A saponin-containing vaccine adjuvant and its preparation method

Abstract

This invention relates to the field of biomedical technology, specifically to a saponin-containing vaccine adjuvant and its preparation method. The saponin-containing vaccine adjuvant is composed of the following raw materials by mass percentage: 0.05-0.2% saponins, 0.5-2.0% phospholipids, 3-7% oil phase, 0.5-2.5% surfactant, 0.05-0.2% antioxidant, 2.5-4.0% trehalose, 0.1-0.5% novel synergistic polymer, and the balance being water for injection. The novel synergistic polymer is a cationic lipid-chitosan polymer. The preparation method of this invention employs an innovative process of "first preparing a blank nanoemulsion, then loading saponins," forming a stable protective layer at the nanoemulsion interface, effectively shielding the hemolysin groups of saponins, significantly reducing their toxic side effects, and protecting their chemical stability. The adjuvant of this invention exhibits good stability, high safety, and strong immunomodulatory effects, showing promising clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a saponin-containing vaccine adjuvant and its preparation method. Background Technology

[0002] Vaccines, as one of the most effective means of preventing and controlling infectious diseases, rely heavily on the use of adjuvants for their efficacy. Adjuvants can non-specifically enhance the body's specific immune response to antigens or alter the type of immune response. Among numerous adjuvant candidates, plant-derived saponins, particularly QS-21 purified from soap bark, have attracted considerable attention due to their superior immunostimulatory capabilities. Unlike traditional aluminum adjuvants, which primarily stimulate humoral immunity, QS-21 can simultaneously and strongly activate both humoral and cellular immune responses. It not only promotes the production of high-titer neutralizing antibodies but also induces a potent CD8+ cytotoxic T lymphocyte (CTL) response, which is crucial for clearing intracellular pathogens (such as viruses) and combating tumors.

[0003] However, saponins, including QS-21, present three major challenges to their clinical application due to their inherent physicochemical properties and strong biological activity. First, there is the issue of chemical stability. The core structure of a saponin molecule consists of a hydrophobic aglycone linked to one or more hydrophilic sugar chains via glycosidic bonds. These glycosidic bonds are highly susceptible to hydrolysis and breakage under acidic, alkaline, thermal, or mechanical stress conditions (such as unavoidable high-speed shearing and high-pressure homogenization during preparation), leading to loss of immunomodulatory activity. In traditional nanoemulsion or liposome preparation processes, saponins typically undergo these drastic physical processes along with other components, resulting in a significant decrease in potency during production and making it difficult to control the consistency of quality between different production batches. Second, there is significant cytotoxicity and hemolytic activity. The amphiphilic structure of saponins allows them to specifically bind to cholesterol molecules on cell membranes, acting like a "detergent," thereby creating pores in the cell membrane and disrupting its integrity. This means that at effective immunization doses, saponins are highly likely to cause red blood cell rupture (i.e., hemolysis) and toxicity to other somatic cells, which severely limits their safe dose window and may even cause local or systemic adverse reactions, thus restricting their widespread use in vaccines.

[0004] To overcome these obstacles, the pharmaceutical community has explored various delivery strategies aimed at maximizing their advantages and minimizing their disadvantages. Common strategies include encapsulating saponins in liposomes, polymer nanoparticles, or water-in-oil emulsions. These strategies reduce the hemolytic activity of saponins to some extent through physical isolation. However, these existing technologies often have limitations. For example, simple encapsulation may not be sufficient to completely shield saponins from membrane-damaging effects, or may lead to premature release during in vivo circulation; while complex preparation processes employed to pursue stability may actually exacerbate saponin degradation during production. More importantly, many existing delivery systems focus only on addressing safety and stability issues, neglecting or failing to effectively improve their immune activation efficiency. Therefore, there is an urgent need in the field to develop a novel technical solution that can simultaneously address the three major issues of saponin stability, safety, and efficiency, in order to fully unleash the enormous potential of saponins as highly effective vaccine adjuvants. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the present invention provides a saponin-containing vaccine adjuvant and its preparation method.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A saponin-containing vaccine adjuvant is composed of the following raw materials by weight percentage: 0.05-0.2% saponins, 0.5-2.0% phospholipids, 3-7% oil phase, 0.5-2.5% surfactant, 0.05-0.2% antioxidant, 2.5-4.0% trehalose, 0.1-0.5% novel synergistic polymer, and the balance being water for injection;

[0008] The novel synergistic polymer is a cationic lipid-chitosan polymer, prepared by a method comprising the following steps:

[0009] (a) Nα-Boc-L-lysine is alkylated with bromododecane in the presence of a first organic solvent and an organic base to obtain Nα-Boc-Nε-dodecyl-L-lysine;

[0010] (b) In the presence of an activator, the product obtained in step (a) is coupled with HOOC-PEG-COOH, and then trifluoroacetic acid is added to remove the Boc protecting group to obtain a cationic lipid with an amino terminus.

