Pickering emulsion adjuvant as well as preparation method and application thereof

Abstract

The invention discloses a Pickering emulsion adjuvant as well as a preparation method and application thereof, belongs to the technical field of immunology, and aims to solve the problems of safety and stability of a common emulsion in the prior art and the problems of mucous membrane residence and mucus penetration and capability of targeted activation of antigen presenting cells. The composition comprises an emulsifier, water, an antigen and an oil phase, the emulsifier is mucoprotein nanoparticles. The vaccine adjuvant is suitable for mucous membrane residence, and can achieve the effects of having excellent ion concentration characteristics, pH stability, temperature stability and storage stability, being capable of protecting the integrity of antigens, being capable of targeting antigen presenting cells, and being capable of residing at mucous membrane parts.

Description

Technical Field

[0001] This invention relates to the field of immunology, specifically to a Pickering emulsion adjuvant, its preparation method, and its application. More particularly, it relates to the preparation and application of a mucosal-resident Pickering emulsion adjuvant prepared using mucin nanoparticles that target antigen-presenting cells as an emulsifier. Background Technology

[0002] Infectious diseases have always been the biggest threat to human and animal health, especially diseases caused by emerging pathogens, which often pose enormous challenges to global health and severely impact human production and lives, sometimes even causing fatalities. Vaccines are recognized as the most economical and effective means of preventing infectious diseases. Adjuvants can reduce vaccine dosage, enhance immunogenicity, and effectively address vaccine production and supply issues. Existing adjuvants mainly include aluminum salts, emulsions, and To11-like receptor agonists. Although traditional aluminum adjuvants have been widely used in various vaccines, they only enhance humoral immunity and not cellular immunity, and their effect on subunit vaccines is limited. Other adjuvants, such as immunomodulators, oligonucleotides, polysaccharides, and cytokines, have not yet achieved clinical application. Therefore, exploring safe and effective new adjuvants remains a key focus of vaccinology research.

[0003] Emulsions have a certain application foundation as vaccine adjuvants. Currently, emulsion adjuvants such as MF59, AS02, AS03, and AFO3 have been approved for human use, making them the most widely used type of adjuvant besides aluminum adjuvants. Emulsions offer several advantages as adjuvants: First, they protect antigens; orally administered emulsion adjuvants exhibit good stability, protecting antigens from protease degradation. Second, they increase antigen surface area and regulate immune responses; the adsorption of antigens by emulsion droplets increases the antigen surface area, facilitating recognition by antigen-presenting cells, thus influencing the intensity and type of immune response. Droplet size, composition, and dispersion uniformity can all affect the adjuvant's effectiveness. Third, they provide sustained antigen release; the adsorption of nanoparticles promotes the slow release of antigens at the injection site, forming sustained immune protection. Fourth, they possess excellent physicochemical properties; emulsion particles are uniformly distributed, can be stored at low or normal temperatures, and their preparation methods are simple and low-cost, making them suitable for large-scale production. However, several issues regarding emulsion adjuvants remain to be addressed. For example, safety needs further improvement; traditional emulsions rely on surfactants to stabilize the oil-water interface, which may trigger strong side effects when injected into the body. Stability also needs improvement; emulsions are thermodynamically unstable and prone to demulsification under high temperature and pressure, affecting particle integrity and adjuvant efficacy, typically requiring sterilization only through filtration. The immune enhancement mechanism is not fully elucidated. While emulsion adjuvants are generally considered to be related to immune cell recruitment and antigen uptake, tending towards Th1-type immune responses and unrelated to TLRs, the deeper immune response mechanisms are still unclear. Specific dosages, immunization schedules, and the degree of antigen conservation are also unclear, making it difficult to measure their adjuvant effect on vaccines and their comparative effects with approved adjuvants.

[0004] Mucosal immunity plays a crucial and unique role in vaccine immunization. Mucosal immunity refers to the immune defense mechanism on the surface of the mucous membranes of the body's cavities that connect to the external environment; it is the first line of defense against pathogen invasion. The mucosa-associated lymphoid tissues of the respiratory, digestive, and genitourinary tracts constitute the mucosal immune system, which produces immune molecules such as secretory IgA (sIgA), exerting local immune effects on the mucosal surface and effectively preventing pathogens from adhering to and invading body tissues. Compared to traditional injectable immunization, mucosal immunity offers numerous advantages, such as eliminating the need for injections, reducing pain and infection risks, and improving recipient compliance; it can stimulate a systemic immune response in mucous membranes, providing broad-based protection; and for pathogens transmitted through the mucosal route, such as influenza viruses and enteroviruses, mucosal immunity provides more direct and effective defense.

