Preparation method and application of a multifunctional biomimetic delivery platform for efficient crossing of mucosal barriers and inducing mucosal immune enhancement

Abstract

The application discloses a preparation method and application of a multifunctional bionic delivery platform for efficiently crossing mucosal barriers and inducing mucosal immune enhancement, and belongs to the technical field of biological medicines. Based on biodegradable PLGA copolymers and mixed lipid components, a mucosal delivery carrier particle capable of simultaneously loading multiple antigens and immune stimulating components is prepared through a double emulsion method, a rotary evaporation film coating method and an ultrasonic method. The PLGA nanoparticle is a loading core, and the outer mixed lipid coating layer can play the functions of mucus penetration, cell uptake and immune activation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing a multifunctional mucosal vaccine delivery system based on polylactic-coated glycolic acid (PLGA) copolymer nanoparticles coated with mixed lipid components, and its application in the vaccine field. Background Technology

[0002] Invasion of respiratory pathogens such as respiratory syncytial virus (RSV), influenza virus, and novel coronavirus seriously endangers life and health. Compared with traditional injection immunization, inhaled mucosal immunization can simultaneously stimulate mucosal, humoral, and cellular immune responses, establishing a lung immune defense to prevent local infection and effectively interrupting pathogen transmission to inhibit disease spread. Furthermore, the non-invasive vaccination method provides strong support for immunization coverage in children and underdeveloped areas. However, mucosal delivery barriers and weak immunogenicity limit the application of mucosal vaccines. Developing a delivery system that can overcome the mucus barrier, the cellular internalization barrier, and induce enhanced mucosal immunity is a necessary step in the development of novel mucosal vaccines.

[0003] Mucus-penetrating nanoparticles (MPPs) are excellent candidates for crossing the mucus barrier. Their hydrophilic and electrically neutral surface properties effectively prevent particle-mucin interactions, thus enabling mucus penetration. This strategy has been widely applied and reported in mucosal diseases of the respiratory, intestinal, and reproductive tracts. Simultaneously, the application of MPP strategies to enhance mucosal immune processes has also attracted significant attention. However, due to the contradiction between the particle surface properties required for mucus penetration and cellular uptake, achieving efficient uptake of carrier particles by epithelial cells or antigen-presenting cells (APCs) after mucus penetration remains a challenge for efficient cross-mucosal delivery. On the other hand, due to delivery barriers and mucosal tolerance-based immune tendencies, simple antigen delivery via the mucosal route often results in weak immunogenicity. The development and introduction of mucosal adjuvants are crucial for enhancing immunogenicity and inducing local mucosal and systemic immune responses. Therefore, based on the needs of cross-mucosal delivery and immune enhancement during mucosal immunity, and combined with the high biocompatibility and clear immune enhancement mechanisms of a series of small molecule adjuvants such as monophospholipid A (MPLA), the design and development of a multifunctional mucosal delivery platform can meet the development requirements of novel mucosal vaccines. Summary of the Invention

[0004] This invention presents a mucosal vaccine delivery platform capable of efficiently crossing the airway mucus barrier, promoting cellular uptake, and inducing immune enhancement. The platform utilizes a biodegradable copolymer (PLGA) as the loading core, with an outer layer of a mixed lipid coating consisting of alveolar surfactant-inspired bio-lipids and lipophilic adjuvant components. This coating enhances the carrier particles' mucus penetration, cellular uptake, and immunogenicity, enabling them to cross the mucosal barrier while simultaneously inducing a mucosal immune response. This lipid-inspired bio-vaccine delivery system, possessing mucus penetration, cellular uptake, and immune activation functions, provides a novel delivery platform and implementation strategy for the design, development, and application of novel mucosal vaccines.

[0005] A method for preparing a mucosal delivery carrier of a mixed lipid-coated copolymer PLGA includes the following steps:

[0006] 1) Using the two-emulsion method, antigen-loaded PLGA nanoparticles were prepared: PLGA was dissolved in dichloromethane to form an organic phase, and an aqueous phase containing dissolved active antigen was added to it. The primary emulsion and the secondary emulsion were prepared sequentially using an ultrasonic method. The secondary emulsion was then continuously stirred at room temperature, and the dichloromethane component was evaporated to prepare antigen-loaded PLGA nanoparticles. The prepared PLGA nanoparticles were dispersed in water or phosphate buffer to obtain a PLGA nanoparticle suspension.

[0007] 2) A chloroform solution containing a mixture of lipids with mucus penetration, cellular uptake, and immune activation functions is added to a container. The chloroform is removed using a rotary evaporation coating method, and the mixture of lipids is coated onto the container wall to obtain a mixed lipid coating. The mixed lipids with mucus penetration, cellular uptake, and immune activation functions are composed of alveolar surfactant-inspired lipids and adjuvant lipids.

[0008] 3) Add the PLGA nanoparticle suspension from step 1) to the container after coating in step 2), and use ultrasound to promote the encapsulation of PLGA nanoparticles by lipid molecules, obtaining a mucosal delivery carrier with a core-shell structure and mixed lipid coating encapsulated copolymer PLGA. The encapsulation rate can be controlled by adjusting the ratio of mixed lipids to PLGA nanoparticles.

[0009] In addition to regulating the lipid formulation of the outer coating of PLGA nanoparticles to achieve multifunctional mucosal delivery, the above-mentioned technical solutions can also be achieved by coating the outer layer of PLGA nanoparticles with alveolar surfactant-inspired biolipids, and co-loading water-soluble antigens and adjuvants into the core PLGA nanoparticles. In step 1), the active antigen is replaced with active antigen and water-soluble adjuvant to prepare PLGA nanoparticles co-loaded with antigen and adjuvant. Simultaneously, in step 2), the mixed lipids for mucus penetration, cellular uptake, and immune activation are replaced with alveolar surfactant-inspired biolipids. That is, when step 1) uses antigen-loaded PLGA nanoparticles, the mixed lipids in step 2) are a mixture of alveolar surfactant-inspired biolipids and adjuvant lipids; when step 1) uses PLGA nanoparticles co-loaded with antigen and water-soluble adjuvant, the mixed lipids in step 2) are only alveolar surfactant-inspired biolipids. The PLGA nanoparticles co-loaded with water-soluble antigen and adjuvant were prepared using the same double emulsion method as in step 1), while the coating of the outer alveolar surfactant biomimetic lipid component of the PLGA nanoparticles was the same as in steps 2) and 3).

[0010] In the above technical solution, the mucosal delivery carrier can simultaneously achieve mucus penetration, cellular uptake, and induce a robust mucosal immune response.

[0011] In the above technical solution, the prepared multifunctional mucosal delivery carrier has a core-shell structure, wherein the shell is a mixed lipid layer and the core is PLGA nanoparticles loaded with either a single antigen or an antigen-adjuvant co-loaded.