[0011] (c) In the presence of MES buffer, the terminal amino-terminated cationic lipid obtained in step (b) is reacted with carboxymethyl chitosan in an aqueous buffer system, and the cationic lipid-chitosan polymer is obtained after purification.

[0012] Although saponin components such as QS-21 are highly effective natural immunostimulants that can simultaneously stimulate strong humoral and cellular immune responses, they still have problems in practical applications: (1) The glycosidic bonds in the saponin molecular structure are easily hydrolyzed and broken under acid, alkali, heat or mechanical stress (such as high pressure homogenization), resulting in loss of immune activity and poor chemical stability. Traditional preparation processes subject it to high-speed shearing or high pressure homogenization with other components, which will accelerate its degradation, resulting in decreased potency and batch-to-batch quality instability; (2) Saponin molecules are both hydrophilic and hydrophobic, and can strongly bind to cholesterol on the cell membrane, thereby destroying the cell membrane integrity at effective concentrations, causing red blood cell rupture (hemolysis) and damage to other somatic cells. Significant cytotoxicity and hemolysis severely limit its safe dose window and scope of use; (3) Although simple saponin molecules can non-specifically stimulate the immune system, they are easily cleared by the body and are difficult to be effectively taken up and presented by antigen-presenting cells, resulting in insufficient immune activation efficiency and insufficient targeting. Therefore, developing a novel delivery system that can simultaneously address the stability, safety, and efficiency of saponins is crucial for promoting their application as vaccine adjuvants.

[0013] This invention introduces a novel synergistic polymer, a cationic lipid-chitosan polymer, whose unique structure precisely addresses the three major problems mentioned above regarding saponin adjuvants. Its mechanism of action is as follows: First, through a three-step synthesis method, the polymer ultimately forms an amphiphilic structure possessing both hydrophobic alkyl chains and a hydrophilic chitosan backbone. Its hydrophobic ends strongly anchor at the oil-phase interface of the nanoemulsion, while the hydrophilic chitosan chains form a dense protective layer on the surface. This physical barrier effectively shields the saponin molecules from damage caused by external aqueous phase and mechanical stress, significantly reducing the risk of hydrolysis of its glycosidic bonds during preparation and storage, thereby ensuring its chemical stability and ultimately stabilizing the interface layer, thus resolving chemical instability. Second, the chitosan backbone in the polymer carries a large amount of positive charge, forming a positively charged hydrophilic barrier on the surface of the nanoemulsion droplets. This generates a strong steric hindrance and electrostatic repulsion effect, physically preventing the hydrophobic ends of the saponin from directly contacting the erythrocyte membrane, neutralizing its membrane-damaging effect. This barrier is effective against ordinary somatic cells without affecting the active uptake by immune cells, thus significantly reducing hemolytic activity and widening the safety window. Finally, the polymer's cationic properties enable multiple positive interactions with antigens and immune cells, which is key to improving vaccine potency. On one hand, its positive surface charge can effectively enrich and concentrate negatively charged antigens through electrostatic adsorption, forming a high concentration of antigen, which helps to continuously provide immune stimulation at the injection site. On the other hand, this positive charge makes it easier for negatively charged antigen-presenting cell membranes to actively recognize and internalize the antigen, greatly improving antigen uptake efficiency. Once inside the acidic environment of the endosomes / lysosomes, the polymer's pH-responsive properties are triggered, rapidly disintegrating to release saponins and antigens, thereby strongly activating the antigen presentation pathway. Synergistically, the immunostimulatory effect of saponins induces higher levels and longer-lasting neutralizing antibodies and effector T cells, significantly improving the vaccine's immunogenicity.

