CN113616786B

CN113616786B - Pickering emulsion, preparation method thereof and application thereof in preparing vaccine immunoadjuvant

Info

Publication number: CN113616786B
Application number: CN202110551382.7A
Authority: CN
Inventors: 赵兰华; 李忠玉; 陈虹亮; 舒明艺
Original assignee: University of South China
Current assignee: University of South China
Priority date: 2021-05-20
Filing date: 2021-05-20
Publication date: 2023-08-25
Anticipated expiration: 2041-05-20
Also published as: CN113616786A

Abstract

The invention relates to the technical field of immunology, in particular to a Pickering emulsion, a preparation method thereof and application thereof as a vaccine immunoadjuvant. The GO-SQPickering emulsion prepared by the invention shows good stability and obviously enhanced adjuvant effect through stability test and in vitro evaluation; the method takes the CtpORF5 recombinant protein vaccine as a model antigen, establishes a mouse genital tract anti-Ct infection model, evaluates the immune protection and safety of the GO-SQPickering adjuvant on organisms, and results show that the GO-SQPickering emulsion provided by the invention can obviously enhance the humoral immunity and the cellular immunity of mice and has higher safety. Therefore, the method has the potential value of enhancing immune response and improving vaccine immunity.

Description

Pickering emulsion, preparation method thereof and application thereof in preparing vaccine immunoadjuvant

Technical Field

The invention relates to the technical field of immunology, in particular to a Pickering emulsion, a preparation method thereof and application thereof in preparing vaccine immunoadjuvant.

Background

Infectious diseases are always the greatest factor threatening human health, and especially diseases caused by novel pathogens often bring great challenges to the global health care industry, bring serious effects to human production and life and even fatal attacks. The number of dead injuries caused by influenza, plague and the like in history is the number of feelings of eye-touch and convulsion, and the new type of coronavirus pneumonia outbreaks again arouse the human. Vaccines are once again becoming a popular focus as the most cost effective means of preventing infectious diseases. The adjuvant can reduce vaccine dosage, enhance immunogenicity, and effectively solve vaccine yield and supply problems during epidemic outbreak. Adjuvants which have been approved for use at present mainly include aluminum salts, emulsions, toll-like receptor agonists, and the like. Although the traditional aluminum adjuvant is widely applied to various vaccines, the traditional aluminum adjuvant can only enhance humoral immunity but not enhance cellular immunity and has limited effect on subunit vaccines, other adjuvants such as liposome, immune regulator, oligonucleotide, polysaccharide, cytokine and the like are not yet applied clinically at present, so the exploration of safe and efficient novel adjuvants is still the key point of vaccine research.

Emulsion (Emulsion) has a certain application foundation AS vaccine adjuvants, and Emulsion adjuvants such AS MF59, AS02, AS03, AF03 and the like are approved for human bodies at present, so that the Emulsion adjuvants are the most widely applied adjuvant types besides aluminum adjuvants. The emulsion has the following characteristics as an adjuvant: (1) The antigen oral emulsion adjuvant can improve the stability of the antigen in the gastrointestinal tract and protect the antigen from protease degradation; the antigen migration can be quickly enhanced during nasal cavity immunization, and the degradation caused by long-time stay in the nasal cavity is avoided; (2) The surface area of the antigen is increased, and the antigen can be increased by regulating the adsorption of the immune response emulsion drops, so that the immune response emulsion drops are recognized by antigen presenting cells and influence the intensity and type of immune response, so that the size, the composition components, the dispersion uniformity and the like of the drops are possibly factors influencing the effect of the adjuvant; (3) The adsorption of the slow-release antigen nano particles can promote the slow release of the antigen at the injection site to form continuous immune protection, and the latest research of the applicant finds that when the nano emulsion adjuvant carries the antigen in different modes, the difference of the antigen release speeds is larger, so that the adjuvant effects are greatly different, and the adjuvant effect of the emulsion is proved to be closely related to the slow release of the antigen; (4) The emulsion with excellent physical and chemical properties has uniform particle distribution and can be stored at low temperature or normal temperature; in addition, the preparation method is simple, has lower cost and is suitable for mass production, thus being considered as an excellent vaccine adjuvant. There are still several problems to be solved:

1. the safety is to be further improved, the traditional emulsion must rely on a surfactant to stabilize an oil-water interface, and the traditional emulsion is used for injecting an adjuvant into an organism to possibly cause stronger side reactions, so that a proper substitute is required to be searched, and the use amount of the surfactant is reduced as much as possible. 2. The stability is to be further improved, emulsion is easy to break to different degrees at high temperature and high pressure due to thermodynamic instability, thus affecting particle integrity and adjuvant effect, and generally only can be sterilized by filtration. 3. The mechanism of immune enhancement is not elucidated, and the emulsion adjuvant is generally related to immune cell recruitment and antigen uptake, and tends to Th1 type immune response, is irrelevant to TLR, but the mechanism of deep immune response is unknown. 4. The specific dosage, immunization program and antigen saving levels are not clear, and it is difficult to measure how much the adjuvant of the emulsion adjuvant has to assist the vaccine, and whether the effect is better than that of the currently approved adjuvants. Therefore, the emulsion system which can avoid the use of a surfactant and stabilize the oil-water interface is provided, and the emulsion system is designed into a novel adjuvant, so that the emulsion system has important practical significance.

Pickering emulsion refers to a special emulsion which adopts solid particles to stabilize an oil-water system. Compared with common emulsion, pickering does not need to add a surfactant, the concentration of introduced solid particles is greatly lower than the dosage of the surfactant, and the toxic action on human bodies and the environment is far lower than that of the surfactant, so that the safety is possibly higher; in addition, the solid particles are irreversibly adsorbed on the oil-water interface, and the formed interface film is firm and can prevent liquid drops from coalescing, so that the Pickering emulsion system is not easily influenced by external acid and alkali, salt concentration, temperature and oil phase composition, and has stronger stability. Xia and the like construct Pickering emulsion stabilized based on PLGA particles, the adjuvant effect of the Pickering emulsion is found to be obviously better than that of a common emulsion adjuvant stabilized by a traditional surfactant, and the optimized Pickering emulsion can activate antigen presenting cells and enhance antigen recruitment, so that humoral and cellular immunity is effectively stimulated.

Chlamydia trachomatis causes infections of the eye or genitalia, the major complications of which include blinding trachoma and reproductive dysfunction, such as urethritis, cervicitis and salpingitis, with more than 1.3 million new cases registered each year according to the latest estimates issued by the world health organization. Resulting in reproductive sequelae such as Pelvic Inflammatory Disease (PID), premature birth and obstructive infertility. It is estimated that 40% -60% of PID cases and 30% of ectopic pregnancy are caused by chlamydia trachomatis infection, causing a huge socioeconomic burden on human healthcare, and that since monitoring in 2001, the reported number of Ct pathologies has exceeded gonorrhea, leading to 8 sexually transmitted pathogens. Therefore, the novel Pickering emulsion is reasonably designed to be used as the Ct vaccine adjuvant to solve the safety and stability problems of the common emulsion, and has important value and significance.

Disclosure of Invention

In view of the above, the invention provides a Pickering emulsion, a preparation method thereof and application thereof as a vaccine immunoadjuvant. The Pickering emulsion provided by the invention has good stability, obviously enhanced immune adjuvant effect and high safety.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of a pickering emulsion, which comprises the following steps:

mixing graphene oxide with water to obtain a water phase;

taking Squalene (SQ) as an oil phase, mixing the oil phase and the water phase, and performing ultrasonic treatment to obtain the pickering emulsion.

In some embodiments, the mass ratio of the aqueous phase to the oil phase is 10: (1-2). In some embodiments, the mass ratio of the aqueous phase to the oil phase is

In some embodiments, the concentration of graphene oxide in the aqueous phase is 1 to 3mg/mL. In some embodiments, the concentration of graphene oxide is 1mg/mL.

In the invention, the thickness of the graphene oxide is 1-3nm, and the sheet diameter is less than 5 mu m. In some embodiments, the graphene oxide may have a sheet diameter of < 15nm, 15nm to 200nm, or 0.5 μm to 5 μm. The source of graphene oxide is not particularly limited, and it can be obtained commercially or can be obtained by a conventional method in the art, such as Hammer method.

In some embodiments, the ultrasound is 5 s-10 s ultrasound per 5 s-15 s interval, the power of the ultrasound is 300-400 w, and the total time of the ultrasound is 5 min-15 min. In some specific embodiments, the ultrasound is 10s ultrasound per 10s interval, the power of the ultrasound is 325w, and the total time of the ultrasound is 10min.