[0005] However, mucosal immunity also has some drawbacks. Firstly, inducing mucosal immunity is challenging. The unique environment of the mucosal surface, such as the presence of the mucus layer, the action of digestive enzymes, and frequent microbial contact, makes it difficult for antigens to effectively reach the immune cells beneath the mucosa, thus affecting the initiation of the immune response. Secondly, the strength and persistence of the mucosal immune response are relatively weak. Compared to injectable immunization, mucosal immunization produces lower antibody levels and a shorter duration, which may lead to less than ideal protection against pathogens. Furthermore, the safety of mucosal immunization is a major concern. Some mucosal immunizing agents may cause local adverse reactions; for example, nasal mucosal immunization may cause nasal congestion and runny nose, while oral immunization may cause gastrointestinal discomfort.

[0006] Pickering emulsions are a special type of emulsion that uses solid particles to stabilize oil-water systems. Compared to ordinary emulsions, Pickering emulsions do not require the addition of surfactants, and the concentration of introduced solid particles is significantly lower than that of surfactants. Therefore, their toxicity to humans and the environment is far less than that of surfactants, potentially resulting in higher safety. Furthermore, the solid particles undergo irreversible adsorption at the oil-water interface, forming a robust interfacial film that prevents droplet aggregation. Thus, Pickering emulsion systems are less susceptible to external factors such as acidity / alkalinity, salt concentration, temperature, and oil phase composition, exhibiting greater stability. Existing technologies have constructed Pickering emulsions stabilized based on PLGA (polylactic acid-glycolic acid copolymer) particles, finding that their adjuvant effect is significantly superior to that of ordinary emulsions stabilized with traditional surfactants. Optimized Pickering emulsions can activate antigen-presenting cells and enhance antigen recruitment, effectively stimulating humoral and cellular immunity. However, current Pickering emulsions cannot specifically target and activate antigen-presenting cells.

[0007] Therefore, the rational design of a novel Pickering emulsion as a vaccine adjuvant to address the safety and stability issues of ordinary emulsions, as well as the problems of mucosal retention and mucus penetration, is of extremely important value and significance. Summary of the Invention

[0008] The purpose of this invention is to provide a Pickering emulsion adjuvant, its preparation method and application, to solve the safety and stability problems of ordinary emulsions, as well as the problems of mucosal retention and mucus penetration, and to target and activate antigen-presenting cells, so as to apply it to the vaccine field of the pharmaceutical industry.

[0009] To solve the above-mentioned technical problems, the present invention is implemented using the following technical solution:

[0010] On one hand, the present invention provides a Pickering emulsion adjuvant comprising an emulsifier, water, an antigen, and an oil phase; wherein the emulsifier is mucin nanoparticles.

[0011] In the above technical solution, the Pickering emulsion adjuvant prepared using mucin nanoparticles as emulsifier has good biocompatibility, excellent ion concentration characteristics, pH stability, temperature stability and storage stability. When encapsulating antigens, it can protect the integrity of the antigens, target antigen-presenting cells, and reside in mucosal sites, thus solving the problems of safety and stability of ordinary emulsions as well as the problems of mucosal retention and mucus penetration.

[0012] Furthermore, the Pickering emulsion has a droplet-like morphology;

[0013] The antigen is encapsulated within mucin nanoparticles, which are adsorbed onto the surface of Pickering emulsion adjuvant droplets; or, the antigen is adsorbed onto the surface of Pickering emulsion adjuvant droplets.

[0014] Optionally, the antigen is ovalbumin.

[0015] In the above technical solution, ovalbumin has stable properties and can induce repeatable adaptive immune responses, which can greatly assist in the development of antigen-targeted Pickering emulsion adjuvants.

[0016] Optionally, the particle size of the mucin nanoparticles is 100~1000 nm; the shape of the mucin nanoparticles includes one or both of spherical and rod-shaped.

[0017] In the above technical solution, the particle size of mucin nanoparticles is similar to that of viruses and bacteria, making them easily recognized and taken up by professional antigen-presenting cells.

[0018] Optionally, the particle size of the Pickering emulsion adjuvant is 1~100 μm.

[0019] Furthermore, the emulsifier accounts for 0.5% to 10% of the mass fraction of the Pickering emulsion adjuvant.

[0020] Optionally, the aqueous phase accounts for 20% to 80% of the volume of the Pickering emulsion adjuvant, and the oil phase accounts for 20% to 80% of the volume of the Pickering emulsion adjuvant.

