Pickering emulsion, its preparation method and application

Abstract

The present application discloses a Pickering emulsion, its preparation method and application. The Pickering emulsion comprises an oil-in-water emulsion composed of graphene oxide quantum dots, an oil phase and an aqueous phase. The preparation method comprises the following steps: mixing a graphene oxide quantum dot solution, an oil phase and water to obtain a mixed solution, and emulsifying the mixed solution under ultrasonic conditions to obtain the Pickering emulsion. The Pickering emulsion uses graphene oxide quantum dots as an emulsifier. Due to the high surface area, biodegradability and biocompatibility of the graphene oxide quantum dots, the Pickering emulsion shows a high immune enhancement efficiency, can slowly release antigens in vivo and in vitro, has biocompatibility, and does not cause damage to the heart, liver and kidney functions of mice. It can be used as a candidate adjuvant with good immune enhancement and biocompatibility. The preparation method of the Pickering emulsion in the present invention is simple and easy to implement, and has low cost. The present invention also discloses the application of the above Pickering emulsion as an adjuvant for a Chlamydia trachomatis vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and particularly relates to a Pickering emulsion and its preparation method and application. Background Art

[0002] A Pickering emulsion is an emulsion prepared by using solid particles instead of surfactants as emulsifiers. The solid particles used in Pickering emulsions mainly include SiO 2 , montmorillonite, and nano-polymer particles, etc. The solid particles are closely arranged together, playing a role of steric hindrance, thereby inhibiting the aggregation between dispersed droplets and enhancing the stability of the emulsion. Therefore, the properties of the solid particles are crucial for the preparation and properties of Pickering emulsions. Summary of the Invention

[0003] The present invention provides a Pickering emulsion and its preparation method and application to solve the problem that there are no solid particles with good properties as emulsifiers in Pickering emulsions in the prior art.

[0004] The technical solutions provided by the present invention are as follows:

[0005] In the first aspect of the present invention, a Pickering emulsion is provided, which comprises an oil-in-water emulsion composed of graphene oxide quantum dots, an oil phase, and a water phase.

[0006] Further, the particle size of the Pickering emulsion is 100 nm to 500 nm.

[0007] Further, the concentration of the graphene oxide quantum dot solution in the raw materials of the Pickering emulsion is 1 mg / mL to 3 mg / mL; the mass ratio of water to oil in the Pickering emulsion is 10:1 to 4.

[0008] Further, the oil phase is squalene.

[0009] In the second aspect of the present invention, a preparation method of the above-mentioned Pickering emulsion is provided, which comprises the following steps: mixing a graphene oxide quantum dot solution, an oil phase, and water to obtain a mixed solution, and emulsifying the mixed solution under ultrasonic conditions to obtain the Pickering emulsion.

[0010] Further, the power of the ultrasonic wave in the ultrasonic conditions is 67.5 W to 325 W.

[0011] Further, the emulsification time is 1 min to 5 min.

[0012] In the third aspect of the present invention, an application of the above-mentioned Pickering emulsion in a Chlamydia trachomatis vaccine is provided.

[0013] Further, the application comprises the following steps: mixing the Pickering emulsion with a Pgp3 protein vaccine solution for injection.

[0014] Furthermore, the mass-volume ratio of the Pgp3 protein vaccine solution to the Pickering emulsion is 1-5:10×10 -3 μg / mL.

[0015] The Pickering emulsion of the present invention uses graphene oxide quantum dots as an emulsifier. Due to the high surface area, biodegradability and biocompatibility of graphene oxide quantum dots, the Pickering emulsion shows a high immune enhancement efficiency, can slowly release antigens in vivo and in vitro, and has biocompatibility, without causing damage to the heart, liver and kidney functions of mice. It can be used as a candidate adjuvant with good immune enhancement and biocompatibility. The preparation method of the Pickering emulsion in the present invention is simple and easy to operate, and has a low cost.

