Intestinal targeting saccharum alhagi polysaccharide-aluminum adjuvant Pickering emulsion as well as preparation method and application of intestinal targeting saccharum alhagi polysaccharide-aluminum adjuvant Pickering emulsion

Abstract

The invention belongs to the technical field of veterinary vaccine adjuvants, and particularly relates to an intestinal tract targeted saccharum alhagi polysaccharide-aluminum adjuvant Pickering emulsion as well as a preparation method and application of the intestinal tract targeted saccharum alhagi polysaccharide-aluminum adjuvant Pickering emulsion. The intestinal tract targeted saccharum alhagi polysaccharide-aluminum adjuvant Pickering emulsion is AHPPE, the AHPPE is formed by mixing an oil phase and a water phase, and the volume ratio of the oil phase to the water phase is 1: (9-13); the oil phase is prepared from the following raw materials: squalene and retinoic acid; the mass ratio of the squalene to the retinoic acid is 1: (0.7-0.8); the water phase is prepared from the following raw materials: saccharum alhagi polysaccharide and aluminum hydroxide; the mass ratio of the saccharum alhagi polysaccharide to the aluminum hydroxide is 1: (10-13). According to the preparation method provided by the invention, the problems that retinoic acid is difficult to dissolve in water and the metabolism speed of saccharum alhagi polysaccharide is high are solved, and meanwhile, an organism can be induced to generate effective systemic immunity and intestinal mucosa immunity by injecting AHPPE.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary vaccine adjuvants, and specifically relates to an intestinal targeted spinosa polysaccharide-aluminum adjuvant Pickering emulsion and a preparation method and application thereof. Background Art

[0002] Vaccination is a key means of preventing and controlling bovine viral diarrhea (BVD). The types of vaccines widely used in the market currently mainly include inactivated vaccines and subunit vaccines. The adjuvants commonly used in these vaccines are ISA201 and ISA206, which mainly focus on enhancing specific cellular immunity and humoral immune responses. However, in terms of adjuvants that can effectively enhance mucosal immune responses, the choices are relatively limited. Mucosa is the first line of defense for the body against pathogen invasion. Its immune mechanism is no less important than specific cellular and humoral immune responses for many vaccines, especially in the defense against mucosal diseases. At present, most vaccine adjuvants that can stimulate mucosal immunity on the market rely on oral routes, but this type of adjuvant is mainly limited to inducing effective mucosal immunity, and cannot significantly stimulate cellular immune responses. Therefore, the cellular and humoral immune effects induced by these adjuvants are not ideal. On the other hand, due to tissue limitations, the injection immunization method cannot induce immune cells to home to mucosa-associated lymphoid tissues, and thus cannot produce effective mucosal immune responses. In view of the above, the development of a new vaccine adjuvant that is low-cost, has few side effects, and can activate both mucosal immunity and systemic immune responses is of great significance for the defense against mucosal diseases.

[0003] Alhagi honey polysaccharide (AHP) is a natural polysaccharide with immunomodulatory activity. It can activate key immune cells such as T cells, B lymphocytes and dendritic cells (DCs), and enhance the body's immune function through multiple pathways. However, as a vaccine adjuvant, alhagi honey polysaccharide has the disadvantages of insufficient targeting and short duration of action. Therefore, it is necessary to develop a new vaccine adjuvant to overcome these problems. Summary of the invention

[0004] In order to solve the shortcomings of spinosa polysaccharide in the prior art, such as insufficient targeting and short action time, the present invention provides an intestinal targeted spinosa polysaccharide-aluminum adjuvant Pickering emulsion and its preparation method and application. To achieve the above purpose, the present invention adopts the following technical scheme to achieve it.

[0005] In a first aspect, the present invention provides an intestinal targeted spinosa polysaccharide-aluminum adjuvant Pickering emulsion.

[0006] The intestinal targeted spiny saccharide-aluminum adjuvant Pickering emulsion is AHPPE.

[0007] The AHPPE is formed by mixing an oil phase and a water phase; the volume ratio of the oil phase to the water phase is 1:9-13.

[0008] The oil phase is made of the following raw materials: squalene and retinoic acid; the mass ratio of the squalene to the retinoic acid is 1:0.7-0.8.

[0009] The aqueous phase is made of the following raw materials: spinosa polysaccharide and aluminum hydroxide; the mass ratio of the spinosa polysaccharide to the aluminum hydroxide is 1:10-13.

[0010] In order to overcome the shortcomings of insufficient targeting and short action time of spiny saccharide in the prior art, the present invention combines spiny saccharide with a carrier with good sustained-release performance, and prepares spiny saccharide and retinoic acid (all-trans retinoic acid) into a new vaccine adjuvant: AHPPE.