[0021] Optionally, the oil phase is squalene.

[0022] In the above technical solution, squalene has good permeability and excellent oxidative stability and safety. As a raw material for emulsion adjuvants, the Pick emulsion adjuvant prepared has good targeted penetration performance.

[0023] In another aspect, the present invention provides a method for preparing Pickering emulsion adjuvant, comprising the following steps:

[0024] Mucin nanoparticles were added to water to bind with antigens to obtain antigen-presenting cell-targeted mucin nanoparticles; the antigen-presenting cell-targeted mucin nanoparticles were dispersed in water to obtain an aqueous phase; the aqueous phase was mixed with the oil phase and emulsified to obtain Pickering emulsion adjuvant.

[0025] Alternatively, it may include the following steps:

[0026] Mucin nanoparticles were dispersed in water to obtain an aqueous phase; the aqueous phase was mixed with an oil phase and emulsified to obtain an emulsion adjuvant; the emulsion adjuvant and antigen were placed in water and stirred to load the antigen to obtain Pickering emulsion adjuvant.

[0027] Furthermore, the mass ratio of the mucin nanoparticles to the antigen is (0.5~1.5):1.

[0028] Furthermore, the binding time between the mucin nanoparticles and the antigen in water is 2-24 hours;

[0029] The emulsion adjuvant and antigen are placed in water and stirred for 2 to 10 hours.

[0030] Optionally, the concentration of the aqueous solution of the antigen is 0.5~10 mg / ml.

[0031] Optionally, the emulsification is performed in a homogenizer or an ultrasonic machine; when performed in a homogenizer, the rotation speed is 9000 rpm to 13000 rpm and the homogenization time is 1 min to 3 min; when performed in an ultrasonic machine, the ultrasonic frequency is 60 to 120 Hz and the ultrasonic time is 1 min to 120 min.

[0032] Furthermore, the preparation of the mucin nanoparticles includes the following steps:

[0033] Mucin and proteolytic enzymes were added to a sample vial and stirred to obtain mucin nanospheres. The mucin nanospheres were then added to a calcium ion solution and stirred to induce the mucin nanospheres into mucin nanotubes. The obtained mucin nanotubes were freeze-dried to obtain nanoparticles.

[0034] Nanoparticles and mannose were added to water in a preset ratio, heated in a water bath, and the heated mixture was freeze-dried to obtain mucin nanoparticles.

[0035] In the above technical solution, mannose can target antigen-presenting cells. After preparing mannose-mucin nanoparticles from nanoparticles and mannose, the mucin nanoparticles have targeting function.

[0036] Optionally, the ratio of the mucin to the proteolytic enzyme is (0.5~1.5) g: 10 mL;

[0037] The stirring time of the mucin and the protein hydrolysate is 1.5~2.5h; the concentration range of the calcium ion solution is 0.05~0.15mol / L; the mass ratio of the mucin nanospheres to the calcium ion solution is (50~100):1; the stirring time of the mucin nanospheres and the calcium ion solution is 1.5~2.5h.

[0038] Furthermore, the mass ratio of the nanoparticles to mannose is 10:1 to 1:10.

[0039] Furthermore, the water bath heating temperature is 50~70℃, and the heating time is 1.5~2.5h.

[0040] Optionally, the mucin nanoparticles are stored at 0-4°C.

[0041] In another aspect, the present invention provides the application of Pickering emulsion adjuvant in the preparation of mucosal-residual vaccine adjuvants.

[0042] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0043] The mucin nanoparticles for targeting antigen-presenting cells prepared by the method provided by this invention, used as emulsifiers, can be used to prepare mucosal-resident Pickering emulsion adjuvants with good biocompatibility, excellent ion concentration characteristics, pH stability, temperature stability and storage stability, can protect the integrity of antigens, can target antigen-presenting cells, and can reside in mucosal sites.

[0044] The Pickering emulsion adjuvant provided by this invention can activate ovalbumin-specific peptides and histocompatibility complexes I and II in bone marrow-derived antigen-presenting cells, thereby activating co-cultured B cells and T cells. Furthermore, it can activate the immune system in mice via nasal drops. The Pickering emulsion adjuvant provided by this invention has the advantages of simple operation, safety, low cost, low energy consumption, and controllable operation, making it suitable for large-scale industrial production and processing. The obtained products have broad application prospects in the vaccine field. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of this disclosure 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 this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 This is a schematic diagram illustrating the characterization of the antigen-presenting cell-targeting mucin nanoparticles prepared in Example 1 of the present invention.