The invention also provides the Pickering emulsion prepared by the preparation method.

Wherein the average particle size of the pickering emulsion is 1049-3239 nm.

The invention also provides application of the pickering emulsion in preparation of vaccine immunoadjuvant.

The vaccine comprises an attenuated/inactivated vaccine, a recombinant protein vaccine or a nucleic acid vaccine.

In some embodiments, the vaccine is a chlamydia trachomatis vaccine. Some embodimentsIn an example, the Chlamydia trachomatis vaccine is specifically _p ORF5 recombinant protein vaccine.

The GO-Pickering emulsion prepared by the invention shows good stability and obviously enhanced adjuvant effect through stability test and in vitro evaluation; the method takes the CtpORF5 recombinant protein vaccine as a model antigen, establishes a mouse genital tract anti-Ct infection model, evaluates the immune protection and safety of the GO-Pickering adjuvant on organisms, and shows that the GO-Pickering emulsion provided by the invention can obviously enhance the humoral immunity of mice and has higher safety.

1. The average particle size of the GO-Pickering emulsion is 1049-3239 nm, wherein the quasi-two-dimensional structure and the huge specific surface area of GO are favorable for adsorbing antigen, slowing down the release of antigen and promoting the recognition and presentation of antigen.

The GO surface is provided with a large number of oxygen-containing functional groups, so that different sheets can be mutually repelled, and the dispersibility in a water-oil system is good; meanwhile, most of the functional groups have hydrophilicity, so that the functional groups are easily combined to an oil emulsion interface to prevent liquid drops from being aggregated, and therefore the GO-Pickering emulsion system is not easily influenced by factors such as pH value, salt concentration, temperature, oil phase composition and the like, can be kept stable after being injected into an organism, and can realize slow release of antigens.

The titer of the GO-SQ Pickering adjuvant for stimulating the antibody production of mice is more than 10 times that of the individual vaccine group, and the cytokine production level is significantly higher than that of the aluminum adjuvant group and the individual vaccine group. Therefore, the compound can be proved to be capable of assisting in inducing mice to generate high-level humoral immunity and cellular immunity at the same time, so that organisms generate good immunity protection, and the adjuvant effect is obviously stronger than that of an aluminum adjuvant.

4. In the GO-Pickering emulsion system, the concentration of GO is far smaller than the dosage of the surfactant, so that the biotoxicity can be reduced to a great extent, and the safety problem of the traditional emulsion adjuvant can be effectively solved.

Drawings

FIG. 1 shows a graph of the morphology observations of the GO-SQ Pickering emulsions of examples 1-3; 1-a are GO-SQ Pickering emulsions from left to right illustrating example 3, example 1 and example 2; 1-b is GO dispersion (sheet diameter 0.5-5 μm) of 1 mg/ml;

FIG. 2 shows microscopic observations of the GO-SQ Pickering emulsions of examples 1-3, 2-a shows the GO-SQ Pickering emulsion of example 2 (water/oil ratio 10:1), 2-b shows the GO-SQ Pickering emulsion of example 1 (water/oil ratio 10:2), 2-c shows the GO-SQ Pickering emulsion of example 3 (water/oil ratio 10:4), 400×;

FIG. 3 is a graph showing the results of particle size and uniformity measurements for the Markov particle sizer of examples 1-3 GO-SQ Pickering emulsions; 3-a shows the GO-SQ Pickering emulsion of example 2, 3-b shows the GO-SQ Pickering emulsion of example 1, and 3-c shows the GO-SQ Pickering emulsion of example 3;

FIG. 4 shows the results of fluorescence analysis of the antigen slow release adsorption of the GO-SQ Pickering emulsion of example 1, wherein 4-a is the observation under fluorescence excitation and 4-b is the observation under bright field;

FIG. 5 shows the results of detection of lgG antibody levels in mice immunized with the GO-SQ Pickering emulsion of example 1 as an immunoadjuvant to the pORF5 vaccine; wherein 5-a is the antibody level of IgG in the body fluid of the mice at weeks 4, 6, 8; 5-b is the antibody level of each group IgG, igG1, igG2a at week 8; 5-c is the antibody level of mouse IgG, igG1, igG2a at different adjuvant doses;

