Saponin liposome immunologic adjuvant and preparation method thereof

Abstract

The invention discloses a saponin liposome immunologic adjuvant and a preparation method thereof, and belongs to the technical field of vaccine adjuvants. The preparation method comprises the following steps: firstly, preparing a blank liposome from 1, 2-dioleoyl-sn-glycerol-3-phosphorylcholine, cholesterol and DSPE-PEG-NHS, and then carrying out surface modification on the liposome by introducing a polyvinyl alcohol-protein graft polymer to form a stable core-shell structure; and finally, encapsulating the QS-21 in the liposome through active drug loading, and freeze-drying to obtain a final product. Through the synergistic effect of the DSPE-PEG-NHS and the polyvinyl alcohol-protein graft polymer, the structural integrity of the liposome in the freeze-drying and storage process is remarkably enhanced, hydrolysis and rearrangement of QS-21 are effectively inhibited, and the problem of chemical instability of QS-21 is solved. The obtained saponin liposome immunologic adjuvant has the advantages of high stability and strong immunogenicity.

Description

Technical Field

[0001] This invention relates to the field of vaccine adjuvant technology, and in particular to a saponin liposome immunoadjuvant and its preparation method. Background Technology

[0002] Saponin liposome adjuvants are novel vaccine delivery systems and adjuvant platforms formed by encapsulating or embedding natural saponins in liposome bilayer membranes. QS-21 is a highly effective saponin adjuvant that can significantly promote cellular and humoral immune responses. However, this technology has drawbacks in practical applications: First, the QS-21 molecule itself is chemically unstable in an aqueous environment, easily undergoing hydrolysis and molecular rearrangement, leading to the loss of its adjuvant activity; second, liposomes, as nanocarriers, inherently have poor physical stability, easily undergoing aggregation, fusion, oxidation, and hydrolysis during storage, resulting in the destruction of the liposome structural integrity, thereby causing leakage of the encapsulated antigen and adjuvant, ultimately affecting the efficacy and safety of the vaccine.

[0003] Currently, although some studies have attempted to improve liposome stability by adding conventional freeze-drying protectants such as sucrose and trehalose or by modifying with common polymers, the effects are limited, and they cannot simultaneously solve the problems of the integrity of the macroscopic structure of liposomes and the chemical stability of the QS-21 micromolecules. Therefore, developing a novel preparation process that can synergistically enhance the structural stability of liposomes and the chemical stability of QS-21 has become an urgent technical challenge to be solved in this field.

[0004] Chinese invention patent publication CN119971022A discloses a QS-21 composite adjuvant, its preparation method, and its application. The QS-21 composite adjuvant comprises the following components in parts by weight: 0.1-0.5 parts QS-21; 4-6 parts liposomes; 0.05-0.1 parts CpG oligonucleotides; 0.1-0.3 parts lyophilization protectant; 0.2-0.5 parts stabilizer; 3-5 parts propylene glycol; 1-2 parts ethanol; 5-15 parts chloroform (organic solvent); and 100-200 parts deionized water. The liposomes include: 3-4 parts distearate phospholipids; and 1-2 parts amino lipids. This formulation contains organic solvents such as propylene glycol, ethanol, and chloroform, whose residues may disrupt the order and integrity of the lipid bilayer, exacerbating the instability of the liposomes.

[0005] Chinese invention patent publication CN120168626A discloses an immune adjuvant composition, its preparation method, and its uses. This immune adjuvant composition comprises an immunoactive agent and liposomes for loading the immunoactive agent. The immunoactive agent comprises saponins and CpG oligodeoxynucleotides. By simultaneously loading QS-21 and CpG oligodeoxynucleotides onto liposomes, they exhibit a good synergistic effect in promoting cellular immunity. However, QS-21 has poor chemical stability and is easily decomposed and inactivated during long-term storage. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a saponin liposome immunoadjuvant and its preparation method. This method, by introducing key components such as DSPE-PEG-NHS and polyvinyl alcohol-protein graft polymer, utilizes their synergistic effect to significantly improve the structural integrity of liposomes during freeze-drying and storage, and effectively stabilizes the chemical structure of QS-21, thereby obtaining a high-performance saponin liposome immunoadjuvant.

[0007] This invention provides a method for preparing a saponin liposome immunoadjuvant, comprising the following steps:

[0008] Step 1: Dissolve 12-15 mg of 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 2.2-2.5 mg of cholesterol, and 0.8-1.2 mg of DSPE-PEG-NHS in 1.5-2.5 mL of chloroform to obtain a mixed solution; transfer the mixed solution to a round-bottom flask and rotary evaporate until a uniform lipid film forms on the flask wall; vacuum dry, then add ammonium sulfate aqueous solution to the dried lipid film for hydration to form a blank liposome suspension; extrude the blank liposome suspension through a polycarbonate membrane, then dialyze the extruded blank liposome suspension to obtain a purified blank liposome suspension;

[0009] Step 2: Take 1.2-1.6 mL of the purified blank liposome suspension and add 0.8-1.2 mL of pH 7.8-8.2 borate buffer pre-dissolved with 9-11 mg of surface modifier. Mix and stir at 22-28℃ and 150-300 rpm for 3-5 hours to obtain a mixture. Purify the mixture to obtain the liposome suspension.

