis of protein expression.

[0071] Figure 9 Particle size distribution and transmission electron microscopy images of the C@DFP / mRNA vaccine.

[0072] Figure 10 Changes in particle size and PDI of C@DFP / mRNA vaccine before and after freeze-drying.

[0073] Figure 11 Gel electrophoresis images of nucleic acid loading capacity and protein expression of C@DFP / mRNA vaccine before and after freeze-drying.

[0074] Figure 12 To detect the protein expression capacity of C@DFP / mRNA vaccines after lyophilization and storage for different times.

[0075] Figure 13 The stability of the C@DFP / mRNA vaccine under different pH conditions.

[0076] Figure 14 Images showing the release effect of C@DFP / mRNA vaccine in different pH environments.

[0077] Figure 15 To detect the efficiency of C@DFP / mRNA vaccine in releasing DFP / mRNA NPs under different pH environments.

[0078] Figure 16 Images showing the mean square displacement and trajectory of released DFP / mRNA NPs in mucus.

[0079] Figure 17 This study investigated the effects of different incubation groups on TEER in a small chamber model that simulates the intestinal epithelial barrier in vitro.

[0080] Figure 18 To simulate the intestinal epithelial barrier in vitro using a small chamber model, the transepithelial transport efficiency and Papp values ​​of different groups were measured.

[0081] Figure 19 Laser confocal microscopy images showing the effect of DC2.4 on DFP / mRNA uptake.

[0082] Figure 20 Laser confocal microscopy image of DFP / mRNA lysosomal escape in DC2.4.

[0083] Figure 21The expression of CD80 and CD86 in BMDCs stimulating DFP / mRNA maturation.

[0084] Figure 22 The expression of MHC-I and MHC-II was investigated to determine how DFP / mRNA promotes antigen presentation by BMDCs.

[0085] Figure 23 To detect the intracellular cytokine nucleic acid levels after DFP / mRNA stimulation of BMDCs maturation.

[0086] Figure 24 To detect the activation of the TLR4 receptor in BMDCs by DFP / mRNA using flow cytometry.

[0087] Figure 25 Western blot analysis was performed to detect the phosphorylation levels of p65 and IRF3 in activated BMDCs.

[0088] Figure 26 The content of sIgA in intestinal lavage fluid (SILF) and fecal extract (FE) 42 days after immunization was analyzed by ELISA.

[0089] Figure 27 To detect the RBD-specific sIgA titer in SILF 42 days after immunization using ELISA.

[0090] Figure 28 To detect the differentiation of IgA+ plasma cells in intestinal lymph nodes (MLNs) by flow cytometry.

[0091] Figure 29 To detect the expression of mature B cells in MLNs by flow cytometry.

[0092] Figure 30 To detect the expression of GC B cells in MLNs by flow cytometry.

[0093] Figure 31 The results of RBD-specific antibody titer detection in serum every 7 days after immunization and the difference in specific antibody titer on day 42 were analyzed for ELISA analysis.

[0094] Figure 32 This study aims to detect serum neutralization titer 42 days post-immunization and to obtain indirect immunofluorescence images of PEDV-infected cells after serum neutralization.

[0095] Figure 33 To detect the differentiation of CD3+CD4+ T cells and CD3+CD8+ T cells in MLNs by flow cytometry.

[0096] Figure 34 To detect the differentiation of Th1 cells and CTL cells in MLNs by flow cytometry.

[0097] Figure 35 To detect the expression levels of nucleic acid of T / B cell differentiation-related cytokines in MLNs using q-PCR.

[0098] Figure 36 To detect the expression of memory B cells in MLNs on day 60 by flow cytometry.

[0099] Figure 37 To detect the expression of long-lived plasma cells in MLNs on day 60 using flow cytometry.

[0100] Figure 38 To analyze the serum RBD-specific IgG antibody titer and SILF-specific sIgA antibody titer at 90 and 120 days post-immunization using ELISA.

[0101] Figure 39 To detect the differentiation of TCM and TEM in MLNs on day 60 by flow cytometry.

[0102] Figure 40 Changes in body weight of mice after oral immunization in different groups (A); H&E images of tissue sections of mouse heart, liver, spleen, lung, kidney, and intestine (B); flow cytometry analysis of intestinal Treg cell differentiation level.

[0103] Figure 41 The levels of relevant blood biochemical indicators in mice after oral immunization. Detailed Implementation

[0104] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0105] In one implementation, tridecafluoroheptyl ethylene oxide and deoxycholic acid are modified onto dendritic polyethyleneimine (PEI), which is then adsorbed onto mRNA encoding the receptor binding domain (RBD) protein of porcine epidemic diarrhea virus to form nanoparticles (DFP / mRNA). These nanoparticles are then coated with calcium alginate gel microspheres to form an oral mRNA vaccine.

[0106] In practice, the mRNA preparation method for RBD protein is as follows:

[0107] The sequence encoding the full-length RBD protein of PEDV HN2021 strain was cloned into the pUC-GW vector containing a 5'UTR, 3'UTR, and a poly(A) tail. After linearization by single enzyme digestion of the plasmid, 5' capping was performed using the T7 in vitro transcription kit, and mRNA was purified using magnetic beads to remove residual DNA. The mRNA concentration was determined using a micro-spectrophotometer.

[0108] The preparation method of DFP / mRNA nanoparticles is as follows:

[0109] A methanol solution of tridecafluoroheptyl ethylene oxide (20 mg / mL) was slowly added dropwise to the dendritic PEI. 1.8K In a methanol solution of (20 mg / mL), at a dropping rate of 0.1 mL / min, tridecafluoroheptyl ethylene oxide and PEI... 1.8K The molar ratio was 20-50:1. The mixture was stirred at room temperature for 48 h. The reactants were dialyzed in 5%–1% methanol solution and deionized water (molecular weight cutoff 500 Da) for 48 h, followed by freeze-drying to obtain F. 25 PEI. Deoxycholic acid, EDC, and NHS were added to DMF at a molar ratio of 1:1.2:1.2 and stirred at room temperature in the dark for 30 min. Then, FPEI was added dropwise to DMF at a molar ratio of 1.7-1.9:1 to deoxycholic acid, with a dropping rate of 0.1 mL / min, and the reaction was carried out at room temperature for 12 h. The reactants were dialyzed in deionized water (molecular weight cutoff 3500 Da) for 48 h and then freeze-dried to obtain DMF. The grafting of fluoride and deoxycholic acid was characterized by NMR and Fourier transform infrared spectroscopy (FT-IR). DFP and mRNA were incubated in DEPC water at a series of different mass ratios at 4 °C for 30 min to obtain DFP / mRNA nanoparticles.