FIG. 6 shows the results of a cellular immunization experiment using the GO-SQ Pickering emulsion of example 1 as an immunoadjuvant to the pORF5 vaccine, with 6-a to 6-c being the expression levels of cytokines IFN-gamma, IL-2 and IL-10 in that order;

FIG. 7 shows the morphology observations of GO-SQ Pickering (10:2 in water-oil ratio) in example 9, wherein the left graph shows the state after 6 months of standing and the right graph shows the state after overnight standing;

FIG. 8 shows the morphology observation results of different plate diameters GO-SQ Pickering, and the results of GO plate diameter particle diameters smaller than 15nm, 15 nm-200 nm and more than 500nm are shown in sequence from 8-a to 8-c; 8-a to 8-c, the results of the water phase/oil phase ratios of 10:1, 10:2 and 10:4 are sequentially from left to right;

FIG. 9 shows microscopic observations of different sheet diameters GO-SQ Pickering, from left to right, at water phase/oil phase ratios of 10:1, 10:2, 10:4;

FIG. 10 shows the results of GO-SQ Pickering safety evaluation.

Detailed Description

The invention provides a Pickering emulsion, a preparation method thereof and application thereof as a vaccine immunoadjuvant. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 preparation of GO-SQ Pickering emulsions of the invention

Mixing Graphene Oxide (GO, with a sheet diameter of 0.5-5 μm) with water to obtain a water phase with a Graphene Oxide concentration of 1 mg/mL;

taking Squalene (SQ) as an oil phase, mixing the water phase and the oil phase according to the mass ratio of 10:2, performing ultrasonic treatment for 10s at 325w power, performing ultrasonic treatment for 10s at intervals, and performing ultrasonic treatment for 10min repeatedly to obtain the GO-SQ Pickering emulsion. The morphology observation result is shown in fig. 1-a, the microscope observation result is shown in fig. 2-b, (400×), the particle size and uniformity detection result by a Markov particle sizer is shown in fig. 3-b, and the embedding rate of GO-SQ Pickering to BSA is determined to be 43%.

EXAMPLE 2 preparation of GO-SQ Pickering emulsions of the invention

Mixing graphene oxide (with a sheet diameter of 0.5-5 mu m) with water to obtain a water phase with the graphene oxide concentration of 1 mg/mL;

taking squalene as an oil phase, mixing the water phase and the oil phase according to the ratio of 10:1, performing ultrasonic treatment for 10s at 325w power, performing ultrasonic treatment repeatedly at intervals of 10s, and performing ultrasonic treatment for 10min to obtain the GO-SQ Pickering emulsion. The morphology is shown in FIG. 1-a, the microscope is shown in FIG. 2-a, (400X), and the particle size and uniformity measurements are shown in FIG. 3-a.

EXAMPLE 3 preparation of GO-SQ Pickering emulsions of the invention

mixing the water phase and the oil phase according to the ratio of 10:4 by taking squalene as the oil phase, performing ultrasonic treatment for 10s at 325w power, performing ultrasonic treatment repeatedly at intervals of 10s, and performing ultrasonic treatment for 10min to obtain the GO-SQ Pickering emulsion.

Example 4 testing and characterization of GO-SQ Pickering emulsions

The morphology observation results of the GO-SQ pickering emulsions of examples 1-3 are shown in FIG. 1, the observation results of a microscope are shown in FIG. 2, and the detection results of the particle size and uniformity of the Markov particle sizer are shown in FIG. 3;

as can be seen from the results in fig. 1, the upper layer is a Pickering emulsion, the lower layer is an unbound GO aqueous dispersion, and the lower layer is lighter in color than the original GO dispersion, indicating that GO sheets have entered the Pickering system. As the oil phase decreases, the upper layer Pickering emulsion also becomes less and the lower layer has a clear difference in transparency.

From FIGS. 2 to 3, it can be seen that the GO-SQ Pickering emulsion prepared in example 1 has smaller and more uniform particle size.

Example 5GO-SQ Pickering antigen adsorption mounting assay

Antigen adsorption experiments: GO-SQpickering was mixed on ice with FITC-BSA for 4h (mixing ratio was consistent with animal experiments, 1ml pickering emulsion contained 500ug BSA), and the emulsion was taken under fluorescent microscope and observed as shown in FIG. 4.

