Emulsifier for preparing Pickering emulsion, Pickering emulsion as well as preparation method and application of Pickering emulsion

Abstract

The invention discloses an emulsifier for preparing a Pickering emulsion, the Pickering emulsion as well as a preparation method and application of the Pickering emulsion. The emulsifier of the Pickering emulsion is obtained by dissolving distearoyl phosphatidylcholine, cholesterol and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 in ethanol, carrying out rotary evaporation to remove ethanol, then adding an antigen aqueous solution containing an antigen, continuing rotary evaporation to obtain liposome nanoparticles, heating the liposome nanoparticles with mannose in a water bath, and carrying out freeze drying. The Pickering emulsion is obtained by dispersing an emulsifier in water as a water phase, taking squalene as an oil phase, mixing the water phase and the oil phase, and homogenizing. The Pickering emulsion disclosed by the invention is prepared by taking antigen presenting cell targeted liposome nanoparticles as a raw material; and the antigen has excellent ion concentration characteristic, pH stability, temperature stability and storage stability, and can protect the integrity of the antigen.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology, in particular to an emulsifier for preparing a Pickering emulsion, a Pickering emulsion, a preparation method and application thereof. BACKGROUND

[0002] Infectious diseases have always been the biggest factor threatening human health, especially diseases caused by new pathogens often bring great challenges to global health, and have a serious impact on human life and even fatal blows. Vaccines are recognized as the most economical and effective means to prevent infectious diseases. Adjuvants can reduce the dosage of vaccines and enhance immunogenicity. The previously approved adjuvants mainly include aluminum salts, emulsions, Toll-like receptor agonists, etc. Although the traditional aluminum adjuvant has been widely used in various vaccines, it can only enhance humoral immunity and has limited effect on subunit vaccines. Other adjuvants such as liposomes, immunomodulators, oligonucleotides, polysaccharides, and cytokines have not yet achieved clinical application, so exploring safe and efficient new adjuvants is still the focus of vaccine research.

[0003] Emulsions as vaccine adjuvants have a certain application basis, and emulsion adjuvants such as MF59, AS02, AS03, and AFO3 have been approved for human use and are the most widely used adjuvant type after aluminum adjuvants. Emulsions as adjuvants have the following characteristics: (1) protection of antigens Oral emulsion adjuvants can improve the stability of antigens in the gastrointestinal tract and protect antigens from protease degradation; when immunized through the nose, it can quickly enhance antigen migration and avoid long-term residence in the nose and degradation; (2) increase the surface area of antigens and regulate immune responses Emulsion droplets adsorbed antigens can increase the surface area of antigens, which can be recognized by antigen-presenting cells and affect the intensity and type of immune response, so the size, composition, and uniformity of droplets may be factors affecting the effect of adjuvants; (3) slow release of antigens Nanoparticle adsorption can promote the slow release of antigens at the injection site, forming sustained immune protection. The applicant's latest research found that when nanometer emulsion adjuvants carry antigens in different ways, the release speed of antigens is quite different, which leads to different effects of adjuvants, proving that the adjuvant effect of emulsion is closely related to the slow release of antigens; (4) excellent physical and chemical properties Emulsion particles are uniformly distributed and can be stored at low or room temperature; combined with simple preparation method and low cost, it is suitable for large-scale production, so it is considered to be an excellent vaccine adjuvant. However, the following problems still need to be solved:

[0004] 1. Safety needs to be further improved, traditional emulsions must rely on surfactants to stabilize the oil-water interface, which may cause strong side effects when injected into the body as adjuvants, so suitable substitutes need to be found to minimize the amount of surfactant used.

[0005] 2. Stability needs to be further improved, emulsion is prone to different program variable demulsification at high temperature and high pressure due to its thermodynamic instability, thereby affecting the particle integrity and adjuvant effect, and usually only filtered to sterilize. 3. The immune enhancement mechanism is not elucidated, and current research shows that emulsion adjuvants are usually related to immune cell recruitment and antigen uptake, and tend to Th1 type immune response, and are not related to TLR, but the deep immune response mechanism is unknown. 4. The specific dose, immunization program and the degree of antigen saving are not clear, and it is difficult to measure the extent to which the emulsion adjuvant assists the vaccine and whether it is better than the currently approved adjuvant. Therefore, it is of great practical significance to provide an emulsion system that can avoid the use of surfactants and stabilize the oil-water interface, and design it into a new adjuvant.