[0014] The specific reaction mechanism is as follows: (a) Using bromododecane and Nα-Boc-L-lysine as the main raw materials and N,N-dimethylformamide as the reaction solvent, under the condition of basic triethylamine, the amino group of the lysine side chain acts as a nucleophile to attack the carbon-bromine bond in bromododecane, replace the bromide ion, form a CN bond, and carry out an alkylation reaction. The product obtained is then carried out in the next step. (b) HOOC-PEG-COOH is first activated and then reacted with the product in step (a). The activated HOOC-PEG-COOH reacts with the amino group in product (a) in an amide reaction. Trifluoroacetic acid is then added to release the terminal primary amino group, resulting in a cationic lipid with an amino group at the end. (c) The cationic lipid with an amino group at the end is dissolved in MES buffer for activation. Carboxymethyl chitosan is added. The amino group in the cationic lipid with an amino group at the end reacts with the carboxyl group in the carboxymethyl chitosan in an amide reaction, thereby grafting multiple cationic lipid molecules onto the chitosan backbone. After dialysis and freeze-drying, the cationic lipid-chitosan polymer is obtained. The polymer in this invention, through its unique cationic properties and amphiphilic structure, can effectively enhance the stability of nanoemulsion systems, promote the uptake by antigen-presenting cells, and improve the intercalation efficiency of saponins at the nanoemulsion interface, thereby significantly reducing the hemolytic activity and cytotoxicity of saponins while enhancing their immune-activating ability. Furthermore, this polymer also possesses pH-responsive properties, allowing for more effective release of the active ingredient in acidic microenvironments, further enhancing the immunomodulatory effect of adjuvants in vaccines.

[0015] Preferably, the preparation method of the novel synergistic polymer is as follows:

[0016] (a) Mix 2-5 parts by weight of bromododecane, 1-3 parts by weight of Nα-Boc-L-lysine and 2-2.8 parts by weight of triethylamine, add 80-150 parts by weight of N,N-dimethylformamide and mix evenly. Stir at 50-70℃ and 200-600 rpm for 6-18 h, remove solvent by rotary evaporation under reduced pressure, pour the product into 80-200 parts by weight of a 1:1 volume ratio of ice-cold diethyl ether / n-hexane mixture and stir to precipitate solid. Wash and vacuum dry for later use.

[0017] (b) Mix 2-4 parts by weight of HOOC-PEG-COOH and 60-100 parts by weight of N,N-dimethylformamide, add 0.5-1.5 parts by weight of EDC·HCl and 0.2-1 parts by weight of NHS, activate at room temperature for 20-50 min, then add 2-5 parts by weight of the above solid, stir at 50-70℃ and 200-600 rpm for 4-10 h, add 3-6 times the volume of trifluoroacetic acid of the reaction solution, stir at room temperature for 2-5 h, remove the solvent by vacuum distillation, and obtain a cationic lipid with an amino terminus.

[0018] (c) Dissolve 1-3 parts by weight of the above-mentioned terminal amino-terminated cationic lipids in 30-80 parts by weight of 0.01 mol / L MES buffer at pH 6.0, wherein the MES buffer comprises 15-30 mM EDC and 30-60 mM sulfo-NHS, and activate at room temperature for 20-50 min; then add 1-4 parts by weight of carboxymethyl chitosan, and react at room temperature in the dark for 12-36 h, dialyze successively with 0.5-2 wt% NaCl solution for 12-36 h, 25 wt% ethanol aqueous solution for 8-24 h, and distilled water for 18-48 h, and then freeze-dry to obtain the cationic lipid-chitosan polymer.

[0019] The saponin components are selected from one or more of purified QS-21, mogrosides, saikosaponins, or their chemically modified derivatives.

[0020] The phospholipid is selected from one or more of hydrogenated soybean lecithin, phosphatidylcholine, and phosphatidylethanolamine.

[0021] The oil phase is selected from one or more of squalene, squalane, and vitamin E acetate.

[0022] The surfactant is selected from one or more of sorbitan trioleate, poloxamer 407, Tween 80, and Span-80.

[0023] The antioxidant is selected from one or more of L-ascorbic acid, acetylcysteine, cysteine, monothiopropyltriglycerol, α-tocopherol, and EDTA-2Na.

[0024] The saponin-containing vaccine adjuvant has a nanoemulsion particle size distribution of 80-200 nm and a polydispersity index of less than 0.2.

[0025] The method for preparing the saponin-containing vaccine adjuvant includes the following steps:

[0026] (1) Preparation of oil phase: Under the protection of inert gas, phospholipids, oil phase and fat-soluble antioxidant are heated and dissolved in a water bath at 50-70℃ and mixed evenly to obtain oil phase;

[0027] (2) Preparation of aqueous phase: Dissolve surfactant, trehalose, water-soluble antioxidant and novel synergistic polymer in water for injection, heat to 50-70℃, stir evenly to obtain aqueous phase;

[0028] (3) Formation of primary emulsion: Under high-speed shearing at 10,000-15,000 rpm, the oil phase is slowly added to the aqueous phase and sheared for 5-15 minutes to form a primary emulsion;

[0029] (4) Formation of nanoemulsion: The above primary emulsion is subjected to a pressure of 500-1500 bar for 5-10 cycles of homogenization to obtain nanoemulsion with a particle size distribution of 100-200 nm;

[0030] (5) Saponin loading: Dissolve the saponin components in water for injection, stir at 32-45℃ for 8-25 min, slowly add to the above nanoemulsion, continue stirring for 30-60 min, filter with a 0.22μm microporous membrane for sterilization, fill with nitrogen under sterile conditions, seal, and obtain the saponin-containing vaccine adjuvant.