[0011] The AHPPE prepared by the present invention is a vaccine adjuvant prepared by using spiny saccharide loaded with aluminum hydroxide as the aqueous phase, and retinoic acid (RA, vitamin A acid) and squalene as the oil phase. The oil phase is used as the inner core, and the aqueous phase is tightly connected as the outer core, which not only enhances the stability of the emulsion, but also improves the adsorption capacity of antigens, thereby enhancing the intestinal targeting of spiny saccharide and prolonging the action time. The AHPPE provided by the present invention can not only solve the shortcomings of spiny saccharide with insufficient targeting and short action time, but also solve the problem that RA has poor water solubility and is not easily absorbed and utilized by the body, facilitates the absorption of drugs, can slowly release drugs, and effectively prolong the efficacy.

[0012] In a second aspect, the present invention provides a method for preparing the intestinal targeted spinosa polysaccharide-aluminum adjuvant Pickering emulsion, comprising the following steps: The aluminum hydroxide is added with water and subjected to ultrasonic treatment to make the aluminum hydroxide evenly dispersed in the water to prepare an aluminum hydroxide solution.

[0013] The aluminum hydroxide solution and the aqueous solution of the spinosa polysaccharide are mixed, and the aluminum hydroxide is used as a particle stabilizer and combined with the spinosa polysaccharide to obtain an AHP-Alum solution. The AHP-Alum solution is used as the aqueous phase.

[0014] The retinoic acid is dissolved in the squalene to obtain a retinoic acid-squalene solution. The retinoic acid-squalene solution serves as the oil phase.

[0015] The AHP-Alum solution (aqueous phase) is added to the retinoic acid-squalene solution (oil phase) and stirred evenly, and then ultrasonic emulsification is performed to construct a drug delivery system to obtain the AHPPE.

[0016] Wherein, the volume ratio of the AHP-Alum solution to the retinoic acid-squalene solution is 9-13:1.

[0017] The present invention uses ultrasonic emulsification to prepare a new emulsion adjuvant, and constructs a vaccine delivery system that simultaneously loads spiny saccharide polysaccharide and RA and can absorb a large amount of antigens. The vaccine adjuvant activates the intestinal mucosal homing mediated by CCR9 receptors through the action of retinoic acid on dendritic cells to produce an effective intestinal targeting effect. Injection of mice induces the body to produce high levels of specific IgG (Immunoglobulin G, IgG) antibodies and specific IgA (Immunoglobulin A, IgA) antibodies.

[0018] Preferably, the aluminum hydroxide is ultrasonically disrupted using a cell ultrasonic pulverizer, with an ultrasonic power of 15 W to 25 W, a variable rod of 6, ultrasonication for 3 s to 5 s, and a stop time of 3 s to 5 s.

[0019] Preferably, the concentration of the spinosa polysaccharide (AHP) in the AHP-Alum solution is 0.4 mg / mL to 0.5 mg / mL; the concentration of the aluminum hydroxide (Alum) in the AHP-Alum solution is 5 mg / mL to 7 mg / mL.

[0020] Preferably, the concentration of the retinoic acid in the retinoic acid-squalene solution is 0.6 mg / mL to 0.7 mg / mL; the mass ratio of squalene in the retinoic acid-squalene solution is 50% to 60%.

[0021] Preferably, the volume ratio of the AHP-Alum solution to the retinoic acid-squalene solution is 9-13:1.

[0022] Preferably, the conditions for ultrasonic emulsification are ultrasonic power of 19 W to 21 W, variable rod 6, ultrasonication for 3.5 s to 4.5 s, and stop for 3.5 s to 4.5 s.

[0023] In a third aspect, the present invention provides the use of the intestinal targeted spiny saccharide-aluminum adjuvant Pickering emulsion in improving the immunity of animal bodies. The intestinal targeted spiny saccharide-aluminum adjuvant Pickering emulsion is the AHPPE, and the AHPPE is used to prepare an adjuvant for animal diarrhea vaccine.

[0024] Preferably, the adjuvant of the animal diarrhea vaccine is prepared by mixing the AHPPE with an antigen and incubating them together.

[0025] The volume ratio of the antigen to the AHPPE is 1:1-4.

[0026] The antigen is animal diarrhea virus.

[0027] Preferably, the animal diarrhea virus comprises bovine viral diarrhea virus.