[0010] Step 3: Add 12-16 μL of QS-21 saponin aqueous solution with a concentration of 0.9-1.1 mg / mL to 1.2-1.6 mL of liposome suspension, mix well, and incubate in a water bath at 58-62℃ for 25-35 min. After drug loading is completed, dialyze to obtain saponin liposome suspension. Add trehalose to the saponin liposome suspension to make the final concentration 48-52 mmol / L, mix well, dispense, and freeze-dry to obtain saponin liposome immunoadjuvant.

[0011] Preferably, in step 1, adding ammonium sulfate aqueous solution to the dried lipid film for hydration specifically involves adding 1.2-1.6 mL of 290-310 mmol / L ammonium sulfate aqueous solution preheated to 48-52°C to the dried lipid film, and placing the flask in a constant temperature water bath at 48-52°C, rotating it at 100-150 rpm for hydration for 40-50 minutes until the lipid film is completely detached.

[0012] Preferably, in step 1, the blank liposome suspension is extruded through polycarbonate membranes of 300-400 nm and 100-200 nm 8-12 times each under a constant temperature of 48-52°C.

[0013] Preferably, in step 1, the blank liposome suspension after extrusion is placed into a dialysis bag and placed in 0.95-1.1L of 8-10% w / v sucrose aqueous solution. Dialysis is performed by magnetic stirring at 1-5℃ and 80-150rpm. The dialysis solution is replaced every 5-7 hours, and the total dialysis time is 22-25 hours.

[0014] Preferably, the surface modifier in step 2 is a polyvinyl alcohol-protein graft polymer.

[0015] Preferably, the method for preparing the polyvinyl alcohol-protein graft polymer includes the following steps:

[0016] S1. Add 1.8-2.2g of PVA-1788 to 140-160mL of water and mix at 90-96℃ and 200-300rpm until PVA-1788 is completely dissolved. Then cool to 48-52℃, add 4-6mL of epichlorohydrin and 9-11mL of 5mol / L NaOH aqueous solution, and mix and stir at 48-52℃ for 3-5h to obtain a mixed solution. Add the mixed solution to 480-520mL of acetone to precipitate, filter, and collect the solid. Wash with acetone 2-3 times, and then vacuum dry at 38-45℃ for 10-13h to obtain epoxidized polyvinyl alcohol.

[0017] S2. Add 18-22 mL of epoxidized polyvinyl alcohol solution dropwise to 48-52 mL of modifier solution, adjust the pH to 8.5-9 with 0.1-0.3 mol / L NaOH aqueous solution, then mix and stir to obtain a mixture; put the mixture into a dialysis bag with a molecular weight cutoff of 8-14 kDa for dialyzing; freeze-dry the dialyzed solution to obtain polyvinyl alcohol-protein graft polymer.

[0018] Preferably, the modifier is selected from one of type I collagen, recombinant human albumin, water-soluble silk fibroin, and gelatin.

[0019] Preferably, after drug loading in step 3, dialysis is performed as follows: the drug is placed in a dialysis bag and immersed in 0.95-1.1L of 8-10% w / v sucrose aqueous solution, and then magnetically stirred for dialysis at 1-5℃ and 80-150rpm for 3-5 hours.

[0020] Preferably, in step 3, freeze drying, the freeze is first pre-frozen at -48°C to -52°C for 1.5-2.5 hours, then dried once at -8°C to -12°C for 22-25 hours, and finally dried a second time at 22-26°C for 5-8 hours.

[0021] The present invention also provides a saponin liposome immunoadjuvant, which is prepared by the above method.

[0022] The beneficial effects of this invention are:

[0023] Compared with existing technologies, this invention creatively combines DSPE-PEG-NHS with a polyvinyl alcohol-protein graft polymer. The active NHS ester groups at the ends of DSPE-PEG-NHS can be covalently linked to the protein portion of the graft polymer, firmly fixing the polymer to the liposome surface and forming a stable core-shell structure. This structure effectively inhibits the aggregation, fusion, and rupture of liposomes during freeze-drying and storage, significantly improving the integrity of the liposome structure. Simultaneously, the introduction of DSPE-PEG-NHS and the polyvinyl alcohol-protein graft polymer also significantly enhances the chemical stability of QS-21 and reduces its degradation. Furthermore, the core-shell structure formed by the combination of DSPE-PEG-NHS and the polyvinyl alcohol-protein graft polymer not only plays a stabilizing role, but recombinant human albumin itself also possesses biological targeting properties, promoting the uptake and processing of antigens by antigen-presenting cells, thereby further enhancing the immunogenicity of the adjuvant. The saponin liposome immunoadjuvant provided by this invention has high stability and strong immunogenicity. Furthermore, it has passed rigorous acute toxicity and local irritation tests, confirming its reliable biosafety and providing a reliable safety basis for its further application. Detailed Implementation

[0024] The parameters and sources of the specific chemical substances used.