[0031] The application of the saponin-containing vaccine adjuvant in the preparation of an inactivated vaccine for porcine reproductive and respiratory syndrome.

[0032] The beneficial effects of this invention are:

[0033] 1. This invention provides a saponin-containing vaccine adjuvant and its preparation method. The unique process of "preparing a blank nanoemulsion first, then loading saponins" completely avoids the harsh mechanical processes of high-pressure homogenization, reducing degradation and loss, and fundamentally preventing the hydrolysis and breakage of its glycosidic bonds, ensuring the complete preservation of its immunomodulatory activity. Simultaneously, the introduced novel synergistic polymer, a cationic lipid-chitosan polymer, forms a dense hydrophilic protective layer at the nanoemulsion interface. Through strong steric hindrance and electrostatic repulsion, this layer effectively shields the hemolytic groups of the saponins, thereby significantly reducing their hemolytic activity and cytotoxicity while retaining their potent immunomodulatory function, thus broadening the safety window for clinical use.

[0034] 2. This invention does not involve simple mixing; instead, it first constructs a nanoemulsion framework stabilized by a cationic lipid-chitosan polymer. Saponin molecules bind to the cationic lipid-chitosan polymer molecules through hydrophobic interactions and hydrogen bonds, embedding themselves into the oil-water interface film of the nanoemulsion. This structure effectively shields the hemolytic groups of saponins, significantly reducing their hemolytic activity and cytotoxicity, while retaining their immune-activating function.

[0035] 3. The vaccine adjuvant provided by this invention forms a nanoemulsion with a uniform particle size distribution (80-200 nm) and a low polydispersity index (PDI < 0.2), maintaining good physical stability under long-term storage and stress conditions. The entire preparation process is mild and the parameters are controllable, making it very suitable for large-scale industrial production and ensuring high batch-to-batch consistency. This adjuvant can effectively improve the immune response level of inactivated vaccines, has few side effects, and the raw materials are readily available, providing a new adjuvant option with excellent performance and broad application prospects for both preventive and therapeutic vaccines. Detailed Implementation

[0036] The invention will now be described in further detail with reference to specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments.

[0037] The raw materials described in this application are partially described; all other raw materials not described are commercially available.

[0038] QS-21 was purchased from Merck Life Sciences (Shanghai) Co., Ltd., item number: S7900.

[0039] Hydrogenated soybean lecithin was purchased from Hubei Xinyuhong Biomedical Technology Co., Ltd., product number xyh001.

[0040] Squalene was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number: S109119-25ml.

[0041] HOOC-PEG-COOH was purchased from Shanghai Yuanye Biotechnology Co., Ltd., model number: S28827.

[0042] Carboxymethyl chitosan was purchased from Wuhan Jiyesheng Chemical Co., Ltd., item number: A25-36-8.

[0043] Example 1

[0044] A saponin-containing vaccine adjuvant is composed of the following raw materials by weight percentage: 0.1% saponin, 1.5% phospholipid, 5% oil phase, 2% surfactant, 0.1% antioxidant, 3% trehalose, 0.3% novel synergistic polymer, and the balance being water for injection.

[0045] The saponin component is QS-21.

[0046] The phospholipid is hydrogenated soybean lecithin.

[0047] The oil phase is a mixture of squalene and vitamin E acetate in a mass ratio of 3:1.

[0048] The surfactant is a mixture of sorbitan trioleate and Tween 80 in a mass ratio of 1:1.

[0049] The antioxidant is a mixture of L-ascorbic acid, acetylcysteine, and α-tocopherol in a mass ratio of 1:1:1.

[0050] The novel synergistic polymer is a cationic lipid-chitosan polymer, and the preparation method of the cationic lipid-chitosan polymer is as follows:

[0051] (a) Mix 3 parts by weight of bromododecane, 2 parts by weight of Nα-Boc-L-lysine and 2.2 parts by weight of triethylamine, add 100 parts by weight of N,N-dimethylformamide and mix well. Stir at 60°C and 400 rpm for 12 h, remove solvent by rotary evaporation under reduced pressure, pour the product into 100 parts by weight of a 1:1 volume ratio of ice-cold diethyl ether / n-hexane mixture and stir to precipitate solid. Wash and vacuum dry for later use.