[0110] The preparation method of C@DFP / mRNA oral vaccine is as follows:

[0111] Take low molecular weight sodium alginate, dissolve it in enzyme-free water to prepare a 2-4% (w / v) alginate solution, add 0.1% (w / v) Tween-80 as a dispersant, stir magnetically until completely dissolved, filter through a 0.22 μm filter membrane to remove impurities, and set aside for later use.

[0112] DFP / mRNA nanoparticles were slowly added to an alginate solution at a volume ratio of 1:(12-18). The mixture was then ultrasonically dispersed in an ice bath (100 W power, 5 min time, 3 s / 3 s cycle) to form a homogeneous suspension, avoiding mRNA degradation caused by ultrasonic heat generation. Microspheres were prepared using an electrostatic spray method with the following parameters: needle inner diameter 0.2 mm, applied voltage 16-19 kV (preferably 18 kV), flow rate 0.6-0.9 mL / h (preferably 0.8 mL / h), and a needle distance of 10 cm from the crosslinking solution surface. The suspension was uniformly extruded and dropped into a 50 mM calcium chloride crosslinking agent solution. The crosslinking reaction was carried out for 30 minutes with magnetic stirring (100 rpm) to ensure uniform microsphere formation and a dense structure.

[0113] 4. Microsphere Modification and Purification: The cross-linked microspheres were collected by centrifugation (8000 rpm, 10 minutes) and washed three times with enzyme-free water to remove free cross-linking agent and unencapsulated DFP / mRNA complexes. The microspheres were then immersed in a 0.1% (w / v) chitosan oligosaccharide solution (molecular weight 2 kDa) for 20 minutes. The chitosan oligosaccharide adsorbed onto the surface of the alginate microspheres via electrostatic attraction, further enhancing the microspheres' mucus penetration ability and cell adhesion. The microspheres were collected by centrifugation again, lyophilized to obtain the finished oral vaccine, and stored in a sealed container at -20℃.

[0114] The reagents involved in this invention are as follows: the pU-IVT vector of the in vitro transcription system was synthesized by Suzhou Genewiz Biotechnology Co., Ltd.; the T7 High RNA Cotranscription kit and Hieff NGS RNA Cleaner were purchased from Shanghai Yisheng Co., Ltd.; the DAPI-containing mounting medium was purchased from Shanghai Beyotime Co., Ltd.; PEI1.8K was purchased from Beijing Bailingwei Technology Co., Ltd., CAS: 9002-98-6; tridecafluoroheptyl ethylene oxide was purchased from Shanghai Myriel Co., Ltd., CAS: 38565-52-5; deoxycholic acid was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., CAT: 83-44-3; FITC and Cy5 labeled mRNA were purchased from APExBIO (Texas, USA); FITC-anti-CD86 antibody, FITC-anti-CD80 antibody, and APC-anti-CD11c antibody were purchased from Abcam (Cambridge Science Park, UK). APC-anti-CD19 antibody, FITC-anti-IgD antibody, APC-anti-CD3 antibody, FITC-anti-CD4 antibody, FITC-anti-CD8 antibody, APC-anti-CD138 antibody, PerCP-anti-CD62 antibody, PE-anti-CD279 (PD-1) antibody, PerCP / Cy5.5-anti-CXCR5 antibody, and PE-anti-GL7 antibody were purchased from Biolegend (California, USA). PE-anti-CD44 antibody, FITC-anti-CD43 antibody, APC / Cy7-anti-B220 antibody, PE-anti-IgA antibody, and PE / CF594-anti-CD38 antibody were purchased from Proteintech Group (Wuhan Sanying Biotechnology Co., Ltd., Wuhan, China). APC-anti-CD69 antibody, PE-anti-CD25 antibody, and APC / Cy7-anti-FoxP3 antibody were purchased from BDBiosciences (New Jersey, USA). Anti-p65 antibody, anti-phosphorylated p65 (p-p65) antibody, anti-NF-κB antibody, and anti-phosphorylated NF-κB (p-NF-κB) antibody were purchased from Cell Signaling Technology (CST) (Boston, Massachusetts, USA).

[0115] The English abbreviations involved in this invention and their meanings are as follows: RBD: receptor-binding domain protein of coronavirus; PEI: dendritic polyethyleneimine; DFP: deoxycholic acid modified polyethyleneimine with fluorocarbon linkages in a molar ratio of 1:25; C: calcium alginate gel microspheres; C@DFP / mRNA: oral mRNA vaccine formed by electrostatic interaction of DFP and mRNA coated with calcium alginate gel microspheres.

[0116] In Examples 1-4 and Verification Example 1 of this invention, all raw materials and reagents used are commercially available. Unless otherwise specified, the raw materials, reagents, consumables, and instruments involved in this invention are all common commercially available products.

[0117] The present invention will be further illustrated below with reference to the embodiments.

[0118] Example 1: Preparation of oral mRNA vaccine

[0119] (I) Preparation of PEDV-RBD mRNA

[0120] Using the receptor-binding domain (RBD) protein of porcine epidemic diarrhea virus (PEDV) strain PEDV / CH / HNPDS / 2025 (GenBank: PZ105934) as a reference amino acid sequence, the corresponding coding sequence is shown in SEQ ID No. 1. This sequence was cloned into the pU-IVT vector containing the 5' UTR shown in SEQ ID No. 2, the 3' UTR shown in SEQ ID No. 3, and the poly (A) tail shown in SEQ ID No. 3, resulting in the recombinant plasmid pU-RBD-IVT.

[0121] After linearization by single enzyme digestion of the pU-RBD-IVT plasmid, mRNA was synthesized via in vitro transcription (IVT) using T7 RNA polymerase, with simultaneous 5' capping. Unreacted DNA template and residual reagents were removed using mRNA purification beads to obtain high-purity RBD-encoded mRNA. RBD The concentration and purity of mRNA were detected using a NanoDrop micro-ultraviolet spectrophotometer, and qualified samples were stored in an ultra-low temperature freezer at -80 ℃ for later use.

[0122] (II) Preparation of DFP carrier

[0123] 1. Synthesis of fluorinated polyethyleneimine (FPEI)

[0124] A methanol solution of tridecafluoroheptyl ethylene oxide (3-(perfluorohexyl-1-yl)-1,2-epoxypropane, 20 mg / mL) was added dropwise to the dendritic PEI at a rate of 0.1 mL / min. 1.8K The reaction was carried out in a methanol solution of (20 mg / mL) with stirring at room temperature for 48 h. Tridecafluoroheptyl ethylene oxide and PEI 1.8KThe molar ratios were 20:1, 25:1, 30:1, 35:1, and 40:1. After the reaction was completed, the reactants were dialyzed sequentially in methanol-water solution and deionized water for 72 h (molecular weight cutoff MWCO = 1000 Da in the dialysis bag). After dialysis, the products were freeze-dried to obtain the fluorinated modified product FPEI.