[0028] The beneficial effects achieved by one or more embodiments of the present invention are as follows: 1. The present invention provides an intestinal targeted spiny saccharide-aluminum adjuvant Pickering emulsion. The intestinal targeted spiny saccharide-aluminum adjuvant Pickering emulsion is AHPPE. The AHPPE prepared by the present invention is a vaccine adjuvant prepared by using spiny saccharide loaded with aluminum hydroxide as the aqueous phase and retinoic acid and squalene as the oil phase. Among them, the oil phase is used as the inner core and the aqueous phase is tightly connected as the outer core, which not only enhances the stability of the emulsion, but also improves the adsorption capacity for antigens, thereby enhancing the intestinal targeting of spiny saccharide and prolonging the action time. The AHPPE prepared by the present invention can not only solve the shortcomings of spiny saccharide, such as insufficient targeting and short action time, but also solve the problem that RA has poor water solubility and is not easily absorbed and utilized by the body, which is convenient for drug absorption, can slowly release the drug, and effectively prolong the efficacy.

[0029] 2. The AHPPE prepared in the present invention can be used as a BVDV vaccine adjuvant, and can induce effective mucosal immunity and systemic immune response in the body through injection.

[0030] 3. The preparation process of the AHPPE preparation method provided by the present invention is mild and simple, and the safe and well-tolerated pharmaceutical excipients give it good biosafety, which can realize large-scale mass production of vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The present invention is a flow chart of the preparation of AHPPE in Example 1 and the optimization of the optimal ratio of adjuvant to antigen; wherein, Figure 1 Figure A in the figure is the preparation flow chart of AHPPE; Figure 1 Figure B shows the optimal mixing ratio of AHPPE and BVDV antigen.

[0032] Figure 2 This is the stability test of AHPPE in Example 1 of the present invention; wherein, Figure 2 Figure A in the figure shows the appearance of AHPPE at 37 °C on day 0; Figure 2 Figure B shows the appearance of AHPPE at 37 °C on the 4th day; Figure 2 Figure C in the figure shows the appearance of AHPPE at 37°C on the 40th day; Figure 2 Figure D in the figure shows the appearance of AHPPE at 37 °C on the 80th day; Figure 2 Figure E in the figure shows the appearance of AHPPE at 37°C on the 100th day.

[0033] Figure 3The graph of particle size, PDI and zeta changes of AHPPE in Example 1 within 91 days of the present invention; wherein, Figure 3 Figure A shows the particle size change of AHPPE, and Figure B shows the PDI and zeta potential change of AHPPE.

[0034] Figure 4 The AHP and BVDV loading rates of AHPPE in Example 1 of the present invention and the in vitro AHP and BVDV release curves are shown; wherein, Figure 4 Figure A shows the AHP and BVDV loading rates of AHPPE, and Figure B shows the in vitro AHP and BVDV release curves of AHPPE.

[0035] Figure 5 This is a laser confocal image (2 μm) of AHPPE in Example 1 of the present invention; wherein, Figure 5 Figure A in the figure is the summation diagram; Figure 5 Figure B in the figure is the staining picture of dextran-FITC; Figure 5 Figure C in the figure is the staining image of squalene-Cy5.

[0036] Figure 6 The effects of each experimental group in Example 2 of the present invention on the content of BVDV-IgG and BVDV-IgA in mice; wherein, Figure 6 Figure A shows specific BVDV-IgG, and Figure B shows specific BVDV-IgA.

[0037] Figure 7 The effects of different experimental groups in Example 2 of the present invention on the secretion of mouse cytokines. Figure 7 Figure A in the figure is tumor necrosis factor-α; Figure 7 Figure B in the figure is interferon-γ; Figure 7 Figure C in the figure is interleukin-4; Figure 7 Figure D shows interleukin-17.

[0038] Figure 8 This is a live and intestinal imaging diagram of the immunized mouse in Example 3 of the present invention; wherein, Figure 8 Figure A in the figure is the in vivo imaging at 0h; Figure 8 Figure B is a 24 h in vivo imaging image; Figure 8 Figure C in the figure is a 3D in vivo imaging image; Figure 8 Figure D in the figure is a 5-day in vivo imaging image; Figure 8 Figure E in the figure is a 10-day in vivo imaging image; Figure 8 Figure F in the figure is a 16-day in vivo imaging image; Figure 8 The G diagram in the figure is a 24-hour intestinal diagram; Figure 8 Figure H in the figure is a 3D intestinal map; Figure 8 Figure I in the figure is the 10-day intestinal map; Figure 8 Figure J is a 16-day intestinal map.

[0039] Fig. 9 is the gene expression level of the intestinal homing receptor in each experimental group in Example 3 of the present invention. Fig. 9 Figure A shows the mRNA expression levels of CCR9 and CCL25, and Figure B shows the mRNA expression levels of MAdCAM1 and Integrinα4β7. DETAILED DESCRIPTION

[0040] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0041] The materials, reagents and experimental animals used in the following examples are as follows: (1) Materials and reagents Alhagi honey polysaccharide (AHP) was purchased from a pharmacy in Urumqi, Xinjiang.