[0052] (b) Mix 2.5 parts by weight of HOOC-PEG-COOH and 80 parts by weight of N,N-dimethylformamide, add 1.8 parts by weight of activator and activate at room temperature for 30 min. The activator is a combination of 1.2 parts by weight of EDC·HCl and 0.6 parts by weight of NHS. Then add 3 parts by weight of the above solid and stir at 60°C and 400 rpm for 6 h. Add 4 times the volume of trifluoroacetic acid of the reaction solution and stir at room temperature for 3 h. Remove the solvent by vacuum distillation to obtain a cationic lipid with an amino terminus.

[0053] (c) Dissolve 2 parts by weight of the above-mentioned terminal amino-terminated cationic lipid in 50 parts by weight of 0.01 mol / L MES buffer at pH 6.0, wherein the MES buffer comprises 20 mM EDC and 50 mM sulfo-NHS, and activate at room temperature for 30 min; then add 2 parts by weight of carboxymethyl chitosan, react at room temperature in the dark for 24 h, dialyze successively with 1 wt% NaCl solution for 24 h, 25 wt% ethanol aqueous solution for 12 h, and distilled water for 24 h, and then freeze dry to obtain the cationic lipid-chitosan polymer.

[0054] The preparation method of the saponin-containing vaccine adjuvant is as follows:

[0055] (1) Preparation of oil phase: Under the protection of inert gas, phospholipids, oil phase and fat-soluble antioxidant are heated and dissolved in a water bath at 65°C and mixed evenly to obtain oil phase;

[0056] (2) Preparation of aqueous phase: Dissolve surfactant, trehalose, water-soluble antioxidant and novel synergistic polymer in water for injection, heat to 60°C and stir evenly to obtain aqueous phase;

[0057] (3) Formation of primary emulsion: Under high-speed shearing at 14000 rpm, the oil phase is slowly added to the aqueous phase and sheared for 12 min to form a primary emulsion;

[0058] (4) Formation of nanoemulsion: The above primary emulsion was subjected to a pressure of 1200 bar for 7 cycles of homogenization to obtain nanoemulsion with a particle size distribution of 120 nm;

[0059] (5) Saponin loading: Dissolve the saponin components in water for injection, stir at 37°C for 20 min, slowly add to the above nanoemulsion, continue stirring for 50 min, filter with a 0.22 μm microporous membrane for sterilization, fill with nitrogen under sterile conditions, seal, and obtain the saponin-containing vaccine adjuvant.

[0060] Example 2

[0061] It is basically the same as Example 1, except that the amount of novel synergistic polymer added to the saponin-containing vaccine adjuvant is 0.1%.

[0062] Example 3

[0063] It is basically the same as Example 1, except that the amount of novel synergistic polymer added to the saponin-containing vaccine adjuvant is 0.5%.

[0064] Comparative Example 1

[0065] A saponin-containing vaccine adjuvant is composed of the following raw materials by weight percentage: 0.1% saponin, 1.5% phospholipid, 5% oil phase, 2% surfactant, 0.1% antioxidant, 3% trehalose, and the balance being water for injection.

[0066] The saponin component is QS-21.

[0067] The phospholipid is hydrogenated soybean lecithin.

[0068] The oil phase is a mixture of squalene and vitamin E acetate in a mass ratio of 3:1.

[0069] The surfactant is a mixture of sorbitan trioleate and Tween 80 in a mass ratio of 1:1.

[0070] The antioxidant is a mixture of L-ascorbic acid, acetylcysteine, and α-tocopherol in a mass ratio of 1:1:1.

[0071] The preparation method of the saponin-containing vaccine adjuvant is as follows:

[0072] (1) Preparation of oil phase: Under the protection of inert gas, phospholipids, oil phase and fat-soluble antioxidant are heated and dissolved in a water bath at 65°C and mixed evenly to obtain oil phase;

[0073] (2) Preparation of aqueous phase: Dissolve surfactant, trehalose and water-soluble antioxidant in water for injection, heat to 60°C and stir evenly to obtain aqueous phase;

[0074] (3) Formation of primary emulsion: Under high-speed shearing at 14000 rpm, the oil phase is slowly added to the aqueous phase and sheared for 12 min to form a primary emulsion;

[0075] (4) Formation of nanoemulsion: The above primary emulsion was subjected to a pressure of 1200 bar for 7 cycles of homogenization to obtain nanoemulsion with a particle size distribution of 120 nm;

[0076] (5) Saponin loading: Dissolve the saponin components in water for injection, stir at 37°C for 20 min, slowly add to the above nanoemulsion, continue stirring for 50 min, filter with a 0.22 μm microporous membrane for sterilization, fill with nitrogen under sterile conditions, seal, and obtain the saponin-containing vaccine adjuvant.