[0012] In the above technical solution, in step 1) of the double emulsion method, dichloromethane is used as the organic phase, and an active antigen solution or a mixed solution of active antigen and water-soluble adjuvant is used as the aqueous phase. The molecular weight of PLGA is 4500-200000, preferably 20000. The molar ratio of lactide to glycolide units in the PLGA used is (25-75):(75-25), preferably 50:50. For example, the mass of PLGA added is 25-200 mg, preferably 50 mg. The prepared PLGA nanoparticles are spherical with a size of 50-500 nm, preferably 100-500 nm, more preferably 200 nm. The active antigens added to the aqueous phase include, but are not limited to, respiratory syncytial virus surface antigen, influenza virus surface antigen, novel coronavirus spike protein receptor-binding domain, pertussis toxin, pneumococcal polysaccharide antigen, and model antigen ovalbumin (OVA). The water-soluble adjuvants added to the aqueous phase include, but are not limited to, CpG oligodeoxynucleotides (CpG-ODN), polyinosinic-polycytidylic acid (Poly(I:C)), and mRNA components with transcriptional function. PLGA can not only load antigens but also co-load antigens with water-soluble adjuvants to achieve co-delivery of antigen-adjuvant components. Based on the types of antigens and adjuvants loaded, PLGA nanoparticles with various types of antigen loading or antigen-adjuvant co-loading can be prepared.

[0013] In the above technical solution, in step 1), the PLGA nanoparticles are prepared using a two-emulsion method, specifically: PLGA is dissolved in dichloromethane to obtain an organic phase; wherein the concentration of PLGA is 10-200 mg / mL, preferably 50 mg / mL; an aqueous phase is added to the organic phase, which is an active antigen solution (an aqueous phase containing dissolved active antigen) or a mixed solution of antigen and water-soluble adjuvant, wherein the concentration of antigen in the aqueous phase is 1-50 mg / mL, preferably 10 mg / mL; when the aqueous phase contains water-soluble adjuvant, the adjuvant concentration in the aqueous phase is 1-50 mg / mL, preferably 10 mg / mL. The volume ratio of the organic phase to the aqueous phase during the preparation of the PLGA-loaded particles is (1-50):1, preferably 5:1. An organic phase and an aqueous phase are mixed and an ultrasonic method is used to prepare a primary emulsion. A surfactant solution is then added to the primary emulsion, and a secondary emulsion is prepared using the same ultrasonic method. The surfactant solution is selected from one or more aqueous solutions of polyvinyl alcohol, sodium cholate, Tween 20, Tween 80, and Pluronic 127, preferably an aqueous solution of polyvinyl alcohol. The volume ratio of the surfactant solution to the primary emulsion is (1-20):1, preferably (1-10):1; the concentration of the surfactant solution is 0.1-5% (w / v), preferably 1% (w / v). The secondary emulsion is stirred and the dichloromethane component is evaporated to prepare PLGA nanoparticles loaded with antigen, or antigen and adjuvant co-loaded. The stirring temperature is 13-43℃, preferably 25℃; the stirring time is 4-15h, preferably 12h. During both emulsion preparations, the ultrasonic intensity is controlled at 10w-110w, preferably 80w; the ultrasonic time for each preparation is controlled at 10s-20min, preferably 1-5min, more preferably 3min. After washing, the prepared PLGA nanoparticles are dispersed in water or phosphate buffer to obtain a PLGA nanoparticle suspension. The concentration range of the PLGA particle suspension is 1-50 mg / mL, preferably 1-20 mg / mL, and more preferably 1-10 mg / mL.

[0014] Regarding the above technical solution, in step 2), the rotary evaporation coating method is used for the preparation of PLGA nanoparticles loaded only with antigen components and without adjuvant loading. The mixed lipids consist of alveolar surfactant biomimetic lipids and adjuvant lipids. For the preparation of PLGA nanoparticles co-loaded with adjuvant, the mixed lipids are alveolar surfactant biomimetic lipids. The alveolar surfactant biomimetic lipids are composed of one or more combinations of phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, and neutral structural lipids. The phosphatidylcholine mentioned is selected from one of the following: 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1-palmitoyl-2-oleoyllecithin (POPC), 1,2-dilauroyl-sn-glycerol-3-phosphate choline (DL). PC), 1,2-diarachido-sn-glycerol-3-phosphatidylcholine (DAPC), 1-stearoyl-2-myristoylphosphatidylcholine (SMPC), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-myristoyl-2-stearoylphosphatidylcholine (MSPC), 1-palmitoyl-2-stearoylphosphatidylcholine Phosphatidylcholine (PSPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), and 1,2-dicarboxy-sn-glycerol-3-phosphate choline (DEPC), preferably 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC); wherein the phosphatidylglycerol is selected from one of the following: 1,2-palmitoylphosphatidylglycerol (DPPG), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPG), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DSPG), etc. Phosphoryl-rac-(1-glycerol) (DOPG), 1,2-dimyristoyl-sn-glycerol-3-phosphate glycerol (DMPG), 1,2-dilauroyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DLPG), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), 1-stearoyl-2-oleoylphosphatidylglycerol (SOPG), preferably 1,2-bishexadecanoyl-sn-glycerol-3-phosphate-(1′rac-glycerol) (DPPG);The phosphatidylinositol is selected from one of the following: 1,2-hexacosanoyl-sn-glycerol-3-phosphate-(1′-myo-inositol) (DPPI), 1,2-bisoctadecanoyl-sn-glycerol-3-phosphate-(1′-myo-inositol) (DSPI), 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphate-(1′-inositol) (DOPI) and 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycerol-3-phosphate-(1′-inositol) (POPI); the phosphatidylserine is selected from one of the following: 1,2-dipalmitoyl-sn-glycerol-3-phosphate-L-serine (DPPS), 1,2-distearatel-sn-glycerol-3-phosphate-L-serine (DSPS), 1,2-dimyristoyl-sn-glycerol-3-phosphate-L-serine (DMPS), 1 2-Dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS) and 1,2-dilauroyl-sn-glycerol-3-phosphate-L-serine (DLPS); the phosphatidylethanolamine is selected from one of the following: 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-distearate-sn-propanetriyl-3-phosphatidylethanolamine (DSPE), 1,2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine (DOPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 1-palmitoyl-2-oleoylethanolamine (POPE), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), 1,2-disarachidonicoyl-glycerol-3-phosphatidylethanolamine (DAPE); the neutral structural lipid is cholesterol. The phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine can also be replaced with corresponding polyethylene glycol (PEG) modified forms, wherein the molecular weight of PEG is 800-100,000. Phosphatidylethanolamine is preferably 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)](DPPE-PEG);The neutral structural lipid is cholesterol. The molecular weight of PEG in DPPE-PEG is 800-100,000, preferably 2,000. The alveolar surfactant biomimetic lipid is preferably a combination of four lipids: phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and neutral structural lipid. When the alveolar surfactant biomimetic lipid is a combination of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and neutral structural lipid, the mass ratio of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and neutral structural lipid is (1-10):(1-5):(1-5):(1-5), preferably 10:1:1:1. Preferably, in the alveolar surfactant biomimetic lipid, the mass ratio of DPPC, DPPG, DPPE-PEG, and cholesterol is (1-10):(1-5):(1-5):(1-5), preferably 10:1:1:1. The adjuvant lipid is monophosphoryl lipid A (MPLA), which is added to the above-mentioned alveolar surfactant biomimetic lipid and then proceeds to step 2). The mass ratio of the adjuvant lipid (MPLA) to the alveolar surfactant biomimetic lipid mentioned above is 0.0001-0.5:1, preferably 0.0001-0.15:1; for example, the mass of MPLA added is 1-3250 μg, preferably 1000 μg. The container is a round-bottom flask or a gaiwan-shaped flask with a volume of 5-50 mL, preferably 10 mL. The conditions for rotary evaporation coating are: rotary evaporation coating temperature: 20-50℃, preferably 25℃; rotary evaporation vacuum degree set to 10-100 mbar, preferably 80 mbar; rotary evaporation speed: 50-300 rpm, preferably 100 rpm.

