阅读说明：本技术 一种pickering乳液及其制备方法和在制备疫苗免疫佐剂中的应用 (Pickering emulsion, preparation method thereof and application thereof in preparation of vaccine immunologic adjuvant ) 是由 赵兰华 李忠玉 陈虹亮 舒明艺 于 2021-05-20 设计创作，主要内容包括：本发明涉及免疫学技术领域,尤其涉及一种pickering乳液及其制备方法和作为疫苗免疫佐剂的应用。本发明制备的GO-SQPickering乳液经稳定性测试和体外评估,显示出良好的稳定性和明显增强的佐剂效果；以CtpORF5重组蛋白疫苗为模式抗原,建立小鼠生殖道抗Ct感染模型,评估了GO-SQPickering佐剂对机体的免疫保护力和安全性,结果显示,本发明提供的GO-SQPickering乳液能够显著增强小鼠体液免疫及细胞免疫,且安全性较高。因此,具有增强免疫反应,提高疫苗免疫保护力的潜在价值。(The invention relates to the technical field of immunology, in particular to pickering emulsion, a preparation method thereof and application of pickering emulsion as a vaccine immunologic adjuvant. The GO-SQPickering emulsion prepared by the invention shows good stability and obviously enhanced adjuvant effect through stability test and in-vitro evaluation; the CtpORF5 recombinant protein vaccine is used as a model antigen to establish a mouse genital tract anti-Ct infection model, and the immune protection and safety of the GO-SQPickering adjuvant on organisms are evaluated, and the result shows that the GO-SQPickering emulsion provided by the invention can obviously enhance the humoral immunity and cellular immunity of mice and has higher safety. Therefore, the vaccine has potential value of enhancing immune response and improving immune protection of the vaccine.)

1. A preparation method of pickering emulsion is characterized by comprising the following steps:

mixing graphene oxide with water to obtain a water phase;

and (3) taking squalene as an oil phase, mixing the water phase and the oil phase, and performing ultrasonic treatment to obtain the pickering emulsion.

2. The method according to claim 1, wherein the mass ratio of the water phase to the oil phase is 10: (1-4).

3. The preparation method according to claim 1, wherein the concentration of graphene oxide in the aqueous phase is 1-3 mg/mL.

4. The preparation method according to claim 1, wherein the graphene oxide has a thickness of 1 to 3nm and a sheet diameter of less than 5 μm.

5. The preparation method according to claim 1, wherein the ultrasound is performed for 5s to 10s every 5s to 15s, the ultrasound power is 300 to 400w, and the total ultrasound time is 5min to 15 min.

6. The pickering emulsion prepared by the preparation method of any one of claims 1 to 5.

7. The pickering emulsion according to claim 6, wherein the pickering emulsion has an average particle size of 1049-3239 nm.

8. Use of the pickering emulsion prepared by the preparation method of any one of claims 1 to 5 or the pickering emulsion of claim 6 or 7 in the preparation of a vaccine.

9. Use according to claim 8, wherein the vaccine is an attenuated/inactivated vaccine, a recombinant protein vaccine or a nucleic acid vaccine.

10. The use according to claim 8, wherein the vaccine is a Chlamydia trachomatis vaccine.

Technical Field

The invention relates to the technical field of immunology, in particular to pickering emulsion, a preparation method thereof and application thereof in preparation of vaccine immunologic adjuvants.

Background

Infectious diseases are always the biggest threat to human health, and especially diseases caused by novel pathogens often bring huge challenges to the global health cause and serious influences or even fatal attacks to human production and life. Historically, the number of injuries from death caused by influenza, plague, etc. was eye-surprised, and the outbreak of this novel coronavirus pneumonia was once again awakened by humans. Vaccines are again the focus of the public as the accepted most cost effective means of preventing infectious diseases. The adjuvant can reduce the dosage of the vaccine, enhance the immunogenicity, and effectively deal with the problems of the yield and the supply of the vaccine during the outbreak of the epidemic situation. The adjuvants which are approved to be used at present mainly comprise 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, cannot enhance cellular immunity and has limited effect on subunit vaccines, and other adjuvants such as liposomes, immunomodulators, oligonucleotides, polysaccharides, cytokines and the like are not clinically applied at present, so that the research of a safe and efficient novel adjuvant is still the key point of vaccinology research.

