Preparation method and application of Pickering emulsion vaccine based on metal organic framework

Abstract

The invention discloses a preparation method and application of a Pickering emulsion vaccine based on a metal organic framework, and the preparation method comprises the following steps: taking Al (NO3) 3.9 H2O and 4, 4 '-diphenyldicarboxylic acid as raw materials, and obtaining white DUT-5 powder through a solvothermal method; adding the prepared DUT-5 into ultrapure water, and carrying out simple ultrasonic uniform mixing, so as to prepare a Pickering emulsion; mixing the obtained Pickering emulsion with PCV2Cap protein, putting the mixture into a shaking table and staying overnight; and carrying out ultrafiltration and centrifugation on the obtained mixed emulsion to obtain DUT-5-PE (at) CAP. The vaccine provided by the invention can effectively transfer an antibody or protein to a specific position and stimulate a strong immune response, provides a new possibility for developing a novel vaccine, provides a new method for preventing and treating the porcine circovirus type II, and has a significant meaning for controlling the disease.

Description

Technical Field

[0001] The invention relates to the technical field of nano material preparation, and in particular to a method for preparing a Pickering emulsion vaccine based on a metal organic framework and an application thereof. Background Art

[0002] The impact of epidemics in animal husbandry is extremely serious. Not only will it cause animals to become sick or die, but it will also have a negative impact on breeding efficiency. Vaccination has become the most cost-effective strategy for preventing a variety of infectious diseases by stimulating or adjusting the host's immune system. It is essential for preventing animal diseases and ensuring the sustainable development of agriculture and animal husbandry. Despite this, existing vaccines still face some challenges, such as insufficient cellular immune response, lack of protection against a variety of pathogens, and antibody-dependent enhancement. Nanotechnology has shown great potential in vaccine development. Nanoparticles as vaccine carriers can enhance the body's immune response and effectively fight viral diseases and cancer.

[0003] In vaccine delivery systems, metal organic frameworks (MOFs) have attracted much attention due to their unique physicochemical properties and biocompatibility. MOFs are composed of organic ligands and metal ions or clusters, and have high loading capacity and diverse functions, making them important in vaccine delivery.

[0004] Researchers are exploring particulate carriers as new adjuvants that mimic the characteristics of pathogens or diseased cells rather than just the mobility of antigens. Pickering emulsions, as particle-stabilized emulsions, have shown potential as vaccine carriers and their effectiveness has been demonstrated. The nanoparticles in these emulsions can be further optimized to achieve more functionality.

[0005] The present invention has developed a MOFs-stabilized Pickering emulsion vaccine. Preliminary studies have shown that this vaccine can effectively deliver antibodies or proteins to specific locations and stimulate a strong immune response, providing new possibilities for the development of new vaccines. From an industrial perspective, this vaccine provides a new method for the prevention and treatment of porcine circovirus type II, which is of significant significance for the control of the disease. Summary of the invention

[0006] In view of this, the present invention provides a method for preparing a Pickering emulsion vaccine based on a metal organic framework and an application thereof.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A method for preparing a Pickering emulsion vaccine based on a metal organic framework comprises the following steps:

[0009] Step 1: Add Al(NO3)3·9H2O to DMF and sonicate until fully dissolved; add 4,4'-biphenyldicarboxylic acid to DMF and sonicate until fully dissolved;

[0010] Step 2: After mixing the above solutions, transfer them into a reactor, place the reactor in an oven for reaction, wash with DMF, then wash with anhydrous ethanol, and then dry under vacuum at room temperature to obtain white DUT-5 powder;

[0011] Step 3: Add the DUT-5 prepared above into ultrapure water, mix by ultrasonication, add squalene thereto, and perform ultrasonication in an ice bath to obtain a Pickering emulsion;

[0012] Step 4: Mix the obtained Pickering emulsion with PCV2 Cap protein and place it in a shaker overnight; Step 5: Take the obtained mixed emulsion for ultrafiltration and centrifugation to obtain DUT-5-PE@CAP.

[0013] Preferably, in step 1, the amount of DMF added is 15 mL; the concentration of Al(NO3)3·9H2O is 1.4 mmol, and the amount added is 0.525 g; the concentration of 4,4'-biphenyldicarboxylic acid is 1.2 mmol, and the amount added is 0.266 g.

[0014] Preferably, in step 2, the reaction kettle is placed in an oven and reacted at 120°C.

[0015] Preferably, in step 2, the mixture is washed with DMF three times, washed with anhydrous ethanol once, and then dried in vacuo at room temperature.

[0016] Preferably, in step 3, the first ultrasonic treatment time is 10 min, and the second ultrasonic treatment time is 20 min.

[0017] Preferably, in step 3, the ultrasonic power is 60W.

[0018] Preferably, in step 3, the amount of squalene added is 200 μL.

[0019] Preferably, in step 4, the obtained Pickering emulsion is mixed with PCV2 Cap protein and placed in a shaker at 4° C. overnight.

[0020] Preferably, in step 5, the centrifugal speed is 1000 rpm and the centrifugal time is 5 min.

[0021] A metal-organic framework-based Pickering emulsion vaccine preparation method for the prevention and treatment of porcine circovirus.