[0125] 2. Synthesis of Deoxycholic Acid Modified FPEI (DFP)

[0126] Deoxycholic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were added to DMF in a molar ratio of 1:1.2:1.2, and the mixture was activated at room temperature in the dark with stirring for 2 h. Nitrogen gas was then introduced into the activation system for protection, and FPEI was added dropwise to DMF at a molar ratio of 1.79:1 to deoxycholic acid, at a dropping rate of 0.1 mL / min. The reaction was allowed to proceed at room temperature for 24 h. After the reaction was complete, the reactants were dialyzed against deionized water for 48 h (molecular weight cutoff MWCO = 10000 Da), and after freeze-drying, the DFP support was obtained. Hydrogen nuclear magnetic resonance (¹H NMR) spectroscopy was performed. Figure 1 Fourier transform infrared spectroscopy (FT-IR) Figure 2 Characterizing the grafting of fluorine groups with deoxycholic acid, see the reaction formula below. Figure 3 .

[0127] (III) Preparation of DFP / mRNA nanoparticles

[0128] DFP and mRNA RBD Each component was dissolved in enzyme-free deionized water to prepare a 0.1 mg / mL single-component solution. The DFP solution was added dropwise to the mRNA solution at a 1:1 mass ratio, and after gentle mixing, the mixture was incubated at 4 °C for 15 min to self-assemble into DFP / mRNA nanoparticles (DFP / mRNA NPs).

[0129] (iv) Preparation of oral C@DFP / mRNA vaccine

[0130] 1. Aqueous phase preparation: Dissolve 2.5 g of low molecular weight sodium alginate in 100 mL of enzyme-free DEPC water, add 0.1 g of Tween-80 as a dispersant, stir magnetically until completely dissolved, filter through a 0.22 μm filter membrane to remove bacteria and impurities, and obtain sodium alginate base solution; slowly add DFP / mRNA NPs solution to 15 mL of sodium alginate solution, and sonicate at 100 W for 5 min under ice-water bath conditions (working cycle 3 s / 3 s) to form a homogeneous aqueous suspension, avoiding mRNA degradation caused by ultrasonic heat generation throughout the process.

[0131] 2. Preparation of oil phase: Measure 100 mL of liquid paraffin into a beaker, add 3 mL of Span-80, stir magnetically until homogeneous, and pre-cool at 4 ℃ for 30 min to obtain the oil phase.

[0132] 3. Emulsification and cross-linking: Transfer the pre-cooled oil phase to a round-bottom flask, and magnetically stir at 300 rpm at 4 ℃. Slowly add the aqueous suspension dropwise. After the addition is complete, adjust the stirring speed to 400 rpm and continue stirring for 30 min to form a stable w / o type emulsion. Slowly add 40 mL of 50 mM calcium chloride solution (prepared with DEPC water) dropwise to the emulsion, adjust the stirring speed to 500 rpm, and cross-link for 120 min to form calcium alginate gel microspheres.

[0133] 4. Demulsification and purification: Add an equal volume of anhydrous ethanol to the emulsion to demulsify, let stand for 10 min, and then centrifuge at 4 ℃ and 8000 rpm for 10 min to collect the microspheres; wash twice with anhydrous ethanol and once with enzyme-free water to remove free cross-linking agent, unencapsulated DFP / mRNA complex and surface impurities to obtain C@DFP / mRNA vaccine microspheres, freeze-dry and seal at -20 ℃ for subsequent experiments.

[0134] Verification Example 1: Basic Characteristics of C@DPF / mRNA Vaccine

[0135] (1) Characterization of particle size and potential of DFP / mRNA NPs

[0136] In Example 1, different molar ratios of tridecafluoroheptyl ethylene oxide and PEI were used. 1.8K The prepared FPEI, after deoxycholic acid modification, bound to mRNA to form nanoparticles. The particle size distribution and zeta potential of the DFP / mRNA NPs were determined using a Malvern Nano ZSE dynamic light scattering (DLS) spectrometer, as shown in Table 1. Characterization analysis revealed that the nanoparticles formed at a DFP:mRNA mass ratio of 25:1 exhibited the most suitable particle size and potential. Therefore, unless otherwise specified, a weighting ratio of 25:1 was used in all studies. Subsequent analysis used transmission electron microscopy (TEM) to observe their microstructure. The results showed that when the DFP:mRNA mass ratio was 1:1, the average particle size of the prepared DFP / mRNA NPs was 149.3 ± 9.3 nm (…). Figure 5 The zeta potential is +19.4 mV, the particles are uniformly spherical, and the dispersion is good. Figure 6 ).

[0137] Table 1: Particle size and potential of nanoparticles prepared with different molar ratios

[0138]

[0139] (2) Cytotoxicity assessment of DFP / mRNA NPs

[0140] DC2.4 cells and IPEC-J2 cells were seeded into 96-well plates at a density of 1×10⁻⁶ cells / well. 4 Cells / wells were cultured overnight, and DFP / mRNA NPs were added to each well at final concentrations of 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, and 30 μg / mL. A blank control group was included, and each group had three replicates. After co-incubation at 37 ℃ and 5% CO2 for 24 h, the relative cell viability was detected using the standard MTT assay. The results showed that when the DFP / mRNA concentration was ≤20 μg / mL, the viability of both DC2.4 and IPEC-J2 cells remained above 90%, indicating that the DFP vector has good biocompatibility and low cytotoxicity. Figure 7 ).

[0141] (3) Detection of transfection efficiency of DFP / mRNA NPs

[0142] DC2.4 cells were seeded in 24-well plates at a density of 1 × 10⁻⁶ cells / well. 5 Cells / well, cultured overnight, and then treated with DFP / mRNA respectively. EGFP NPs (containing 2 μg / mL EGFP-encoded mRNA), PEI 25k / mRNA EGFP Positive control group, free mRNA EGFP Cells were treated in groups, with three replicates per group. Twenty-four hours after transfection, the expression of green fluorescence within the cells was observed using a fluorescence microscope. Figure 8 A).

[0143] Using the same transfection protocol, DFP / mRNA NPs encoding RBD protein mRNA were transfected into DC2.4 cells. Twelve h after transfection, cells were collected using a cell scraper, and 200 μL of freshly prepared lysis buffer (RIPA lysis buffer: PMSF: phosphatase inhibitor volume ratio = 200:1:2) was added. Lysis was performed at 4 °C for 30 min; followed by centrifugation at 10,000 rpm for 10 min at 4 °C. The supernatant was collected, and SDS loading buffer was added. After vortexing, the mixture was heated at 95 °C for 10 min to completely denature the protein. Western blotting was used to detect intracellular RBD protein expression. Figure 8 B). The results showed that DFP could efficiently mediate mRNA entry into DC2.4 cells and achieve efficient expression of target proteins, with transfection efficiency significantly superior to commercial PEI. 25k Positive control.