[0015] In the above technical solution, step 2) specifically involves: adding a chloroform solution containing the dissolved mixed lipids into a round-bottom flask or a mantle flask; removing the chloroform using a rotary evaporation coating method; and coating the mixed lipids onto the container wall to obtain a mixed lipid coating. The concentration of the mixed lipids in chloroform is 1-10 mg / mL, preferably 4.2-8.3 mg / mL; wherein, when the mixed lipids are only alveolar surfactant biomimetic lipids, the concentration of the four total lipids (alveolar surfactant biomimetic lipids) in chloroform is preferably 8.3 mg / mL; when the mixed lipids are a mixture of alveolar surfactant biomimetic lipids and adjuvant lipids, the concentration of the five total lipids (alveolar surfactant biomimetic lipids and adjuvant lipids) in chloroform is preferably 4.2 mg / mL.

[0016] Regarding the above technical solution, in step 3), the mass ratio of the mixed lipids to PLGA nanoparticles is 1:1 to 9:1, preferably 3:1. The ultrasonic intensity is 10-50 W, preferably 30 W. The ultrasonic time is 1-20 min, preferably 5-20 min, more preferably 20 min.

[0017] The above-mentioned technical solution further includes: dispersing the multifunctional mucosal delivery carrier of PLGA coated with a mixed lipid coating in physiological saline or phosphate buffer for storage, with a concentration range of 0.5-20 mg / mL, preferably 1-10 mg / mL. Preferably, after the multifunctional mucosal delivery carrier of PLGA coated with a mixed lipid coating is prepared, it is dispersed and stored in a buffer solution with pH=7.4, and the buffer solution is replaced with physiological saline for inhalation immunization before animal immunization.

[0018] The above technical solution also includes: preparing a lyophilized dosage form of a multifunctional mucosal delivery carrier encapsulating a copolymer PLGA with a mixed lipid coating, which is beneficial for long-distance transportation and long-term storage. Regarding the preparation of the lyophilized dosage form, the multifunctional mucosal delivery carrier prepared in step 3) is mixed with a lyophilization protectant, pre-frozen, and then lyophilized using a freeze dryer to obtain the lyophilized dosage form. A lyophilization protectant is used in the preparation of the lyophilized dosage form; the lyophilization protectant is selected from trehalose, sucrose, lactose, and inulin, with trehalose being the preferred protectant. The lyophilization protectant is an aqueous solution of the lyophilization protectant with a mass concentration of 0.5%-20%, preferably 5%; the multifunctional mucosal carrier material is a dispersion of the multifunctional mucosal carrier material, obtained by dispersing the multifunctional mucosal carrier material in a phosphate buffer solution, with a concentration of 0.5-20 mg / mL, preferably 1-10 mg / mL; the volume ratio of the lyophilization protectant to the multifunctional mucosal carrier material is (1-10):1, preferably 2:1; the pre-freezing temperature is -20 to -80°C, preferably -80°C; the pre-freezing time is 2-15 h, preferably 4 h; the lyophilization time is 2-72 h, preferably 24 h; and the lyophilization temperature is -20 to -80°C, preferably -80°C.

[0019] The PLGA nanoparticles of this invention, after being coated with a mixed lipid coating, can significantly enhance mucus penetration and cellular uptake, thereby crossing the physical delivery barrier of the mucosa.

[0020] The beneficial effects of this invention are:

[0021] This invention utilizes biodegradable PLGA copolymers and mixed lipid components to prepare mucosal delivery carrier particles capable of simultaneously loading multiple antigens and immunostimulatory components via a two-emulsion method, a rotary evaporation coating method, and an ultrasonic method. The PLGA nanoparticles serve as the loading core, while the outer mixed lipid coating enables mucus penetration, cellular uptake, and subsequent immune activation. Furthermore, this invention is also applicable to the co-loading and delivery of various water-soluble small molecule adjuvants, such as CpG-ODN and Poly(I:C). Therefore, the multifunctional mucosal delivery carrier particles of this invention can enhance the resistance to protease degradation of antigen molecules during mucosal delivery, overcome the barriers of mucus and epithelial cells, and induce antigen-specific mucosal and systemic immune responses. These capabilities have been validated through cellular and mouse in vivo experiments. Attached Figure Description

[0022] Figure 1 Transmission electron microscopy (TEM) images show PLGA nanoparticles without a lipid-mixed coating (Pristine-PLGA) and PLGA nanoparticles with a lipid-mixed coating (BLVD). The blue dashed boxes represent magnified views of PLGA nanoparticles without a lipid coating. The red dashed boxes represent magnified views of PLGA nanoparticles successfully coated with a lipid coating; the BLVD with the successful lipid-mixed coating exhibits a core-shell structure. The scale bar is 200 nm.

[0023] Figure 2 To study the mean square displacement of BLVD nanoparticles in mucus through the penetration and adhesion behavior in mucus using multi-particle tracking technology. <msd>The quantitative evaluation results were presented. The recognized slime-adhesive particles, namely unmodified PLGA nanoparticles (Pristine-PLGA), and the recognized slime-penetrating particles, namely PEG-modified PLGA nanoparticles (PLGA-PEG), were used as control materials.

[0024] Figure 3 This study evaluated the cytotoxicity of BLVD nanoparticles against antigen-presenting cells, namely mouse dendritic cell line (DC 2.4) and primary mouse bone marrow-derived dendritic cells (BMDC). Unmodified PLGA nanoparticles (Pristine-PLGA) and PEG-modified PLGA nanoparticles (PLGA-PEG) served as control materials, while the BLVD nanoparticles were BLVD-3 nanoparticles. Figure 3 (a) shows the cytotoxicity evaluation of BLVD nanoparticles on DC 2.4 cells; Figure 3 (b) shows the cytotoxicity evaluation of BLVD nanoparticles on BMDC cells.