[0022] Compared with the prior art, the present invention has achieved the following technical effects:

[0023] (1) The present invention prepares nano-scale Al-based MOFs materials by a solvothermal method. Based on Al-MOFs nano-vaccines, antigens can be encapsulated on the surface of the emulsion by a one-step ultrasound, providing a stable environment for the antigens and enhancing the stability of the antigens;

[0024] (2) The present invention is a treatment method for porcine circovirus type 2, based on a MOFs-based Pickering emulsion vaccine, which combines the emulsion with MOFs materials to enhance the delivery and sustained release of vaccine antigens, thereby inducing a more efficient immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the infrared spectrum of DUT-5 of the present invention;

[0026] Figure 2 is the X-ray diffraction spectrum of DUT-5 of the present invention;

[0027] Figure 3 It is the particle size analysis diagram of DUT-5 of the present invention;

[0028] Figure 4 This is the SEM image of DUT-5 of the present invention;

[0029] Figure 5 This is a Pickering emulsion diagram based on DUT-5 of the present invention;

[0030] Figure 6 This is an inverted microscope image of the Pickering emulsion based on DUT-5 of the present invention;

[0031] Figure 7 This is a particle size analysis diagram of the Pickering emulsion based on DUT-5 of the present invention;

[0032] Figure 8 This is a zeta potential analysis diagram of the Pickering emulsion based on DUT-5 of the present invention;

[0033] Fig. 9 The calibration curve corresponding to the absorbance of different concentrations of BSA protein of the present invention;

[0034] Fig.10 This is a standard BCA protein test result diagram of the present invention;

[0035] Fig.11 This is a particle size analysis diagram of the Pickering emulsion after protein loading in the present invention;

[0036] Fig.12 This is a cytotoxicity analysis diagram of DUT-5 of the present invention;

[0037] Fig.13 This is a graph showing the weight changes of mice in each experimental group after immunization according to the second embodiment of the present invention;

[0038] Fig.14 This is a graph showing the total antibody levels of mice in the PE@Cap group 0-14 days after immunization in the second embodiment of the present invention;

[0039] Fig.15 This is a comparison chart of the total antibody levels of mice in each experimental group 14 days after immunization in the second embodiment of the present invention. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The present invention discloses a method for preparing a Pickering emulsion vaccine based on a metal organic framework, comprising the following steps:

[0042] A method for preparing a Pickering emulsion vaccine based on a metal organic framework, characterized in that it comprises the following steps:

[0043] Step 1: Add 0.525 g of Al(NO3)3·9H2O with a concentration of 1.4 mmol to 15 mL of DMF and sonicate until fully dissolved; add 0.266 g of 4,4'-biphenyldicarboxylic acid with a concentration of 1.2 mmol to 15 mL of DMF and sonicate until fully dissolved;

[0044] Step 2: After mixing the above solutions, transfer them to a reactor, put the reactor into an oven, react at 120°C, wash with DMF three times, wash with anhydrous ethanol once, and then dry under vacuum at room temperature to obtain white DUT-5 powder;

[0045] like Figure 1 As shown in the infrared spectrum of DUT-5, the change of the characteristic stretching vibration peak proves that 4,4'-biphenyldicarboxylic acid and Al 3+ Coordination occurred, and DUT-5 material was successfully synthesized;

[0046] XRD powder diffraction spectrum Figure 2 As shown, it is proved that DUT-5 has a higher crystallinity;

[0047] The particle size analysis and SEM images of DUT-5 show that DUT-5 is a cake-shaped nanomaterial with an average particle size of about 200 nm. Figure 3 and Figure 4 shown.

[0048] Step 3: Add the DUT-5 prepared above into ultrapure water, mix by ultrasonic for 10 min, add 200 μL of squalene, and ultrasonicate at 60W for 20 min in an ice bath to obtain a Pickering emulsion;

[0049] Step 4: Mix the obtained Pickering emulsion with PCV2 Cap protein and place in a shaker at 4°C overnight;

[0050] Step 5: The obtained mixed emulsion was ultrafiltered and centrifuged at 1000 rpm for 5 min to obtain DUT-5-PE@CAP.

[0051] like Figure 5 and Figure 6 Shown are the Pickering emulsion image and the inverted microscope image;

[0052] The particle size analysis of the Pickering emulsion showed that the emulsion size was about 2.0 μm, which fully met the injection conditions, such as Figure 7 As shown;

[0053] like Figure 8 Shown is the zeta potential analysis of the Pickering emulsion;

[0054] A calibration curve of BSA protein concentration and absorbance was established using the BCA protein quantitative analysis kit. Fig. 9 As shown;

[0055] It was found that the Pickering emulsion loading efficiency for 2 mg / mL Cap protein was about 99%. Fig.10 As shown;

[0056] The particle size analysis of the Pickering emulsion after protein loading confirmed that the emulsion did not undergo significant changes, such as Fig.11 As shown;

[0057] The invention also discloses a method for preparing a metal organic framework-based Pickering emulsion vaccine and its application in the prevention and treatment of porcine circovirus.