[0042] Aluminum hydroxide, referred to as alum, hydrogel suspension, its English name is Alum, which was purchased from InvivoGen.

[0043] Retinoic acid, also known as vitamin A acid (RA), was purchased from MacLean Biotechnology Co., Ltd.

[0044] Squalene was purchased from MacLean Biotechnology Co., Ltd.

[0045] BVDV was purchased from Tiankang Biotechnology.

[0046] BCA protein quantification kit was purchased from Solebio Biotech Co., Ltd.

[0047] PBS was purchased from Sevier Biotech Co., Ltd. with a concentration of 1 mol / L and a pH of 7.3.

[0048] FITC-dextran (Dextran-FITC) was purchased from Solebio Biotech Co., Ltd.

[0049] ELISA kits were purchased from Shanghai Fankewei Biotechnology Co., Ltd.

[0050] The reverse transcription kit was purchased from Novazon Biotech Co., Ltd.

[0051] CY5.5 carboxylic acid (CY5.5) was purchased from APExBIO.

[0052] (2) Experimental animals All 5-week-old mice were obtained from Xinjiang Medical University.

[0053] (3) Antigen The source of bovine viral diarrhea virus (BVDV) antigen is Tiankang Biotechnology Co., Ltd.

[0054] Example 1: Preparation and characterization of AHPPE 1. Preparation of AHPPE (1) Dissolve the spinosa polysaccharide in distilled water to prepare an aqueous solution of the spinosa polysaccharide with a concentration of 2.5 mg / mL, and set aside.

[0055] (2) After the aluminum hydroxide is subjected to ultrasonic treatment, an aluminum hydroxide solution with a concentration of 10 mg / mL is obtained for later use. The method of ultrasonic treatment is as follows:

[0056] The aluminum adjuvant was sonicated using a cell ultrasonic pulverizer, with an ultrasonic power of 20 W, a variable speed of 6, sonication for 4 s, and a stop time of 4 s.

[0057] (3) Mix the aluminum hydroxide solution and the AHP aqueous solution to obtain an AHP-Alum solution. The concentration of Alum in the AHP-Alum solution is 6 mg / mL. The concentration of AHP in the AHP-Alum solution is 0.458 mg / mL and is set aside.

[0058] It should be noted that the prepared AHP-Alum solution is used as the aqueous phase (W phase).

[0059] (4) Dissolve retinoic acid in squalene to obtain a retinoic acid-squalene solution with a concentration of 0.625 mg / mL for later use.

[0060] It should be noted that the prepared retinoic acid-squalene solution is used as the oil phase (O phase).

[0061] (5) Add the AHP-Alum solution obtained in step (3) to the retinoic acid-squalene solution obtained in step (4), place in an ice box for ultrasonic emulsification for 3 minutes, and obtain AHPPE. The prepared AHPPE can be used to prepare an adjuvant for animal diarrhea vaccine.

[0062] The conditions for ultrasonic emulsification were as follows: using a cell ultrasonic crusher, ultrasonic power 20 W, variable rod 6, ultrasonic 4 s, and stop 4 s.

[0063] The volume ratio of the water phase to the oil phase is 11:1, that is, the volume ratio of the AHP-Alum solution to the retinoic acid-squalene solution is 11:1.

[0064] (6) The AHPPE obtained in step (5) is mixed with bovine viral diarrhea virus (BVDV) at a volume ratio of 3:1 for 30 minutes to obtain AHPPE / BVDV. AHPPE / BVDV is the prepared adjuvant for animal diarrhea vaccine. The adjuvant for animal diarrhea vaccine can be used to improve the immunity of animal body.

[0065] (7) The AHPPE / BVDV obtained in step (6) is mixed with Cy5.5 to obtain AHPPE / BVDV-Cy5.5.

[0066] The preparation flow chart of AHPPE is as follows Figure 1 shown.

[0067] 2. Stability test of AHPPE The AHPPE prepared above was placed in a 4 ℃ refrigerator and a 37 ℃ incubator, respectively, and samples were taken at 0 h, 4 d, 40 d, 80 d and 100 d to observe whether there was precipitation in the AHPPE and to detect its stability.

[0068] Test results such as Figure 2 As shown in the figure, AHPPE in the 4 ℃ refrigerator and 37 ℃ incubator showed no precipitation and stratification within 100 days.

[0069] The above results show that AHPPE has a long-term stability.

[0070] 3. Detection of AHPPE particle size, PDI (polymer dispersibility index) and zeta potential The AHPPE prepared as above was tested by using a particle size analyzer at 37 °C to detect the changes in particle size, PDI and Zeta potential at 1 d, 4 d, 7 d, 14 d, 20 d, 40 d, 80 d and 100 d.