[0025] Figure 4 The uptake capacity of BLVD nanoparticles by respiratory epithelial cells (BEAS-2B) and antigen-presenting cells (DC 2.4) was evaluated. Unmodified PLGA nanoparticles (Pristine-PLGA), PEG-modified PLGA nanoparticles (PLGA-PEG), and culture medium (ctrl) served as control groups. The BLVD nanoparticles used were BLVD-3 nanoparticles. Figure 4 (a) shows the qualitative evaluation of the uptake capacity of BLVD nanoparticles by BEAS-2B cells; Figure 4 (b) shows the qualitative evaluation of the cellular uptake capacity of BLVD nanoparticles on DC2.4 cells; Figure 4 (c) shows the flow cytometry quantitative evaluation of the uptake capacity of BLVD nanoparticles by BEAS-2B cells.

[0026] Figure 5 This study evaluated the cell activation and cytokine release of primary mouse bone marrow-derived dendritic cells (BMDCs) induced by BLVD nanoparticles. Unmodified PLGA nanoparticles (Pristine-PLGA), PEG-modified PLGA nanoparticles (PLGA-PEG), culture medium (ctrl), and lipopolysaccharide (LPS) served as control groups. The BLVD nanoparticles used were BLVD-3 nanoparticles. BLVD promoted the activation of (a)CD80 on the surface of BMDC cells. + (b)CD86 + and (c)MHCⅡ + The expression of BLVD promotes the further secretion of (d)IL-6, (e)IL-12 and (f)TNF-α cytokines by BMDC cells.

[0027] Figure 6 This study evaluated the immune response induced by BLVD nanoparticles in a respiratory syncytial virus (RSV) inhalation model mice. Saline, pure RSV antigen, RSV antigen with MPLA adjuvant (antigen+MPLA), unmodified PLGA nanoparticles with MPLA adjuvant (Pristine-PLGA+MPLA), PEG-modified PLGA nanoparticles with MPLA adjuvant (PLGA-PEG+MPLA), and intramuscular BLVD injection (BLVD(IM)) were used as control groups. The BLVD used in BLVD(IN) and BLVD(IM) was BLVD-3 nanoparticles. Figure 6 Evaluation of IgA antibody titer levels in mouse nasal lavage fluid (NPLF) as an immune endpoint; Figure 6 Evaluation of IgA antibody titer levels in bronchoalveolar lavage fluid (BALF) of mice as an immune endpoint (b); Figure 6 Evaluation of IgG antibody titer levels in mouse serum at the immunization endpoint (c); Figure 6 (d) Immunological endpoint: CD4 levels in mouse spleen + Evaluation of T cell activation levels; Figure 6 (e) Immunological endpoint: CD8 in mouse spleen + Evaluation of T cell activation levels; Figure 6 Evaluation of B cell activation levels in the spleen of mice at the midpoint (f) immune endpoint.

[0028] Figure 7 This study evaluated the induction of immune response by BLVD lyophilized formulation (BLVD-FD) in a mouse respiratory syncytial virus (RSV) inhalation model. Saline and purified RSV antigen were used as control materials. Both the BLVD nanoparticles (BLVD(IN)) and the BLVD lyophilized formulation (BLVD-FD(IN)) used BLVD-3 nanoparticles. Figure 7 (a) represents the evaluation of IgA antibody titer levels in bronchoalveolar lavage fluid (BALF) of mice as an immune endpoint; Figure 7 Evaluation of IgG antibody titer levels in mouse serum at the immune endpoint (b). Detailed Implementation

[0029] The following non-limiting embodiments are not intended to limit the invention in any way. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; and the reagents and materials involved are conventional reagents unless otherwise specified.

[0030] Example 1

[0031] A method for preparing a multifunctional transmucosal delivery carrier particle loaded with a mixture of lipids (alveolar surfactant biomimetic lipids and adjuvant lipids MPLA) for carrying a model antigen OVA includes the following steps:

[0032] 1) Preparation of antigen-loaded PLGA nanoparticles:

[0033] 50 mg of PLGA material (purchased from Sigma-Aldrich, molecular weight 20,000) was weighed and dissolved thoroughly in dichloromethane to obtain an organic phase with a PLGA concentration of 50 mg / mL. Then, 200 μL of an aqueous solution of the model antigen OVA (purchased from Sigma-Aldrich) was added to the organic phase, with an antigen concentration of 10 mg / mL. An oil-in-water primary emulsion (OW) was prepared by sonication at 80 W for 30 s. The primary emulsion was added to 10 mL of an aqueous solution of polyvinyl alcohol (1% w / v). A water-in-oil-in-water (WOW) secondary emulsion was prepared by sonication at 80 W for 3 min. The secondary emulsion was stirred overnight at room temperature to obtain antigen-loaded PLGA nanoparticles. The PLGA nanoparticles were washed with ultrapure water and dispersed in 10 mL of ultrapure water to obtain a PLGA nanoparticle suspension.

[0034] 2) Preparation of alveolar surfactant-inspired biomimetic lipid membranes combined with adjuvant-based lipid membranes:

[0035] 1,2-Dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dihexadecanoyl-sn-glycerol-3-phosphate-(1′rac glycerol) (DPPG), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)] (DPPE-PEG) 2000 Cholesterol and adjuvant lipid MPLA were dissolved in chloroform, with a concentration of 10 mg / mL for each lipid component. The solutions of the first four alveolar surfactant biomimetic lipids and the MPLA solution were added to a 10 mL round-bottom flask. Among the first four alveolar surfactant biomimetic lipids, DPPC, DPPG, and DPPE-PEG... 2000 The ratio of cholesterol to DPPC, DPPG, and DPPE-PEG is 10:1:1:1. 2000 The total mass of cholesterol was 6.6 mg; the mass ratio of MPLA to alveolar surfactant biomimetic lipids was 0.15:1. Then, the solvent chloroform was removed by rotary evaporation at 25 °C, 80 mbar vacuum, and 100 rpm for 4 h, resulting in a mixed lipid film coated on the wall of a round-bottom flask.

[0036] 3) Preparation of the mucosal delivery carrier for the PLGA nanoparticles in step 1) encapsulated in step 2):

[0037] Following the mass ratios of the five mixed lipids to PLGA in step 2) of 1:1, 3:1, and 9:1, the antigen and adjuvant-loaded PLGA nanoparticles prepared in step 1) were added to a 10 mL round-bottom flask after coating. The flask was ultrasonicated at 30 W for 20 min to obtain multifunctional mucosal delivery carrier particles (the multifunctional mucosal delivery carrier particle material prepared with a mass ratio of 1:1 of the five mixed lipids to PLGA is designated as BLVD-1; the multifunctional mucosal delivery carrier particle material prepared with a mass ratio of 3:1 of the five mixed lipids to PLGA is designated as BLVD-3; and the multifunctional mucosal delivery carrier particle material prepared with a mass ratio of 9:1 of the five mixed lipids to PLGA is designated as BLVD-9).