[0006] Pickering emulsion refers to a special emulsion stabilized by solid particles. Compared with ordinary emulsion, Pickering does not need to add surfactant, and the concentration of solid particles introduced is much lower than the amount of surfactant, and the toxic effect on human body and environment is much smaller than that of surfactant, so the safety may be higher; in addition, the solid particles are irreversibly adsorbed on the oil-water interface to form a firm interface film, which can prevent droplet coalescence, so the Pickering emulsion system is not easily affected by external acid-base, salt concentration, temperature and oil phase composition, and has stronger stability. Xia et al. constructed a Pickering emulsion stabilized by PLGA particles, and found that the adjuvant effect was obviously better than that of ordinary emulsion adjuvant stabilized by traditional surfactant, and the optimized Pickering emulsion could activate antigen presenting cells and enhance antigen recruitment, and effectively stimulate humoral and cellular immunity, but the current Pickering emulsion cannot target the activation of antigen presenting cells.

[0007] Therefore, it is of great value and significance to rationally design a new Pickering emulsion as a vaccine adjuvant to solve the safety and stability problems of ordinary emulsion. SUMMARY

[0008] In order to solve the problems in the prior art, the present application aims to provide an emulsifier for preparing Pickering emulsion, Pickering emulsion, its preparation method and application. The Pickering emulsion provided by the present application has good stability and significantly enhanced immune adjuvant effect, and high safety.

[0009] The technical scheme of the present application is as follows:

[0010] In a first aspect, the present application provides an emulsifier for preparing a Pickering emulsion, the emulsifier being an antigen-presenting cell-targeted liposome nanoparticle, which is prepared by the following method: dissolving distearoylphosphatidylcholine, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in ethanol, removing ethanol by rotary evaporation, then adding an aqueous antigen solution containing an antigen, continuing rotary evaporation to obtain a liposome nanoparticle, and then heating the liposome nanoparticle with mannose in a water bath, and freeze-drying to obtain the emulsifier.

[0011] Further, the molar ratio of the distearoylphosphatidylcholine, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 is 2:1:0.192;

[0012] And / or, the concentration of the aqueous antigen solution is 5-100 μg;

[0013] And / or, the mass ratio of the liposome nanoparticle to mannose is 10:1-1:1;

[0014] And / or, the particle size of the antigen-presenting cell-targeted liposome nanoparticle is 100-1000 nm.

[0015] Further, the antigen includes but is not limited to ovalbumin.

[0016] Further, the rotary evaporation time is 2-10 h;

[0017] And / or, the water bath heating temperature is 60-65°C, and the time is 2-2.5 h.

[0018] In a second aspect, the present application provides a preparation method of a Pickering emulsion, the preparation method comprising: dispersing the emulsifier in water as an aqueous phase, using squalene as an oil phase, mixing the aqueous phase with the oil phase, and obtaining a Pickering emulsion after homogenization.

[0019] Further, the particle size of the Pickering emulsion is 2-10 μm.

[0020] Further, the volume content of water in the emulsion is 25%-75%, and the volume content of squalene in the emulsion is 75%-25%;

[0021] Preferably, the volume content of water in the emulsion is 30%-70%, and the volume content of squalene in the emulsion is 70%-30%;

[0022] And / or, the mass percentage of the antigen-presenting cell-targeted liposome nanoparticle in the aqueous phase is 0.5%-3%.

[0023] Further, the rotation speed is 9000 rpm-13000 rpm and the homogenization time is 1 min-3 min.

[0024] In a third aspect, the present application provides the Pickering emulsion prepared by the preparation method.

[0025] In a fourth aspect, the present application provides the use of the Pickering emulsion in any one of the following:

[0026] 1) preparing a vaccine adjuvant;

[0027] 2) preparing a medicine for preventing and / or treating bacterial infection;

[0028] 3) preparing a medicine for preventing and / or treating tumor;

[0029] 4) preparing a medicine for preventing and / or treating viral infection.

[0030] Compared with the prior art, the present application can achieve the following technical effects:

[0031] The Pickering emulsion provided by the present application is prepared by using antigen-presenting cell-targeted liposome nanoparticles as raw materials, and the antigen-presenting cell-targeted liposome nanoparticles have a small particle size and are simple to prepare. The Pickering emulsion prepared by the present application has good biocompatibility, excellent ion concentration characteristics, pH stability, temperature stability and storage stability, and can protect the integrity of antigens.