[0025] PVA-1788, degree of polymerization 1700, degree of alcoholysis 88%, brand: Sinopec;

[0026] DSPE-PEG-NHS, where PEG is PEG2000, catalog number: R-0042-2K, is from Xi'an Ruixi Biotechnology Co., Ltd.

[0027] QS-21, item number: B01002, sourced from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd.

[0028] Recombinant human albumin, catalog number: ART101S, is sourced from Beijing Anruit Biotechnology Co., Ltd.

[0029] Type I collagen, code: C9791, is sourced from the Merck Group in Darmstadt, Germany.

[0030] Water-soluble silk fibroin, product number: W293432, brand: Aladdin;

[0031] Gelatin, item number: G108394, brand: Aladdin.

[0032] Example 1

[0033] A method for preparing a saponin liposome immunoadjuvant includes the following steps:

[0034] Step 1: Dissolve 14 mg of 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 2.4 mg of cholesterol, and 1 mg of DSPE-PEG-NHS in 2 mL of chloroform to obtain a mixed solution. Transfer the mixed solution to a round-bottom flask and evaporate it using a rotary evaporator at 40 °C and 100 rpm for 30 min until a uniform lipid film forms on the flask wall. Then, transfer the flask to a vacuum desiccator and dry it at 25 °C for 8 h. Add 1.5 mL of 300 mmol / L ammonium sulfate aqueous solution preheated to 50 °C to the dried lipid film, and place the flask in a 50 °C constant temperature water bath at 120 rpm. The lipid membrane was completely detached by rotational hydration for 45 min to form a blank liposome suspension. The blank liposome suspension was extruded through 400 nm and 100 nm polycarbonate membranes 10 times each under constant temperature of 50 °C. The extruded blank liposome suspension was placed in a dialysis bag with a molecular weight cutoff of 12000 and placed in 1 L of 9% w / v sucrose aqueous solution. Dialysis was performed with magnetic stirring at 4 °C and 100 rpm. The dialysis solution was changed every 6 h and dialyzed for a total of 24 h to obtain a purified blank liposome suspension.

[0035] Step 2: Take 1.5 mL of the purified blank liposome suspension and add 1.0 mL of pH 8.0 borate buffer pre-dissolved with 10 mg of surface modifier. Mix and stir at 25 °C and 200 rpm for 4 h to obtain a mixture. Purify the mixture using a Sepharose CL-4B gel column with pH 7.4 PBS as the elution buffer and a flow rate controlled at 0.5 mL / min. Collect the first milky white liposome component to elute to obtain the liposome suspension.

[0036] Step 3: Add 15.0 μL of QS-21 saponin aqueous solution with a concentration of 1.0 mg / mL to 1.5 mL of liposome suspension, mix well, and incubate in a 60℃ water bath for 30 min. After drug loading, place the mixture into a dialysis bag with a molecular weight cutoff of 12000, place it in 1 L of 9% w / v sucrose aqueous solution, and dialyze with magnetic stirring at 4℃ and 100 rpm for 4 h to obtain saponin liposome suspension. Add trehalose to the saponin liposome suspension to make the final concentration 50 mmol / L, mix well, and dispense into 2 mL neutral borosilicate glass vials, 1.0 mL per vial. Freeze-dry the vials, first placing them in an ultra-low temperature freezer at -50℃ for 2 h, then transferring them to a freeze dryer for drying at -10℃ for 24 h, and finally drying them again at 25℃ for 6 h to obtain saponin liposome immunoadjuvant.

[0037] In step 2, the surface modifier is a polyvinyl alcohol-protein grafted polymer, and the preparation method of the polyvinyl alcohol-protein grafted polymer includes the following steps:

[0038] S1. Add 2.0 g of PVA-1788 to 150 mL of water and mix at 95 °C and 300 rpm until PVA-1788 is completely dissolved. Then cool to 50 °C, add 5 mL of epichlorohydrin and 10 mL of 5 mol / L NaOH aqueous solution, and mix and stir at 50 °C for 4 h to obtain a mixed solution. Add the mixed solution to 500 mL of acetone to precipitate, filter, and collect the solid. Wash with acetone 3 times, and then dry under vacuum at 40 °C for 12 h to obtain epoxidized polyvinyl alcohol.