[0144] (4) Characterization of particle size, encapsulation efficiency and morphology of C@DFP / mRNA microspheres

[0145] The particle size distribution of the microspheres was determined using a Malvern Mastersizer 3000 laser particle size analyzer; the microstructure of the microspheres was observed using TEM. Figure 9 The results showed that the prepared C@DFP / mRNA microspheres had an average particle size of 1.94 μm, uniform particle size, complete spherical morphology, dense structure, and no obvious damage.

[0146] The encapsulation efficiency of the microspheres was determined using a fluorescent labeling method: C@DFP / mRNA microspheres loaded with Cy5-labeled mRNA were prepared using the same method described above. The microspheres were dispersed in 0.2 M sodium citrate solution and incubated at 37 °C in the dark for 6 h to allow the microspheres to completely dissolve and release their contents. After centrifugation, the supernatant was collected, and the fluorescence intensity of Cy5-labeled mRNA was quantitatively detected using a fluorescence spectrophotometer. The encapsulation efficiency of the microspheres for DFP / mRNA NPs was calculated to be 93.5%.

[0147] (5) Testing of vaccine stability

[0148] The prepared DFP / mRNA NPs were placed in DMEM medium containing 10% fetal bovine serum (FBS) and PBS buffer, respectively, and incubated at room temperature. Samples were taken continuously for 1–7 days, and particle size changes were detected by DLS. The results showed that the DFP / mRNA nanoparticles did not show significant changes in particle size or obvious aggregation after continuous incubation in PBS and 10% FBS for 7 days, exhibiting good in vitro stability (Tables 2 and 3).

[0149] Table 2: Particle size monitoring results (nm) after 7 consecutive days in PBS

[0150] Table 3: Particle size monitoring results (nm) after 7 consecutive days in FBS

[0151] The freeze-dried C@DFP / mRNA was reconstituted with enzyme-free water. DLS was used to detect changes in particle size and PDI before freeze-drying and after reconstitution. The results are as follows: Figure 10 The integrity of the encapsulated mRNA was verified by agarose gel retardation assay; simultaneously, C@DFP / mRNA before and after lyophilization was transfected into 293T cells, and cells were collected 24 h after transfection. The expression level of RBD protein in the cells was detected by Western blotting. Figure 11As shown in A and B. The above results indicate that C@DFP / mRNA can be preserved in lyophilized powder form, and the particle size is not affected after reconstitution. Furthermore, the lyophilization process does not destroy the integrity of the mRNA within the microspheres. There is no significant difference in the expression level of RBD protein after cell transfection with the microspheres before and after lyophilization, indicating that the microspheres have good lyophilization stability.

[0152] The C@DFP / mRNA vaccine was stored as a lyophilized powder at 4°C for 30, 60, and 90 days, and then transfected into cells again. Western blotting was used to verify the vaccine activity. Results are as follows: Figure 12 The C@DFP / mRNA vaccine maintains good activity for 60 days at temperature. This indicates that the vaccine delivery system has good nucleic acid protection, and the vaccine can significantly reduce the cost pressure associated with vaccine preservation and transportation.

[0153] Example 2: Preparation of oral mRNA vaccine

[0154] (I) Preparation of PEDV-RBD mRNA

[0155] Using the protein binding domain (RBD) protein of porcine epidemic diarrhea virus (PEDV) strain PEDV / CH / HNPDS / 2025 (GenBank: PZ105934) as a reference amino acid sequence, the corresponding coding sequence was codon optimized. The optimized RBD coding sequence was cloned into the pU-IVT vector containing 5'UTR, 3'UTR and poly (A) tail to obtain the recombinant plasmid pU-RBD-IVT.

[0156] After linearization by single enzyme digestion of the pU-RBD-IVT plasmid, mRNA was synthesized via in vitro transcription (IVT) using T7 RNA polymerase, with simultaneous 5' capping. Unreacted DNA template and residual reagents were removed using mRNA purification beads to obtain high-purity RBD-encoded mRNA. RBD The concentration and purity of mRNA were detected using a NanoDrop micro-ultraviolet spectrophotometer, and qualified samples were stored in an ultra-low temperature freezer at -80 ℃ for later use.

[0157] (II) Preparation of DFP carrier

[0158] 1. Synthesis of fluorinated polyethyleneimine (FPEI)

[0159] A methanol solution of tridecafluoroheptyl ethylene oxide (3-(perfluorohexyl-1-yl)-1,2-epoxypropane, 20 mg / mL) was added dropwise to the dendritic PEI at a rate of 0.1 mL / min. 1.8K The reaction was carried out in a methanol solution of (20 mg / mL) with stirring at room temperature for 48 h. Tridecafluoroheptyl ethylene oxide and PEI 1.8K The molar ratio was 25:1. After the reaction was completed, the reactants were dialyzed sequentially in methanol-water solution and deionized water for 72 h (molecular weight cutoff MWCO = 1000 Da in the dialysis bag). After dialysis, the product was freeze-dried to obtain the fluorinated modified product FPEI.

[0160] 2. Synthesis of Deoxycholic Acid Modified FPEI (DFP)

[0161] Deoxycholic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were added to DMF in a molar ratio of 1:1.2:1.2, and the mixture was activated at room temperature in the dark with stirring for 2 h. Nitrogen gas was then introduced into the activation system for protection, and FPEI was added dropwise to DMF at a molar ratio of 1.79:1 to deoxycholic acid, at a dropping rate of 0.1 mL / min. The reaction was allowed to proceed at room temperature for 24 h. After the reaction was complete, the reactants were dialyzed against deionized water for 48 h (molecular weight cutoff MWCO = 10000 Da), and after freeze-drying, the DFP support was obtained. Hydrogen nuclear magnetic resonance (¹H NMR) spectroscopy was performed. Figure 1 Fourier transform infrared spectroscopy (FT-IR) Figure 2 Characterizing the grafting of fluorine groups with deoxycholic acid, see the reaction formula below. Figure 3 .

[0162] (III) Preparation of DFP / mRNA nanoparticles

[0163] DFP and mRNA RBD Each component was dissolved in enzyme-free deionized water to prepare a 0.1 mg / mL single-component solution. The DFP solution was added dropwise to the mRNA solution at DFP:mRNA mass ratios of 1:4, 1:2, 1:1, and 1:0.5. After gentle mixing, the mixture was incubated at 4 °C for 15 min to self-assemble into DFP / mRNA nanoparticles (DFP / mRNA NPs).