[0071] Detection Figure 3 As shown in Figure A, the particle size of AHPPE at 37 °C is 2615 ± 78.39 nm. Figure 3 As shown in Figure B, the polymer dispersibility index (PDI) and Zeta potential of AHPPE at 37 °C are 0.27±0.10 and 20.4±1.15 mV, respectively. The particle size of AHPPE did not change significantly within 42 days. The potential is positive and the PDI is less than 0.3, showing good stability.

[0072] The above results show that AHPPE has good stability.

[0073] 4. AHPPE / BVDV loading rate and in vitro release AHPPE / BVDV was centrifuged at 12000 r / min for 30 min, and the supernatant was taken. The free AHP concentration in AHPPE / BVDV was determined by phenol-sulfuric acid method, and the free BVDV content was detected by BCA protein quantification kit.

[0074] The concentrations of total AHP and total BVDV in the centrifuged AHPPE / BVDV were determined in the same manner as described above, and the loading rates of AHP and BVDV in the AHPPE were detected. The calculation formulas for the antigen and polysaccharide loading rates are as follows:

[0075] Load rate = (C t -C f ) / C t ×100%; The English name of the load rate in the above formula is Loading efficiency, C f is the concentration of free drug, C t is the concentration of total drug. The unit of loading rate is %.

[0076] After 9 mL of AHPPE and BVDV were fully mixed at a volume ratio of 3:1, they were added to a dialysis bag with a molecular weight of less than 50,000 Da, placed in a bottle of PBS with a pH of 7.3, sealed, and placed in a shaker at 100 rpm. AHPPE / BVDV was taken out of the centrifuge tube at regular intervals at 3 h, 6 h, 9 h, 12 h, 1 d, 2 d, 3 d, 5 d, 10 d, 15 d, 20 d, 25 d, and 30 d.

[0077] In a 1.5 mL ep tube, add an equal amount of 2 M NaCl and place at 4 °C overnight. The BVDV content in AHPPE / BVDV was detected using the BCA protein quantification kit, and the cumulative release percentage of BVDV was calculated; the AHP content was detected using the phenol-sulfuric acid method, and the cumulative release percentage of AHP was calculated.

[0078] Among them, phenol-sulfuric acid method: take 200 μL of supernatant and add water to 300 μL. Then add 150 μL of 5% phenol solution and 750 μL of sulfuric acid solution in sequence, shake well, place in an ice water bath to cool down, and then place in a 37 ℃ water bath to heat for 20 min after cooling, take out and cool under running water. Take 200 μL and put it into a 96-well plate, and measure the absorbance at 490 nm with a UV spectrophotometer.

[0079] Depend on Figure 4 From the results of Figure A in Figure 1, we can see that the AHP loading rate of AHPPE / BVDV is 73.2%; the BVDV antigen loading rate is 66.8%, showing good AHP and BVDV loading effects.

[0080] The results of in vitro release of AHPPE / BVDV are shown in Figure 4 As shown in Figure B.

[0081] Depend on Figure 4 As shown in Figure B, BVDV was released rapidly in the first 24 hours, reaching about 22.36±1.62%, and then began to release slowly, lasting for more than 30 days, with a cumulative release of about 83.78±1.24%. The release rate of AHP was similar to that of BVDV, also releasing faster on the first day, reaching about 24.09±0.16%, and then slowly accelerating, with a cumulative release of about 80.24±0.18% on the 30th day.

[0082] The above results showed that AHPPPE / BVDV had good sustained-release ability.

[0083] 5. AHPPE laser confocal detection The AHPPE emulsion was prepared by replacing spinosad polysaccharide with FITC-dextran (Dextran-FITC). The specific steps are as follows: (1) Take FITC-dextran (Dextran-FITC) instead of AHP and dissolve it in distilled water to prepare a FITC-dextran solution with a concentration of 1 mg / mL for later use.

[0084] (2) After the aluminum hydroxide is subjected to ultrasonic treatment, an aluminum hydroxide solution with a concentration of 10 mg / mL is obtained for later use. The method of ultrasonic treatment is as follows:

[0085] The aluminum adjuvant was sonicated using a cell ultrasonic pulverizer, with an ultrasonic power of 20 W, a variable speed of 6, sonication for 4 s, and a stop time of 4 s.

[0086] (3) Mix the aluminum hydroxide solution and the FITC-dextran solution to obtain a FITC-dextran-Alum solution with a concentration of 6 mg / mL.

[0087] It should be noted that the prepared FITC-dextran-Alum solution serves as the aqueous phase (W phase).

[0088] (4) Take Cy5.5 instead of RA and dissolve it in squalene to obtain a Cy5.5-squalene solution with a concentration of 0.5 mg / mL, which is set aside.

[0089] It should be noted that the prepared Cy5.5-squalene solution is used as the oil phase (O phase).