[0047] Figure 2This is a schematic diagram illustrating the characterization of mannose binding in the antigen-presenting cell-targeting mucin nanoparticles prepared in Example 1 of the present invention.

[0048] Figure 3 This is a schematic diagram illustrating the storage stability of the Pickering emulsion adjuvant prepared using mucin nanoparticles as an emulsifier, as obtained in Example 1 of the present invention.

[0049] Figure 4 This is a schematic diagram illustrating the characterization of the Pickering emulsion adjuvant prepared using mucin nanoparticles as an emulsifier, obtained in Example 1 of the present invention.

[0050] Figure 5 This is a schematic diagram of the antigen loading rate detection results in Example 1 of the present invention;

[0051] Figure 6 This is a schematic diagram of the cell viability detection results in Example 1 of the present invention;

[0052] Figure 7 This is a graph showing the mucosal retention detection results of Example 2 of the present invention;

[0053] Figure 8 This is a lysosomal escape diagram of Example 2 of the present invention;

[0054] Figure 9 This is a schematic diagram showing the antigen retention rate of Pickering emulsion adjuvant prepared using mucin nanoparticles as emulsifier in Example 3 of the present invention after storage for 7 days under different conditions.

[0055] Figure 10 The particle size distribution of the Pickering emulsion adjuvant prepared using mucin nanospheres as an emulsifier in Example 3 of this invention after storage for 7 days under different conditions is shown.

[0056] Figure 11 The particle size distribution of Pickering emulsion adjuvant prepared using mucin nanotubes as emulsifier in Example 3 of the present invention after storage for 7 days under different conditions;

[0057] Figure 12 This is a schematic diagram of the immune activation effect detection results in Example 4 of the present invention;

[0058] Figure 13 This is a schematic diagram of the detection results of the nasal mucosal dendritic cell activation effect in Example 5 of the present invention;

[0059] Figure 14 CD3 in nasal lymph node tissue on days 7, 14, and 21 of Example 6 of the present invention. + CD3 + CD4 + CD3 + CD8+ CD19 + CD27 + Percentage diagram;

[0060] Figure 15 CD3 in the spleen on the seventh, fourteenth, and twenty-first days of Example 6 of this invention. + CD3 + CD4 + CD3 + CD8 + CD19 + CD27 + Percentage diagram;

[0061] Figure 16 CD11c in nasal lymphoid tissue three days after the second immunization in Example 6 of this invention + CD80 + CD86 + MHC class I + MHC class II + Percentage diagram;

[0062] Figure 17 CD11c in the spleen three days after the second immunization in Example 6 of this invention + CD80 + CD86 + MHCclass I + MHC class II + Percentage diagram;

[0063] Figure 18 CD3 in the spleen three days after the second immunization in Example 6 of this invention + CD3 + CD4 + CD3 + CD8 + CD19 + CD27 + Percentage diagram;

[0064] Figure 19 This is a schematic diagram of the in vivo antibody activation effect detection results in Example 7 of the present invention; Detailed Implementation

[0065] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0066] It should be understood that all experimental procedures not detailed in the experiment are routine experimental procedures well known to those skilled in the art.

[0067] Example 1

[0068] This embodiment provides a method for preparing a Pickering emulsion adjuvant, comprising the following steps:

[0069] 1g of mucin and 10mL of proteolytic enzyme were added to a sample vial and stirred for 2h to obtain mucin nanospheres. Then, the mucin nanospheres were added to 180μL of 0.05mol / L calcium ion solution and stirred for 2h to induce them into mucin nanotubes. The obtained mucin nanotubes were freeze-dried to obtain mucin nanoparticles.

[0070] Mucin nanoparticles and mannose were mixed in an aqueous solution at a mass ratio of 10:1, heated in a water bath at 60°C for 2 h, and then freeze-dried to obtain mannose-mucin nanoparticles.

