Liposome vaccine adjuvant and preparation method thereof

Abstract

The invention discloses a liposome vaccine adjuvant and a preparation method thereof, and belongs to the technical field of vaccine adjuvants. The adjuvant comprises hydrogenated soybean phospholipids, cholesterol, a saponin adjuvant QS-21, a membrane function lipid and a stabilizer. The preparation method comprises the following steps: dissolving a lipid component in an organic solvent to form a film, hydrating by using a buffer solution containing a stabilizer, carrying out high-pressure homogenization to form blank liposome, purifying, incubating with a QS-21 solution for drug loading, and finally purifying and sterilizing to obtain the product. Through synergistic cooperation of the components and the process, the technical problems of low encapsulation efficiency, poor storage stability, high hemolytic property and the like when QS-21 is encapsulated by traditional liposome are successfully solved, and the prepared liposome vaccine adjuvant passes strict acute toxicity and local irritation tests, is good in biological safety and has good application prospects. The important clinical application value is realized.

Description

Technical Field

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

[0002] Vaccine adjuvants, as important excipients for enhancing antigen immunogenicity, have received widespread attention in the development of novel vaccines in recent years. QS-21 is a highly effective saponin-based vaccine adjuvant that can strongly stimulate cellular and humoral immunity and is widely used in the development of novel vaccines. However, it suffers from poor chemical stability, easily hydrolyzing or aggregating and becoming inactive in aqueous solutions. Furthermore, QS-21 exhibits strong hemolytic toxicity, severely limiting its clinical safety.

[0003] Currently, liposome technology is one of the ideal strategies for encapsulating QS-21, but existing technologies still have shortcomings: First, the encapsulation efficiency is low, leading to significant drug waste and potential toxicity; second, stability is poor, with the drug easily leaking from the liposomes during storage, resulting in significant burst release; finally, conventional lipid membranes cannot completely shield the hemolytic activity of QS-21, and components such as polyethylene glycol introduced to improve stability may introduce new immunogenic risks. Therefore, there is an urgent need to develop a QS-21 liposome that can simultaneously achieve high encapsulation, high stability, and high safety to improve its adaptability in complex vaccine environments and its prospects for industrial transformation.

[0004] WO2025124295A1 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 a saponin and a CpG oligodeoxynucleotide. By simultaneously loading QS-21 and CpG oligodeoxynucleotides onto liposomes, they exhibit a better synergistic effect in promoting cellular immunity, inducing a stronger cellular immune response compared to commercially available vaccines. However, liposome-loaded QS-21 has poor storage stability, is prone to drug leakage or liposome aggregation, affecting product efficacy and shelf life.

[0005] 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: QS-21: 0.1-0.5 parts; liposomes: 4-6 parts; CpG oligonucleotides: 0.05-0.1 parts; lyophilization protectant: 0.1-0.3 parts; stabilizer: 0.2-0.5 parts; propylene glycol: 3-5 parts; ethanol: 1-2 parts; chloroform (organic solvent): 5-15 parts; deionized water: 100-200 parts. The liposomes include: distearate phospholipids: 3-4 parts; aminolipids: 1-2 parts. This invention, through the combination of liposome carrier and QS-21, the addition of CpG oligonucleotides, and the optimization of the lyophilization protectant, improves the loading efficiency, immune effect stability, and post-lyophilization recovery of the immune adjuvant, thereby enhancing the persistence and efficacy of the immune response. However, this compound adjuvant cannot effectively shield against the hemolytic toxicity of QS-21, and its clinical safety needs further evaluation. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and address the common problems of traditionally prepared QS-21 liposome adjuvants, such as low encapsulation efficiency leading to drug waste and inaccurate dosage; poor storage stability, prone to drug leakage or liposome aggregation affecting product efficacy and shelf life; and potential risks of high hemolysis and insufficient biosafety due to the release of free adjuvants. This invention provides a liposome vaccine adjuvant with high encapsulation efficiency, good stability, and effective shielding against QS-21 hemolytic toxicity by optimizing lipid composition and preparation process.

[0007] This invention provides a method for preparing a liposomal vaccine adjuvant, comprising the following steps:

[0008] Step 1: Accurately weigh 550-600 mg of hydrogenated soybean lecithin, 180-220 mg of membrane-functional lipids, and 250-350 mg of cholesterol. Then, add them to a mixed solvent of 16-24 mL of anhydrous ethanol and 8-12 mL of chloroform to dissolve them. Heat the mixture in a water bath at 45-55°C for 1-2 minutes to obtain a mixed solution. Transfer the mixed solution to a round-bottom flask and evaporate the organic solvent under reduced pressure using a rotary evaporator in a water bath at 40-50°C until a uniform lipid film forms on the flask wall. Place the lipid film in a vacuum drying oven overnight.