[0090] (5) The aqueous solution obtained in step (3) was added to the Cy5.5-squalene solution obtained in step (4), and the mixture was placed in an ice box for ultrasonic emulsification for 3 min to obtain a picking emulsion (FITC-Alum / Cy5.5) with FITC and Cy5.5 fluorescence for simulating the morphological structure of AHPPE.

[0091] The morphological structure of the FITC-Alum / Cy5.5 prepared above was observed under a laser confocal microscope, where Dextran-FITC was illuminated in green light and Cy5.5 in red light.

[0092] Confocal results are as follows Figure 5 As shown, it can be seen that the green Dextran-FITC can be attached to the red squalene, which shows that the AHPPE is a Picking emulsion with squalene as the core and aluminum hydroxide as the outer core, and the surface is strawberry-shaped.

[0093] Example 2: Immunoadjuvant activity of AHPPE 1. Preparation process of PE / BVDV and Alum / BVDV (1) After subjecting aluminum hydroxide to ultrasonic treatment, an aluminum hydroxide solution with a concentration of 10 mg / mL is obtained for later use. The method of ultrasonic treatment is as follows:

[0094] The aluminum adjuvant was sonicated using a cell ultrasonic pulverizer, with an ultrasonic power of 20 W, a variable speed of 6, sonication for 4 s, and a stop time of 4 s.

[0095] (2) Mix the aluminum hydroxide solution and the aqueous solution to obtain an aluminum hydroxide solution with a concentration of 6 mg / mL for later use.

[0096] It should be noted that the prepared aluminum hydroxide solution is used as the water phase (W phase).

[0097] (3) Take squalene and set aside.

[0098] It should be noted that the prepared squalene solution is used as the oil phase (O phase).

[0099] (4) Add the aluminum hydroxide solution obtained in step (2) to the squalene solution obtained in step (3), place the mixture in an ice box for ultrasonic emulsification for 3 minutes, and obtain a picking emulsion (picking emulsion is also called PE).

[0100] (5) The PE obtained in step (4) was mixed with bovine viral diarrhea virus (BVDV) at a ratio of 3:1 for 30 min to obtain PE / BVDV.

[0101] (6) The aluminum hydroxide obtained in step (2) is mixed with bovine viral diarrhea virus (BVDV) at a volume ratio of 3:1 for 30 minutes to obtain Alum / BVDV.

[0102] (7) Mixing the PE / BVDV obtained in step (5) with Cy5.5 to obtain PE / BVDV-Cy5.5.

[0103] (8) Mix the Alum / BVDV obtained in step (6) with Cy5.5 to obtain Alum / BVDV-Cy5.5.

[0104] (9) BVDV and PBS are mixed with Cy5.5 respectively to obtain BVDV-Cy5.5 and PBS-Cy5.5.

[0105] 2. The AHPPE prepared in Example 1 was used as the research object, the PE / BVDV and AHPPE / BVDV prepared in this example were used as the control group, and the Alum / BVDV group was used as the positive control group, and their effects on the content of BVDV-specific IgG antibody, BVDV-specific IgA antibody and cytokines IFN-γ, TNF-α, IL-4, IL-10, and IL-17 were measured respectively. The specific method is as follows:

[0106] (1) Animal grouping and treatment Five-week-old mice were rested for 1 week without any drug injection and randomly divided into five experimental groups (n=20): AHPPE / BVDV group, PE / BVDV group, Alum / BVDV group, BVDV group and PBS group. Among them, AHPPE / BVDV group and PE / BVDV group were experimental control groups, Alum / BVDV group was positive control group, BVDV group was vaccine control group, and PBS group was blank control group.

[0107] Each mouse in the AHPPE / BVDV group was immunized by intramuscular injection of 0.1 mL of AHPPE / BVDV; each mouse in the PE / BVDV group was immunized by intramuscular injection of 0.1 mL of PE / BVDV; each mouse in the Alum / BVDV group was immunized by intramuscular injection of 0.1 mL of Alum / BVDV; each mouse in the BVDV group was immunized by intramuscular injection of 0.1 mL of BVDV; each mouse in the PBS group was immunized by intramuscular injection of 0.1 mL of PBS.

[0108] All mice in the above experimental groups were killed on the 3rd, 7th, 14th and 35th days after immunization.