[0038] Comparative Example 1

[0039] A method for preparing surface-unmodified PLGA nanoparticles (Pristine-PLGA) includes the following steps:

[0040] 50 mg of PLGA material (purchased from Sigma-Aldrich, molecular weight 20,000) was weighed and dissolved thoroughly in dichloromethane to obtain an organic phase with a PLGA concentration of 50 mg / mL. Then, 200 μL of an aqueous solution of the model antigen OVA (10 mg / mL) was added to the organic phase. An oil-in-water primary emulsion (OW) was prepared by sonication at 80 W for 30 s. The primary emulsion was added to 10 mL of an aqueous solution of polyvinyl alcohol (1% w / v). A water-in-oil (WOW) secondary emulsion was prepared by sonication at 80 W for 3 min. The secondary emulsion was stirred overnight at room temperature to obtain antigen-loaded PLGA nanoparticles. The PLGA nanoparticles were washed with ultrapure water and dispersed in 10 mL of ultrapure water to obtain a Pristine-PLGA nanomaterial suspension.

[0041] Comparative Example 2

[0042] Preparation of PLGA nanoparticles modified with polyethylene glycol-5000 (PLGA-PEG(5000)):

[0043] PLGA-PEG (5000) material (purchased from Sigma-Aldrich) was weighed and fully dissolved in dichloromethane, with a PLGA-PEG concentration of 50 mg / mL, to obtain an organic phase. Then, 200 μL of an aqueous solution of the model antigen OVA (10 mg / mL) was added to the organic phase. An oil-in-water primary emulsion (OW) was prepared by sonication at 80 W for 30 s. The primary emulsion was added to 10 mL of an aqueous solution of polyvinyl alcohol (1% w / v). A water-in-oil-in-water (WOW) secondary emulsion was prepared by sonication at 80 W for 3 min. The secondary emulsion was stirred overnight at room temperature to obtain antigen-loaded PLGA-PEG nanoparticles. The PLGA-PEG nanoparticles were washed with ultrapure water and dispersed in 10 mL of ultrapure water to obtain a PLGA-PEG nanomaterial suspension.

[0044] Comparative Example 3

[0045] A method for preparing alveolar surfactant-inspired liposome materials includes the following steps:

[0046] 1,2-Dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dihexadecanoyl-sn-glycerol-3-phosphate-(1′rac glycerol) (DPPG), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)] (DPPE-PEG) 2000 The lipids (DPPC, DPPG, DPPE-PEG) and cholesterol were dissolved in chloroform, with each lipid component having a concentration of 10 mg / mL. The four lipid solutions were then processed according to the following formulas: DPPC, DPPG, DPPE-PEG. 2000 The mass ratio of cholesterol to 10:1:1:1, and the four lipids (DPPC, DPPG, DPPE-PEG) are also present. 2000 A total of 6.6 mg of a mixture (including cholesterol) was added to a 10 mL round-bottom flask. The solvent chloroform was then removed by rotary evaporation at 25 °C for 4 h, yielding a mixed lipid membrane coated on the wall of the round-bottom flask. After coating, 2 mL of phosphate buffer (pH 7.4) was added to the round-bottom flask for further hydration for 12 h. The hydrated lipid solution was then extruded through a 200 nm pore size membrane to obtain liposomes with a size of 200 nm.

[0047] Example 2

[0048] Physicochemical properties of the transmucosal delivery carrier particles prepared in Example 1 and Comparative Examples 1-3 were tested.

[0049] The BLVD series materials prepared in Example 1 were negatively stained, and their morphology was examined using transmission electron microscopy. The results are shown in the figure. Figure 1 Electron microscopy analysis showed that both the unencapsulated PLGA (Pristine-PLGA) and the encapsulated BLVD were spherical nanoparticles with sizes ranging from 200 to 300 nm. After lipid encapsulation, the BLVD particles exhibited a transparent lipid shell on their surface. With increasing lipid ratio, the lipid encapsulation rate of the BLVD particles significantly increased, as did the proportion of core-shell structured nanoparticles.

[0050] The hydrodynamic dimensions and zeta potentials of the series of materials prepared in Example 1 and Comparative Examples 1-3 were further examined, and the results are shown in Table 1. The hydrodynamic dimensions of the series of materials prepared in Example 1 were between 200-300 nm. As the lipid ratio increased, the zeta potential of BLVD gradually changed from neutral to negative, and remained consistent with the potential of liposomes prepared using only mixed lipids, which further proves the successful encapsulation of lipid coatings.

[0051] Table 1. Hydrodynamic particle size, PDI, and Zeta potential of the series of particles prepared in Example 1 and Comparative Examples 1-3

[0052]

[0053] Example 3

[0054] Detection of the mucus penetration ability of transmucosal delivery carrier particles prepared in Example 1 and Comparative Examples 1-2

[0055] The mucus-penetrating ability of BLVD transmucosal delivery carrier particles was evaluated using multi-particle tracking technology. The results are shown in [Table / Reference]. Figure 2 Compared with the control groups Pristine-PLGA nanoparticles and PLGA-PEG particles, quantitative analysis showed that BLVD had a higher mean square displacement in mucus (…). <msd>Furthermore, as the proportion of lipid added increases, the mobility of BLVD particles in mucus increases.

[0056] Example 4

[0057] Cytotoxicity assay of the transmucosal delivery carrier particles prepared in Example 1 and Comparative Examples 1-2 is shown in the figure. Figure 3 The cell viability of mouse dendritic cell line (DC2.4) and primary mouse bone marrow-derived dendritic cells (BMDC) treated with different concentrations of BLVD material was determined using the MTS method. The results showed that BLVD did not exhibit concentration-dependent cytotoxicity in either cell line, indicating that the material possesses good biosafety properties.

[0058] Example 5

[0059] The cellular uptake capacity of the transmucosal delivery carrier particles prepared in Example 1 and Comparative Examples 1-2 was detected by immunofluorescence assay. The results are shown in [Figure Number]. Figure 4 The cellular uptake capacity of the delivery materials in Examples 1 and Comparative Examples 1-2 was investigated using the human respiratory epithelial cell line (BEAS-2B) and the mouse dendritic cell line (DC2.4), respectively. Qualitative analysis using confocal microscopy showed that BLVD significantly promoted uptake by respiratory epithelial cells and dendritic cells, as shown in the results below. Figure 4 As shown in (ab). Flow cytometry analysis further validated that BLVD significantly promotes BEAS-2B cell uptake, as shown in the results. Figure 4 As shown in (c).