Emulsion (Emulsion) has a certain application basis AS a vaccine adjuvant, Emulsion adjuvants such AS MF59, AS02, AS03, AF03 and the like are approved to be used for human bodies at present, and the Emulsion is the most widely applied adjuvant type except for aluminum adjuvants. The emulsion as an adjuvant has the following characteristics: (1) the oral emulsion adjuvant for protecting the antigen can improve the stability of the antigen in the gastrointestinal tract and protect the antigen from being degraded by protease; when the nasal cavity is immunized, the antigen migration can be rapidly enhanced, and the degradation caused by long-time staying in the nasal cavity is avoided; (2) the surface area of the antigen is increased, and the surface area of the antigen can be increased by adjusting the antigen adsorption of the immune response emulsion droplets, so that the antigen is identified by antigen presenting cells and the immune reaction strength and type are influenced, therefore, the size, the composition, the dispersion uniformity and the like of the droplets are possible factors influencing the adjuvant effect; (3) the adsorption of the sustained-release antigen nanoparticles can promote the antigen to be slowly released at an injection part to form continuous immune protection, and the latest research of an applicant finds that when the nano-emulsion adjuvant carries the antigen in different modes, the antigen release speed difference is large, so that the adjuvant effect is greatly different, and the adjuvant effect of the emulsion is proved to be closely related to the antigen sustained release; (4) the emulsion with excellent physicochemical properties has uniform particle distribution and can be stored at low temperature or normal temperature; in addition, the preparation method is simple, has low cost and is suitable for large-scale production, so the vaccine is considered to be an excellent vaccine adjuvant. There are still several problems to be solved:

1. the safety needs to be further improved, and the traditional emulsion needs to rely on a surfactant to stabilize an oil-water interface, so that the traditional emulsion can cause strong side reaction when being used for adjuvant injection to a body, and therefore a suitable substitute needs to be searched, and the using amount of the surfactant needs to be reduced as much as possible. 2. The stability needs to be further improved, and due to thermodynamic instability of the emulsion, emulsion breaking is easy to occur to different degrees at high temperature and high pressure, so that the particle integrity and the adjuvant effect are influenced, and generally only sterilization can be performed in a filtration mode. 3. Immune enhancement mechanisms are not elucidated, and current studies suggest that emulsion-type adjuvants are usually associated with immune cell recruitment, antigen uptake, and favor Th 1-type immune responses, independent of TLRs, but the underlying immune response mechanism is unknown. 4. The specific dose, immunization schedule and degree of antigen conservation are not clear, and it is difficult to determine how much the adjuvant effect of the emulsion adjuvant on the vaccine is, and whether it is more effective than the currently approved adjuvants. Therefore, it is important to provide an emulsion system which can not only avoid the use of a surfactant but also stabilize an oil-water interface, and to design the emulsion system as a novel adjuvant.

The Pickering emulsion is a special emulsion adopting solid particles to stabilize an oil-water system. Compared with the common emulsion, the Pickering does not need to add a surfactant, the concentration of the introduced solid particles is greatly lower than the dosage of the surfactant, and the toxic action on human bodies and the environment is far less than that of the surfactant, so the safety is possibly higher; in addition, the solid particles are irreversibly adsorbed on an oil-water interface, and an interface film is formed firmly and can prevent liquid drops from coalescence, so that the Pickering emulsion system is not easily influenced by the external acidity and alkalinity, the salt concentration, the temperature and the oil phase composition and has stronger stability. Xia et al constructed stable Pickering emulsion based on PLGA particles, found that the adjuvant effect is obviously superior to that of the common emulsion adjuvant stabilized by the traditional surfactant, and the optimized Pickering emulsion can activate antigen presenting cells and enhance antigen recruitment, and effectively stimulate humoral and cellular immunity.