[0164] (iv) Preparation of oral C@DFP / mRNA vaccine (refer to Example 1).

[0165] (v) Detection of the effect of DFP on mRNA loading

[0166] After incubating the DFP prepared in Example 1 with mRNA at different mass ratios, the ability of the DFP to load nucleic acid was detected using a nucleic acid gel retardation assay. The results are as follows: Figure 4 As shown, a DFP to mRNA mass ratio of 1:1 completely blocks the electrophoretic migration of mRNA, achieving full loading of mRNA. Therefore, unless otherwise stated, all studies used a weighting ratio of 1:1.

[0167] Example 3: Preparation of oral mRNA vaccine

[0168] (I) Preparation of PEDV-RBD mRNA

[0169] Using the protein binding domain (RBD) protein of porcine epidemic diarrhea virus (PEDV) strain PEDV / CH / HNPDS / 2025 (GenBank: PZ105934) as a reference amino acid sequence, the corresponding coding sequence was codon optimized. The optimized RBD coding sequence was cloned into the pU-IVT vector containing 5'UTR, 3'UTR and poly (A) tail to obtain the recombinant plasmid pU-RBD-IVT.

[0170] After linearization by single enzyme digestion of the pU-RBD-IVT plasmid, mRNA was synthesized via in vitro transcription (IVT) using T7 RNA polymerase, with simultaneous 5' capping. Unreacted DNA template and residual reagents were removed using mRNA purification beads to obtain high-purity RBD-encoded mRNA. RBD The concentration and purity of mRNA were detected using a NanoDrop micro-ultraviolet spectrophotometer, and qualified samples were stored in an ultra-low temperature freezer at -80 ℃ for later use.

[0171] (II) Preparation of DFP carrier

[0172] 1. Synthesis of fluorinated polyethyleneimine (FPEI)

[0173] A methanol solution of tridecafluoroheptyl ethylene oxide (3-(perfluorohexyl-1-yl)-1,2-epoxypropane, 20 mg / mL) was added dropwise to the dendritic PEI at a rate of 0.1 mL / min. 1.8KThe reaction was carried out in a methanol solution of 20 mg / mL at room temperature with stirring for 48 h. The molar ratio of tridecafluoroheptylethylene oxide to PEI1.8K was 25:1. After the reaction was completed, the reactants were dialyzed sequentially in methanol-water solution and deionized water for 72 h (molecular weight cutoff MWCO = 1000 Da in the dialysis bag). After dialysis, the product was freeze-dried to obtain the fluorinated modified product FPEI.

[0174] 2. Synthesis of Deoxycholic Acid Modified FPEI (DFP)

[0175] Deoxycholic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were added to DMF in a molar ratio of 1:1.2:1.2, and the mixture was activated at room temperature in the dark with stirring for 2 h. Nitrogen gas was then introduced into the activation system for protection, and FPEI was added dropwise to DMF at a molar ratio of 1:1 to deoxycholic acid at a dropping rate of 0.1 mL / min. The reaction was allowed to proceed at room temperature for 24 h. After the reaction was complete, the reactants were dialyzed against deionized water for 48 h (molecular weight cutoff MWCO = 10000 Da), and after freeze-drying, the DFP support was obtained. Hydrogen magnetic resonance (¹H NMR) spectroscopy was performed. Figure 1 Fourier transform infrared spectroscopy (FT-IR) Figure 2 Characterizing the grafting of fluorine groups with deoxycholic acid, see the reaction formula below. Figure 3 .

[0176] (III) Preparation of DFP / mRNA nanoparticles (refer to Example 1).

[0177] (iv) Preparation of oral C@DFP / mRNA vaccine (refer to Example 1).

[0178] Example 1: Evaluation of C@DFP / mRNA pH responsiveness and transepithelial transport efficiency

[0179] (a) pH-responsiveness detection of C@DFP / mRNA

[0180] To evaluate the pH responsiveness and release rate of the oral C@DFP / mRNA vaccine prepared in Example 3, the specific procedures are as follows.

[0181] (1) Detection of C@DFP / mRNA response at different pH levels

[0182] C@DFP / mRNA was incubated in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.4), respectively. Samples were taken at preset time points for particle size analysis, and TEM samples were prepared for image observation. This was to evaluate the structural stability of the microspheres in the simulated gastrointestinal environment. The results are as follows: Figure 13 C@DFP / mRNA maintains good stability with no significant change in particle size in acidic environments; however, in weakly alkaline environments, the particle size increases significantly, and calcium alginate swells in response to pH changes.

[0183] (2) Detection of C@DFP / mRNA release rate in a weakly alkaline environment

[0184] Further investigation was conducted to assess the release effect of C@DFP / mRNA in a weakly alkaline environment. C@DFP / mRNA microspheres loaded with FITC-labeled mRNA were dispersed in 5 mL of simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4), respectively, and then incubated at 37 °C for 120 min. Starting from 0 min of incubation, the samples were vortexed and mixed every 30 min for imaging acquisition. The results are shown below. Figure 14 Simultaneously, at each time point, 100 μL of medium was removed from each sample vial, and an equal volume of preheated fresh medium was immediately added to maintain a constant total volume. The release efficiency of DFP / mRNA NPs from calcium alginate microspheres was detected by measuring the fluorescence intensity of the samples. The results are as follows: Figure 15 The results show that the C@DFP / mRNA oral delivery system remains stable in the acidic environment of the stomach, effectively protecting the vaccine particles it encapsulates. Furthermore, it can rapidly release the DFP / mRNA NPs it encapsulates in the weakly alkaline environment of the intestine, effectively preventing the vaccine from being excreted from the body through intestinal peristalsis during digestion.

[0185] (II) In vitro mucus penetration ability test

[0186] (1) Anti-adhesive protein aggregation experiment

[0187] The C@DFP / mRNA vaccine prepared by the inventors releases internally encapsulated DFP / mRNA NPs in the intestinal environment. To evaluate the ability of DFP / mRNA NPs to resist the adhesion of intestinal mucins, the specific operation steps are as follows.