[0077] Comparative Example 2

[0078] The method is basically the same as in Example 1, except that the preparation method of the saponin-containing vaccine adjuvant is as follows:

[0079] (1) Preparation of oil phase: Under the protection of inert gas, phospholipids, oil phase and fat-soluble antioxidant are heated and dissolved in a water bath at 65°C and mixed evenly to obtain oil phase;

[0080] (2) Preparation of aqueous phase: Saponin components, surfactants, trehalose, water-soluble antioxidants, and novel synergistic polymers are dissolved in water for injection, heated to 60°C, and stirred evenly to obtain the aqueous phase;

[0081] (3) Formation of primary emulsion: Under high-speed shearing at 14000 rpm, the oil phase is slowly added to the aqueous phase and sheared for 12 min to form a primary emulsion;

[0082] (4) Formation of nanoemulsion: The above-mentioned primary emulsion was subjected to a pressure of 1200 bar for 7 cycles of homogenization to obtain nanoemulsion with a particle size distribution of 120 nm; it was sterilized by filtration with a 0.22 μm microporous membrane, and then filled with nitrogen under sterile conditions and sealed to obtain the saponin-containing vaccine adjuvant.

[0083] Comparative Example 3

[0084] This is essentially the same as Example 1, except that the saponin-containing vaccine adjuvant does not contain the novel synergistic polymer, and the preparation method of the saponin-containing vaccine adjuvant is as follows:

[0085] (1) Preparation of oil phase: Under the protection of inert gas, phospholipids, oil phase and fat-soluble antioxidant are heated and dissolved in a water bath at 65°C and mixed evenly to obtain oil phase;

[0086] (2) Preparation of aqueous phase: Saponin components, surfactants, trehalose, and water-soluble antioxidants are added to water for injection, heated to 60°C, and stirred until homogeneous to obtain the aqueous phase;

[0087] (3) Formation of primary emulsion: Under high-speed shearing at 14000 rpm, the oil phase is slowly added to the aqueous phase and sheared for 12 min to form a primary emulsion;

[0088] (4) Formation of nanoemulsion: The above-mentioned primary emulsion was subjected to a pressure of 1200 bar for 7 cycles of homogenization to obtain nanoemulsion with a particle size distribution of 120 nm; it was sterilized by filtration with a 0.22 μm microporous membrane, and then filled with nitrogen under sterile conditions and sealed to obtain the saponin-containing vaccine adjuvant.

[0089] Comparative Example 4

[0090] Similar to Example 1, except that a saponin-containing vaccine adjuvant is composed of the following raw materials by mass percentage: 0.1% saponin, 1.5% phospholipid, 5% oil phase, 2% surfactant, 0.1% antioxidant, 3% trehalose, 0.3% carboxymethyl chitosan, and the balance being water for injection.

[0091] Comparative Example 5

[0092] The process is basically the same as in Example 1, except that a simple mixture of 2 parts by weight of chitosan and 2 parts by weight of cationic lipid is used instead of the novel synergistic polymer. The cationic lipid is 2,3-dioleoxypropyltrimethylammonium chloride.

[0093] Comparative Example 6

[0094] The method is basically the same as in Example 1, except that Nα-Boc-L-lysine is replaced with threonine in the preparation method of the cationic lipid-chitosan polymer.

[0095] Comparative Example 7

[0096] The method is basically the same as in Example 1, except that the novel synergistic polymer is a cationic lipid-chitosan polymer, and the preparation method of the cationic lipid-chitosan polymer is as follows:

[0097] (a) Mix 3 parts by weight of bromododecane, 2 parts by weight of Nα-Boc-L-lysine and 2.2 parts by weight of triethylamine, add 100 parts by weight of N,N-dimethylformamide and mix well. Stir at 60°C and 400 rpm for 12 h, remove solvent by rotary evaporation under reduced pressure, pour the product into 100 parts by weight of a 1:1 volume ratio of ice-cold diethyl ether / n-hexane mixture and stir to precipitate solid. Wash and vacuum dry for later use.

[0098] (b) Mix 2.5 parts by weight of HOOC-PEG-COOH and 80 parts by weight of N,N-dimethylformamide, add 1.8 parts by weight of activator and activate at room temperature for 30 min. The activator is a combination of 1.2 parts by weight of EDC·HCl and 0.6 parts by weight of NHS. Then add 3 parts by weight of the above solid and stir at 60°C and 400 rpm for 6 h. Add 4 times the volume of trifluoroacetic acid of the reaction solution and stir at room temperature for 3 h. Remove the solvent by vacuum distillation to obtain a cationic lipid with an amino terminus.