Chlamydia trachomatis causes ocular or genital infections, the major complications of which include blinding trachoma and reproductive dysfunction such as urethritis, cervicitis and salpingitis, with more than 1.3 billion new cases registered each year according to recent estimates promulgated by the world health organization. Resulting in reproductive sequelae such as Pelvic Inflammation (PID), premature labor and obstructive infertility. It is estimated that 40% -60% of PID cases and 30% of ectopic pregnancies are caused by chlamydia trachomatis infection, which causes huge socio-economic burden for human healthcare since 2001 monitoring in China, the number of Ct pathologies reported has exceeded that of gonorrhea and is the first to live in 8 sexually transmitted pathogens. Therefore, the novel Pickering emulsion is reasonably designed to be used as the Ct vaccine adjuvant to solve the problems of safety and stability 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 immunologic adjuvant. 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 pickering emulsion, which comprises the following steps:

mixing graphene oxide with water to obtain a water phase;

squalene (SQ) is used as an oil phase, and the oil phase and the water phase are mixed and subjected to 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 water phase to the oil phase is

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

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

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

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 immunologic adjuvants.

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

In some embodiments, the vaccine is a chlamydia trachomatis vaccine. In some embodiments, the Chlamydia trachomatis vaccine is specifically Chlamydia trachomatis_pORF5 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 CtpORF5 recombinant protein vaccine is used as a model antigen to establish a mouse genital tract anti-Ct infection model, and the immune protection and safety of the GO-Pickering adjuvant on organisms are evaluated, and the result 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 large specific surface area of GO are beneficial to adsorbing antigens, slowing down antigen release and promoting antigen recognition and presentation.

2, a large number of oxygen-containing functional groups are arranged on the surface of GO, so that different sheet layers can be mutually repelled, and the dispersity in a water-oil system is good; meanwhile, most of the functional groups have hydrophilicity, so that the functional groups are easy to combine with an oil emulsion interface to prevent liquid drops from aggregating, and therefore the GO-Pickering emulsion system disclosed by the invention is not easy to be 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 realizes the slow release of antigens.

The titer of the antibody generated by the GO-SQ Pickering adjuvant stimulated mice is more than 10 times that of the single vaccine group, and the level of the stimulation of the cytokine generation is obviously higher than that of the aluminum adjuvant group and the single vaccine group. Therefore, the compound can be proved to be capable of assisting in inducing a mouse to simultaneously generate high-level humoral immunity and cellular immunity, so that an organism generates good immune 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 less than the dosage of the surfactant, so that the biotoxicity can be greatly reduced, and the safety problem of the traditional emulsion adjuvant can be effectively solved.

Drawings

FIG. 1 is a graph showing the appearance observation results of GO-SQ pickering emulsions of examples 1-3; 1-a GO-SQ pickering emulsions of example 3, example 1 and example 2, in order from left to right; 1-b is 1mg/ml GO dispersion (sheet diameter is 0.5-5 μm);

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

FIG. 3 is a graph showing the results of particle size and uniformity measurements performed by a Marvens particle sizer for GO-SQ pickering emulsions of examples 1-3; 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 fluorescence analysis results of antigen sustained release adsorption of GO-SQ pickering emulsion of example 1, wherein 4-a is the observation result under fluorescence excitation and 4-b is the observation result under bright field;

FIG. 5 shows the results of the detection of lgG antibody levels when the GO-SQ pickering emulsion of example 1 was used as an immunoadjuvant for pORF5 vaccine to immunize mice; wherein 5-a is the antibody level of IgG in mouse body fluid at weeks 4, 6 and 8; 5-b is the antibody levels of groups IgG, IgG1, IgG2a at week 8; 5-c is the antibody levels of mouse IgG, IgG1, IgG2a at different adjuvant doses;

FIG. 6 shows the results of cellular immunization experiments with GO-SQ pickering emulsion of example 1 as an immunological adjuvant for pORF5 vaccine, 6-a to 6-c being the expression levels of cytokines IFN-. gamma., IL-2 and IL-10 in order;

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

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

FIG. 9 shows the microscopic observation results of GO-SQ Pickering with different sheet diameters, from left to right, the results are sequentially the results with the water phase/oil phase ratio of 10:1, 10:2 and 10: 4;

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

Detailed Description

The invention provides a pickering emulsion, a preparation method thereof and application of the pickering emulsion as a vaccine immunologic adjuvant. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention 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 emulsion of the invention

Mixing Graphene Oxide (GO with the 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 (SQ) as an oil phase, mixing the water phase and the oil phase according to a mass ratio of 10:2, carrying out ultrasonic treatment for 10s at a power of 325w at intervals of 10s, and carrying out ultrasonic treatment repeatedly for 10min to obtain the GO-SQ pickering emulsion. The appearance observation result is shown in figure 1-a, the microscope observation result is shown in figure 2-b, (400 x), the particle size and uniformity detection result of a Malvern particle sizer is shown in figure 3-b, and the embedding rate of GO-SQ Pickering on BSA is determined to be 43%.