[0060] Example 6

[0061] The cell activation and cytokine secretion capabilities of the delivery materials in Example 1 and Comparative Examples 1-2 were investigated using mouse primary bone marrow-derived dendritic cells (BMDC) (enzyme-linked immunosorbent assay). Results are shown in [Figure number missing]. Figure 5 (ac). BLVD significantly promoted the activity of CD80, CD86, and major histocompatibility complex type II (MHCII) at CD11. C + Upregulation of BLVD expression in cells demonstrates that BLVD can promote the activation of BMDCs. Furthermore, BLVD can also significantly promote the secretion of Th1 and Th2 cytokines by BMDCs, namely IL-6, IL-12, and TNF-α. (See attached results). Figure 5 (df), which further verified that BLVD has the ability to induce a robust immune response.

[0062] Example 7

[0063] Six- to eight-week-old C57BL / 6 mice were selected, and the local mucosal and systemic immune response capabilities of a series of antigen delivery carrier particles prepared in Example 1 and Comparative Examples 1-2 were evaluated using an RSV inhalation model. The results are shown in [Figure 1]. Figure 6 The immunization procedure and method of use included the following steps: Mice were immunized by nasal inhalation of the vaccine system on days 0 and 14. The vaccine system consisted of three groups: PLGA carrier particle group (Pristine-PLGA+MPLA, i.e., each mouse inhaled 30 μL of saline containing 380 μg PLGA particles, 10 μg RSV antigen, and 4 μg MPLA), PLGA-PEG carrier particle group (Pristine-PLGA+MPLA, i.e., each mouse inhaled 30 μL of saline containing 350 μg PLGA-PEG particles, 10 μg RSV antigen, and 4 μg MPLA), and BLVD group (BLVD(IN), i.e., each mouse inhaled 30 μL of saline containing 320 μg BLVD-3 particles and 10 μg RSV antigen). In addition, the following groups were set up as controls: a saline group (30 μL of saline per mouse), a pure RSV antigen group (30 μL of saline containing 10 μg RSV antigen per mouse), an RSV antigen plus MPLA adjuvant group (10 μg RSV antigen and 4 μg MPLA per mouse inhaled in 30 μL of saline), and a BLVD intramuscular injection group (BLVD(IM) per mouse intramuscularly injected with 320 μg of BLVD-3 particles and 10 μg RSV antigen). Five mice were immunized in each group. Nasal lavage fluid, bronchoalveolar lavage fluid, and blood were collected from mice on day 28 for antibody titer evaluation (results are shown in [link to results]). Figure 6 (ac)). BLVD significantly induced the production of IgA and IgG in local mucosal cavities and systemic serum. Mouse spleens were further collected, ground, and single-cell suspensions were prepared. Cells were then restimulated with RSV antigen (2 μg / mL), and the responses of various immune cell types were analyzed. The results showed that BLVD significantly induced the production of CD4+ in the lungs and spleen. + T cells, CD8 + The production of T cells and B cells, results are shown in […]. Figure 6 (df). In summary, BLVD can significantly enhance the specific immune response to local mucosa and systemic antigens.

[0064] Example 8

[0065] The lyophilized formulation (BLVD-FD) of the multifunctional mucosal delivery carrier particles in Example 1 was prepared as follows: The multifunctional mucosal delivery carrier particles (BLVD-3 particles) prepared in Example 1 were resuspended in phosphate buffer (pH 7.4) at a concentration of 1 mg / mL. This resuspended particle was then mixed with a trehalose aqueous solution at a ratio of 1:2 (v / v), wherein the mass concentration of the trehalose aqueous solution was 5%. The mixture was pre-frozen at -80°C for 4 h and then lyophilized at -80°C for 24 h using a freeze dryer to prepare BLVD-FD. Further investigation was conducted using an RSV inhalation model on 6-8 week old C57BL / 6 mice to investigate the immune response induced by BLVD-FD. The results are shown in […]. Figure 7 The immunization procedure and methods included the following steps: Mice were immunized via nasal inhalation of the vaccine system on days 0 and 14. The vaccine systems included a BLVD group (BLVD(IN), each mouse inhaling 30 μL of saline containing 320 μg BLVD-3 particles and 10 μg RSV antigen) and a BLVD-FD group (BLVD-FD(IN), each mouse inhaling 30 μL of saline containing 320 μg BLVD-FD particles and 10 μg RSV antigen). Additionally, a saline group (each mouse inhaling 30 μL of saline) and an RSV antigen group (each mouse inhaling 30 μL of saline containing 10 μg RSV antigen) served as controls. Five mice were immunized in each experimental group. Blood and bronchoalveolar lavage fluid were collected on day 28 for antibody titer evaluation. The results showed that the lyophilized form of BLVD also induced significant IgA and IgG production in local mucosal cavities and systemic serum.

[0066] Example 9

[0067] BLVD, as a mucosal immune delivery platform, can also achieve co-loading of antigens and water-soluble adjuvants. Specifically, using OVA as the model antigen, BLVD can co-load CpG-ODN, Poly(I:C) adjuvant, and GFP-mRNA; these materials are named BLVD-C, BLVD-P, and BLVD-G, respectively. The preparation methods for BLVD materials with different loading components are as follows:

[0068] 1. Preparation of transmucosal delivery carrier particles co-loaded with alveolar surfactant biomimetic lipid model antigen OVA and CpG oligodeoxynucleotides (CpG-ODN):

[0069] 1) Preparation of PLGA nanoparticles co-loaded with model antigen OVA and CpG-ODN:

[0070] 50 mg of PLGA material (purchased from Sigma-Aldrich, molecular weight 20,000) was weighed and dissolved thoroughly in dichloromethane to obtain an organic phase with a PLGA concentration of 50 mg / mL. Then, 200 μL of an aqueous solution containing model antigen OVA (purchased from Sigma-Aldrich) and CPG-ODN (purchased from Sangon Biotech, model CPG-1018) was added to the organic phase, with OVA and CPG-ODN concentrations of 10 mg / mL each. A water-in-oil primary emulsion (OW) was prepared by sonication at 80 W for 30 s. The primary emulsion was added to 10 mL of a 1% (w / v) aqueous solution of polyvinyl alcohol. A water-in-oil (WOW) secondary emulsion was prepared by sonication at 80 W for 3 min. The secondary emulsion was stirred overnight at room temperature to obtain PLGA nanoparticles co-loaded with antigen and adjuvant. After washing the PLGA nanoparticles with ultrapure water, they were dispersed in 10 mL of ultrapure water to obtain a PLGA nanoparticle suspension.

