CN116115565A - PLGA nanoemulsion and preparation method and application thereof in paratuberculosis vaccine - Google Patents

PLGA nanoemulsion and preparation method and application thereof in paratuberculosis vaccine Download PDF

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CN116115565A
CN116115565A CN202211671784.1A CN202211671784A CN116115565A CN 116115565 A CN116115565 A CN 116115565A CN 202211671784 A CN202211671784 A CN 202211671784A CN 116115565 A CN116115565 A CN 116115565A
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plga
nanoemulsion
prepared
dopamine hydrochloride
retinoic acid
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周向梅
刘一朵
王元智
葛昕
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China Agricultural University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
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Abstract

The invention provides a preparation method of novel PLGA nanoemulsion co-loaded with various immunostimulants and antigens, wherein PLGA is used for encapsulating all-trans retinoic acid, and after dopamine hydrochloride is plated on the surfaces of nanoparticles, recombinant antigen proteins and CPG oligonucleotides are successfully loaded on the surfaces of the nanoparticles, so that a novel method for preventing and treating intestinal pathogens and paratuberculosis is provided.

Description

PLGA nanoemulsion and preparation method and application thereof in paratuberculosis vaccine
Technical Field
The application belongs to the field of vaccines in the field of biotechnology, and particularly provides PLGA nanoemulsion co-loaded with CPG (CPG-complementary g-oligonucleotide-all-trans-retinoic acid-antigen protein) and preparation and application thereof in paratuberculosis vaccines.
Background
Paratuberculosis (Paratuberculosis) is a chronic digestive tract disease of common domestic animals and ruminants caused by mycobacterium Paratuberculosis (Mycobacterium aviumsubsp Paratuberculosis, MAP) of mycobacterium avium subspecies, and seriously affects the development of animal husbandry, and development of a new method for preventing and treating mycobacterium Paratuberculosis is needed. The traditional vaccination modes such as subcutaneous injection, intramuscular injection and intradermal injection have been repeatedly proven to be incapable of realizing the activation of mucosal immunity due to tissue restriction, and in order to improve the clinical use convenience, the research aims at inventing a novel nano vaccine which has simple and convenient immunization path and can activate intestinal mucosal immunity so as to prevent and treat livestock paratuberculosis infection.
CpG ODN is artificially synthesized oligodeoxynucleotide with unmethylated cytosine and guanine nucleotide as core, can simulate bacterial DNA to stimulate immune cells of various mammals including human, is also a Toll-like receptor 9 agonist, directly activates antigen presenting cells such as B cells, macrophages and dendritic cells, can indirectly activate NK cells and T cells, stimulates the immune cells to secrete cytokines such as TNF-alpha, IFN-gamma, IL-12, and the like, induces Th1 type immune response, and generates stronger humoral immunity and cellular immunity. In recent years, the important role and mechanism of all-trans retinoic acid (all-trans retinoic acid, atRA) in mucosal immune responses has been revealed: it can induce lymphocyte homing to intestinal tract by regulating alpha 4 beta 7 integrin, cytokine CCR9, thymic Stromal Lymphopoietin (TSLP), lactoferrin and other ways, and raise the secretion level of sIgA in intestinal mucosa. However, the atRA has poor water solubility and poor photostability, severely restricting its use in adjuvants.
PLGA is a degradable high molecular organic compound formed by randomly polymerizing two monomers of lactic acid and glycolic acid, and has the advantages of no toxicity, good biocompatibility and capability of forming capsules and films. PLGA has been approved by the food and drug administration (Food and Drug Administration, FDA) as a delivery vehicle for vaccines and drugs due to its excellent safety in the human and animal body.