[0188] Intestinal specimens were aseptically collected from healthy piglets. Natural intestinal mucus was gently scraped from the surface of the intestinal epithelium and equilibrated at 37 °C for 15 min to ensure uniform dispersion. Free FITC-mRNA, PEI / FITC-mRNA, and DFP / FITC-mRNA were mixed separately with the equilibrated intestinal mucus and incubated at 37 °C for 30 min. The incubated samples were then dropped onto glass slides, and time-series images were acquired using a laser confocal microscope at 10 s acquisition times and a time resolution of 30 ms / frame. Data analysis was performed using ImageJ software, and the mean square displacement (MSD) was calculated using the following formula:

[0189] MSDτ =(x (t+τ) − xt) 2+(y (t+τ) − yt) 2

[0190] The results are as follows Figure 16 In groups A and B, the MSD and motility of DFP / mRNA NPs in natural intestinal mucus were significantly higher than those of the free mRNA and PEI / mRNA groups, indicating that DFP modification can significantly enhance the penetration ability of nanoparticles in intestinal mucus.

[0191] (II) In vitro transepithelial transport analysis

[0192] Transepithelial transport experiments were conducted using a Transwell chamber model inoculated with Caco-2 cells. Caco-2 cell monolayers with a transepithelial resistance (TEER) of 500–1000 Ω·cm² were selected for the experiments. The cell monolayers were washed three times with preheated Hank's balanced salt solution (HBSS) at 37 °C. Free FITC-mRNA, PEI / FITC-mRNA NPs, and DFP / FITC-mRNA NPs were diluted with HBSS to a final FITC-mRNA concentration of 50 μg / mL and added to the upper chamber of the Transwell chamber. Preheated HBSS solution was added to the lower chamber, and the chambers were incubated at 37 °C.

[0193] At preset time points, 100 μL samples were taken from the lower chamber, and an equal volume of preheated fresh HBSS solution was added simultaneously. The TEER value of the cell monolayer was monitored throughout the process to ensure barrier integrity. Results are as follows: Figure 17 The concentration of FITC-mRNA in the sample was detected by a fluorescence spectrophotometer, and the apparent permeability coefficient (Papp) and transport efficiency were calculated using the following formulas:

[0194] Papp = (dQ / dt) × 1 / (A×C0);

[0195] Where dQ / dt is the linear slope of the FITC-mRNA cumulative transport-time curve, A is the surface area of ​​the cell monolayer (cm²), and C0 is the initial concentration in the upper chamber.

[0196] Transfer efficiency (%) = (Xt / X0) × 100%;

[0197] Where Xt is the cumulative amount of FITC-mRNA transported at the preset time point, and X0 is the initial amount of FITC-mRNA added to the upper chamber.

[0198] The results are as follows Figure 18 A and B show that the apparent permeability coefficient of DFP / mRNA NPs is more than 2 times higher than that of the PEI / mRNA group, and the transport efficiency is significantly improved, demonstrating excellent transepithelial transport capability. Moreover, this phenomenon is not caused by damage to epithelial cells.

[0199] Example 2: Investigation into the intracellular delivery and immune response mechanism of C@DFP / mRNA vaccine

[0200] Based on the aforementioned experimental tests, in order to accurately evaluate the immune response mechanism of the nanovaccine prepared in this application, the inventors conducted further tests, the specific process and results of which are briefly described below.

[0201] (a) Intracellular uptake and lysosomal escape experiment

[0202] (1) Intracellular uptake detection

[0203] DC2.4 cells were seeded in 24-well plates at a density of 2 × 10⁻⁶ cells / well. 5 Cells per well were cultured overnight to form a confluent monolayer. FITC-mRNA, PEI / FITC-mRNA, and DFP / FITC-mRNA (containing 0.02 mg / mL FITC-mRNA) were co-incubated with DC2.4 cells. After 6 h of incubation, the culture medium was discarded, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 20 min, and stained with DAPI. Intracellular fluorescence distribution was observed using a confocal laser microscope (CLSM). Simultaneously, cells were collected after 4 h of incubation, and the uptake efficiency of FITC-mRNA was quantitatively detected by flow cytometry. Results are as follows: Figure 19 As shown in Figures A and B, significant green fluorescence was observed in the cells of the DFP / FITC-mRNA group, while the fluorescence of the free FITC-mRNA group was negligible. The flow cytometry results were consistent with the fluorescence imaging results. DCs increased the uptake of DFP / FITC-mRNA, and DFP significantly enhanced the delivery of mRNA into the dendritic cell cytoplasm.

[0204] 2. Lysosomal escape experiment

[0205] FITC-mRNA, PEI / FITC-mRNA, and DFP / FITC-mRNA were co-incubated with DC2.4 cells for 12 h, washed three times with PBS, and stained sequentially with LysoTracker Red (lysosomal tracer) and DAPI. Intracellular fluorescence co-localization was observed using CLSM. Results are as follows: Figure 20 The above results indicate that DFP / mRNA NPs can be efficiently endocytosed by DC2.4 cells and can effectively escape from lysosomes, avoiding mRNA degradation by lysosomes and thus ensuring the translation of cytoplasmic mRNA.

[0206] (II) TLR4-mediated activation mechanism of DC innate immune system

[0207] 1. The effect of DFP / mRNA on DC maturation

[0208] In the in vitro DC maturation experiment, BMDCs were seeded in 24-well plates at a seeding density of 2 × 10⁶ cells / well. 5 Cells / well were cultured overnight. Cells were incubated with Free mRNA, PEI / mRNA, and DFP / mRNA (RBD mRNA, 2 μg / mL), respectively. After 24 hours of incubation, cells were collected and labeled with anti-CD86-FITC, anti-CD80-FITC, anti-CD11c-APC, anti-MHC-I-PE, anti-MHC-II-FITC, and anti-CD11c-APC antibodies to assess DC maturation and antigen presentation. After 45 minutes, cells were washed with PBS and analyzed by flow cytometry. Results are as follows: Figure 21 , Figure 22 DFP / mRNA effectively stimulated dendritic cell (DC) maturation, with superior maturation effects compared to unmodified PEI. Furthermore, DFP / mRNA significantly enhanced DC antigen presentation, providing a foundation for subsequent induction of adaptive immunity and T cell differentiation. Cell samples were further collected, and cells were thoroughly lysed using an RNA extraction kit, following the manufacturer's instructions to obtain total RNA. 2 μg of total RNA was used as a template, and a reverse transcription kit was used. The mRNA was reverse transcribed into cDNA using reverse transcriptase. q-PCR was used to detect the expression of relevant cytokine genes, and the results are shown below. Figure 23 DFP / mRNA promotes the maturation of dendritic cells (DCs) and the expression of related cytokine genes, which can effectively enhance the innate immune response.

[0209] 2. Research on the mechanism of DFP / mRNA-induced DC innate immune activation

[0210] After treating BMDCs with DFP / mRNA NPs for 12 h, cells were collected and incubated with anti-TLR4-FITC and anti-CD11c-APC antibodies at 4 °C in the dark for 45 min. After washing with PBS, the expression level of TLR4 on the DC surface was detected by flow cytometry. The results are as follows: Figure 24 .