Example 2 preparation of GO-SQ Pickering emulsion of the invention

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

and (2) taking squalene as an oil phase, mixing the water phase and the oil phase according to a ratio of 10:1, performing ultrasonic treatment for 10s at a power of 325w at intervals of 10s, and performing ultrasonic treatment repeatedly for 10min to obtain the GO-SQ pickering emulsion. The appearance observation result is shown in figure 1-a, the microscope observation result is shown in figure 2-a, (400 ×), and the particle size and uniformity detection result of the Malvern particle sizer is shown in figure 3-a.

Example 3 preparation of GO-SQ Pickering emulsion of the invention

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

and (2) taking squalene as an oil phase, mixing the water phase and the oil phase according to a ratio of 10:4, performing ultrasonic treatment for 10s at a power of 325w at intervals of 10s, and performing ultrasonic treatment repeatedly for 10min to obtain the GO-SQ pickering emulsion.

Example 4 testing and characterization of GO-SQ pickering emulsions

The appearance observation result of the GO-SQ pickering emulsion in the embodiment 1-3 is shown in figure 1, the microscope observation result is shown in figure 2, and the particle size and uniformity detection result of the Malvern particle sizer is shown in figure 3;

from the results in fig. 1, it can be seen that the upper layer is Pickering emulsion, the lower layer is unbound GO aqueous dispersion, and the color of the lower layer is lighter than that of the original GO dispersion, indicating that the GO sheet layer has entered the Pickering system. With the reduction of the oil phase, the pickering emulsion on the upper layer is also reduced, and the difference of the transparency of the lower layer is obvious.

As can be seen from FIGS. 2 to 3, the GO-SQ pickering emulsion prepared in example 1 has smaller and more uniform particle size.

Example 5GO-SQ Pickering antigen adsorption Loading test

Antigen adsorption experiments: GO-SQPickering and FITC-BSA are mixed for 4h on ice (the mixing ratio is consistent with the animal experiment, 500ug BSA is contained in 1ml Pickering emulsion), and the emulsion is taken to be observed under a fluorescence microscope, and the result is shown in figure 4.

The result shows that the protein is adsorbed on the surface of GO-SQ Pickering emulsion liquid drop, and the antigenicity of the carried vaccine can be enhanced by the adsorption mode, so that the carried vaccine can be easily identified and presented by an immune system.

Example 6 humoral immunoassay

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, and 4 weeks with pORF5 protein alone and an aluminum adjuvant group as controls. The antibody levels of IgG were determined by indirect ELISA using mouse serum at weeks 4, 6, and 8 (FIG. 5-a), and groups of IgG, IgG1, and IgG2a at week 8 (FIG. 5-b) were compared with the antibody levels of mouse IgG, IgG1, and IgG2a at different adjuvant doses (FIG. 5-c), and the results are shown in FIG. 5.

According to the results, the IgG antibodies of all groups of mice change along with the immunization time, and the IgG antibodies of all groups of mice basically increase along with the increase of the immunization times, so that the recombinant protein vaccine has good immunogenicity, the IgG antibody of the GO-SQ Pickering adjuvant group is higher than that of the single vaccine group and the normal saline group, and the IgG antibody titer is about 10-100 times that of the single vaccine group. Therefore, the GO-SQ Pickering adjuvant group can obviously enhance the humoral immunity of the mice.

IgG and IgG subtype IgG1 and IgG2a in serum of mice in each group are detected 8 weeks after priming, namely 4 weeks after tertiary immunization, and the IgG, IgG1 and IgG2a in the GO-SQ Pickering adjuvant group mice are all higher than those in the single vaccine (p is more than 0.05), and data show that the IgG antibody titer is more than 10 times that of the single vaccine group. The GO-SQ Pickering adjuvant can enhance the humoral immunity, and the effect is basically equivalent to that of an aluminum adjuvant group. The average value of IgG1 in each group of mice is higher than that of IgG2a, so that it can be roughly inferred that the adjuvant and the pORF5 recombinant protein vaccine in each group of mice stimulate the body to generate an immune response which is inclined to Th1 type.