[0039] S2. Add 1g of recombinant human albumin to 50mL of 0.1mol / L pH 8.5 borate buffer and mix and stir at 25℃ and 200rpm for 5h to obtain a recombinant human albumin solution. Add 0.5g of epoxidized polyvinyl alcohol to 20mL of water and mix and dissolve to obtain an epoxidized polyvinyl alcohol solution. Add the epoxidized polyvinyl alcohol solution dropwise to the recombinant human albumin solution at a rate of 1 drop / s while stirring at 25℃ and 200rpm. Adjust the pH to 8.5 with 0.1mol / L NaOH aqueous solution, and then mix and stir at 25℃ and 200rpm for 24h to obtain a mixture. Dialyze the mixture into a dialysis bag with a molecular weight cutoff of 10kDa. Freeze-dry the dialyzed solution to obtain PVA-g-recombinant human albumin, i.e., polyvinyl alcohol-protein graft polymer.

[0040] Example 2

[0041] The method for preparing a saponin liposome immunoadjuvant differs from Example 1 only in that the method for preparing the polyvinyl alcohol-protein graft polymer includes the following steps:

[0042] S1. Add 2.0 g of PVA-1788 to 150 mL of water and mix at 95 °C and 300 rpm until PVA-1788 is completely dissolved. Then cool to 50 °C, add 5 mL of epichlorohydrin and 10 mL of 5 mol / L NaOH aqueous solution, and mix and stir at 50 °C for 4 h to obtain a mixed solution. Add the mixed solution to 500 mL of acetone to precipitate, filter, and collect the solid. Wash with acetone 3 times, and then dry under vacuum at 40 °C for 12 h to obtain epoxidized polyvinyl alcohol.

[0043] S2. Add 1g of water-soluble silk fibroin to 50mL of water and mix and stir at 25℃ and 200rpm for 1h to obtain a silk fibroin solution; add 0.5g of epoxidized polyvinyl alcohol to 20mL of water and mix and dissolve to obtain an epoxidized polyvinyl alcohol solution; add the epoxidized polyvinyl alcohol solution dropwise to the silk fibroin solution at a rate of 1 drop / s while stirring at 50℃ and 200rpm, adjust the pH to 9 with 0.1mol / L NaOH aqueous solution, and then mix and stir at 50℃ and 300rpm for 12h to obtain a mixture; put the mixture into a dialysis bag with a molecular weight cutoff of 10kDa for dialyzing; freeze-dry the dialyzed solution to obtain PVA-g-silk fibroin, i.e., polyvinyl alcohol-protein graft polymer.

[0044] Example 3

[0045] The method for preparing a saponin liposome immunoadjuvant differs from Example 1 only in that the method for preparing the polyvinyl alcohol-protein graft polymer includes the following steps:

[0046] S1. Add 2.0 g of PVA-1788 to 150 mL of water and mix at 95 °C and 300 rpm until PVA-1788 is completely dissolved. Then cool to 50 °C, add 5 mL of epichlorohydrin and 10 mL of 5 mol / L NaOH aqueous solution, and mix and stir at 50 °C for 4 h to obtain a mixed solution. Add the mixed solution to 500 mL of acetone to precipitate, filter, and collect the solid. Wash with acetone 3 times, and then dry under vacuum at 40 °C for 12 h to obtain epoxidized polyvinyl alcohol.

[0047] S2. Add 0.5g of type I collagen to 50mL of 0.1mol / L acetic acid aqueous solution at 4℃ and stir at 150rpm for 12h to obtain a collagen solution. Add 0.5g of epoxidized polyvinyl alcohol to 20mL of water and dissolve to obtain an epoxidized polyvinyl alcohol solution. Add the epoxidized polyvinyl alcohol solution to the collagen solution at a rate of 1 drop / s while stirring at 200rpm at 4℃. Adjust the pH to 8.5 with 0.1mol / L NaOH aqueous solution, and then stir at 150rpm at 4℃ for 48h to obtain a mixture. Dialyze the mixture into a dialysis bag with a molecular weight cutoff of 10kDa. Freeze-dry the dialyzed solution to obtain PVA-g-collagen, i.e., polyvinyl alcohol-protein graft polymer.

[0048] Example 4

[0049] The method for preparing a saponin liposome immunoadjuvant differs from Example 1 only in that the method for preparing the polyvinyl alcohol-protein graft polymer includes the following steps:

[0050] S1. Add 2.0 g of PVA-1788 to 150 mL of water and mix at 95 °C and 300 rpm until PVA-1788 is completely dissolved. Then cool to 50 °C, add 5 mL of epichlorohydrin and 10 mL of 5 mol / L NaOH aqueous solution, and mix and stir at 50 °C for 4 h to obtain a mixed solution. Add the mixed solution to 500 mL of acetone to precipitate, filter, and collect the solid. Wash with acetone 3 times, and then dry under vacuum at 40 °C for 12 h to obtain epoxidized polyvinyl alcohol.