[0009] Step 2: Hydrate the lipid film from Step 1 with 18-22 mL of preheated citrate buffer containing 0.5-2 wt% stabilizer and 4-6 wt% sucrose at a concentration of 280-320 mmol / L. Shake at 150-300 rpm for 50-80 min in a water bath at 50-60°C to obtain the colostrum. Homogenize the obtained colostrum to obtain the blank liposome suspension.

[0010] Step 3: Replace the external aqueous phase of the blank liposome suspension with pre-cooled PBS buffer via ultrafiltration centrifugation, repeating the replacement 5-8 times, and bring the volume to a final level to obtain the liposome suspension; add 28-33 mg of QS-21 to 1-2 mL of PBS buffer to dissolve and obtain the QS-21 solution, add the QS-21 solution to the liposome suspension while stirring at 80-150 rpm, mix, and then incubate in the dark to obtain the drug-loaded liposomes;

[0011] Step 4: Centrifuge the drug-loaded liposomes at 12000-15000×g for 25-35 min at 2-5℃, discard the filtrate, and add PBS buffer pre-cooled to 2-5℃ to the concentrated liposomes to the original volume and mix. Repeat the above operation 1-3 times and make up the volume. Then filter through a 0.22-0.45μm sterile filter membrane and store at 1-8℃ in the dark to obtain the liposomal vaccine adjuvant.

[0012] Preferably, the membrane functional lipid is selected from one of phytosphingosine, sphingomyelin, and DSPE-PEG.

[0013] Preferably, the stabilizer is selected from one of sulfobutyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and dextran sulfate.

[0014] Preferably, step 2 involves hydration using a citrate buffer solution preheated to 50-60°C.

[0015] Preferably, in step 2, the obtained colostrum is first homogenized 5-8 times at a pressure of 800-1000 bar, and then homogenized again 5-8 times at a pressure of 1000-1200 bar to obtain a blank liposome suspension.

[0016] Preferably, step 3, which involves replacing the external aqueous phase by ultrafiltration centrifugation, involves transferring the blank liposome suspension to an ultrafiltration centrifuge tube pre-cooled to 2-5°C, centrifuging at 12000-15000×g for 25-35 min at 2-5°C, discarding the filtrate, and adding PBS buffer pre-cooled to 2-5°C to the concentrated liposomes until the original volume is reached and mixed.

[0017] Preferably, step 3, the light-protected incubation, involves placing the container in a water bath at 45-55°C and incubating it in the dark for 50-70 minutes.

[0018] The present invention also provides a liposomal vaccine adjuvant prepared by the above method.

[0019] The beneficial effects of this invention are:

[0020] Compared with existing technologies, this invention achieves a synergistic enhancement of multiple beneficial effects by optimizing lipid composition and preparation process. This invention introduces key components such as membrane-functional lipids and stabilizers into the preparation method of liposomal vaccine adjuvants, where the membrane-functional lipids are selected from phytosphingosine, sphingomyelin, and DSPE-PEG, and the stabilizers are selected from sulfobutyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and dextran sulfate. This not only significantly increases the encapsulation efficiency of QS-21 to over 90%, effectively reducing drug loss, but also enhances lipid membrane rigidity and provides a surface stability barrier, enabling the product to exhibit excellent stability under accelerated storage conditions while reducing hemolysis rate to a low level. Furthermore, the liposomal vaccine adjuvant provided by this invention, based on high encapsulation efficiency, excellent stability, and low hemolysis, has passed rigorous acute toxicity and local irritation tests, confirming its reliable biosafety and providing strong technical support for the development of safe and efficient vaccine products. Detailed Implementation

[0021] The parameters and sources of the specific chemical substances used.

[0022] Phytosphingosine, pharmaceutical grade, purity ≥98%, CAS No.: 554-62-1;

[0023] Sphingomyelin, pharmaceutical grade, purity ≥98%, CAS No.: 85187-10-6;

[0024] DSPE-PEG, where PEG is PEG2000, catalog number: R-1028-2K, is sourced from Xi'an Ruixi Biotechnology Co., Ltd.

[0025] Hydrogenated soybean lecithin, model: HSPC-95A, sourced from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd.