[0071] Mannose-mucin nanoparticles and ovalbumin (OVA, model antigen) were mixed at a mass ratio of 1:1 on a shaker. The antigen was then loaded into the mannose-mucin nanoparticles to obtain antigen-presenting cell-targeted mucin nanoparticles. These mucin nanoparticles included two particle size morphologies: antigen-presenting cell-targeted mucin nanospheres (NS) and antigen-presenting cell-targeted mucin nanotubes (NT). The characterization results of the mucin nanoparticles are as follows: Figure 1 As shown, the morphology of the two types of mucin nanoparticles, as determined by scanning electron microscopy, is as follows: Figure 1 As shown in Figure a, the particle sizes of the two types of mucin nanoparticles, as determined by a nanolaser particle size analyzer, are as follows: Figure 1 As shown in b, the zeta potentials of the two types of mucin nanoparticles, measured by a zeta potential meter, are as follows: Figure 1 As shown in c. The characterization results of the binding of mucin nanoparticles to mannose are as follows. Figure 2 As shown in the figure, the spectral results of the binding of two types of mucin nanoparticles to mannose, measured by ultraviolet spectrophotometer, are as follows: Figure 2 a and Figure 2 As shown in b, the grafting rates of the two types of mucin nanoparticles with mannose are as follows:Figure 2 As shown in Figure c, the appearance of the two types of mucin nanoparticles bound to mannose was determined by fluorescence microscopy. Figure 2 As shown in d. The loading efficiency of the two mucin nanoparticles with the antigen was determined by an enzyme-linked immunosorbent assay (ELISA) reader, and the results are as follows. Figure 5 As shown in a.

[0072] The storage conditions for antigen-presenting cell-targeted mucin nanoparticles are 4°C.

[0073] The preparation of Pickering emulsion adjuvants using mucin nanoparticles targeting antigen-presenting cells as emulsifiers includes the following steps:

[0074] The prepared antigen-presenting cell-targeting mucin nanoparticles were dispersed in water as the aqueous phase.

[0075] Squalene was used as the oil phase;

[0076] The aqueous and oil phases were mixed and homogenized at 13,000 rpm for 1 min using a high-speed disperser to obtain a mucosal-residing Pickering emulsion adjuvant containing mucin nanoparticles targeting antigen-presenting cells. Based on the particle size and morphology of the prepared mucin nanoparticles, two types of Pickering emulsion adjuvants were prepared: one using mucin nanospheres as emulsifiers (NS Pickering emulsion, also known as NS Pickering load OVA, NSMPO) and the other using mucin nanotubes as emulsifiers (NT Pickering emulsion, also known as NT Pickering load OVA, NSMPO). Both are collectively referred to as Pickering emulsion adjuvants prepared using mucin nanoparticles as emulsifiers. The loading efficiency of the two Pickering emulsion adjuvants with antigen was determined using an enzyme-linked immunosorbent assay (ELISA) reader, and the results are as follows: Figure 5 As shown in b.

[0077] In this embodiment, the mass percentage of mucin nanoparticles targeting antigen-presenting cells in the aqueous phase is 0.5%wt. In other embodiments, it may also be set to 1%wt, 2%wt, or 3%wt.

[0078] The squalene content in the total emulsion system is 30% by volume, and in other embodiments, it can also be set to 40%, 50%, 60%, or 70%.

[0079] Pickering emulsion adjuvants, prepared using antigen-presenting cell-targeting mucin nanoparticles with different aqueous and oil phases as emulsifiers, were bottled and subjected to storage stability experiments at 4°C. Photos were taken on the first and thirtieth days. The stability results are shown below. Figure 3 As shown, Figure 3a represents the stability result of NSMPO. Figure 3 b represents the stability result of NTMPO.

[0080] A 1 μL sample of the Pickering emulsion adjuvant prepared using the prepared antigen-presenting cell-targeting mucin nanoparticles as an emulsifier was observed under an optical microscope. The characterization results are as follows: Figure 4 As shown in Figure a, mucin nanoparticles targeting antigen-presenting cells were stained with fluorescent dyes and observed under a fluorescence microscope. The characterization results are as follows. Figure 4 As shown in b.

[0081] Mouse bone marrow cells were extracted and induced to become mouse bone marrow-derived dendritic cells (BMDCs) using community-stimulating factor (CSGF) and interleukin-4 (IL-4). Pickering emulsion adjuvants, prepared using antigen-presenting cell-targeting mucin nanoparticles as an emulsifier, were added to the BMDCs at concentrations of 0.1, 0.3, 0.5, 0.7, 1, 3, 5, 7, 10, and 20 μL, and cultured for 24 h. Cell viability was then measured. The cell viability of the two Pickering emulsion adjuvants on BMDCs was determined using a microplate reader. The results are shown below. Figure 6 As shown, Figure 6 a represents the NSMPO determination result. Figure 6 b represents the NTMPO determination result.

[0082] The results show that the mucosal-residing Pickering emulsion adjuvant prepared by using the antigen-presenting cell-targeting mucosal protein obtained in the embodiments of the present invention as an emulsifier exhibits a stable emulsion system.

[0083] Example 2

[0084] This embodiment provides a method and results for detecting mucosal retention and lysosomal escape of the Pickering emulsion adjuvant prepared in Example 1.