Disclosure of Invention
According to the method, the fat-soluble atRA is coated in the PLGA nano particles, after active groups are introduced into the surface coating of the nano particles through dopamine hydrochloride, the antigen and the CPG oligonucleotide are successfully loaded on the surfaces of the nano particles, so that the common loading of the atRA, the CPG oligonucleotide and antigen proteins is realized, the humoral immunity, the cellular immunity and the activation of the mucosal immunity can be simultaneously induced through the conventional injection immune mode, and the feasibility and the advantages of the common loading nano microsphere as an auxiliary tuberculosis vaccine adjuvant are proved. The application research provides a preparation method of novel PLGA nanoemulsion co-loaded with various immunostimulants and antigens, wherein PLGA is used for encapsulating all-trans retinoic acid, dopamine hydrochloride is plated on the surface of nanoparticles, and recombinant antigen proteins and CPG oligonucleotides are loaded on the surfaces of the nanoparticles, so that a novel method for preventing and treating intestinal pathogens and paratuberculosis is provided.
In one aspect, the present application provides a PLGA nanoemulsion loaded with a CPG oligonucleotide-all-trans retinoic acid-antigen protein.
Further, the PLGA nanoemulsion is prepared by loading fat-soluble all-trans retinoic acid and water-soluble CPG oligonucleotide and antigen protein on PLGA.
Further, the antigen protein is a mycobacterium paratuberculosis antigen.
Further, the antigen protein is fusion protein HBHA-Ag85B-Bfra.
In another aspect, the present application provides a method of preparing the PLGA nanoemulsion, comprising:
1) Weighing a proper amount of PLGA and all-trans retinoic acid, dissolving in an oil phase, and oscillating and uniformly mixing until the PLGA and the all-trans retinoic acid are completely dissolved;
2) Dropwise adding the solution prepared in the step 1) into PVA solution, and performing ultrasonic emulsification under ice bath conditions;
3) Pouring the solution prepared in the step 2) into PVA solution, stirring for 4 hours, and volatilizing an oil phase;
4) Centrifugally washing the PLGA nanoemulsion coated with the atRA prepared in the step 3), and then, re-suspending in Tris buffer solution containing dopamine hydrochloride, and mixing and reacting at room temperature;
5) After the prepared nanoemulsion with the surface plating dopamine hydrochloride is centrifugally washed, the nanoemulsion is resuspended in PBS solution containing CPG oligonucleotide and antigen protein, and the mixture is mixed at room temperature;
6) And (3) centrifugally washing the prepared nanoparticle solution in a high-speed refrigerated centrifuge, and discarding the supernatant after washing, namely drying the nanoparticle solution at the temperature of minus 80 ℃ by using a vacuum refrigerated dryer to obtain microsphere powder.
Further, the mass ratio of PLGA to trans-retinoic acid was 0.5%.
Further, PLGA was passed through the coating of dopamine hydrochloride to enable adsorption of antigen and CPG.
Further, the coated dopamine hydrochloride nanoemulsion was resuspended in PBS containing 100. Mu.g/mL CPG oligonucleotide and 40. Mu.g/mL antigen protein.
Further, the oil phase is ethyl acetate or dichloromethane.
Further, in Tris buffer containing dopamine hydrochloride, the concentration of dopamine hydrochloride is 2mg/mL, the concentration of Tris is 10M, and the pH is 8.5.
On the other hand, the application provides application of the PLGA nanoemulsion or the PLGA nanoemulsion prepared according to the method in preparation of paratuberculosis vaccines.
Further, the vaccine can reduce the bacterial load of the vaccinated subjects after infection of the mycobacterium paratuberculosis.
Further, the vaccine can promote TNF-alpha, IL-10, IFN-gamma, antibody IgG and intestinal mucosa IgA secretion after immunization of a vaccinated subject.
In another aspect, the present application provides a paratuberculosis vaccine comprising the above PLGA nanoemulsion or PLGA nanoemulsion prepared according to the above method.
The antigen protein in the present application is not limited to HBHA-Ag85B-Bfra, and other antigen proteins of MAP may be used.
The CPG oligonucleotide is a B class CpG ODN, is a linear CpG ODN with complete thio modification, has strong immunostimulatory activity on B cells, and can be obtained commercially or by self-made by a person skilled in the art.
The nanoparticles/vaccines herein are preferably used orally, but it is not excluded that the nanoparticles/vaccines are administered by injection, nasal spray, etc. after selection of the appropriate carrier and formulation.