[0026] QS-21, item number: B01002, is from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd.

[0027] Example 1

[0028] A method for preparing a liposomal vaccine adjuvant includes the following steps:

[0029] Step 1: Accurately weigh 600 mg of hydrogenated soybean lecithin, 200 mg of phytosphoprotein, and 300 mg of cholesterol. Then, add them to a mixed solvent of 20 mL of anhydrous ethanol and 10 mL of chloroform and mix to dissolve. Heat the mixture in a 50°C water bath for 2 minutes to obtain a mixed solution. Transfer the mixed solution to a round-bottom flask and evaporate it under reduced pressure in a 45°C water bath using a rotary evaporator until a uniform lipid film forms on the flask wall. Place the lipid film in a vacuum drying oven overnight.

[0030] Step 2: The lipid membrane from Step 1 was hydrated with 20 mL of citrate buffer (300 mmol / L, pH 4.0) containing 1.5 wt% sulfobutyl-β-cyclodextrin and 5 wt% sucrose, preheated to 55°C. The membrane was then shaken at 200 rpm for 60 min in a 55°C water bath to obtain the colostrum. The colostrum was homogenized 6 times at 800 bar, followed by 6 times at 1000 bar to obtain a blank liposome suspension.

[0031] Step 3: Transfer the blank liposome suspension to an ultrafiltration centrifuge tube pre-cooled to 4°C. Centrifuge at 14000×g for 30 min at 4°C, discard the filtrate, and add pre-cooled PBS buffer (pH 7.4) to the concentrated liposomes until the original volume is reached. Repeat the displacement process 5 times, and then bring the volume up to 100 mL with PBS buffer (pH 7.4) to obtain the liposome suspension. Dissolve 30 mg of QS-21 in 1.5 mL of PBS buffer (pH 7.4) to obtain the QS-21 solution. Add the QS-21 solution to the liposome suspension while stirring at 100 rpm, and then incubate in a 50°C water bath in the dark for 60 min to obtain the drug-loaded liposomes.

[0032] Step 4: Centrifuge the drug-loaded liposomes at 14000×g for 30 min at 4°C, discard the filtrate, and add PBS buffer pre-cooled to 4°C and pH 7.4 to the concentrated liposomes to the original volume and mix. Repeat the above operation twice, and bring the volume to 100 mL with PBS buffer at pH 7.4. Then filter through a 0.22 μm sterile filter membrane and store at 4°C in the dark to obtain the liposome vaccine adjuvant.

[0033] Example 2

[0034] The method for preparing a liposomal vaccine adjuvant differs from Example 1 only in that step 2 is:

[0035] The lipid membrane from step 1 above was hydrated with 20 mL of citrate buffer (300 mmol / L, pH 4.0) containing 1.5 wt% hydroxypropyl-β-cyclodextrin and 5 wt% sucrose, preheated to 55 °C. The membrane was then shaken at 200 rpm for 60 min in a 55 °C water bath to obtain the colostrum. The colostrum was homogenized 6 times at 800 bar, followed by 6 times at 1000 bar to obtain a blank liposome suspension.

[0036] Example 3

[0037] The method for preparing a liposomal vaccine adjuvant differs from Example 1 only in that step 2 is:

[0038] The lipid membrane from step 1 above was hydrated with 20 mL of citrate buffer (300 mmol / L, pH 4.0) containing 1.5 wt% dextran sulfate and 5 wt% sucrose, preheated to 55°C. The membrane was then shaken at 200 rpm for 60 min in a 55°C water bath to obtain the colostrum. The colostrum was homogenized 6 times at 800 bar, followed by 6 times at 1000 bar to obtain a blank liposome suspension.

[0039] Example 4

[0040] The method for preparing a liposomal vaccine adjuvant differs from Example 1 only in that step 1 is:

[0041] Accurately weigh 600 mg of hydrogenated soybean lecithin, 200 mg of DSPE-PEG2000, and 300 mg of cholesterol. Then, add them to a mixed solvent of 20 mL of anhydrous ethanol and 10 mL of chloroform and mix to dissolve. Heat the mixture in a 50 °C water bath for 2 min to obtain a mixed solution. Transfer the mixed solution to a round-bottom flask and evaporate the organic solvent under reduced pressure using a rotary evaporator in a 45 °C water bath until a uniform lipid film forms on the flask wall. Place the lipid film in a vacuum drying oven overnight.