[0211] Simultaneously, the processed BMDCs were collected and lysed on ice with RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined by the BCA method. Protein samples were separated by SDS-PAGE electrophoresis and then electroblotted onto PVDF membranes. The membranes were incubated overnight at 4°C with primary antibodies against anti-NF-κB, antiphospho-NF-κB (Ser536), antiphospho-TBK1 (Ser172), antiphospho-IRF3 (Ser396), and anti-β-actin, respectively. After washing, horseradish peroxidase-labeled secondary antibody was added, and the membranes were visualized using a Tanon 4600 SF imaging system to detect the expression and phosphorylation levels of pathway-related proteins. The results are shown below. Figure 25 A and B.

[0212] The results show that DFP / mRNA can significantly upregulate the expression of TLR4 on the surface of DCs, activate the TLR4 signaling pathway, promote the phosphorylation of NF-κB and IRF3 transcription factors, and thus initiate the secretion of downstream pro-inflammatory cytokines and chemokines, thereby achieving efficient activation of innate immunity.

[0213] Example 3: Evaluation of the immune response efficacy of C@DPF / mRNA vaccine

[0214] Based on the aforementioned experimental tests, in order to accurately evaluate the immune response effect of the C@DPF / mRNA vaccine prepared in this application, the inventors conducted further animal experiments, the specific process and results of which are briefly described below.

[0215] (I) C@DPF / mRNA vaccine immunization procedure and sample collection

[0216] Female BALB / c mice (6-8 weeks old) were randomly divided into 5 groups (n=6 / group). On days 0, 14, and 28, they were orally immunized by gavage with PBS, DFP / mRNA, blank C@DF, C@FP / mRNA, and C@DFP / mRNA (20 μg mRNA). RBD / mouse). Mice were given free access to water before each immunization, and were fasted for 12 hours prior to immunization and for 4 hours prior to immunization. Mouse weight was monitored every 6 days throughout the experiment. Tail tip blood was collected on days 7, 14, 21, 28, 35, 42, 90, and 120 after the initial immunization. Serum was separated by centrifugation and stored at -80 ℃ for later use. Fecal extract and intestinal lavage fluid (ILF) were collected on day 42 after the initial immunization, and mesenteric lymph nodes (MLNs) were aseptically isolated to prepare single-cell suspensions for subsequent immunological detection. MLNs were isolated on day 60 after the initial immunization, and single-cell suspensions were prepared for memory immune cell detection.

[0217] (ii) C@DPF / mRNA vaccine enhances intestinal mucosal immune response

[0218] Intestinal lavage fluid and feces were collected on day 42, and sIgA content and specific sIgA titer were detected by ELISA. The results showed that the sIgA content in the intestinal lavage fluid (SILF) and fecal extract (FE) of the C@DFP / mRNA group was significantly increased, being 2.64 times and 2.45 times that of the DFP / mRNA group, respectively. Figure 26 A and B). The RBD-specific sIgA titer in SILF was also detected; C@DFP / mRNA was 76.1 times that of the DFP / mRNA group ( Figure 27 ).

[0219] The differentiation effect of IgA+ plasma cells in MLNs was further analyzed using flow cytometry. C@DFP / mRNA significantly induced the differentiation of IgA+ plasma cells. Figure 28 ).

[0220] This indicates that the C@DFP / mRNA vaccine can resist the damage of gastric acid, reach the small intestine, effectively activate intestinal mucosal immune cells, and induce high levels of sIgA production.

[0221] (III) C@DPF / mRNA vaccine enhances adaptive immune response

[0222] To assess the adaptive immune response induced by the vaccine, single cells of MLNs were collected from immunized mice on day 42 and analyzed by flow cytometry. Figure 29 The C@DFP / mRNA shown contains IgD + CD19 + The proportion of B cells (40.03%) was significantly higher than that of DFP / mRNA (8.33%), C@DFP (13.08%), and C@FP / mRNA (27.12%); the proportion of germinal center B cells in C@DFP / mRNA (30.56%) was also significantly higher than that in other control groups. Figure 30This indicates that C@DFP / mRNA has a stronger ability to promote B cell activation.

[0223] Subsequently, serum RBD-specific IgG titers were further measured using ELISA. Compared with other control groups, RBD-specific IgG titers in C@DFP / mRNA were significantly increased, reaching a peak 42 days after primary immunization. Figure 31 A). Among them, the RBD-specific IgG titer of C@DFP / mRNA was 1.6 to 786.9 times higher than that of other controls ( Figure 31 B) indicates that C@DFP / mRNA effectively protects the vaccine from digestion and degradation, and after releasing the encapsulated nanoparticles, it can effectively cross the intestinal epithelial barrier and effectively promote the production of IgG by stimulating B cell activation.

[0224] In addition, serum was collected on day 42 to determine the neutralizing antibody titer, a key indicator of the protective efficacy of the porcine epidemic diarrhea vaccine. The results showed that the C@DFP / mRNA group had a significantly higher neutralizing antibody titer compared to other groups. Figure 32 A), which were 6.53-fold and 1.65-fold higher than those in the DFP / mRNA and C@FP / mRNA groups, respectively; further, indirect immunofluorescence was used to detect the infection status of PEDV on susceptible cells after co-incubation with serum. Figure 32 The results showed that serum neutralization of PEDV in the C@DFP / mRNA group significantly reduced infection of susceptible cells. This indicates that C@DFP / mRNA can induce effective protective humoral immunity.

[0225] To assess T cell-mediated immune responses, single-cell MLNs were collected, and flow cytometry analysis showed that CD3+ was present in C@DFP / mRNA. + CD4 + and CD3 + CD8 + The proportion of T cells was significantly higher than that of other control groups, reaching 43.7% and 33.8%, respectively. Figure 33 A and B). Further analysis was performed on Th1 cells (CD3+) from differentiated T cells. + CD4 + IFN-γ + ) and CTL (CD3) + CD8 + IFN-γ + The degree of differentiation of ) was significantly enhanced by C@DFP / mRNA, which significantly increased the response of effector T cells, and Th1 and CTL showed a significant increase ( Figure 34 (A and B).

[0226] In addition, q-PCR was used to detect the gene levels of cytokines IL-21, IL-17A, IL-6, IL-4, and TGF-β1 in MLNs, and the results were as follows: Figure 35 In the C@DFP / mRNA group, IL-21, IL-6, and IL-17A were significantly increased, while IL-4 and TGF-β1 were slightly increased.