Adjuvants in this application may be selected by those skilled in the art from known or developed varieties according to conventional knowledge in the vaccine art, including, but not limited to, adjuvants, solvents, co-solvents, buffers, antioxidants, preservatives.
Drawings
FIG. 1 shows recombinant fusion protein HBHA-Ag85B-Bfra purified by SDS-PAGE and Western blot verification;
FIG. 2 is a representation of novel co-loaded nanoparticles;
FIG. 3 is an immune evaluation of nanoparticles;
FIG. 4 shows liver load of mice.
Detailed Description
EXAMPLE 1 expression of recombinant fusion proteins
The E.coli transformed with HBHA-AG85B-Bfra-pET-30a (+) prokaryotic expression plasmid was recovered, the bacteria were cultured in LB medium containing kanamycin (final concentration: 50. Mu.g/mL) to logarithmic phase (OD 600 nm: 0.6-0.8), IPTG (final concentration: 1 mM) was added, shaking table at 30℃and 160rpm, induced expression was performed for 4 hours, 4℃and centrifugation at 8000rpm was performed for 3 minutes to collect the cells, and washing with pre-chilled PBS was performed twice, the cells were resuspended and sonicated with PBS, lysed, and centrifugation at 10000rpm for 10 minutes to obtain a lysate. Purifying the cleavage supernatant by Ni column affinity chromatography to obtain recombinant fusion protein HBHA-AG85B-Bfra.
The nucleic acid sequence of the recombinant fusion protein is as follows
CTTAGAGGGATTCATTCCCTCTAGAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAGGCCATGGCTGATGATATCGGCGGAGGTGGAGGATCAACAGATCTGAGCGAGAAGGTCCGGGCCTGGGGGCGCCGGCTTTTGGTCGGCGCAGCGGCGGCTGTAACCCTGCCGGGCCTGATCGGTCTTGCCGGCGGCGCGGCGACCGCGAATGCGTTTTCGCGTCCGGGCCTGCCCGTCGAGTACCTGCAGGTGCCCTCCGCGGGAATGGGCCGCGACATCAAGGTCCAGTTCCAGAGCGGCGGCAACGGCTCCCCCGCGGTGTATCTGCTGGACGGCCTGCGGGCTCAGGACGACTACAACGGCTGGGACATCAACACCCCGGCCTTCGAGTGGTACTACCAGTCCGGCCTGTCGGTGATCATGCCCGTCGGCGGACAGTCCAGCTTCTACGCGGACTGGTACCAGCCCGCGTGCGGCAAGGCCGGTTGCTCCACTTATAAGTGGGAGACCTTCCTGACCAGCGAGCTGCCGTCGTACCTGGCCTCCAACAAGGGTGTGAAGCGCACCGGCAGCGCCGCAGTCGGCATTTCGATGTCCGGATCCTCGGCGATGATCCTGGCCGTCAACCATCCCGACCAATTCATCTATGCCGGATCGCTCTCGGCGCTGCTCGACCCGTCCCAGGGCATGGGGCCGTCGCTGATCGGTCTGGCGATGGGTGACGCCGGCGGCTACAAGGCCGACGCCATGTGGGGCCCGTCCAGCGACCCGGCCTGGCAGCGCAACGACCCGAGCCTGCACATCCCCGAGCTGGTCGGCCACAACACCCGGCTGTGGGTGTACTGCGGTAACGGGACACCGTCGGAGCTGGGTGGCGCCAACATGCCCGCCGAGTTCCTGGAGAACTTCGTGCGCAGCAGCAACCTGAAGTTCCAGGACGCCTACAACGGCGCCGGCGGCCACAACGCCGTGTTCAACTTCAACGCCAACGGAACGCACAGCTGGGAGTACTGGGGAGCCCAGCTCAACGCCATGAAGCCCGACCTGCAGGGCACCCTGGGCGCGTCCCCGGGCGGCGGCGGAGGAATTCATGGCGGAAAACCCGAACATCGACGACTTGCGAGCCCCGCTGCTCGCGGCCCTGGGCGCGGCCGACCTGGCCCTGGCCACGGTCAACGACCTGATCGCCAACCTGCGCGAGCGGGCCGAGGAGACCCGCGCCGAGACCCGCACCCGGGTCGAGGAGCGCCGCGCCCGGCTGACCAAGTTCCAGGAGGACCTGCCCGAGCAGTTCACCGAGCTGCGCGACAAGTTCACCACCGAGGAGCTGCGCAAGGCGGCCGAGGGCTACCTGGAGGCGGCGACCAACCGGTACAACGAGCTGGTCGAGCGCGGCGAGGCGGCCCTGCAGCGGCTGCGCAGCCAGACCGCCTTCGAGGACGCCTCCGCGCGCGCCGAGGGCTACGTGGACCAGGCCGTCGAGCTGACCCAGGAGGCGCTGGGCACCGTCGCGTCGCAGACCCGCGCGGTCGGTGAGCGCGCCGCCAAGCTGGTGGGCATCGAGCTGCCGGGCAAGGCCGAGGCCGCCGGCAAGAAGGCCCAGAAGGCCATCGCCAAGGCCCCCGCCAAGAAGGCGCCGGCCAAGAAGGTCACCCAGAAGGAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGAAGCGTAGGTCCCG(SEQ ID NO.1)