[0016] The Pickering emulsion of the present invention, as a Chlamydia trachomatis vaccine adjuvant, efficiently recruits dendritic cells at the injection site, can promote lymphocyte proliferation, stimulate the production of higher cytokines and antibodies, improves the vaccine effect, and has high safety; can greatly reduce the vaccine dosage; when the Pickering emulsion is co-immunized with the Chlamydia trachomatis vaccine, it significantly enhances the immune response intensity of the Pgp3 protein vaccine. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is the TEM image of the Pickering emulsion prepared in Example 1 of the present application;

[0019] Figure 2 It is the particle size distribution diagram of the Pickering emulsion prepared in Example 1 of the present application;

[0020] Figure 3 It is the change diagram of the size of the Pickering emulsion prepared in Example 1 of the present application with the storage time;

[0021] Figure 4 It is the change diagram of the zeta potential of the Pickering emulsion prepared in Example 1 of the present application with time;

[0022] Figure 5 It is the in vitro release diagram of the Pgp3 protein in Example 2 of the present application;

[0023] Figure 6 It is the antigen fluorescence intensity at the subcutaneous injection site of BALB / c mice in Example 2 of the present application;

[0024] Figure 7 It is the graph of the change of the quantitative fluorescence intensity at the injection site over time in Example 2 of this application, n = 3;

[0025] Figure 8 It is the titers of IgG, IgG1 and IgG2a in the sera of mice in each group on the 14th day after the completion of three immunizations in Example 3 of this application;

[0026] Figure 9 It is the graph of the change of the IgG titer over time in mice in each group after the completion of three immunizations in Example 3 of this application;

[0027] Figure 10 It is the sIgA antibody titers in the intestinal and vaginal mucosae of mice in each group at different times after inoculation in Example 3 of this application, n = 6;

[0028] Figure 11 It is the lymphocyte proliferation graph of mice in each group in Example 4 of this application;

[0029] Figure 12 It is the level graph of the cytokine IL-4 secreted by the spleen of mice in each group in Example 4 of this application;

[0030] Figure 13 It is the level graph of the cytokine IFN-γ secreted by the spleen of mice in each group in Example 4 of this application;

[0031] Figure 14 It is the level graph of the cytokine TNF-α secreted by the spleen of mice in each group in Example 4 of this application;

[0032] Figure 15 It is the flow cytometry graph of dendritic cells at the injection site of mice in each group in Example 5 of this application;

[0033] Figure 16 It is the statistical comparison of dendritic cells of mice in each group after injection in Example 5 of this application;

[0034] Figure 17 It is the graph of the serum ALT test results of mice in each group in Example 6 of this application;

[0035] Figure 18 It is the graph of the serum AST test results of mice in each group in Example 6 of this application;

[0036] Figure 19 It is the graph of the serum ALP test results of mice in each group in Example 6 of this application;

[0037] Figure 20 It is the graph of the serum BUN test results of mice in each group in Example 6 of this application;

[0038] Figure 21This is the detection result graph of serum LDH-L of each group of mice in Example 6 of this application. Detailed implementation manners

[0039] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the scope of protection of this application.

[0040] It should be noted that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those skilled in this technology to understand and read, and are not used to limit the limited conditions under which this application can be implemented. Therefore, they do not have substantial technical significance. Any modification of the structure, change of the proportional relationship or adjustment of the size, without affecting the effects that this application can produce and the purposes that can be achieved, should still fall within the scope covered by the technical content disclosed in this application.

[0041] In the first aspect of the embodiments of this application, a Pickering emulsion is provided. The Pickering emulsion includes an oil-in-water emulsion composed of graphene oxide quantum dots, an oil phase, and an aqueous phase.