[0099] (c) Dissolve 2 parts by weight of the above-mentioned terminal amino-terminated cationic lipid in 50 parts by weight of 0.01 mol / L MES buffer at pH 6.0, wherein the MES buffer comprises 20 mM EDC and 50 mM sulfo-NHS, and activate at room temperature for 30 min; then add 2 parts by weight of chitosan, react at room temperature in the dark for 24 h, dialyze successively with 1 wt% NaCl solution for 24 h, 25 wt% ethanol aqueous solution for 12 h, and distilled water for 24 h, and then freeze dry to obtain the cationic lipid-chitosan polymer.

[0100] Comparative Example 8

[0101] The method is basically the same as in Example 1, except that the novel synergistic polymer is a cationic lipid-chitosan polymer, and the preparation method of the cationic lipid-chitosan polymer is as follows:

[0102] Two parts by weight of 2,3-dioleoxypropyltrimethylammonium chloride were dissolved in 50 parts by weight of 0.01 mol / L MES buffer at pH 6.0, wherein the MES buffer contained 20 mM EDC and 50 mM sulfo-NHS. The mixture was activated at room temperature for 30 min. Then, two parts by weight of carboxymethyl chitosan were added, and the mixture was reacted at room temperature in the dark for 24 h. The mixture was then dialyzed against 1 wt% NaCl solution for 24 h, against 25 wt% ethanol aqueous solution for 12 h, and against distilled water for 24 h. Finally, the mixture was freeze-dried to obtain the cationic lipid-chitosan polymer.

[0103] Test Example 1

[0104] Physical stability test: The particle size of the saponin-containing vaccine adjuvants prepared in the examples and comparative examples was measured on day 0 (d0), day 30 (d30), and day 60 (d60), respectively, in nm. The stability was tested after heating at room temperature and at 50°C for 2 h. Each group was tested 5 times and the average value was taken. The results are shown in Table 1 below.

[0105] Table 1. Stability Performance Test Results

[0106]

[0107] Test Example 2

[0108] Performance testing: Using the saponin-containing vaccine adjuvants from the examples and comparative examples as vaccine adjuvants, and porcine reproductive and respiratory syndrome (PRRSV) inactivated vaccine as antigen, 250 μl of the saponin-containing vaccine adjuvants from the examples and comparative examples were mixed with 50 μg of PRRSV antigen; sterile PBS buffer (pH 7.5) was added to bring the solution to 500 μl. Under light-protected conditions, the mixture was subjected to adsorption by pipetting to obtain the vaccine composition. The potency of the prepared vaccine composition was then tested. The potency was tested according to the 2020 edition of the Chinese Pharmacopoeia, and the specific test results are shown in Table 2.

[0109] Table 2. Test results of potency

[0110]

[0111] The results above show that the saponin-containing vaccine adjuvant prepared by this invention has good stability and can effectively improve antigen titer. Table 1 provides a detailed analysis: Examples 1-3 exhibited optimal and stable particle size and polydispersity index at different time points and under thermal stress conditions, attributed to the cationic lipid-chitosan polymer prepared via a specific three-step synthesis method. This polymer is strongly embedded in the oil phase interface and phospholipid layer of the nanoemulsion through hydrophobic interactions; simultaneously, its hydrophilic chitosan backbone forms a dense three-dimensional barrier on the outside. This unique amphiphilic structure greatly enhances the strength and integrity of the interfacial film, effectively resisting instability phenomena such as Ostwald ripening and particle aggregation. In contrast, the nanoemulsion in Comparative Example 1, lacking this crucial protective barrier, exhibited poor interfacial stability, leading to a significant increase in particle size and eventual precipitation. Comparative Example 2, using a conventional process, showed better stability than Comparative Example 1 but far inferior to Example 1, indicating that the polymer itself provides a certain degree of physical stability, but the degradation of saponins during high-pressure homogenization may disrupt the orderly molecular arrangement at the interface, partially offsetting its stabilizing effect. Crucially, the stability of Comparative Example 4 (using ordinary carboxymethyl chitosan), Comparative Example 5 (using only physical mixtures), and Comparative Example 6 (using threonine monomers) was significantly worse than that of Example 1. This strongly demonstrates that Comparative Example 4, with only a hydrophilic framework, or Comparative Example 5 (using simple physical mixing of components), and Comparative Example 6 (failing to successfully construct a complete hydrophobic anchor), cannot achieve the comb-like polymer structure with both rigid anchoring and flexible protection functions constructed through chemical bonding as described in this invention. The polymer of Example 1 forms a regular comb-like structure through a three-step synthesis. Its hydrophobic chains strongly anchor the interface, and the hydrophilic chitosan framework forms a dense protective layer, significantly improving emulsion stability. Comparative Example 7, using unmodified chitosan, resulted in a low grafting rate and defects in the protective layer; Comparative Example 8, using 2,3-dioleoxypropyltrimethylammonium chloride, had a mismatched structure and weak interfacial anchoring force. Neither of these methods effectively prevented droplet aggregation and fusion, resulting in a significant decrease in stability.