[0109] (2) Effect of AHPPE on BVDV-IgA and BVDV-IgG antibody levels Immunization was performed as described above. On the 3rd, 7th, 14th and 35th days, 1 mL of blood was randomly collected from 5 mice in each group and centrifuged at 3000 r / min for 10 min to separate the serum. 96-well ELISA plates (100 μL / well) were coated with carbonate buffer containing BVDV and incubated overnight at 4°C, where the final concentration of BVDV was 2 μg / mL; the plates were washed three times with washing buffer, the 96-well plates were patted dry, and BSA blocking solution (200 μL / well) with a concentration of 0.01 g / mL was prepared with PBS and incubated at 37°C for 2 h; the plates were washed three times with washing buffer and patted dry; the test serum diluted with PBS (100 μL / well) was added, and the plates were incubated at 37°C for 1 h, then washed three times with washing buffer and patted dry; HRP-labeled goat anti-mouse secondary antibodies including IgG and IgA (100 μL / well) were added, and the plates were incubated at 37°C for 1 h, washed and patted dry; TMB solution (100 μL / well) was added, and after incubation at room temperature for 10 min, 2 M H2SO4 (50 μL / well) was added to terminate the reaction, and the OD was measured using an enzyme reader. 450 value.

[0110] Specific antibody results such as Figure 6 It can be seen that the levels of BVDV-IgG and BVDV-IgA in the AHPPE / BVDV group, PE / BVDV group, Alum / BVDV group and BVDV group were significantly higher than those in the PBS group ( P <0.05), the levels of IgA and IgG induced in the AHPPE group were significantly higher than those in the BVDV group ( P <0.05).

[0111] In summary, AHPPE vaccine adjuvant can continuously and effectively induce BVDV-specific antibodies and effectively improve the immune enhancement effect of BVDV.

[0112] (3) Effect of AHPPE on serum cytokines On the 35th day after immunization of mice, the blood of mice in each experimental group was centrifuged at 4 ℃ 3000 rpm to separate the serum, and the ELISA kit was used to determine the secretion levels of Th1 cytokines (IFN-γ and TNF-α), Th2 cytokines (IL-4), and Th17 cytokines (IL-17) in the serum of mice in each experimental group.

[0113] Cytokine results Figure 7 As shown in Figure 2, the AHPPE / BVDV group significantly increased TNF-α, IFN-γ, IL-17, and IL-4 compared with the BVDV group, Alum / BVDV group, and PE-BVDV group ( P<0.05), further revealing that AHPPE / BVDV induces mixed immunity of Th1, Th2 and Th17 types.

[0114] Example 3: AHPPE enhances intestinal immunity in mice (1) In vivo fluorescence detection of AHPPE / BVDV Six-week-old mice were injected with 0.1 mL of Cy5.5-loaded AHPPE / BVDV, PE / BVDV, Alum / BVDV and BVDV into the left leg muscle of each mouse. The mice were anesthetized at 0 h, 24 h, 3 d, 5 d, 10 d and 16 d after immunization, and the migration of AHPPE / BVDV-Cy5.5, PE / BVDV-Cy5.5, Alum / BVDV-Cy5.5 and BVDV-Cy5.5 in the mice was observed with a small animal in vivo imaging device; the intestines of the immunized mice were collected at 24 h and 3 d, 10 d and 16 d to observe the migration of the above four drugs in the intestine.

[0115] Intestinal targeting results such as Figure 8 As shown, 24 hours after mouse immunization, AHPPE / BVDV-Cy5.5 had reached the intestine; 3 days after mouse immunization, the intestinal fluorescence intensity of AHPPE-Cy5.5 was higher than that of Alum-Cy5.5 and PE-Cy5.5; 10 and 16 days after mouse immunization, the intestinal fluorescence intensity of AHPPE-Cy5.5 remained high and higher than that of the Alum-Cy5.5 group, indicating that AHPPE has good intestinal targeting and long-lasting immune response.

[0116] (2) Detection of chemokine receptor mRNA expression in mouse intestine On the 21st day after mice were immunized, 6 mice were randomly selected from each group, and the small intestine was collected. The total RNA of the small intestine was extracted using Trizol reagent, and the RNA concentration and 260 / 280 and 230 / 260 values ​​were detected using a single drop spectrometer.

[0117] Total RNA from small intestine was reverse transcribed into cDNA using a reverse transcription kit and stored at -20 ºC. qPCR was performed on a 7500 Software system.

[0118] qPCR reaction conditions: 95 ºC pre-denaturation for 30 s, microcycle reaction for 95 ºC 10 s, 60 ºC 30 s, 40 cycles; melting curve for 95 ºC, 15 s, 60 ºC, 60 s, 95 ºC, 15 s. Three replicates were performed for each sample and normalized with the internal reference gene GAPDH. -ΔΔCtThe relative mRNA expression levels of CCR9, CCL25, MAdCAM1, and Integrin α4β7 were calculated by the method and expressed as the ratio of the experimental group to the GAPDH group.