[0042] Graphene oxide quantum dots (abbreviated as GOQDs) are graphene-based nanoparticles with significant quantum confinement effects, tunneling effects, and edge effects. Compared with graphene oxide (abbreviated as GO), the high surface area, biodegradability, and reasonable biocompatibility of GOQDs make it more potential to become a loading platform for various molecules such as drugs and proteins. GOQDs with appropriate sizes can change the drug delivery process by reducing the translocation free energy penetrating into the biomembrane. However, there are few potential reports on using GOQDs to prepare Pickering emulsions (abbreviated as GQPE) and further enhance the immune response.

[0043] Safety is an important issue in biological applications. The toxicity data of graphene oxide are still insufficient to indicate its side effects on different organisms. Some studies have shown that graphene oxide quantum dots are more suitable as carriers than graphene oxide because it has a lower toxicity level, weaker inflammatory response, and better cell uptake. Because large-sized graphene oxide is easy to absorb proteins, mainly aggregates in the pulmonary blood vessels, activates platelet clots, and causes pathological changes in lung tissues, while GOQDs do not aggregate and block in capillaries and have no obvious toxic side effects in vivo. This study preliminarily confirmed the biosafety of the GQPE adjuvant through the liver, kidney, and myocardial function indexes after 3 injections, which has great advantages in medical applications.

[0044] The Pickering emulsion of the embodiment of the present application uses graphene oxide quantum dots as an emulsifier. Due to the high surface area, biodegradability and biocompatibility of graphene oxide quantum dots, the Pickering emulsion shows a higher immune enhancement effect, can slowly release antigens in vivo and in vitro, and has biocompatibility and will not cause other damage to mice. It can be used as an adjuvant candidate drug with good immune enhancement and biocompatibility.

[0045] The particle size of the Pickering emulsion of the present application example is 100nm to 500nm. The Pickering emulsion has a small particle size and is evenly distributed. Compared with the emulsion with a large size, the internalization rate is effectively increased; and the small-sized Pickering emulsion shows a higher immune enhancement effect.

[0046] The second aspect of the embodiments of the present application provides a method for preparing the above-mentioned Pickering emulsion, comprising the following steps: mixing a graphene oxide quantum dot solution, an oil phase and water to obtain a mixed solution, and emulsifying the mixed solution under ultrasonic conditions to obtain a Pickering emulsion.

[0047] The reagents used in the following examples are all commercially available. Specifically: GOQDs solution (1 mg / mL, size <15 nm, monolithic) was purchased from Nanjing Xianfeng Nanotechnology Co., Ltd. Squalene was purchased from McLean Biochemical Co., Ltd. Pgp3 recombinant protein was obtained by Escherichia coli BL21 expression and purified by removing the GST tag. Mouse cytokine ELISA detection kit was provided by Biolegend Co., Ltd. for the detection of cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-10). Specific pathogen-free BALB / c mice (SYXK-2020-0002) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. Cell counting kit-8 (CCK8) and HRP-labeled goat anti-mouse IgG, IgG1, IgG2a, and IgA antibodies were purchased from Solarbio Technology Co., Ltd. Cy7-BSA was purchased from Xi'an Qiyue Biological Co., Ltd. Fluorescent labeled antibodies CD45-PE, CD11c-APC, and CD11b-PerCP Cy5.5 were purchased from Biolegend, USA. All animal experiments were authorized by the Animal Ethics Committee of the University of South China. All procedures were performed in accordance with the Regulations on the Administration of Laboratory Animals in China.

[0048] In the following examples, unless otherwise stated, all results are shown as mean ± SD of at least three experiments. Unless otherwise stated, statistical differences were determined using one-way ANOVA and analyzed using GraphPad Prism 7.0. All analyses were compared with the control group, and the significance of the differences was *P<0.05, **P<0.01 or ***P<0.001, respectively.

[0049] Preparation of Pickering Emulsion in Example 1

[0050] A graphene oxide quantum dot solution with a concentration of 1 mg / mL, squalene, and water were mixed to obtain a mixed solution, where the water-oil ratio of the mixed solution was 10:2; the mixed solution was emulsified for 1 min under ultrasonic conditions with a power of 325 W to obtain a Pickering emulsion.