[0051] S2. Add 1g of gelatin to 50mL of 0.1mol / L pH 9.0 phosphate buffer at 50℃ and stir at 250rpm for 5h to obtain a gelatin solution; add 0.5g of epoxidized polyvinyl alcohol to 20mL of water and dissolve to obtain an epoxidized polyvinyl alcohol solution; add the epoxidized polyvinyl alcohol solution to the gelatin solution at a rate of 1 drop / s while stirring at 200rpm at 50℃, adjust the pH to 9 with 0.1mol / L NaOH aqueous solution, and then stir at 300rpm at 50℃ for 12h to obtain a mixture; put the mixture into a dialysis bag with a molecular weight cutoff of 10kDa for dialyzing; freeze-dry the dialyzed solution to obtain PVA-g-gelatin, i.e., polyvinyl alcohol-protein graft polymer.

[0052] Comparative Example 1

[0053] The method for preparing a saponin liposome immune adjuvant differs from that in Example 1 only in that the surface modifier in step 2 is polyvinyl alcohol.

[0054] Comparative Example 2

[0055] The method for preparing a saponin liposome immune adjuvant differs from that in Example 1 only in that the surface modifier in step 2 is recombinant human albumin.

[0056] Comparative Example 3

[0057] The method for preparing a saponin liposome immunoadjuvant differs from that in Example 1 only in step 2, which specifically involves:

[0058] Step 2: Take 1.5 mL of the purified blank liposome suspension and add 1.0 mL of pH 8.0 borate buffer. Mix and stir at 25℃ and 200 rpm for 4 h to obtain a mixture. Purify the mixture using a Sepharose CL-4B gel column with pH 7.4 PBS as the elution buffer and a flow rate controlled at 0.5 mL / min. Collect the first milky white liposome component to elute to obtain the liposome suspension.

[0059] Comparative Example 4

[0060] The method for preparing a saponin liposome immune adjuvant differs from that in Example 1 only in that DSPE-PEG-NHS is not added in step 1.

[0061] Test Example 1

[0062] Average particle size test

[0063] Saponin liposome immunoadjuvants were prepared according to the preparation methods of Examples 1-4 and Comparative Examples 1-4, respectively. Using a Malvern Zetasizer Nano ZS instrument, the average particle size (nm) of the saponin liposome immunoadjuvants of each example and comparative example was measured at 25°C before freeze-drying and after reconstitution. Reconstitution was performed using 1 mL of pH 7.4 PBS buffer. Three replicates were set up, and the average value was taken. The particle size change rate was calculated according to the following formula:

[0064] Particle size change rate (%) = (Particle size after reconstitution - Particle size before lyophilization / Particle size before lyophilization) × 100%

[0065] The test results are shown in Table 1 below;

[0066] Encapsulation efficiency test

[0067] Saponin liposome immunoadjuvants were prepared according to the preparation methods of Examples 1-4 and Comparative Examples 1-4, respectively. Free QS-21 was separated by ultrafiltration centrifugation using Amicon ultrafiltration tubes with a molecular weight cutoff of 100 kDa, and the concentration of QS-21 was measured by high performance liquid chromatography. Three parallel tests were set up and the average value was taken. The encapsulation efficiency (EE) before and after lyophilization was calculated according to the following formula 1, and then the encapsulation retention rate was calculated according to the encapsulation efficiency before and after lyophilization. The encapsulation retention rate was calculated according to the following formula 2.

[0068] Formula 1: Encapsulation efficiency (EE) = (Total concentration of QS-21 - Free concentration of QS-21) / Total concentration of QS-21 × 100%

[0069] Formula 2: Encapsulation retention rate (%) = (EE after freeze-drying / EE before freeze-drying) × 100%.

[0070] The test results are shown in Table 1 below;

[0071] The specific test results are taken as average values, and the specific results are shown in Table 1 below;

[0072] Table 1

[0073]

[0074] As shown in Table 1, comparing Examples 1-4 and Comparative Examples 1-4, the particle size change rate of Examples 1-4 is lower than that of Comparative Examples 1-4, while the encapsulation retention rate is higher. Example 1 exhibits the lowest particle size change rate (3.1%) and the highest encapsulation retention rate (98.5%), demonstrating good structural integrity. This may be because during liposome preparation, the primary amino group in the introduced PVA-g-recombinant human albumin structure undergoes a covalent reaction with the active NHS ester at the DSPE-PEG-NHS terminus to form a highly stable amide bond. This grafts PVA-g-recombinant human albumin onto the liposome surface, forming a uniform and dense protective layer. The introduction of PVA-g-recombinant human albumin not only prevents liposomes from approaching each other during freeze-drying and reduces mechanical damage to the liposome membrane caused by ice crystal puncture and compression, but also specifically binds to QS-21 saponin molecules, stabilizing them and reducing leakage. Compared to Example 1, the structures of silk fibroin, collagen, and gelatin in Examples 2-4 may be more prone to denaturation during freeze-drying and have weaker interaction with QS-21, resulting in poorer structural integrity.