[0032] The Pickering emulsion provided by the present application can activate the ovalbumin-specific peptide segment and the histocompatibility complex I and II of the antigen-presenting cells derived from bone marrow, thereby activating the co-cultured B cells and T cells, and can activate the immune system of mice through intraperitoneal injection. The adjuvant provided by the present application has the advantages of simple operation, safety, low cost, low energy consumption, controllable operation, and is suitable for large-scale industrial production and processing. The obtained product has a broad application prospect in the field of vaccines. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Figure 1 is a characterization diagram of the antigen-presenting cell-targeted liposome prepared in Example 1 of the present application;

[0034] Figure 2 Figure 2 is a diagram of the stability and biological safety of the Pickering emulsion adjuvant prepared by using the antigen-presenting cell-targeted liposome nanoparticles prepared in Example 2 of the present application as an emulsifying agent;

[0035] Figure 3Figure of the immune activation effect of the Pickering emulsion adjuvant prepared by taking the antigen-presenting cell-targeted liposome nanoparticle of the present application embodiment 3 as the emulsifier on the dendritic cells;

[0036] Figure 4 Figure of the activation effect of the Pickering emulsion adjuvant prepared by taking the antigen-presenting cell-targeted liposome nanoparticle of the present application embodiment 4 as the emulsifier on the co-cultured B cells and T cells;

[0037] Figure 5 Figure of the activation effect of the Pickering emulsion adjuvant prepared by taking the antigen-presenting cell-targeted liposome nanoparticle of the present application embodiment 5 as the emulsifier on the mouse immune system. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the following described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0039] It should be understood that the experimental operations not described in detail in the experiment are all conventional experimental operations well known to those skilled in the art.

[0040] Embodiment 1

[0041] The present embodiment provides a preparation method of an antigen-presenting cell-targeted liposome, specifically as follows:

[0042] 1. Preparation of PEG-liposome: PEG-liposome is prepared by taking distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol (2000) (2:1:0.192, molar ratio) as raw materials.

[0043] Distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol (2000) are dissolved in anhydrous ethanol in proportion, and stirred in a 60℃ water bath until clear and transparent. Then the solution is transferred to a 250mL round-bottom flask, and the organic solvent is removed by a rotary evaporator under the condition of a 60℃ water bath. After a uniform film is formed on the wall of the flask, the rotary evaporation is continued for 30min. Then the mixture is hydrated in a 8℃ water bath with 0.9mL 0.10% sucrose and 4mM lactic acid buffer (pH 0.60) containing 100μg of ova, and the crude dispersed liposome can be obtained after the mixture is hydrated for about 30min. The obtained liposome is further homogenized on a high-pressure homogenizer (or a membrane extruder) for 3min. Finally, all the liposomes are stored at 4℃.

[0044] 2. Preparation of antigen-presenting cell-targeted liposomes: PEG-liposomes and mannose are mixed in an aqueous solution at a mass ratio of 10:1, heated in a water bath at 60 DEG C for 2 h, and then freeze-dried to obtain antigen-presenting cell-targeted liposomes.

[0045] The appearance morphology (A), particle size (B), zeta potential (C), mannose binding (D), and grafting rate of mannose (E) of the obtained antigen-presenting cell-targeted liposomes are measured by a scanning electron microscope, a nano-laser particle size analyzer, a zeta potential meter, a fluorescence microscope, and an enzyme-labeled instrument. Figure 1 A), particle size (B), zeta potential (C), mannose binding (D), and grafting rate of mannose (E) of the obtained antigen-presenting cell-targeted liposomes are measured by a scanning electron microscope, a nano-laser particle size analyzer, a zeta potential meter, a fluorescence microscope, and an enzyme-labeled instrument. Figure 1 Figure 1 C), mannose binding (D), and grafting rate of mannose (E) of the obtained antigen-presenting cell-targeted liposomes are measured by a scanning electron microscope, a nano-laser particle size analyzer, a zeta potential meter, a fluorescence microscope, and an enzyme-labeled instrument. Figure 1 Figure 1

[0046] The results show that the antigen-presenting cell-targeted liposomes prepared in the embodiment of the application exhibit a stable nano system.

[0047] Example 2

[0048] In this embodiment, the prepared antigen-presenting cell-targeted liposomes are used as emulsifiers to prepare Pickering emulsion adjuvants, and the stability and biological safety of the Pickering emulsion adjuvants are determined.

[0049] The preparation method of the Pickering emulsion adjuvant specifically includes: dispersing the antigen-presenting cell-targeted liposomes prepared in Example 1 in water as an aqueous phase; using squalene as an oil phase; mixing the aqueous phase and the oil phase, and homogenizing (13000 rpm, 1 min) by a high-speed disperser to obtain a Pickering emulsion containing the antigen-presenting cell-targeted liposomes.