[0051] The above Pickering emulsion was diluted with double-distilled water, and after dilution 10 times, it was observed under an optical microscope (Nikon Ts2r). The droplet size and zeta potential were measured with a Malvern Nano ZS90. Transmission electron microscopy (TEM) was used for photography and recording. To evaluate the stability of GQPE, we measured the droplet size and zeta potential after storing for different times at room temperature. The TEM image of the prepared Pickering emulsion was referred to Figure 1 and the particle size distribution was referred to Figure 2 The size at different storage times was referred to Figure 3 and the change of zeta potential with time was referred to Figure 4 .

[0052] The encapsulation efficiency test was carried out after placing GQPE overnight, and the specific steps were as follows: 50 μg of Pgp3 protein was co-incubated with 100 μL of GQPE for 4 h, centrifuged at 3000 r / min for 20 min in a 100 kD ultrafiltration tube, and the free protein was collected and the encapsulation efficiency was calculated. The results showed that in the GQPE system, the encapsulation efficiency of Pgp3 protein was 63.74 ± 4%.

[0053] The morphology of the above Pickering emulsion was observed by transmission electron microscopy to determine its basic characteristics. Referring to Figure 1 , the droplets in the selected area showed a regular spherical morphology, Figure 2 and it was shown in Figure 3 that its diameter varied between 200 nm and 500 nm. Referring to 4 , the average droplet size of the Pickering emulsion prepared by the above method was 422 nm, and the zeta potential was -52.7 mV; after different storage times, the particle size and zeta potential did not change significantly (P > 0.05). The above characterizations all indicated that the prepared Pickering emulsion maintained relative stability within 3 weeks.

[0054] Example 2 Controlled Release In Vivo and In Vitro

[0055] Using PBS as a control, the in vitro release of Pgp3 protein was detected.

[0056] The GQPE and Pgp3 protein mixture was placed in a dialysis microtube (50 kD MW), and the microtube was placed in 2 mL of PBS for dialysis. Every 24 h, 50 μL of the external fluid sample was taken, and the protein concentration in the mixture was detected using a UV-visible spectrophotometer (DeNovix DS-11, DeNovix, USA). Each sample was measured 6 times. At the same time, 50 μL of PBS was added to the dialysis fluid to keep the volume of the external fluid unchanged. The above test results were referred to Figure 5 , Figure 5 which was the in vitro release profile of Pgp3 protein.

[0057] In in vivo imaging observation, since Pgp3 protein was difficult to be efficiently labeled with Cy7, Cy7-BSA was used as a model antigen. PBS was mixed with Cy7-BSA, and GQPE was mixed with Cy7-BSA respectively. 100 μL of each of the above mixtures was taken and subcutaneously injected into BALB / c mice, with 3 female mice (7 weeks old) in each group. Using the Vilber Fusion FX spectroscopy, images were taken at 2 h, 12 h, 24 h, 34 h, 36 h, and 48 h respectively, and the excitation wavelength was 740 nm. The fluorescence intensity at the injection site was analyzed to compare their antigen depot effects. The above test results were referred to Figure 6 , Figure 7 , Figure 6 which was the fluorescence intensity of the antigen at the skin injection site of BALB / c mice, Figure 7 and Figure 7

[0058] was the graph of the change in quantitative fluorescence intensity over time, n = 3. Figure 6 After subcutaneous injection of the mixture of PBS and Cy7-BSA and the mixture of GQPE and Cy7-BSA for 2 h to 48 h, the intensity of Cy7-BSA at the injection site was detected, referring to Figure 7 . The results showed that the accumulation amount of BSA in the GQPE group was higher than that in the PBS group, and the quantitative data confirmed the above conclusion (P < 0.001), referring to Figure 5 . If Pgp3 protein was mixed with GQPE, in a continuous experiment of 96 h, Pgp3 protein was slowly released in vitro (P < 0.001), referring to Figure 5 . The in vivo and in vitro experimental results showed that GQPE interacted with Pgp3 protein, could produce an antigen depot effect in vivo, and showed a controlled release effect in vitro.