[0075] Compared with Example 1, in Comparative Example 1, polyvinyl alcohol could not form covalent bonds with DSPE-PEG-NHS and could only adhere to the surface through weak physical adsorption, which was very easy to fall off during freeze-drying and reconstitution; in Comparative Example 2, the lack of long polyvinyl alcohol chains could not effectively prevent physical compression and fusion between liposomes during freeze-drying, resulting in large changes in particle size; in Comparative Example 3, the lack of PVA-g-recombinant human albumin could not form an effective three-dimensional protective network, resulting in poor protection; in Comparative Example 4, no DSPE-PEG-NHS was added, and the lack of strong covalent anchoring resulted in poor stability of the physically adsorbed PVA-g-recombinant human albumin, which was very easy to fall off, resulting in a significant deterioration in the structural integrity of the liposomes.

[0076] Test Example 2

[0077] Accelerated stability testing

[0078] Equal amounts of the saponin liposome immunoadjuvants prepared in Examples 1-4 and Comparative Examples 1-4 were reconstituted with 1 mL of PBS buffer (pH 7.4) and dispensed into vials as samples. Each sample was stored at 40°C and 75% relative humidity for 4 weeks, and these samples were recorded as Examples 1-4 and Comparative Examples 1-4, with 5 replicates for each group. Samples were taken weekly, and the concentration of QS-21 was measured using high-performance liquid chromatography (HPLC). The residual rate of QS-21 was calculated using the following formula:

[0079] QS-21 residual rate (%) = (QS-21 concentration after storage / initial QS-21 concentration) × 100%

[0080] The specific test results are shown in Table 2 below.

[0081] Table 2

[0082]

[0083] As shown in Table 2, comparing Examples 1-4 and Comparative Examples 1-4, the QS-21 residue rate in Examples 1-4 was higher than that in Comparative Examples 1-4 after accelerated testing. In Example 1, the QS-21 residue rate reached 95.2%, and under accelerated conditions of high temperature and humidity, less than 5% degraded within 4 weeks. This demonstrates that the formulation can extremely effectively inhibit the hydrolysis, oxidation, or other chemical decomposition pathways of QS-21, exhibiting good chemical stability. The reason for this may be that the introduction of PVA-g-recombinant human albumin forms a uniform and dense protective layer on the liposome surface, effectively blocking water molecules and free radicals from directly and extensively contacting and attacking QS-21 inside the liposome. Simultaneously, recombinant human albumin can specifically bind to QS-21 molecules, significantly increasing the activation energy for its chemical degradation and significantly reducing the degradation of QS-21 molecules. Compared with Example 1, the interaction between silk fibroin, collagen and gelatin and QS-21 in Examples 2-4 is weaker, and they cannot provide the same effective stabilization mechanism. Therefore, their effect on resisting QS-21 degradation is slightly worse.

[0084] Compared with Example 1, Comparative Example 1 lacks covalent linkages, and the protective layer may be more prone to failure under accelerated conditions, resulting in a weakened barrier effect; Comparative Example 2 liposomes lack a dense physical barrier, and QS-21 molecules are easily attacked by external moisture and oxygen; Comparative Example 3 lacks PVA-g-recombinant human albumin, and cannot form an effective barrier to block water molecules; while Comparative Example 4 lacks DSPE-PEG-NHS, and the physically adsorbed PVA-g-recombinant human albumin will fall off in large quantities from the liposome surface, causing its protective mechanism to fail, QS-21 degradation rate to accelerate, and residual amount to be low.

[0085] Test Example 3

[0086] Biosafety testing

[0087] To assess the in vivo safety of the saponin liposome immunoadjuvant of the present invention, the saponin liposome immunoadjuvants prepared in Examples 1-4 were used as experimental samples, and the following experiments were conducted in accordance with the Good Laboratory Practice (GLP) for non-clinical studies of pharmaceuticals.

[0088] 1. Experimental Materials and Methods

[0089] Experimental animals: SPF grade BALB / c mice, 6-8 weeks old, weighing 18-22 g, half male and half female; all animals were acclimatized for one week in a barrier environment with a temperature of 22±2℃, humidity of 50±10% and a 12 h / 12 ​​h light-dark cycle.