The result shows that the protein is adsorbed on the surface of the GO-SQ Pickering emulsion liquid drop, and the antigenicity of the GO-SQ Pickering emulsion liquid drop can be enhanced by carrying the vaccine in an adsorption mode, so that the GO-SQ Pickering emulsion liquid drop is easy to recognize and present by an immune system.

Example 6 humoral immunity detection

Each female Balb/c mouse was injected with 0.1ml of GO-SQ Pickering emulsion containing 50. Mu.g of pORF5 protein, and immunized three times at 0, 2, 4 weeks with the individual pORF5 protein and aluminum adjuvant group as controls. The antibody levels of IgG were determined by indirect ELISA using mice serum from weeks 4, 6 and 8 (FIG. 5-a), and the antibody levels of each group of IgG, igG1 and IgG2a from week 8 (FIG. 5-b) were compared with the antibody levels of mice IgG, igG1 and IgG2a from different adjuvant doses (FIG. 5-c), and the results are shown in Table 5.

From the above results, the IgG antibodies of the mice in each group changed with the immunization time, and it can be seen that the mice antibodies in each group basically tended to increase with the increase of the immunization times, which indicates that the recombinant protein vaccine has good immunogenicity, the IgG antibodies in the GO-SQ Pickering adjuvant group are higher than those in the individual vaccine group and the physiological saline group, and the IgG antibody titer is about 10-100 times that of the individual vaccine group. Therefore, the GO-SQ Pickering adjuvant group can obviously enhance the humoral immunity of mice.

IgG and its subtype IgG1, igG2a were detected in the serum of each group of mice at week 8 after priming, i.e. at week 4 after three priming, and the GO-SQ Pickering adjuvant group of mice were found to be higher than the individual vaccine (p > 0.05) for IgG1, igG2a, and the data showed that the IgG antibody titer was more than 10 times that of the individual vaccine group. The GO-SQ Pickering adjuvant can enhance humoral immunity, and the effect is basically equivalent to that of an aluminum adjuvant group. The average value of IgG1 was higher for each group of mice than for IgG2a, from which it could be roughly deduced that each group of adjuvants elicited a Th 1-type-biased immune response with the pORF5 recombinant protein vaccine.

The adjuvant has no obvious difference from the original level of IgG produced by the adjuvant after 5-fold dilution, so that the 5-fold dilution Pickering adjuvant can be used for subsequent experiments based on safety and cost.

Example 7 cytokine detection

(1) Collecting spleen lymphocytes of the mice after the last immunization for 2 weeks, adding 3ml of erythrocyte lysate, fully blowing and mixing, standing at room temperature for 5 minutes, adding 6ml of incomplete 1640 culture medium for stopping, and centrifuging at 4 ℃ for 5 minutes at 1000 g; discarding the supernatant, adding 6ml Hanks solution (1:100) containing double antibody (penicillin + streptomycin), fully blowing and mixing uniformly, and centrifuging at 4 ℃ for 5min at 1000 g;

(2) Taking 1ml of spleen lymphocyte suspension with the density of 2X 106/ml into a 24-hole culture plate, adding 10 mu g of pORF5 protein for stimulating each hole, fully mixing, and placing in a 5% CO2 incubator at 37 ℃ for 48 hours;

(3) After the stimulation is completed, fully blowing and beating uniformly mixed cells, transferring the uniformly mixed cells to an EP tube, centrifuging for 10min at 4 ℃ and 1200g, collecting supernatant, and preserving the supernatant in a refrigerator at-20 ℃;

the expression level of cytokines (IFN-. Gamma., IL-10, IL-2) in the culture supernatant was examined according to the kit instructions. The results are shown in FIG. 6.

The results showed IFN-. Gamma.: the GO-SQ Pickering adjuvant stimulated the body to produce high levels of IFN-gamma, significantly higher than the vaccine alone and the aluminum adjuvant vaccine, essentially equivalent to the GO group (see FIG. 6-a).

IL-2: GO-SQPickering has a remarkable stimulatory effect, and can stimulate the body to produce high levels of IL-2, the average value of which is about 4 times that of the individual vaccine group, and is remarkably higher than that of the GO group (see FIG. 6-b).