[0059] Example 3 Animal vaccination and antibody detection

[0060] Eighteen 5-week-old female BALB / c mice were randomly divided into three groups (6 mice in each group). Mice in group 1 were injected with PBS, those in group 2 were injected with the Pgp3 protein vaccine solution, and those in group 3 were injected with a mixture of GQPE and the Pgp3 protein vaccine solution. The total injection volume for each mouse was 100 μL. The Pgp3 protein vaccine solution in groups 2 and 3 was 50 μg each, and the injection was subcutaneously administered in the abdomen. All mice were immunized on days 0, 14, and 28. Serum was collected 2 weeks after each immunization to detect antibody titers.

[0061] The indirect enzyme-linked immunosorbent assay (ELISA) was used to quantitatively calculate the addition of IgG, IgG1, and IgG2a antibodies. Each well of a 96-well plate was coated with 10 μg of recombinant Pgp3 protein and incubated overnight at 4°C. It was blocked with PBST containing 5% skim milk for 2 hours. Serum samples were serially diluted, and 50 μL of each dilution was added to the reaction wells and incubated at 37°C for 2 hours. After washing four times, it was incubated with a 1:5000 dilution of HRP-labeled goat anti-mouse antibody at 37°C for 1 hour. The color development reaction included adding 100 μL of 3,3',5,5'-tetramethylbenzidine solution and then incubating at 37°C for 15 min; adding 50 μL of 2M H 2 SO 4 to terminate the color development, and the OD value was read at 450 nm within 10 min. The antibody titer judgment criterion was the maximum dilution factor that made the OD value greater than twice the average value of the negative serum.

[0062] Five mice in each group were sacrificed on the 14th day after the third immunization, and 3 cm of the small intestine and large intestine were collected separately, added with 1 mL of PBS for tissue homogenization. The vaginal swab was vortexed in 1 mL of PBS to obtain the vaginal lavage fluid, and the sIgA titer was detected by indirect ELISA. Except for using HRP-labeled goat anti-mouse IgA antibody as the secondary antibody, the other methods were the same as those for IgG detection.

[0063] The indirect ELISA method was used to detect IgG and subtype antibodies in the serum. As Figure 8 the results showed, on the 14th day after each vaccination, the IgG antibody titer in group 3 was 10 - 15 times higher than that in group 2. The IgG1 and IgG2a antibody titers were also detected on the 14th day after the third immunization, and the IgG1 and IgG2a antibodies in group 3 were significantly higher than those in group 2. This indicated that GQPE had an obvious adjuvant effect, greatly improving the level of antibody production and effectively stimulating the humoral immune response. Figure 9 showed the change of IgG titer over time, where days 0, 14, and 28 were the time points of mouse immunization.

[0064] In addition, indirect ELISA was used to detect sIgA in intestinal and vaginal secretions. As Figure 10As shown, the sIgA expressed in the large intestine and small intestine of the 3 groups was significantly higher than that of the 2 groups, with a good intestinal mucosal response. However, there was no difference in vaginal sIgA secretion (p>0.05). It can be inferred that injecting GQPE can stimulate the intestinal mucosal immune response, but this stimulation has no effect on the lower genital tract.

[0065] As can be seen from the above detection, GQPE helps the Pgp3 vaccine stimulate the production of higher levels of IgG and sIgA in mice.

[0066] Example 4 Spleen lymphocyte proliferation experiment

[0067] Take the spleen cells of the mice in each group in Example 3 to detect cytokines, and treat them with red blood cell lysis buffer for 5 min. The spleen cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin-l-glutamine. The cells were stimulated with 10 μg Pgp3 for 48 h, and the supernatant was collected according to the kit instructions for ELISA detection to detect TNF-α, IFN-γ, IL-4, IL-10, and IL-2 respectively.