[0085] The Pickering emulsion adjuvant prepared in Example 1 was stained with Nile Red, and 5 μL was dripped into the nasal cavity of mice. The mice were photographed using a small animal in vivo imaging system at 0 h and 24 h, and the fluorescence intensity was counted.

[0086] Mucosal retention assays were randomly divided into three groups: the MF59 group (fluorescence detection was performed using the existing emulsion adjuvant MF59); the NSMPO group (the morphology of the mucin nanoparticles in the Pickering emulsion was mucin nanospheres); and the NTMPO group (the morphology of the mucin nanoparticles in the Pickering emulsion was mucin nanotubes). The mucosal retention assay results are as follows: Figure 7 As shown, Figure 7 a is a live image of a small animal. Figure 7b is a fluorescence statistical graph of in vivo imaging of small animals.

[0087] Mouse bone marrow cells were extracted and induced to become mouse bone marrow-derived dendritic cells using community-stimulating factor and interleukin-4. The antigen contained in the Pickering emulsion adjuvant prepared by targeting antigen-presenting cells with mucin nanoparticles as emulsifier was stained with FITC (fluorescein isothiocyanate) and lysosomes in BMDC were stained. The images were taken using a fluorescence scanning confocal microscope.

[0088] The experiment was randomly divided into four groups: the Ctr (control) group (Pickering emulsion adjuvant was replaced with PBS); the OVA group (Pickering emulsion adjuvant was replaced with OVA); the NSMPO group (Pickering emulsion adjuvant prepared with mucin nanospheres as emulsifier); and the NTMPO group (Pickering emulsion adjuvant prepared with mucin nanotubes as emulsifier). Immunosome escape detection results are as follows: Figure 8 As shown.

[0089] The results showed that the Pickering emulsion adjuvant prepared by using the antigen-presenting cell-targeting mucin nanoparticles obtained in the embodiments of the present invention as an emulsifier could remain on the nasal mucosa for 24 hours and still exhibit fluorescence, and as... Figure 8 This can protect antigens from phagocytosis by lysosomes.

[0090] Example 3

[0091] This embodiment provides a method and results for testing the stability of the Pickering emulsion adjuvant prepared in Example 1.

[0092] Pickering emulsion adjuvant was placed in water with ion concentrations of 100, 300, 500, 700, and 1000 mmol / L, pH values ​​of 1, 5.5, 6.9, and 7.4, and temperatures of 4, 25, and 37°C, respectively. Particle size and OVA retention after seven days were measured, and stability was determined using an enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figures 9-11 As shown, the antigen retention rates of Pickering emulsion adjuvants after 7 days of storage under different pH conditions are as follows: Figure 9 As shown in Figure a, the antigen retention rates under different ion concentration conditions are as follows: Figure 9 As shown in b, the antigen retention rates after 7 days of storage under different temperature conditions are as follows: Figure 9 As shown in c; the particle size changes of the two Pickering emulsion adjuvants after 7 days of storage under different pH conditions are shown in the figure. Figure 10 a, Figure 11As shown in Figure a, the particle size changes after 7 days of storage under different ion concentrations are as follows: Figure 10 b、 Figure 11 As shown in b, the particle size changes after 7 days of storage under different temperature conditions are as follows: Figure 10 c. Figure 11 As shown in c.

[0093] The results show that the mucosal-residing Pickering emulsion adjuvant prepared by the antigen-presenting cell-targeting mucin nanoparticles prepared in the embodiments of the present invention as emulsifiers has good antigen protection rate and stable particle size under different ion concentrations, pH and temperature conditions. Therefore, the mucosal-residing Pickering emulsion adjuvant prepared by the antigen-presenting cell-targeting mucin nanoparticles as emulsifiers has good ion concentration, pH and temperature stability and biosafety.

[0094] Example 4

[0095] This embodiment provides a method for detecting the immune activation effect of the Pickering emulsion adjuvant prepared in Example 1 and the results thereof:

[0096] The Pickering emulsion adjuvant prepared in Example 1 was added to induced mouse bone marrow-derived dendritic cells and cultured for 24 h. The cells were then co-cultured with mouse spleen cells, and flow cytometry was performed after 48 h.