[0112] The immunopotency results in Table 2 are positively correlated with the stability trend, further highlighting the innovativeness of the novel synergistic polymer in biological function. The highest immunopotency in Example 1 is due to the synergistic effect of its stable nanodelivery system and the active polymer function. First, the system maximizes the preservation of the immunopotency of saponin QS-21 through a post-loading process. Second, and more importantly, the specific polymer in Example 1, with its abundant positive surface charge, can effectively enrich and concentrate negatively charged PRRSV antigen through electrostatic adsorption, forming a high-concentration antigen reservoir, which is beneficial for continuous immune stimulation; this cationic property allows it to be actively recognized and efficiently internalized by negatively charged antigen-presenting cell membranes. In contrast, Comparative Example 1, without polymer, retained saponin activity but lacked active targeting and intelligent release functions, resulting in a generally low immunopotency; Comparative Example 2, using a traditional process, suffered from significant saponin degradation during preparation, limiting its immunopotency even with polymer-assisted delivery due to insufficient active ingredients; Comparative Example 3, which did not add the novel synergistic polymer and used pre-loading of saponin, had the lowest immunopotency. The potency of Comparative Examples 4-6 was only comparable to or slightly higher than that of Comparative Example 1, demonstrating that ordinary polysaccharides, physical mixtures, or polymers with structural defects could not achieve effective antigen-presenting cell targeting, and their immune-enhancing effects were weak. The specific polymer in Example 1 could efficiently adsorb antigens and had excellent pH responsiveness, rapidly releasing the active ingredient within cells. Comparative Example 7 suffered from insufficient antigen adsorption and targeting efficiency due to its low grafting rate; the 2,3-dioleoxypropyltrimethylammonium chloride structure of Comparative Example 8 lacked effective pH responsiveness, resulting in poor content release efficiency and a significant reduction in immune activation effect and potency.

Claims

1. A saponin-containing vaccine adjuvant, characterized in that, It is composed of the following raw materials by weight percentage: 0.1% saponins, 1.5% phospholipids, 5% oil phase, 2% surfactant, 0.1% antioxidant, 3% trehalose, 0.1-0.5% novel synergistic polymer, and the balance being water for injection; The saponin component is QS-21; The phospholipid is hydrogenated soybean lecithin; The oil phase is a mixture of squalene and vitamin E acetate in a mass ratio of 3:1; The surfactant is a mixture of sorbitan trioleate and Tween 80 in a mass ratio of 1:

1. The antioxidant is a mixture of L-ascorbic acid, acetylcysteine, and α-tocopherol in a mass ratio of 1:1:

1. The novel synergistic polymer is a cationic lipid-chitosan polymer, prepared by a method comprising the following steps: (a) Nα-Boc-L-lysine is alkylated with bromododecane in the presence of a first organic solvent and an organic base to obtain Nα-Boc-Nε-dodecyl-L-lysine; (b) In the presence of an activator, the product obtained in step (a) is coupled with HOOC-PEG-COOH, and then trifluoroacetic acid is added to remove the Boc protecting group to obtain a cationic lipid with an amino terminus. (c) In the presence of MES buffer, the terminal amino-terminated cationic lipid obtained in step (b) is reacted with carboxymethyl chitosan in an aqueous buffer system, and the cationic lipid-chitosan polymer is obtained after purification. The method for preparing the saponin-containing vaccine adjuvant includes the following steps: (1) Preparation of oil phase: Phospholipids, oil phase and fat-soluble antioxidant are dissolved by heating to obtain oil phase; (2) Preparation of aqueous phase: Dissolve surfactant, trehalose, water-soluble antioxidant, and novel synergistic polymer in water for injection and heat to obtain aqueous phase; (3) Formation of primary emulsion: Under high-speed shearing, the oil phase is slowly added to the aqueous phase to form a primary emulsion; (4) Formation of nanoemulsion: The above primary emulsion is homogenized under high pressure to obtain nanoemulsion; (5) Saponin loading: Saponin components are dissolved in water for injection and then mixed with the nanoemulsion to obtain the saponin-containing vaccine adjuvant.

2. The saponin-containing vaccine adjuvant as described in claim 1, characterized in that, Its nanoemulsion has a particle size distribution of 80-200 nm and a polydispersity index of less than 0.

2.

3. The use of the saponin-containing vaccine adjuvant as described in claim 1 in the preparation of an inactivated vaccine for porcine reproductive and respiratory syndrome.

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