[0090] Test sample and reference sample:

[0091] Test samples: The lyophilized powder of saponin liposome immunoadjuvant prepared in Examples 1-4 was reconstituted with 0.9% sodium chloride injection 20 min before injection, and temporarily stored at 4℃. These samples were recorded as the groups of Examples 1-4.

[0092] Reference standard: 0.9% sodium chloride injection was used as a blank control;

[0093] 2. Acute toxicity test protocol

[0094] One hundred healthy mice were randomly divided into five groups of 20 mice each, with half males and half females, corresponding to groups 1-4 in Examples and a blank control group. The mice in each group were then subjected to experiments, and the specific experimental methods are as follows:

[0095] Administration: Mice from Examples 1-4 were randomly divided into two groups: a high-dose group and a low-dose group, with 10 mice in each group (half male and half female). Each group was administered the drug, with the mice in Examples 1-4 receiving the test product from Examples 1-4. The dosage for the mice was based on the total mass of the lyophilized saponin liposome adjuvant powder. The high-dose and low-dose groups were injected with 1000 mg / kg (approximately 8 times the clinical dose) and 250 mg / kg (approximately 2 times the clinical dose), respectively. The blank control group was injected with an equal volume of 0.9% sodium chloride injection. Administration was via subcutaneous injection at a volume of 20 mL / kg.

[0096] Observation and testing:

[0097] Clinical observation: Closely observe the animals for 2 hours after administration, and then record their general behavior, signs, coat condition, eye and nasal secretions and mortality for 14 consecutive days.

[0098] Weight and food intake: Record changes in weight and food intake daily;

[0099] Pathological examination: At the end of the experiment, all animals were euthanized, and the macroscopic morphology of major organs such as the heart, liver, spleen, lungs, and kidneys was observed by dissection and macroscopic observation. The organs were accurately weighed and their coefficients were calculated.

[0100] Experimental Results: Acute toxicity tests were conducted on the lyophilized powders of saponin liposome immunoadjuvants prepared in Examples 1-4. The results showed that no mice died during the entire 14-day observation period, and no acute toxic symptoms such as tremors, convulsions, or respiratory distress were observed. The weight gain trend and average daily food intake of the mice in each group were basically consistent with those of the blank control group. After dissection, the color and morphology of the major organs were normal, and no abnormal changes such as congestion, edema, or necrosis were observed. The organ coefficients were not statistically different from those of the control group. The results indicate that the saponin liposome immunoadjuvants of the present invention did not exhibit acute toxicity at the experimental dose.

[0101] 3. Local irritation test

[0102] Sixty healthy mice were randomly divided into 5 groups of 12 mice each, with half males and half females, corresponding to groups 1-4 in Examples 1-4 and a blank control group. Experiments were performed on each group of mice using the following specific methods:

[0103] Administration: Mice in Examples 1-4 were administered the test product from Examples 1-4 by subcutaneous injection at 1000 mg / kg. The dosage for mice was calculated based on the total mass of the lyophilized saponin liposome adjuvant powder. The blank control group was injected with an equal volume of 0.9% sodium chloride injection as a control.

[0104] Evaluation method:

[0105] Stimulation response score: Erythema and induration were scored at the injection site at 1, 24, 48 and 72 h after administration; the scoring criteria are shown in Table 3.

[0106] Histopathological analysis: 72 hours later, skin and subcutaneous tissue from the injection site were taken, fixed with 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin (HE), and observed under an optical microscope to assess the integrity of the tissue structure, inflammatory cell infiltration, and fibrin exudation.

[0107] Table 3

[0108]

[0109] Experimental Results: Local irritation tests were conducted on the lyophilized saponin liposome immunoadjuvant powders prepared in Examples 1-4. The results showed that at all observation time points, no obvious erythema or induration was observed at the injection sites in the experimental group animals, and the irritation response scores were all 0 points. Histopathological examination showed that the epidermal structure of the skin in the experimental group was intact, and only a very small number of scattered inflammatory cells were observed in the dermis and subcutaneous tissue, with no significant difference compared to the control group. No pathological changes such as edema, hemorrhage, or fibrin exudation were observed. The results indicate that the formulation of this invention is non-irritating to the injection site.

[0110] Conclusion: In summary, through systematic acute toxicity and local irritation evaluation, the saponin liposome immunoadjuvant prepared in this invention has been confirmed to have good biocompatibility under the set experimental conditions, and meets the safety requirements for clinical application of vaccine adjuvants.