[0068] For the lymphocyte proliferation assay, the cells were stimulated with Pgp3 or ConA and cultured for 48 h. The negative control was treated with an equivalent medium. 10 μL of CCK8 was added to each well and incubated for 3 h, and the OD value was obtained at 450 nm.

[0069] The CCK-8 method was used to detect the proliferation ability of spleen lymphocytes in each group. The comparison of the stimulation indexes in each group showed that the 3 groups were significantly higher than the 2 groups and the 1 group. Figure 11 It shows that the GQPE adjuvant can enhance the proliferation of spleen lymphocytes and directly enhance cellular immunity. ELISA kits were used to detect the cytokines IL-4, IFN-γ, and TNF-α secreted by the spleen, Figures 12 to 14 The results showed that the GQPE adjuvant could effectively stimulate the production of IL-4, IFN-γ, and TNF-α, and the secretion levels of these cytokines were significantly higher than those of the 2 groups. The above results showed that GQPE could not only enhance the proliferation of spleen cells but also enhance the production of their cytokines, suggesting that it has a strong immune protection ability in anti-infection immunity.

[0070] Example 5 Recruitment of dendritic cells at the injection site

[0071] Four 7-week-old female BALB / c mice were in each group. Four groups were subcutaneously injected with normal saline, five groups were injected with Pgp3 protein vaccine, and six groups were injected with a mixture of GQPE and Pgp3 protein vaccine. The total injection volume was 100 μL, and the injection amount of Pgp3 protein vaccine was 50 μg. The subcutaneous tissue at the injection site was taken, chopped, and then each tissue sample was digested with 3 mL of DMEM containing 1 mg / mL type VI collagenase D, 0.25 mg / mL ribonuclease, and 10% fetal bovine serum at 37 °C for 40 min. The digested tissue was filtered through a 70-μm cell strainer to obtain a single-cell suspension, which was fluorescently labeled with CD11c-APC, CD45-PE, and CD11b-PerCP-Cy5.5, and detected by BD FACSCalibur flow cytometry. The FlowJo V10.0 software was used to detect the expression of surface markers.

[0072] Refer to Figure 15 、 16 , in groups 4 and 5, due to injection-induced tissue trauma or antigen stimulation, dendritic cell recruitment at the injection site was shown. In group 6, the percentage of dendritic cells was nearly 3 times that of group 2 (P < 0.001). Overall, dendritic cells were effectively recruited by the GQPE adjuvant, which may play an important role in antigen recognition and presentation, enhancing the innate immune response.

[0073] Example 6 Safety Assessment

[0074] For the mice in Example 3, 100 μL of serum was collected from each mouse on the 14th day after the third immunization. An animal biochemical analyzer was used to detect multiple key biochemical items such as ALT, AST, ALP, BUN, and LDH-L to evaluate liver, kidney, and myocardial functions. The detection results are as Figures 17 to 21 shown. The results showed that there were no differences in the main biochemical indexes of the sera of the mice in groups 2 and 3 (ALT P = 0.152, AST P = 0.828, ALP P = 0.294, BUN P = 0.1157, LDH P = 0.681, all P > 0.05), and the relevant indexes were within the normal range (refer to Table 1). Thus, it can be seen that GQPE did not cause liver, kidney, or myocardial damage in mice and has good biocompatibility.

[0075] Table 1 Main Biochemical Indexes of Mice after Three Vaccinations

[0076]

[0077] The embodiments of the present application disclose a Pickering emulsion and a preparation method thereof. Using graphene oxide quantum dots as an emulsifier, the obtained Pickering emulsion has a particle size between 100 nm and 500 nm. The Pickering emulsion shows a high immune enhancement effect, can enhance humoral immunity and cellular immunity, can slowly release antigens in vivo and in vitro, and has biocompatibility, without causing damage to the heart, liver and kidney functions of mice. It can be used as a candidate adjuvant drug with good immune enhancement and biocompatibility. The preparation method of the Pickering emulsion in the present invention is simple and easy to implement, and has low cost.