[0097] The experiment was randomly divided into 8 groups: Ctr group: Pickering emulsion adjuvant was replaced with PBS; OVA group: Pickering emulsion adjuvant was replaced with OVA; NSMP group: Pickering emulsion adjuvant prepared without antigen loading was used, and the morphology of the mucin nanoparticles was mucin nanospheres; NSPO group: Pickering emulsion adjuvant prepared without mannose binding was used, and the morphology of the mucin nanoparticles was mucin nanospheres; NSMP Group O: Pickering emulsion adjuvant prepared using mucin nanospheres as emulsifier was used; Group NTMP: Pickering emulsion adjuvant prepared without antigen loading was used, and the morphology of the mucin nanoparticles was mucin nanotubes; Group NTPO: Pickering emulsion adjuvant prepared without mannose binding was used, and the morphology of the mucin nanoparticles was mucin nanotubes; Group NTMPO: Pickering emulsion adjuvant prepared using mucin nanotubes as emulsifier was used.

[0098] Cells from each group were collected separately and stained using flow cytometry. The results of the immune activation effect assay are as follows: Figure 12 As shown, where Figure 12 a is CD80 + Percentage in BMDC, 12b for SINFKEL + The percentage in BMDC, 12c is MHC class I + The percentage in BMDC, 12d for MHC class II + The percentage in BMDC, 12e is CD8 T + The percentage of lymphocytes, 12f is the percentage of activated B cells in lymphocytes.

[0099] After antigens are presented by dendritic cells (DCs), early memory B cells (CD19)... + CD27 + ) and CD8 T cells (CD8 + It has a good activation effect, and the results are as follows: Figure 12 As shown. This experiment validated in vitro that Pickering emulsion adjuvant prepared using antigen-presenting cell-targeting mucin nanoparticles as emulsifiers can enhance humoral immunity (early memory B cells and T cells) through DC cell activation.

[0100] The results show that the Pickering emulsion adjuvant prepared by using the antigen-presenting cell-targeting mucin nanoparticles provided by the present invention as an emulsifier has a good immune activation effect on mouse-derived dendritic cells.

[0101] Example 5

[0102] A method and results for detecting the nasal mucosal dendritic cell activation effect of the Pickering emulsion adjuvant prepared in Example 1;

[0103] The mucosal-residing Pickering emulsion adjuvant prepared by using the antigen-presenting cell-targeting mucin nanoparticles obtained in Example 1 as an emulsifier was dripped into the nasal cavity of mice. The nasal mucosa of mice was extracted on the first and third days, and the dendritic cells in the nasal mucosa of mice were stained with flow cytometry antibodies.

[0104] Results of nasal mucosal dendritic cell activation assay: Figure 13 As shown, where Figure 13 a is CD11c + Percentage in nasal lymphoid tissue Figure 13 b is CD80 + CD11c + Percentage in nasal lymphoid tissue Figure 13 c is cDC1+ Percentage in nasal lymphoid tissue Figure 13 d is cDC2 + The percentage of antigen-presenting cell-targeting mucin nanoparticles in nasal lymphoid tissue indicated that Pickering emulsion adjuvants prepared using mucin nanoparticles as emulsifiers could significantly activate dendritic cells in mouse nasal mucosa.

[0105] Example 6

[0106] A method and results for detecting the in vivo immune activation effect of the Pickering emulsion adjuvant prepared in Example 1:

[0107] Pickering emulsion adjuvant, prepared using the antigen-presenting cell-targeting mucin nanoparticles obtained in Example 1 as an emulsifier, was dripped into the nasal cavity of mice. On the seventh day, fourteenth day, twenty-first day, and three days after the second immunization, nasal mucosa, nasal lymphoid tissue, and spleen of mice were extracted. B cells and T cells in the nasal mucosa, nasal lymphoid tissue, and spleen of mice were stained with flow cytometry antibodies.

[0108] Results of in vivo immune activation effect test: Figures 14-18 As shown, CD3 levels were found in the nasal lymph nodes and spleen on days 7, 14, and 21. + Percentage diagram as follows Figure 14 As shown in a and 15a, CD3 + CD4 + Percentage diagram as follows Figure 14 As shown in b and 15b, CD3 + CD8 + Percentage diagram as follows Figure 14 As shown in c and 15c, CD19 + CD27 + Percentage diagram as follows Figure 14 As shown in d and 15d; CD11c levels in nasal lymphoid tissue and spleen three days after secondary immunization. + Percentage diagram as follows Figure 16 As shown in a and 17a, CD80 + CD86 + Percentage diagram as follows Figure 16 As shown in b and 17b, MHC class I + Percentage diagram as follows Figure 16 As shown in c and 17c, MHC class II + Percentage diagram as follows Figure 16 As shown in d and 17d; CD3 in the spleen three days after secondary immunization + Percentage diagram as follows Figure 18 As shown in a, CD3 + CD4+ Percentage diagram as follows Figure 18 As shown in b, CD3 + CD8 + Percentage diagram as follows Figure 18 As shown in c, CD19 + CD27 + Percentage diagram as follows Figure 18 As shown in d. The results indicate that mucosal-resident Pickering emulsions prepared using antigen-presenting cell-targeting mucin nanoparticles as emulsifiers can significantly enhance the activation of the immune system in mice.

