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

Abstract

The present invention discloses a method for preparing a metal organic framework-based Pickering emulsion vaccine and its application, comprising the following steps: using Al(NO3)3·9H2O and 4,4'-diphenyldicarboxylic acid as raw materials, obtaining white DUT-5 powder by a solvothermal method; adding the prepared DUT-5 to ultrapure water, and mixing by simple ultrasound to obtain a Pickering emulsion; mixing the obtained Pickering emulsion with PCV2Cap protein and placing it in a shaking table overnight; ultrafiltration and centrifugation of the obtained mixed emulsion to obtain DUT-5-PE@CAP. The vaccine of the present invention can effectively deliver antibodies or proteins to specific locations and stimulate a strong immune response, providing new possibilities for the development of new vaccines, and providing a new method for the prevention and treatment of porcine circovirus type II, which is of great significance for controlling the disease.

Description

Technical Field

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

[0002] Epidemics have a devastating impact on the livestock industry, not only causing illness or death in animals but also negatively impacting farming efficiency. Vaccination, by stimulating or modulating the host immune system, has become the most cost-effective strategy for preventing a wide range of infectious diseases. It is crucial for preventing animal diseases and ensuring the sustainable development of agriculture and animal husbandry. Despite this, existing vaccines still face challenges, such as insufficient cellular immune responses, lack of protection against multiple pathogens, and antibody-dependent enhancement. Nanotechnology holds great potential in vaccine development. Nanoparticles, as vaccine carriers, can enhance the body's immune response and effectively combat viral diseases and cancer.

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

[0004] Researchers are exploring particulate carriers as novel adjuvants. These carriers mimic the characteristics of pathogens or diseased cells, rather than simply the mobility of antigens. Pickering emulsions, as particle-stabilized emulsions, have shown potential as vaccine carriers, with proven efficacy. The nanoparticles in these emulsions can be further optimized to achieve even more functionalities.

[0005] This study developed a Pickering emulsion vaccine based on MOFs. Preliminary studies have shown that this vaccine can effectively deliver antibodies or proteins to specific locations and stimulate a strong immune response, offering new possibilities for the development of novel vaccines. From an industrial perspective, this vaccine offers a new approach for the prevention and treatment of porcine circovirus type 2, with significant implications for disease control. 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 its application.

[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 to 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 to ultrapure water, mix by ultrasonication, add squalene thereto, and ultrasonicate in an ice bath to obtain a Pickering emulsion;

[0012] Step 4: The obtained Pickering emulsion was mixed with PCV2 Cap protein and placed in a shaker overnight; Step 5: The obtained mixed emulsion was ultrafiltered and centrifuged to obtain DUT-5-PE@CAP.

[0013] Preferably, in step 1, the amount of DMF added is 15 mL; the concentration of Al(NO 3 ) 3 ·9H 2 O 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 reactor is placed in an oven and reacted at 120°C.

[0015] Preferably, in step 2, the product 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 ultrasonication time is 10 min, and the second ultrasonication 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 the Al-MOFs nanovaccine, the antigen can be encapsulated on the surface of the emulsion through a one-step ultrasound, providing a stable environment for the antigen and achieving enhanced antigen stability.

[0024] (2) The present invention is a treatment method for porcine circovirus type 2, which is based on a MOFs-based Pickering emulsion vaccine. The emulsion is combined 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 This is a 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] Figure 9 The figure is a calibration curve corresponding to the absorbance of BSA protein at different concentrations according to the present invention;

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

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

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

[0037] Figure 13 Figure 2 is a graph showing changes in body weight of mice in each experimental group after immunization according to the present invention;

[0038] Figure 14 This is a graph showing the total antibody levels of mice in the PE@Cap group 0-14 days after immunization;

[0039] Figure 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 clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts 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 by comprising the following steps:

[0043] Step 1: Add 0.525 g of Al(NO3)3·9H2O at 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 at 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, place the reactor in an oven, react at 120°C, wash with DMF three times, then 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 to ultrapure water, mix by ultrasonication for 10 min, add 200 μL of squalene, and ultrasonicate at 60 W for 20 min in an ice bath to obtain a Pickering emulsion;

[0049] Step 4: Mix the resulting 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 Pickering emulsion;

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

[0055] It was found that the loading efficiency of Pickering emulsion for 2 mg / mL Cap protein was about 99%. Figure 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 Figure 11 As shown;

[0057] The present invention also discloses an application of a metal organic framework-based Pickering emulsion vaccine preparation method 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] The prepared DUT-5 mother solution was dispersed and mixed, and diluted with 2% DMEM to a concentration of 100-1000 μg / mL. RAW264.7 cells were seeded into 96-well plates and cultured in an incubator for 24 hours (37°C, 5% CO2). Washed 3 times with PBS, and different concentrations of DUT-5 (100 μL) were added and cultured again. Then 10 μL of CCK-8 solution was added and incubated for another 4 hours, and the absorbance was detected using an enzyme reader (450 nm). The data obtained were compared with the viable cell rate of the blank group and expressed as a percentage. Five parallel experiments were performed in each group.

[0061] like Figure 12 As shown in the results, when DUT-5 was applied to RAW 264.7 cells, the cell viability of the 800 μg / mL experimental group began to decline, significantly different from the untreated control group. The results indicate that DUT-5 is cell-safe within the 500 μg / mL range.

[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). 100 μL of PBS, PCV2 Cap (50 μg), a mixture of PCV2 Cap (50 μg) solution and Pickering emulsion, and Pickering emulsion vaccine loaded with 50 μg 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 curve was recorded after the second immunization.

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

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

[0066] like Figure 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 merely a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any minor 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: The solutions in step 1 were mixed and transferred to a reactor, which was placed in an oven for reaction, washed with DMF, then washed with anhydrous ethanol, and then dried under vacuum at room temperature to obtain white DUT-5 powder; Step 3: Add the DUT-5 prepared above to ultrapure water, mix by ultrasonication, add squalene thereto, and ultrasonicate in an ice bath to obtain a Pickering emulsion; Step 4: The obtained Pickering emulsion was mixed with PCV2 Cap protein and placed in a shaker overnight; Step 5: The obtained mixed emulsion was ultrafiltered and centrifuged to obtain DUT-5-PE@CAP; 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; In step 2, the reactor is placed in an oven and reacted at 120°C; In step 3, the ultrasonic power is 60W; In step 5, the centrifugal speed is 1000 rpm and the centrifugal time is 5 min; In step 3, the amount of squalene added is 200 μL.

2. The method for preparing a metal organic framework-based Pickering emulsion vaccine according to claim 1, wherein: In the step 2, the product was washed with DMF three times, washed with anhydrous ethanol once, and then dried in vacuo at room temperature.

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

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

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