Claims

1. A method for preparing a saponin liposome immunoadjuvant, characterized in that, Includes the following steps: Step 1: Dissolve 12-15 mg of 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 2.2-2.5 mg of cholesterol, and 0.8-1.2 mg of DSPE-PEG-NHS in 1.5-2.5 mL of chloroform to obtain a mixed solution; transfer the mixed solution to a round-bottom flask and rotary evaporate until a uniform lipid film forms on the flask wall; vacuum dry, then add ammonium sulfate aqueous solution to the dried lipid film for hydration to form a blank liposome suspension; extrude the blank liposome suspension through a polycarbonate membrane, then dialyze the extruded blank liposome suspension to obtain a purified blank liposome suspension; Step 2: Take 1.2-1.6 mL of the purified blank liposome suspension and add 0.8-1.2 mL of pH 7.8-8.2 borate buffer pre-dissolved with 9-11 mg of surface modifier. Mix and stir at 22-28℃ and 150-300 rpm for 3-5 hours to obtain a mixture. Purify the mixture to obtain the liposome suspension. Step 3: Add 12-16 μL of QS-21 saponin aqueous solution with a concentration of 0.9-1.1 mg / mL to 1.2-1.6 mL of liposome suspension, mix well, and incubate in a water bath at 58-62℃ for 25-35 min. After drug loading is completed, dialyze to obtain saponin liposome suspension. Add trehalose to the saponin liposome suspension to make the final concentration 48-52 mmol / L, mix well, dispense, and freeze-dry to obtain saponin liposome immunoadjuvant.

2. The method for preparing the saponin liposome immunoadjuvant according to claim 1, characterized in that, In step 1, the hydration of the dried lipid film by adding an ammonium sulfate aqueous solution specifically involves adding 1.2-1.6 mL of a 290-310 mmol / L ammonium sulfate aqueous solution preheated to 48-52℃ to the dried lipid film, and placing the flask in a constant temperature water bath at 48-52℃, rotating it at 100-150 rpm for 40-50 minutes until the lipid film is completely detached.

3. The method for preparing the saponin liposome immunoadjuvant according to claim 1, characterized in that, In step 1, the blank liposome suspension is extruded through polycarbonate membranes of 300-400 nm and 100-200 nm 8-12 times each under constant temperature conditions of 48-52℃.

4. The method for preparing the saponin liposome immunoadjuvant according to claim 1, characterized in that, In step 1, the extruded blank liposome suspension is placed in a dialysis bag and then in 0.95-1.1L of 8-10% w / v sucrose aqueous solution. Dialysis is performed with magnetic stirring at 1-5℃ and 80-150rpm. The dialysis solution is replaced every 5-7 hours, and the total dialysis time is 22-25 hours.

5. The method for preparing the saponin liposome immunoadjuvant according to claim 1, characterized in that, In step 2, the surface modifier is a polyvinyl alcohol-protein graft polymer.

6. The method for preparing the saponin liposome immunoadjuvant according to claim 5, characterized in that, The preparation method of the polyvinyl alcohol-protein grafted polymer includes the following steps: S1. Add 1.8-2.2g of PVA-1788 to 140-160mL of water and mix at 90-96℃ and 200-300rpm until PVA-1788 is completely dissolved. Then cool to 48-52℃, add 4-6mL of epichlorohydrin and 9-11mL of 5mol / L NaOH aqueous solution, and mix and stir at 48-52℃ for 3-5h to obtain a mixed solution. Add the mixed solution to 480-520mL of acetone to precipitate, filter, and collect the solid. Wash with acetone 2-3 times, and then vacuum dry at 38-45℃ for 10-13h to obtain epoxidized polyvinyl alcohol. S2. Add 18-22 mL of epoxidized polyvinyl alcohol solution dropwise to 48-52 mL of modifier solution, adjust the pH to 8.5-9 with 0.1-0.3 mol / L NaOH aqueous solution, then mix and stir to obtain a mixture; put the mixture into a dialysis bag with a molecular weight cutoff of 8-14 kDa for dialyzing; freeze-dry the dialyzed solution to obtain polyvinyl alcohol-protein graft polymer.

7. The method for preparing the saponin liposome immunoadjuvant according to claim 6, characterized in that, The modifier is selected from one of type I collagen, recombinant human albumin, water-soluble silk fibroin, and gelatin.

8. The method for preparing the saponin liposome immunoadjuvant according to claim 1, characterized in that, After drug loading is completed in step 3, dialysis is performed as follows: the drug is placed in a dialysis bag and immersed in 0.95-1.1L of 8-10% w / v sucrose aqueous solution. The mixture is then magnetically stirred at 1-5℃ and 80-150rpm for 3-5 hours.

9. The method for preparing the saponin liposome immunoadjuvant according to claim 1, characterized in that, Step 3, freeze drying, consists of: pre-freezing at -48℃ to -52℃ for 1.5-2.5 hours, followed by drying at -8℃ to -12℃ for 22-25 hours, and finally drying at 22-26℃ for 5-8 hours.

10. A saponin liposome immunoadjuvant, characterized in that: Prepared by the method described in any one of claims 1-9.

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