[0109] Example 7

[0110] A method and results for detecting the in vivo antibody activation effect of the Pickering emulsion adjuvant prepared in Example 1:

[0111] Pickering emulsion adjuvant, prepared using the antigen-presenting cell-targeting mucin obtained in Example 1 as an emulsifier, was administered into the nasal cavity of mice. Serum and nasal wash were extracted from mice on day 14, day 21, and day 3 after the second immunization, and the antibody concentration was detected using enzyme-linked immunosorbent assay (ELISA).

[0112] Results of antibody activation effect test in vivo: Figure 19 As shown, where Figure 19 Figure a shows the production of IgG1 in serum by using OVA as an antigen-vaccine adjuvant. Figure 19 Figure b shows the production of IgG2a in serum from OVA as an antigen-vaccine adjuvant. Figure 19 c shows the IgG1 produced by nasal wash solution using OVA as an antigen-vaccine adjuvant. Figure 19 Figure d shows the IgG2a produced by nasal wash OVA as an antigen vaccine adjuvant. Pickering emulsion adjuvant prepared by mucin nanoparticles targeting antigen-presenting cells as emulsifier can significantly increase antibody levels in mice.

Claims

1. A Pickering emulsion adjuvant, characterized in that, Includes emulsifiers, water, antigens, and oil phase; The emulsifier is mucin nanoparticles.

2. The Pickering emulsion adjuvant according to claim 1, characterized in that, The Pickering emulsion has a droplet-like morphology; The antigen is encapsulated within mucin nanoparticles, which are adsorbed onto the surface of Pickering emulsion adjuvant droplets; or, the antigen is adsorbed onto the surface of Pickering emulsion adjuvant droplets.

3. The Pickering emulsion adjuvant according to claim 1, characterized in that, The emulsifier accounts for 0.5% to 10% of the mass fraction of the Pickering emulsion adjuvant.

4. A method for preparing the Pickering emulsion adjuvant according to any one of claims 1-3, characterized in that, Includes the following steps: Mucin nanoparticles were added to water to bind with antigens to obtain antigen-presenting cell-targeted mucin nanoparticles; the antigen-presenting cell-targeted mucin nanoparticles were dispersed in water to obtain an aqueous phase; the aqueous phase was mixed with the oil phase and emulsified to obtain Pickering emulsion adjuvant. Alternatively, it may include the following steps: Mucin nanoparticles were dispersed in water to obtain an aqueous phase; the aqueous phase was mixed with an oil phase and emulsified to obtain an emulsion adjuvant; the emulsion adjuvant and antigen were placed in water and stirred to load the antigen to obtain Pickering emulsion adjuvant.

5. The method for preparing Pickering emulsion adjuvant according to claim 4, characterized in that, The mass ratio of the mucin nanoparticles to the antigen is (0.5~1.5):

1.

6. The method for preparing Pickering emulsion adjuvant according to claim 4, characterized in that, The binding time between the mucin nanoparticles and the antigen in water is 2-24 hours. The emulsion adjuvant and antigen are placed in water and stirred for 2 to 10 hours.

7. The method for preparing Pickering emulsion adjuvant according to claim 4, characterized in that, The preparation of the mucin nanoparticles includes the following steps: Mucin and proteolytic enzymes were added to a sample vial and stirred to obtain mucin nanospheres. The mucin nanospheres were then added to a calcium ion solution and stirred to induce the mucin nanospheres into mucin nanotubes. The obtained mucin nanotubes were freeze-dried to obtain nanoparticles. Nanoparticles and mannose were added to water in a preset ratio, heated in a water bath, and the heated mixture was freeze-dried to obtain mucin nanoparticles.

8. The method for preparing Pickering emulsion adjuvant according to claim 7, characterized in that, The mass ratio of the nanoparticles to mannose is 10:1 to 1:

10.

9. The method for preparing Pickering emulsion adjuvant according to claim 7, characterized in that, The water bath heating temperature is 50~70℃, and the heating time is 1.5~2.5h.

10. The use of the Pickering emulsion adjuvant according to claims 1-3 in the preparation of mucosal-residual vaccine adjuvants.

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