The purified recombinant protein was subjected to SDS-PAGE gel electrophoresis and Western blot identification using MAP positive serum from mice infected, and the result showed that a single band was visible on NC membrane, conforming to the expected size (FIG. 1).
Example 2 preparation of Co-Supported nanoparticles
1) Weighing 0.1g of PLGA and 500 mug of all-trans retinoic acid, dissolving in 5ml of dichloromethane, and shaking and mixing until the PLGA and the 500 mug of all-trans retinoic acid are completely dissolved;
2) Dropwise adding the solution prepared in the previous step into 10mL of 1% PVA, performing ultrasonic emulsification under ice bath conditions, wherein the ultrasonic conditions are 300W, the total time is 6min, working is 2s, and stopping for 3s;
3) Pouring the solution prepared in the second step into 10mL of 1% PVA solution, stirring at 300-400rpm at room temperature for 4 hours, and volatilizing an oil phase;
4) And (3) centrifugally washing the PLGA nanoemulsion coated with the atRA, which is prepared in the third step, and then re-suspending in Tris buffer containing 2mg/mL dopamine hydrochloride, and mixing and reacting for 3 hours at room temperature. The method comprises the steps of carrying out a first treatment on the surface of the
5) After the prepared nanoemulsion with the surface plating dopamine hydrochloride is centrifugally washed, the nanoemulsion is resuspended in PBS solution containing 100 mug/mL CPG oligonucleotide and 40 mug/mL antigen protein, and the mixture is mixed for 3 hours at room temperature;
6) Centrifuging and washing the obtained nanoparticle solution in a high-speed refrigerated centrifuge, cleaning for 3 times, discarding supernatant, and preserving at 4deg.C for several days or vacuum lyophilizing at-80deg.C to obtain microsphere powder
Example 3 characterization of Co-loaded nanoparticles
And dispersing a proper amount of microsphere powder with a small amount of deionized water, uniformly spreading on a corresponding metal plate of the instrument, drying at normal temperature, spraying gold, and observing the morphology of the microspheres under a scanning electron microscope. Taking a small amount of microsphere powder, re-dissolving with deionized water to uniformly disperse the microsphere powder, placing the microsphere powder into a dynamic optical particle analyzer according to the specification, and analyzing data by using Malvern Instrument software.
As shown in FIG. 2, the average particle size of the prepared nanoparticles was about 700nm, and the potential was-29.8 mV. The scanning electron microscope result shows that the size of the co-loaded nano particles is uniform and the co-loaded nano particles are spherical with smoother surfaces.