[0119] The mRNA expression results are as follows Fig. 9 As shown in Figure A, AHPPE / BVDV, PE / BVDV, and Alum / BVDV can all increase the mRNA expression of CCR9 and CCL25 in the duodenum of mice. The mRNA expression of CCR9 and CCL25 in the AHPPE / BVDV group was significantly higher than that in the other experimental groups ( P <0.05).

[0120] The mRNA expression results are as follows Fig. 9 As shown in Figure B, AHPPE / BVDV, PE / BVDV and Alum / BVDV can increase the mRNA expression of MAdCAM1 and Integrin α4β7 in the duodenum of mice. The mRNA expression of MAdCAM1 and Integrin α4β7 in the AHPPE / BVDV group was significantly higher than that in the other experimental groups ( P <0.05).

[0121] From the above experimental results, it can be seen that AHPPE as a BVDV vaccine adjuvant can induce the body to produce long-lasting and strong immune protection through intestinal targeting, which provides a good choice for the development of BVDV vaccine adjuvants.

[0122] The present invention provides an intestinal targeted spiny saccharide-aluminum adjuvant Pickering emulsion. The intestinal targeted spiny saccharide-aluminum adjuvant Pickering emulsion is AHPPE. The AHPPE prepared by the present invention can not only solve the shortcomings of spiny saccharide with insufficient targeting and short action time, but also solve the problem that RA has poor water solubility and is not easily effectively absorbed and utilized by the body, facilitates the absorption of drugs, can slowly release drugs, and effectively prolongs the efficacy.

[0123] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0124] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. An intestinal targeted spinosa polysaccharide-aluminum adjuvant Pickering emulsion, characterized in that The intestinal targeted spiny polysaccharide-aluminum adjuvant Pickering emulsion is AHPPE; The AHPPE is formed by mixing an oil phase and a water phase: the volume ratio of the oil phase to the water phase is 1:9-13; The oil phase is made of the following raw materials: squalene and retinoic acid; the mass ratio of the squalene to the retinoic acid is 1:0.7-0.8; The aqueous phase is made of the following raw materials: spinosa polysaccharide and aluminum hydroxide; the mass ratio of the spinosa polysaccharide to the aluminum hydroxide is 1:10-13.

2. The method for preparing the intestinal targeted spinosa polysaccharide-aluminum adjuvant Pickering emulsion according to claim 1, characterized in that: The steps include: Adding water to the aluminum hydroxide and subjecting it to ultrasonic treatment to uniformly disperse the aluminum hydroxide in the water to prepare an aluminum hydroxide solution; The aluminum hydroxide solution and the aqueous solution of the spinosa polysaccharide are mixed, wherein the aluminum hydroxide acts as a particle stabilizer and combines with the spinosa polysaccharide to obtain an AHP-Alum solution; the AHP-Alum solution serves as the aqueous phase; The retinoic acid is dissolved in the squalene to obtain a retinoic acid-squalene solution; the retinoic acid-squalene solution is used as the oil phase; The AHP-Alum solution is added to the retinoic acid-squalene solution, mixed, and subjected to ultrasonic emulsification to construct a drug delivery system to obtain the AHPPE; Wherein, the volume ratio of the AHP-Alum solution to the retinoic acid-squalene solution is 9-13:

1.

3. The preparation method according to claim 2, characterized in that: The conditions of the ultrasonic treatment are as follows: Ultrasonic power 15 W ~ 25 W, change rod 6, ultrasonic 3 s ~ 5 s, stop 3 s ~ 5 s.

4. The preparation method according to claim 2, characterized in that: The concentration of the spinosa polysaccharide in the AHP-Alum solution is 0.4 mg / mL to 0.5 mg / mL; the concentration of the aluminum hydroxide in the AHP-Alum solution is 5 mg / mL to 7 mg / mL.

5. The preparation method according to claim 2, characterized in that: The concentration of the retinoic acid in the retinoic acid-squalene solution is 0.6 mg / mL to 0.7 mg / mL; the mass ratio of squalene in the retinoic acid-squalene solution is 50% to 60%.

6. The preparation method according to claim 2, characterized in that: The conditions of ultrasonic emulsification are as follows: ultrasonic power 19 W~21 W, variable rod 6, ultrasonic 3.5 s~4.5 s, and stop 3.5 s~4.5 s.

7. The use of the intestinal targeted spiny saccharide-aluminum adjuvant Pickering emulsion according to claim 1 in improving the immunity of an animal body, characterized in that: The AHPPE is used for preparing an adjuvant for animal diarrhea vaccine.

8. The use according to claim 7, characterized in that: The adjuvant of the animal diarrhea vaccine is prepared by mixing the AHPPE with an antigen and incubating them together: The volume ratio of the antigen to the AHPPE is 1:1-4; The antigen is animal diarrhea virus.

9. The use according to claim 8, characterized in that: The animal diarrhea virus includes bovine viral diarrhea virus.

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