[0050] In this embodiment, the antigen-presenting cell-targeted liposomes with different aqueous phases and oil phases are prepared as emulsifiers to prepare Pickering emulsion adjuvants. The mass percentage of the antigen-presenting cell-targeted liposomes in the aqueous phase is 0.5%wt, 1%wt, 2%wt, and 3%wt, respectively. The volume content of squalene in the total emulsion system is 30%, 40%, 50%, 60%, and 70%, respectively.

[0051] The Pickering emulsion adjuvants prepared by using the prepared antigen-presenting cell-targeted liposomes with different aqueous phases and oil phases as emulsifiers are loaded into a Schlenk flask, and a storage stability experiment is performed at 4 DEG C. Photographs are taken on the first day and the thirtieth day, and the results are shown in Figure 2 A. 1 μL of the prepared Pickering emulsion adjuvant prepared by using the antigen-presenting cell-targeted liposomes as emulsifiers is placed under an optical microscope for observation, and the results are shown in Figure 2 ​​​The antigen-presenting cell-targeted liposome prepared in Example 2 was used as an emulsifier to prepare a Pickering emulsion adjuvant, which was added to the induced mouse bone marrow-derived dendritic cells, and the cells were cultured for 24 h, and then the SIINFEKL, MHC class I and MHC class II of the cells were stained using a flow cytometer, and the results are shown in Figs. Figure 2 The mouse bone marrow cells were extracted, induced into mouse bone marrow-derived dendritic cells using colony stimulating factor and interleukin 4, and the prepared antigen-presenting cell-targeted liposome nanoparticles were added to the mouse bone marrow-derived dendritic cells as an emulsifier to prepare a Pickering emulsion adjuvant in an amount of 0.1, 0.3, 0.5, 0.7, 1, 3, 5, 7, 10, 20 μL, and the cells were cultured for 24 h, and then the cell activity was determined, and the results are shown in Fig. Figure 2 The emulsion was placed in an environment with an ion concentration of 100, 300, 500, 700, 1000 mM, a pH of 1, 5.5, 6.9, 7.4, and a temperature of 4, 25, 37°C, and the particle size was determined, the appearance was photographed, and the microstructure was photographed using an optical microscope, and the results are shown in Figs. Figure 2 E, F, G, H, L, I, G, K, L, M.

[0052] The results show that the antigen-presenting cell-targeted liposome nanoparticles prepared in the embodiment of the application as an emulsifier to prepare a Pickering emulsion adjuvant have good ion concentration, pH, temperature stability and biological safety.

[0053] Example 3

[0054] The antigen-presenting cell-targeted liposome prepared in Example 2 was used as an emulsifier to prepare a Pickering emulsion adjuvant, which was added to the induced mouse bone marrow-derived dendritic cells, and the cells were cultured for 24 h, and then the SIINFEKL, MHC class I and MHC class II of the cells were stained using a flow cytometer, and the results are shown in Figs.

[0055] The antigen-presenting cell-targeted liposome prepared in Example 2 was used as an emulsifier to prepare a Pickering emulsion adjuvant, which was added to the induced mouse bone marrow-derived dendritic cells, and the cells were cultured for 24 h, and then the SIINFEKL, MHC class I and MHC class II of the cells were stained using a flow cytometer, and the results are shown in Figs. Figure 3 A, B, C, D, E, F.

[0056] The results show that the antigen-presenting cell-targeted liposome prepared in the embodiment of the application as an emulsifier to prepare a Pickering emulsion adjuvant has good immune activation effect on mouse-derived dendritic cells.

[0057] Example 4

[0058] The antigen-presenting cell-targeted liposome prepared in Example 2 was used as an emulsifier to prepare a Pickering emulsion adjuvant, which was added to the induced mouse bone marrow-derived dendritic cells, and the cells were cultured for 24 h, and then the SIINFEKL, MHC class I and MHC class II of the cells were stained using a flow cytometer, and the results are shown in Figs.

[0059] The Pickering emulsion adjuvant prepared by the antigen-presenting cell-targeted liposome prepared in Example 2 as an emulsifying agent is added to the induced mouse bone marrow-derived dendritic cells, incubated for 24 h, and co-cultured with mouse spleen cells, and then flow cytometry is performed after 48 h. The results show that, after the antigen is presented by the DC cells, the early memory B cells (CD19+CD27+) and CD4 T and CD8 T cells (CD3+CD4+, CD3+CD8+) have a good activation effect. Figure 3 The experiment in vitro verifies that the Pickering emulsion adjuvant prepared by the antigen-presenting cell-targeted liposome as an emulsifying agent can mediate more significant humoral immunity (early memory B cells and T cells) through the enhancement of DC cell activation.