[0058] Example 1: Cytotoxicity experiment of DUT-5

[0059] The cytotoxicity of DUT-5 was determined using the CCK-8 kit (Wuhan Abotek Biotechnology Co., Ltd.).

[0060] Disperse and mix the prepared DUT-5 mother solution, and dilute it with 2% DMEM to a concentration of 100-1000 μg / mL. Inoculate RAW264.7 cells into a 96-well plate and place in an incubator for 24 hours (37°C, 5% CO2). Wash with PBS three times, and add different concentrations of DUT-5 (100 μL) for further culture. Then add 10 μL of CCK-8 solution and continue incubation for 4 hours, and use an enzyme reader to detect the absorbance (450nm). The data obtained are all compared with the live cell rate of the blank group, expressed as a percentage, and 5 parallel experiments are taken for each group.

[0061] like Fig.12 As shown in the figure, when DUT-5 acted on RAW 264.7 cells, the cell activity of the 800μg / mL experimental group began to decline, which was significantly different from the control group without any treatment. The results showed that DUT-5 was cell-safe within 500μg / mL.

[0062] Example 2: Immunity Level Test of DUT-5-PE@Cap

[0063] In order to further detect the specific immune level of DUT-5-PE@Cap. 16 BALB / c female mice (6-8 weeks) were selected as models and randomly divided into 4 groups (4 mice in each group). 100μL of PBS, PCV2 Cap (50μg), a mixture of PCV2 Cap (50μg) solution and Pickering emulsion, and Pickering emulsion vaccine loaded with 50μg PCV2 Cap were subcutaneously injected into the mice, and recorded as PBS, Cap, PE+Cap and PE@Cap groups. 14 days later, the mice were immunized for the second time in the same way. After immunization, blood was collected from the tail vein of the mice, and the antibody level in the serum was analyzed using an ELISA kit. The weight of the mice in the experimental group was tested daily, and their growth curves were recorded after the second immunization.

[0064] like Fig.13 As shown, there was almost no difference in body weight among the mice in each group, proving that the experimental procedure had no impact on the health of the mice.

[0065] like Fig.14 As shown, the antibody level of the PE@Cap group after the second immunization was first positively correlated with time, and then the antibody level gradually increased over time.

[0066] like Fig.15 As shown, 14 days after the second immunization, the immune level of the PE@Cap group was significantly higher than that of the other experimental groups.

[0067] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a Pickering emulsion vaccine based on a metal organic framework, characterized in that: The steps include: Step 1: Add Al(NO3)3·9H2O to DMF and sonicate until fully dissolved; add 4,4'-biphenyldicarboxylic acid to DMF and sonicate until fully dissolved; Step 2: After mixing the above solutions, transfer them into a reactor, place the reactor in an oven for reaction, wash with DMF, then wash with anhydrous ethanol, and then dry under vacuum at room temperature to obtain white DUT-5 powder; Step 3: Add the DUT-5 prepared above into ultrapure water, mix by ultrasonication, add squalene thereto, and perform ultrasonication in an ice bath to obtain a Pickering emulsion; Step 4: Mix the obtained Pickering emulsion with PCV2 Cap protein and place in a shaker overnight; Step 5: The obtained mixed emulsion is ultrafiltered and centrifuged to obtain DUT-5-PE@CAP.

2. The method for preparing a metal organic framework-based Pickering emulsion vaccine according to claim 1, characterized in that: In the step 1, the amount of DMF added is 15 mL; the concentration of Al(NO3)3·9H2O is 1.4 mmol, and the amount added is 0.525 g; the concentration of 4,4'-biphenyldicarboxylic acid is 1.2 mmol, and the amount added is 0.266 g.

3. The method for preparing a metal organic framework-based Pickering emulsion vaccine according to claim 1, characterized in that: In the step 2, the reaction kettle is placed in an oven and reacted at 120°C.

4. The method for preparing a metal organic framework-based Pickering emulsion vaccine according to claim 1, characterized in that: In the step 2, the mixture is washed with DMF three times, washed with anhydrous ethanol once, and then dried in vacuum at room temperature.

5. The method for preparing a metal organic framework-based Pickering emulsion vaccine according to claim 1, characterized in that: In step 3, the first ultrasonic treatment time is 10 min, and the second ultrasonic treatment time is 20 min.

6. The method for preparing a metal organic framework-based Pickering emulsion vaccine according to claim 1, characterized in that: In step 3, the ultrasonic power is 60W.

7. The method for preparing a metal organic framework-based Pickering emulsion vaccine according to claim 1, characterized in that: In the step 3, the amount of squalene added is 200 μL.

8. The method for preparing a metal organic framework-based Pickering emulsion vaccine according to claim 1, characterized in that: In the step 4, the obtained Pickering emulsion is mixed with the PCV2 Cap protein and placed in a shaker at 4° C. overnight.

9. The method for preparing a metal organic framework-based Pickering emulsion vaccine according to claim 1, characterized in that: In step 5, the centrifugal speed is 1000 rpm and the centrifugal time is 5 min.

10. Application of the metal organic framework-based Pickering emulsion vaccine preparation method according to claims 1-9 in the prevention and treatment of porcine circovirus.

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