[0078] The Pickering emulsion of the embodiments of the present application, as a Chlamydia trachomatis vaccine adjuvant, efficiently recruits dendritic cells at the injection site, can promote lymphocyte proliferation, stimulate the production of higher cytokines and antibodies, improves the vaccine effect, and has high safety. It can greatly reduce the vaccine dosage. The combined immunization of the Pickering emulsion and the Chlamydia trachomatis vaccine significantly enhances the immune response intensity of the Pgp3 protein vaccine.

[0079] The widely approved aluminum adjuvant cannot effectively stimulate the cellular immune response. The stimulation of cytokine levels by GQPE indicates that this is a promising strategy for stimulating cellular and humoral immunity, because clinical data show that the immune-related factors for protecting against Chlamydia trachomatis infection are cell-mediated responses, and GQPE can provide effective immune protection against Chlamydia trachomatis, which is crucial for Chlamydia clearance.

[0080] For innate immunity, the recruitment and activation of myeloid cells and lymphocytes at the injection site are important mechanisms for emulsion adjuvants to enhance the immune response against antigens. Our research results show that the percentage of dendritic cells (CD11b + CD11c + ) is significantly higher at the GQPE injection site, which means that effective antigen-presenting cells can first report and transmit danger signals to the immune system. The subcutaneous injection site is adjacent to the dermis of the skin, which contains a large number of specialized dermal dendritic cells. We observed that GQPE encapsulates the Pgp3 protein in vesicles in the subcutaneous tissue, which can recruit dendritic cells, recognize and internalize antigens, and may be transported to the draining lymph nodes. In addition, the controlled release and storage effects of antigens may play a key role. The extended release of the immunogen by the nano-droplets continuously increases the recruitment of dendritic cells at the injection site. The antigen exposed for a long time at the injection site is then endocytosed by local and newly recruited antigen-presenting cells, generating a strong immune response.

[0081] In the embodiments of the present application, Pickering emulsions stabilized by graphene oxide quantum dots were prepared, which can slowly release antigens in vivo and in vitro. Mouse immunization experiments showed that, with the Ct Pgp3 recombinant protein as the model antigen, GQPE can significantly activate humoral immunity, cellular immunity, and intestinal mucosal immunity. Safety assessments showed that GQPE has biocompatibility and does not cause liver, kidney, or myocardial damage in mice. In summary, GQPE can be used as a candidate adjuvant drug with good immune enhancement and biocompatibility.

[0082] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of Pickering emulsion as an adjuvant in the preparation of Chlamydia trachomatis vaccine, Characterized in that, The Pickering emulsion includes an oil-in-water emulsion composed of graphene oxide quantum dots, an oil phase, and an aqueous phase; the particle size of the Pickering emulsion is 100nm to 500nm; the mass ratio of water to oil in the Pickering emulsion is 10:1 to 4; the oil phase is squalene; The preparation method of the Pickering emulsion includes the following steps: mixing a graphene oxide quantum dot solution, an oil phase, and water to obtain a mixed solution, and emulsifying the mixed solution under ultrasonic conditions to obtain the Pickering emulsion.

2. The application according to claim 1, Characterized in that, The concentration of the graphene oxide quantum dot solution in the raw materials of the Pickering emulsion is 1mg / mL to 3mg / mL.

3. The application according to claim 1, Characterized in that, The power of the ultrasonic wave in the ultrasonic conditions is 67.5W to 325W.

4. The application according to claim 1, Characterized in that, The emulsification time is 1min to 5min.

5. The application according to any one of claims 1 to 4, Characterized in that, Includes the following steps: Mixing the Pickering emulsion with the Pgp3 protein vaccine solution for injection.

6. The application according to claim 5, Characterized in that, The mass-volume ratio of the Pgp3 protein vaccine solution to the Pickering emulsion is 1 to 5:10×10 -3 μg / mL.

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