[0071] 2) Preparation of alveolar surfactant-inspired biomimetic lipid membrane:

[0072] 1,2-Dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dihexadecanoyl-sn-glycerol-3-phosphate-(1′rac glycerol) (DPPG), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)] (DPPE-PEG) 2000 The lipids (DPPC, DPPG, DPPE-PEG) and cholesterol were dissolved in chloroform, with each lipid component having a concentration of 10 mg / mL. The four lipid solutions were then processed according to the following formulas: DPPC, DPPG, DPPE-PEG. 2000 The mass ratio of cholesterol to 10:1:1:1, and the four lipids (DPPC, DPPG, DPPE-PEG) are also present. 2000 A total of 6.6 mg of the mixture (including cholesterol) was added to a 10 mL round-bottom flask. Then, the solvent chloroform was removed by rotary evaporation at 25 °C, 80 mbar vacuum, and 100 rpm for 4 h, resulting in a mixed lipid film coated on the wall of the round-bottom flask.

[0073] 3) Preparation of BLVD carrier for mucosal delivery of PLGA nanoparticles from step 1) by encapsulating alveolar surfactants with biomimetic lipids in step 2):

[0074] At a mass ratio of 3:1 for alveolar surfactant biomimetic lipids (4 types of lipids) to PLGA, PLGA nanoparticles co-loaded with the antigen and CPG-ODN adjuvant prepared in step 1) were added to a round-bottom flask that had been coated. The flask was sonicated for 20 minutes using an ultrasonic intensity of 30 W to obtain multifunctional mucosal delivery carrier particles (denoted as BLVD-C).

[0075] 2. Preparation of transmucosal delivery carrier particles co-loaded with alveolar surfactant-mixed lipid model antigen OVA and polyinosinic acid (Poly(I:C)):

[0076] 1) Preparation of PLGA nanoparticles co-loaded with model antigen OVA and Poly(I:C):

[0077] 50 mg of PLGA material (purchased from Sigma-Aldrich, molecular weight 20,000) was weighed and dissolved thoroughly in dichloromethane, resulting in a PLGA concentration of 50 mg / mL. Next, 200 μL of an aqueous solution containing the model antigen OVA (purchased from Sigma-Aldrich) and Poly(I:C) (purchased from InvivoGen, model number tirl-picw) was added to the dichloromethane organic phase, with an OVA concentration of 10 mg / mL and a Poly(I:C) concentration of 2.5 mg / mL. An oil-in-water primary emulsion (OW) was prepared by sonication at 80 W for 30 s. The primary emulsion was added to 10 mL of a 1% (w / v) aqueous solution of polyvinyl alcohol. A water-in-oil-in-water (WOW) secondary emulsion was prepared by sonication at 80 W for 3 min. The secondary emulsion was stirred overnight at room temperature to obtain PLGA nanoparticles co-loaded with antigen and adjuvant. After washing the PLGA nanoparticles with ultrapure water, they were dispersed in 10 mL of ultrapure water to obtain a PLGA nanoparticle suspension.

[0078] 2) Preparation of alveolar surfactant-inspired biomimetic lipid membrane:

[0079] 1,2-Dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dihexadecanoyl-sn-glycerol-3-phosphate-(1′rac glycerol) (DPPG), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)] (DPPE-PEG) 2000 The lipids (DPPC, DPPG, DPPE-PEG) and cholesterol were dissolved in chloroform, with each lipid component having a concentration of 10 mg / mL. The four lipid solutions were then processed according to the following formulas: DPPC, DPPG, DPPE-PEG. 2000 The mass ratio of cholesterol to 10:1:1:1, and the four lipids (DPPC, DPPG, DPPE-PEG) are also present. 2000 A total of 6.6 mg of the mixture (including cholesterol) was added to a 10 mL round-bottom flask. Then, the solvent chloroform was removed by rotary evaporation at 25 °C, 80 mbar vacuum, and 100 rpm for 4 h, resulting in a mixed lipid film coated on the wall of the round-bottom flask.

[0080] 3) Preparation of BLVD carrier for mucosal delivery of PLGA nanoparticles from step 1) by encapsulating alveolar surfactants with biomimetic lipids in step 2):

[0081] At a mass ratio of 3:1 for alveolar surfactant biomimetic lipids (4 types of lipids) to PLGA, PLGA nanoparticles co-loaded with the antigen and Poly(I:C) adjuvant prepared in step 1) were added to a round-bottom flask that had been coated. The flask was then sonicated for 20 minutes using an ultrasonic intensity of 30 W to obtain multifunctional mucosal delivery carrier particles (denoted as BLVD-P).

[0082] 3. Preparation of transmucosal delivery carrier particles co-loaded with alveolar surfactant biomimetic lipid model antigen OVA and GFP-mRNA:

[0083] 1) Preparation of PLGA nanoparticles co-loaded with model antigen OVA and GFP-mRNA:

[0084] 50 mg of PLGA material (purchased from Sigma-Aldrich, molecular weight 20,000) was weighed and dissolved thoroughly in dichloromethane, resulting in a PLGA concentration of 50 mg / mL. Next, 200 μL of an aqueous solution containing model antigen OVA (purchased from Sigma-Aldrich) and GFP-mRNA (purchased from Bestco Biotechnology (Shenzhen) Co., Ltd.) was added to the dichloromethane organic phase, with an OVA concentration of 10 mg / mL and a GFP-mRNA concentration of 1.1 mg / mL. An oil-in-water primary emulsion (OW) was prepared by sonication at 80 W for 30 s. The primary emulsion was added to 10 mL of a 1% (w / v) aqueous solution of polyvinyl alcohol. A water-in-oil-in-water (WOW) secondary emulsion was prepared by sonication at 80 W for 3 min. The secondary emulsion was stirred overnight at room temperature to obtain PLGA nanoparticles co-loaded with antigen and adjuvant. The PLGA nanoparticles were washed with ultrapure water and dispersed in 10 mL of ultrapure water to obtain a PLGA nanoparticle suspension.

[0085] 2) Preparation of alveolar surfactant-inspired biomimetic lipid membrane:

[0086] 1,2-Dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dihexadecanoyl-sn-glycerol-3-phosphate-(1′rac glycerol) (DPPG), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)] (DPPE-PEG) 2000 The lipids (DPPC, DPPG, DPPE-PEG) and cholesterol were dissolved in chloroform, with each lipid component having a concentration of 10 mg / mL. The four lipid solutions were then processed according to the following formulas: DPPC, DPPG, DPPE-PEG. 2000 The mass ratio of cholesterol to 10:1:1:1, and the four lipids (DPPC, DPPG, DPPE-PEG) are also present. 2000 A total of 6.6 mg of the mixture (including cholesterol) was added to a 10 mL round-bottom flask. Then, the solvent chloroform was removed by rotary evaporation at 25 °C, 80 mbar vacuum, and 100 rpm for 4 h, resulting in a mixed lipid film coated on the wall of the round-bottom flask.