EXAMPLE 4 evaluation of the immunogenicity and protective Properties of Co-loaded nanoparticles
The C57BL/6 mice were randomly divided into 4 groups of 12, each of PBS group (control), antigenic protein+atRA group (Ag-atRA), CPG+antigenic protein+atRA group (Ag-atRA-CPG), aluminum adjuvant+Ag+atRA group (Ag-atRA-ALum) and co-loaded nanoparticle group (Ag-PLPCa). The immunization mode is intramuscular injection, each group is immunized three times, 10mg each time, two weeks each time interval, and 3 mice are randomly selected for detection of related immune indexes after the last immunization. The toxin attacking mode is as follows: two weeks after the last immunization, each group of mice was intraperitoneally injected with 100 μl of 10 8 CFU/MAP only (2015 WD-1 strain); after 8 weeks of challenge, 5 mice per group were randomly selected for follow-up testing by dissecting samples.
The results in FIG. 3 show that Bfra-PLGA can significantly promote secretion of intestinal mucosal IgA and proliferation of spleen T cells after immunization (FIG. 3).
The liver load of mice was detected 8 weeks after MAP challenge, and the results (FIG. 4) show that intramuscular injection of co-loaded nanoparticles can significantly reduce liver load of mice.

Claims (12)

1. A PLGA nanoemulsion, characterized in that the PLGA nanoemulsion is loaded with a CPG oligonucleotide-all-trans retinoic acid-antigen protein.
2. The PLGA nanoemulsion according to claim 1, wherein the PLGA nanoemulsion is prepared by co-loading fat-soluble all-trans retinoic acid with a water-soluble CPG oligonucleotide and an antigen protein on PLGA.
3. The PLGA nanoemulsion of claim 2, wherein the antigenic protein is a mycobacterium paratuberculosis antigen.
4. The PLGA nanoemulsion of claim 3, wherein the antigenic protein is the fusion protein HBHA-Ag85B-Bfra.
5. The method of preparing PLGA nanoemulsion according to any one of claims 1-4, wherein the method comprises:
1) Weighing a proper amount of PLGA and all-trans retinoic acid, dissolving in an oil phase, and oscillating and uniformly mixing until the PLGA and the all-trans retinoic acid are completely dissolved;
2) Dropwise adding the solution prepared in the step 1) into PVA solution, and performing ultrasonic emulsification under ice bath conditions;
3) Pouring the solution prepared in the step 2) into PVA solution, stirring for 4 hours, and volatilizing an oil phase;
4) Centrifugally washing the PLGA nanoemulsion coated with the atRA prepared in the step 3), and then, re-suspending in Tris buffer solution containing dopamine hydrochloride, and mixing and reacting at room temperature;
5) After the prepared nanoemulsion with the surface plating dopamine hydrochloride is centrifugally washed, the nanoemulsion is resuspended in PBS solution containing CPG oligonucleotide and antigen protein, and the mixture is mixed at room temperature;
6) And (3) centrifugally washing the prepared nanoparticle solution in a high-speed refrigerated centrifuge, and discarding the supernatant after washing, namely drying the nanoparticle solution at the temperature of minus 80 ℃ by using a vacuum refrigerated dryer to obtain microsphere powder.
6. The method of claim 5, wherein the mass ratio of PLGA to trans-retinoic acid is 0.5%.
7. The method of claim 5, wherein the PLGA is coated with dopamine hydrochloride to adsorb antigen and CPG.
8. The method according to claim 5, wherein the dopamine hydrochloride coated nanoemulsion is resuspended in PBS containing 100. Mu.g/mL CPG oligonucleotide and 40. Mu.g/mL antigen protein.
9. The method of claim 5 or 6, wherein the oil phase is ethyl acetate or methylene chloride.
10. The method of any one of claims 5-9, wherein the concentration of dopamine hydrochloride in the Tris buffer containing dopamine hydrochloride is 2mg/mL, the concentration of Tris is 10m, and the ph is 8.5.
11. Use of PLGA nanoemulsion according to any one of claims 1-5 or prepared using the method according to any one of claims 5-10, in the preparation of a paratuberculosis vaccine.
12. A paratuberculosis vaccine, characterized in that the vaccine comprises PLGA nanoemulsion according to any one of claims 1-4 or prepared using the method according to any one of claims 5-11.
CN202211671784.1A 2022-12-26 2022-12-26 PLGA nanoemulsion and preparation method and application thereof in paratuberculosis vaccine Pending CN116115565A (en)

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