After the adjuvant is diluted 5 times, the level of IgG generated by the adjuvant is not obviously different from that generated by the original adjuvant, so that the Pickering adjuvant diluted 5 times can be used for subsequent experiments based on safety and cost.

Example 7 cytokine assay

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

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

(3) after the stimulation is finished, fully and uniformly mixing the cells by blowing, transferring the cells to an EP tube, centrifuging for 10min at the temperature of 4 ℃ and 1200g, collecting supernatant, and storing the supernatant in a refrigerator at the temperature of-20 ℃;

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

The results show that IFN-. gamma.: the GO-SQ Pickering adjuvant stimulates the body to generate high-level IFN-gamma which is obviously higher than that of a single vaccine group and an aluminum adjuvant vaccine group and is basically equivalent to that of the GO group (see figure 6-a).

IL-2: GO-SQPickering has obvious stimulation effect, can stimulate the organism to generate high level IL-2, and the average value is about 4 times of that of the single vaccine group and is obviously higher than that of the GO group (see figure 6-b).

IL-10: the comparison between GO-SQ Pickering and the single vaccine group is not obviously different, and the generation of an immunosuppressive factor IL-10 by the GO-SQ Pickering group is lower than that by the aluminum adjuvant group, which shows that the immune effect of the GO-SQ Pickering is obviously stronger than that of the aluminum adjuvant (see figure 6-c).

The 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 a single GO group and an aluminum adjuvant group.

Example 8 Properties of different sheet diameters GO-SQ Pickering

GO-SQ Pickering is prepared by using GO with different sheet diameters, the preparation method is the same as that of the embodiment 1, and the obtained appearance observation result of the GO-SQ Pickering is shown in a figure 8; the microscopic observation results are shown in FIG. 9.

The result shows that GO-SQ Pickering formed by GO with the plate diameter less than 15nm is milk white (9-a), the Pickering emulsion gradually presents brown with the increase of the GO plate diameter (9-b shows that the GO plate diameter is 50-200nm, and 9-c shows that the GO plate diameter is more than 500nm), and the proportion of the Pickering emulsion on the upper layer gradually increases with the increase of the oil phase. Microscopic observations revealed that emulsion droplet size increased with increasing oil phase and increased with increasing GO sheet diameter.

Example 9 stability testing

(1) Centrifugal test

Centrifuging at 10000g for 10min, and accelerating the liquid drop to float on the upper layer without obvious change of size

(2) High temperature testing

Standing in boiling water bath for 20min, the emulsion has unchanged appearance, particle size and uniformity

(3) Freeze drying test

After vacuum freeze drying, the mixture is shaken and evenly mixed in water, and the particle size is not changed.

(4) Long term stability observations

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

And (4) conclusion: from the observation results of centrifugation, high temperature, freeze-drying and long-term stability, GO-SQ Pickering has good stability and is convenient for long-term storage in special environment.

Example 10 evaluation of safety

Balb/c mice (average weight of 30g) are fasted for 8h, 0.6ml of GO-SQ Pickering (maximum administration) in example 1-3 is injected into the abdominal cavity of each mouse at one time, the activity state of the mice is observed, the mice are found to be normal and have no death, the mice are dissected after 7 days to observe organs, and the distribution of GO is observed. The results of GO-SQ Pickering of example 1 are shown in FIG. 10.

The result shows that after the Pickering emulsion is injected into the abdominal cavity at high dose, the heart, the liver and the lung of the Pickering emulsion have no obvious appearance change, the large intestine still has unabsorbed black GO, and a sac block is formed under the skin of a mouse to wrap the Pickering emulsion which cannot absorb metabolism in time, so that the Pickering emulsion is prevented from migrating to the abdominal organs to cause damage, and on the other hand, a slow release effect can be formed. The pickering emulsion disclosed by the invention greatly reduces the dosage of GO, avoids the burden of excessive GO on the metabolism of an organism, has high safety and simultaneously reduces the preparation cost.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.