[0087] 3) Preparation of BLVD carrier for mucosal delivery of PLGA nanoparticles from step 1) by encapsulating alveolar surfactants with biomimetic lipids in step 2):

[0088] At a mass ratio of 3:1 for alveolar surfactant biomimetic lipids (4 types of lipids) to PLGA, the PLGA nanoparticles co-loaded with the antigen and GFP-mRNA prepared in step 1) were added to a round-bottom flask that had been coated. The flask was sonicated for 20 minutes using an ultrasonic intensity of 30 W to obtain multifunctional mucosal delivery carrier particles (denoted as BLVD-G).

[0089] The encapsulation efficiency test results of BLVD materials with different loading components are shown in Table 2.

[0090] Table 2. Encapsulation efficiency of BLVD materials co-loaded with multiple immunologically active components and model antigen OVA

[0091] < / msd> < / msd>

Claims

1. A method for preparing a multifunctional mucosal delivery carrier of PLGA coated with a mixed lipid coating, comprising the following steps: 1) PLGA was dissolved in dichloromethane to form an organic phase. An aqueous phase containing dissolved active antigen was added to the organic phase. The primary emulsion and secondary emulsion were prepared sequentially using an ultrasonic method. The secondary emulsion was then continuously stirred at room temperature to evaporate and remove the dichloromethane component, thereby preparing antigen-loaded PLGA nanoparticles. These nanoparticles were then dispersed in water or a buffer solution to obtain a PLGA nanoparticle suspension. 2) adding a chloroform solution of mixed lipids having mucous penetration, cell uptake and immune activation functions into the container, removing the chloroform using a rotary evaporation film coating method to coat the container wall with the mixed lipids, to obtain a mixed lipid coating; wherein, The mixed lipids with mucus penetration, cellular uptake and immune activation functions are composed of alveolar surfactant biomimetic lipids and adjuvant lipids; 3) Add the PLGA nanoparticle suspension prepared in step 1) to the container after coating in step 2), and use the ultrasonic method to complete the encapsulation of the PLGA nanoparticles by the mixed lipid coating, thus preparing a multifunctional mucosal delivery carrier particle with a core-shell structure. In step 1), the antigen is at least one of respiratory syncytial virus surface antigen, influenza virus surface antigen, SARS-CoV-2 spike protein receptor binding domain, pertussis toxin, and pneumococcal polysaccharide antigen. The alveolar surfactant biomimetic lipids are a combination of four lipids: phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and neutral structural lipids. The mass ratio of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and neutral structural lipids is (1-10):(1-5):(1-5):(1-5). The adjuvant lipids are monophosphatidyllipid A (MPLA), and the mass ratio of adjuvant lipids to alveolar surfactant biomimetic lipids is 0.15-0.5:

1. In step 3), the mass ratio of the mixed lipids with mucus penetration, cellular uptake, and immune activation functions to PLGA nanoparticles is (3-9):

1. The mucosal delivery carrier described above can simultaneously achieve mucus penetration, cellular uptake, and induce a robust mucosal immune response.

2. The preparation method according to claim 1, characterized in that, In step 1), the molecular weight of the PLGA is 4500-200000; the molar ratio of lactide and glycolide units in the PLGA used is (25-75):(75-25); the size of the formed PLGA nanoparticles is 50-500 nm; and the concentration of the PLGA nanoparticle suspension is 1-50 mg / mL.

3. The preparation method according to claim 1, characterized in that, The prepared multifunctional mucosal delivery carrier has a core-shell structure, with the shell being a mixed lipid layer and the core being PLGA nanoparticles loaded with a single antigen.

4. The preparation method according to claim 1, characterized in that, In step 1), the volume ratio of the organic phase to the aqueous phase in the preparation of the PLGA loaded particles is (1-50):1, the concentration of PLGA in the organic phase is 10-200 mg / mL, and the concentration of antigen in the aqueous phase is 1-50 mg / mL; the ultrasonic intensity used in the ultrasonic method is 10w-110w.

5. The preparation method according to claim 1, characterized in that, In step 1), the PLGA nanoparticles are prepared using a two-emulsion method, specifically as follows: PLGA is dissolved in dichloromethane to obtain an organic phase; an aqueous phase containing dissolved active antigen is added to the organic phase; the organic phase and the aqueous phase are mixed and a primary emulsion is prepared using an ultrasonic method; a surfactant solution is added to the primary emulsion and a secondary emulsion is prepared using an ultrasonic method; the secondary emulsion is stirred and the dichloromethane is evaporated to prepare PLGA nanoparticles loaded with antigen. The surfactant solution is selected from one or more aqueous solutions of polyvinyl alcohol, sodium cholate, Tween 20, Tween 80 and Prönnicke 127. The volume ratio of surfactant to primary emulsion is (1-20):

1. The concentration of surfactant solution is 0.1%-5%. The ultrasonic time is controlled at 1-20 min each time. The stirring temperature is 13-43°C and the time is 4-15 h.

6. The preparation method according to claim 1, characterized in that, The phosphatidylcholine is 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC); the phosphatidylglycerol is 1,2-palmitoylphosphatidylglycerol (DPPG); and the phosphatidylethanolamine is 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine. N [Methoxy(polyethylene glycol)] (DPPE-PEG); the neutral structural lipid is cholesterol.

7. The preparation method according to claim 6, characterized in that, 1,2-Dipalmitoyl-sn-glycerol-3-phosphate ethanolamine N The molecular weight of PEG in [methoxy(polyethylene glycol)] (DPPE-PEG) is 800-2000.

8. The preparation method according to claim 1, characterized in that, Step 2), the conditions for the rotary evaporation coating are: temperature 20-50 °C, vacuum degree 10-100 mbar, rotation speed 50-300 rpm; the coating container is a round-bottom flask or an eggplant-shaped flask with a volume of 5-50 mL.

9. The method for preparing the multifunctional mucosal delivery carrier of PLGA coated with a mixed lipid coating according to claim 1, characterized in that, In step 3), the ultrasonic intensity is 10w-50w and the ultrasonic time is 1-20min.

10. The preparation method according to claim 1, further comprising: The prepared multifunctional mucosal delivery carrier was dispersed in physiological saline or phosphate buffer for storage.

11. The preparation method according to claim 1, further comprising: The obtained multifunctional mucosal delivery carrier was prepared into a lyophilized dosage form. A lyophilization protectant was used during the preparation of the lyophilized dosage form. The lyophilization protectant was selected from one or more of trehalose, sucrose, lactose, and inulin. The lyophilization protectant was an aqueous solution with a mass concentration of 0.5%-20%. The multifunctional mucosal carrier material was a dispersion obtained by dispersing the multifunctional mucosal carrier material in a phosphate buffer solution with a concentration of 0.5-20 mg / mL. The volume ratio of the lyophilization protectant to the multifunctional mucosal delivery carrier material was (1-10):

1. The pre-freezing temperature was -20 °C to -80 °C, and the pre-freezing time was 2-15 h. The lyophilization temperature was -45 °C to -80 °C, and the lyophilization time was 2-72 h.

Images