[0227] In summary, the results indicate that C@DFP / mRNA strongly drives Tfh cell differentiation by regulating IL-6 and IL-21, resulting in a large number of germinal centers and helper T cells; low concentrations of TGF-β1 and IL-6 mildly drive Th17 differentiation, assisting intestinal mucosal immunity; IL-21 synergistically promotes Th1 differentiation and enhances the killing function of CD8+CTLs without excessive inflammation or immunosuppression; and induces the formation of a large number of plasma cells and memory B cells, driving IgA conversion and enhancing intestinal mucosal immunity.

[0228] It effectively triggers a strong humoral and cellular immune response, thereby achieving comprehensive systemic immunity.

[0229] (III) C@DPF / mRNA induces long-term immune response

[0230] The formation of long-term immune memory is essential for vaccine-induced adaptive immunity, as it provides durable protection against re-challenge by pathogens through rapid recall responses of memory B cells and T cells. To assess the durability of the humoral immune response induced by C@DPF / mRNA, memory B cells and memory T cells in mesenteric lymph nodes were analyzed by flow cytometry on day 60. Figure 36 As shown, compared with DFP / mRNA and C@FP / mRNA, memory B cells (CD3+) in mesenteric lymph nodes with C@DPF / mRNA had significantly higher levels of DFP / mRNA. - B220 + CD38 + CD138 + The proportions of [specific cells] increased significantly, by 3.8-fold and 1.32-fold, respectively. Furthermore, the proportion of long-lived plasma cells in MLNs was further analyzed, and it remained the highest in the C@DPF / mRNA group, reaching 34.2% ([specific percentages not provided in the original text]). Figure 37 This indicates that C@DPF / mRNA promotes the activation of memory B cells and induces long-lived plasma cells to secrete antibodies for an extended period. Furthermore, serum antibody titers were continuously monitored at different time points. The RBD-specific IgG titer induced by C@DPF / mRNA was significantly higher than that in other groups, slightly decreasing over time, while maintaining a high antibody titer for up to 120 days. Figure 38A). Furthermore, RBD-specific sIgA in SILF was detected and remained detectable at days 90 and 120, with the C@DPF / mRNA group showing a 53.9-fold higher titer of RBD-specific sIgA compared to the DPF / mRNA group. Figure 38 B). This indicates that C@DPF / mRNA induces durable and robust humoral immunity. Additionally, memory T cells, another key component of long-term adaptive immunity, were subsequently analyzed. Consistent with the above findings, the proportions of central memory T cells (TCM, CD3+CD8α+CD44+CD62L+) and effector memory T cells (TEM, CD3+CD8α+CD44+CD62L-) in C@DPF / mRNA were 1.2–6.1 times higher than in other control groups. Figure 39 (A, B, and C). These results collectively demonstrate that C@DPF / mRNA can effectively induce robust and durable humoral and cellular immune responses.

[0231] Example 4: Safety Assessment of Oral Vaccines

[0232] Based on the aforementioned experimental tests, the inventors evaluated the safety of the oral vaccine. The inventors tested changes in mouse body weight during oral vaccine immunization, HE sections of major organs after immunization, thymus index, and post-immunization blood biochemical analysis. The specific process and results are briefly described below.

[0233] Six- to eight-week-old BALB / c mice were intranasally immunized with the aforementioned different components (20 μg mRNA / mouse, n = 3). On day 14 post-immunization, the heart, liver, spleen, lung, and kidney of the mice were harvested. The collected organs were paraffin-embedded, sectioned, and stained with H&E to evaluate toxicity. Blood samples were collected to detect the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CR), and blood urea nitrogen (BUN). Single cells of MLNs were collected to detect the expression of Treg cells, regulatory cells of the intestinal mucosa, and to analyze the homeostasis of intestinal mucosal immunity. The results showed that the body weight of mice immunized with C@DFP / mRNA steadily increased throughout the immunization period. Figure 40 A), no obvious pathological changes in major organs ( Figure 40 B), Treg cell expression levels increased ( Figure 40 C). Hematological analysis further confirmed that there was no statistically significant difference between C@DFP / mRNA and PBS in key biochemical indicators such as BUN, CR, AST, and ALT. Figure 41 This indicates that oral immunization with C@DFP / mRNA did not damage the intestinal microenvironment and maintained the integrity of the intestinal barrier. These results collectively demonstrate that C@DFP / mRNA has good in vivo safety.

[0234] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An mRNA delivery vector, the structural formula of which is shown in (I): （I）； Where n = any integer between 35 and 58.

2. The mRNA delivery vector according to claim 1, characterized in that: It also includes pharmaceutically acceptable salts, stereoisomers, or solvates of polymers as shown in formula (I).

3. The method for preparing the mRNA delivery vector according to claim 1, characterized in that, The steps are as follows: grafting tridecylfluoroheptyl ethylene oxide with polyethyleneimine, and then reacting the grafted product with deoxycholic acid, EDC, and NHS in an organic solvent to obtain an mRNA delivery vector.

4. The method for preparing the mRNA delivery vector according to claim 3, characterized in that: The polyethyleneimine is PEI. 1.8K The structural formula is shown in (II): (II); Where n is any integer between 35 and 58.

5. The method for preparing the mRNA delivery vector according to claim 3, characterized in that: The molar ratio of the reaction between tridecafluoroheptyl ethylene oxide and polyethyleneimine is 20-50:1; the molar ratio of the reaction between the grafted product and deoxycholic acid, EDC, and NHS is 0.5-2:1:1.2:1.2; and the organic solvent is N,N-dimethylformamide or dimethyl sulfoxide.

6. A method for preparing an oral vaccine, characterized in that, The steps are as follows: the mRNA delivery vector of claim 1 and the mRNA encoding the receptor binding domain protein are mixed and combined, and then combined with acceptable excipients or adjuvants to obtain an oral vaccine.

7. The method for preparing an oral vaccine according to claim 6, characterized in that: The mRNA is an mRNA encoding the receptor-binding domain protein of the target virus.

8. The method for preparing an oral vaccine according to claim 6, characterized in that, The steps are as follows: (1) A solution containing the mRNA delivery vector of claim 1 is added dropwise to an mRNA solution, and nanoparticles are obtained by self-assembly after incubation; (2) The nanoparticles from step (1) were added to a sodium alginate solution and microspheres were obtained by ice bath sonication and electrostatic spraying. (3) After washing, the microspheres from step (2) are immersed in chitosan oligosaccharide solution for incubation. The microspheres are collected and freeze-dried to obtain the oral vaccine.

9. The method for preparing an oral vaccine according to claim 8, characterized in that: In step (1), the mass ratio of mRNA delivery vector to mRNA is 1:0.5-4; in step (2), the mass ratio of nanoparticles to sodium alginate is 8:1; and in step (3), the concentration of chitosan oligosaccharide solution is 0.1% w / v.

10. An oral vaccine prepared using the method of claim 7, 8, or 9.

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