[0060] Example 5

[0061] In this embodiment, the in vivo immune activation effect of the Pickering emulsion adjuvant prepared by the antigen-presenting cell-targeted liposome as an emulsifying agent is detected, and the specific process is as follows:

[0062] The Pickering emulsion adjuvant prepared by the antigen-presenting cell-targeted liposome prepared in Example 2 as an emulsifying agent is loaded with ovalbumin and injected into the mouse body through intraperitoneal injection, the mouse spleen is extracted on the seventh day, the dendritic cells in the mouse spleen are stained by flow cytometry antibodies, the mouse spleen and lymph nodes are extracted on the fourteenth day, and the spleen and lymph node cells are stained by flow cytometry antibodies, and the results are shown in Figure 5 .

[0063] The results are shown in Figure 5 , and the Pickering emulsion adjuvant prepared by the antigen-presenting cell-targeted liposome as an emulsifying agent can significantly improve the antigen presentation effect (SIINFEKL+) and immune activation (MHC-class I+ / MHC-class II+) of the mouse immune system.

[0064] In summary, the Pickering emulsion adjuvant prepared by the antigen-presenting cell-targeted liposome as an emulsifying agent has a uniform appearance, good ion concentration, pH, temperature stability and biological safety. The immune activation experiments in Examples 3 and 4 show that the Pickering emulsion adjuvant prepared by the antigen-presenting cell-targeted liposome as an emulsifying agent can better activate the immune response, and the Pickering emulsion adjuvant prepared by the antigen-presenting cell-targeted liposome as an emulsifying agent provided in the present application has a strong immune activation effect, especially T cell activation, which is of great significance for improving the efficacy of the adjuvant.

[0065] In the above embodiments, the description of each embodiment has its own focus, and the parts not described in detail in a certain embodiment can be referred to the relevant description of other embodiments. The above is a specific implementation of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of various equivalent modifications or replacements within the technical range disclosed by the present application, and these modifications or replacements should be covered within the protection scope of the present application.

Claims

1. An emulsifier for preparing Pickering emulsions, characterized in that, The emulsifier is an antigen-presenting cell-targeting liposome nanoparticle, which is prepared by the following method: distearylphosphatidylcholine, cholesterol, and distearylphosphatidylethanolamine-polyethylene glycol 2000 are dissolved in ethanol, the ethanol is removed by rotary evaporation, an antigen aqueous solution containing the antigen is added, and rotary evaporation is continued to obtain liposome nanoparticles. Then, the liposome nanoparticles are heated with mannose in a water bath and freeze-dried to obtain the emulsifier.

2. The emulsifier according to claim 1, characterized in that, The molar ratio of distearylphosphatidylcholine, cholesterol, distearylphosphatidylethanolamine-polyethylene glycol 2000 is 2:1:0.192; And / or, the concentration of the aqueous antigen solution is 5-100 μg; And / or, the mass ratio of the liposome nanoparticles to mannose is 10:1 to 1:1; And / or, the particle size of the antigen-presenting cell-targeted liposome nanoparticles is 100-1000 nm.

3. The emulsifier according to claim 1, characterized in that, The antigens include, but are not limited to, ovalbumin.

4. The emulsifier according to claim 1, characterized in that, The rotary evaporation time is 2-10 hours; And / or, the water bath heating temperature is 60-65℃, and the time is 2-2.5h.

5. A method for preparing a Pickering emulsion, characterized in that, The preparation method includes: dispersing the emulsifier of claim 1 in water as an aqueous phase, using squalene as an oil phase, mixing the aqueous phase and the oil phase, and obtaining a Pickering emulsion after homogenization.

6. The preparation method according to claim 5, characterized in that, The Pickering emulsion has a particle size of 2-10 μm.

7. The preparation method according to claim 5, characterized in that, The water content of the emulsion is 25%-75% by volume, and the squalene content of the emulsion is 75%-25% by volume. Preferably, the water accounts for 30%-70% of the volume of the emulsion, and the squalene accounts for 70%-30% of the volume of the emulsion; And / or, the mass percentage of the antigen-presenting cell-targeting liposome nanoparticles in the aqueous phase is 0.5%-3%.

8. The preparation method according to claim 5, characterized in that, During homogenization, the rotation speed is 9000rpm-13000rpm, and the homogenization time is 1min-3min.

9. The Pickering emulsion prepared by the preparation method according to any one of claims 5-8.

10. The use of the Pickering emulsion of claim 4 in any of the following: 1) Preparation of vaccine adjuvants; 2) To prepare drugs for the prevention and / or treatment of bacterial infections; 3) To prepare drugs for the prevention and / or treatment of tumors; 4) Prepare drugs for the prevention and / or treatment of viral infections.

Images