IL-10: there was no obvious difference in the comparison of GO-SQ Pickering with the individual vaccine groups, and the GO-SQ Pickering group produced a lower immunosuppressive factor IL-10 than the aluminum adjuvant group, indicating that the immune effect of GO-SQ Pickering was significantly stronger than that of the aluminum adjuvant (see FIG. 6-c).

Comparison of the cytokine levels shows that the GO-SQ Pickering adjuvant stimulates the organism to generate good cellular immunity, and the effect is stronger than that of the GO group and the aluminum adjuvant group.

Example 8 Properties of different sheet paths GO-SQ Pickering

The morphology observation results of the GO-SQ Pickering prepared by using GO with different sheet diameters are shown in FIG. 8, wherein the preparation method is the same as that of example 1; the microscopic observation results are shown in FIG. 9.

The result shows that the GO-SQ Pickering formed by the GO with the plate diameter smaller than 15nm is milky (9-a), the Pickering emulsion gradually presents a slightly brown color along with the increase of the GO plate diameter (9-b shows that the GO plate diameter is 50-200nm,9-c shows that the GO plate diameter is greater than 500 nm), and the proportion of the Pickering emulsion at the upper layer gradually increases along with the increase of the oil phase. Microscopic observation revealed that emulsion droplet size increased with increasing oil phase and with increasing GO sheet diameter.

Example 9 stability test

(1) Centrifugal test

10000g centrifuging for 10min, and dripping to float on the upper layer without obvious change in size

(2) High temperature testing

Placing in boiling water bath for 20min, without change in appearance of emulsion, and without change in particle size and uniformity

(3) Freeze-drying test

After vacuum freeze drying, shake and mix in water, the particle size is unchanged.

(4) Long-term stability observations

After the ultrasonic treatment, GO still does not completely enter the emulsion layer, the lower layer is brown, and after standing for 6 months, GO is dispersed into the upper layer Pickering emulsion, so that the lower layer is lighter in color and even is basically completely clear, but the volume and the morphology of the upper layer emulsion are not obviously changed but the color is darker, as shown in fig. 7.

Conclusion: the GO-SQ Pickering has good stability and is convenient for long-term storage in special environments as the result of centrifugation, high temperature, freeze-drying and long-term stability observation.

Example 10 safety evaluation

Balb/c mice (average body weight 30 g) were fasted for 8h, and each mouse was intraperitoneally injected with 0.6ml of each of examples 1-3 GO-SQ Pickering (maximum administration), and the active state was observed, and the mice were found to be normal without death, and after 7 days, the mice were dissected to observe the viscera and the distribution of GO was observed. The results of example 1GO-SQ Pickering are shown in FIG. 10.

The result shows that after the Pickering emulsion is injected into the abdominal cavity at a high dosage, the heart, the liver and the lung have no obvious appearance change, unabsorbed black GO is still remained at the large intestine, the Pickering emulsion which is wrapped by the capsule block and cannot be absorbed and metabolized in time is formed under the skin of the mouse, the damage caused by the fact that the Pickering emulsion runs in the abdominal cavity organ is avoided, and on the other hand, a slow release effect can be formed. The pickering emulsion greatly reduces the usage amount of GO, avoids the formation of excessive GO on the metabolism of organisms, and reduces the preparation cost while having high safety.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

Application of Pickering emulsion in preparing vaccine;

the vaccine is chlamydia trachomatis vaccine;

the preparation method of the pickering emulsion comprises the following steps:

mixing graphene oxide with water to obtain a water phase;

taking squalene as an oil phase, mixing the water phase with the oil phase, and performing ultrasonic treatment to obtain a pickering emulsion;

the mass ratio of the water phase to the oil phase is 10: (1-2);

in the water phase, the concentration of graphene oxide is 1-3 mg/mL;

the thickness of the graphene oxide is 1-3nm, and the sheet diameter is smaller than 5 mu m;

the ultrasonic is carried out for 5s to 10s at intervals of 5s to 15s, the ultrasonic power is 300 w to 400w, and the total ultrasonic time is 5min to 15min;

the average particle size of the Pickering emulsion obtained by the preparation method is 1049-3239 nm.
2. The use according to claim 1, wherein the vaccine is an attenuated/inactivated vaccine, a recombinant protein vaccine or a nucleic acid vaccine.