[0042] Example 5

[0043] The method for preparing a liposomal vaccine adjuvant differs from Example 1 only in that step 1 is:

[0044] Accurately weigh 600 mg of hydrogenated soybean lecithin, 200 mg of sphingomyelin, and 300 mg of cholesterol, then add them to a mixed solvent of 20 mL of anhydrous ethanol and 10 mL of chloroform to dissolve them. Heat the mixture in a 50 °C water bath for 2 min to obtain a mixed solution. Transfer the mixed solution to a round-bottom flask and evaporate the organic solvent under reduced pressure using a rotary evaporator in a 45 °C water bath until a uniform lipid film forms on the flask wall. Place the lipid film in a vacuum drying oven overnight.

[0045] Comparative Example 1

[0046] The method for preparing a liposomal vaccine adjuvant differs from Example 1 only in that step 2 is:

[0047] The lipid membrane from step 1 above was hydrated with 20 mL of citrate buffer (300 mmol / L, pH 4.0) containing 1.5 wt% L-glutamine and 5 wt% sucrose, preheated to 55 °C. The membrane was then shaken at 200 rpm for 60 min in a 55 °C water bath to obtain the colostrum. The colostrum was homogenized 6 times at 800 bar, followed by 6 times at 1000 bar to obtain a blank liposome suspension.

[0048] Comparative Example 2

[0049] The method for preparing a liposomal vaccine adjuvant differs from Example 1 only in that step 2 is:

[0050] The lipid membrane from step 1 above was hydrated with 20 mL of citrate buffer containing 5 wt% sucrose at a concentration of 300 mmol / L and a pH of 4.0, preheated to 55°C. The membrane was then shaken at 200 rpm for 60 min in a 55°C water bath to obtain the colostrum. The colostrum was homogenized 6 times at 800 bar and then 6 times at 1000 bar to obtain a blank liposome suspension.

[0051] Comparative Example 3

[0052] The method for preparing a liposomal vaccine adjuvant differs from Example 1 only in that step 1 is:

[0053] Accurately weigh 800 mg of hydrogenated soybean lecithin and 300 mg of cholesterol, then add them to a mixed solvent of 20 mL of anhydrous ethanol and 10 mL of chloroform to dissolve them. Heat the mixture in a 50 °C water bath for 2 min to obtain a mixed solution. Transfer the mixed solution to a round-bottom flask and evaporate the organic solvent under reduced pressure using a rotary evaporator in a 45 °C water bath until a uniform lipid film forms on the flask wall. Place the lipid film in a vacuum drying oven overnight.

[0054] Test Example 1

[0055] Encapsulation efficiency test

[0056] 1.0 mL of the liposomal vaccine adjuvants prepared in Examples 1-5 and Comparative Examples 1-3 were accurately measured as samples, with five replicates for each. Each sample was placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa and centrifuged at 4°C and 10,000 rpm for 15 min. Free QS-21 permeated through the filter membrane, while encapsulated QS-21 was retained in the liposomes. The filtrate was accurately measured, and the concentration of free QS-21 was determined by high-performance liquid chromatography (HPLC) and recorded as C. 游离 Separately, accurately measure 1.0 mL of the liposomal vaccine adjuvant stock solution, demulsify it with 10% Triton X-100 aqueous solution to completely dissolve the liposomal vaccine adjuvant and release all QS-21, then dilute to 5 mL with methanol and determine the total QS-21 concentration using high-performance liquid chromatography (HPLC). Record this concentration as C. 总 Then calculate the encapsulation efficiency (EE%) of QS-21, using the following formula:

[0057] Encapsulation efficiency (EE%) = (1-C) 游离 / C 总 ) × 100%

[0058] The specific test results are taken as average values, and the specific results are shown in Table 1 below;

[0059] Accelerated stability testing

[0060] 1 mL of the liposomal vaccine adjuvants prepared in Examples 1-5 and Comparative Examples 1-3 were aliquoted into vials, sealed, and stored in constant temperature incubators at 4°C, 25°C, and 60°C, respectively, protected from light. These were recorded as Examples 1-5 and Comparative Examples 1-3, with five replicates for each group. Samples were taken on day 0 (initial) and day 28 after storage. The encapsulation efficiency was then measured at each time point according to the test method described above. The decrease in encapsulation efficiency on day 28 compared to the initial encapsulation efficiency was calculated using the following formula:

[0061] Encapsulation rate decrease (%) = (Initial encapsulation rate - Encapsulation rate on day 28) / Initial encapsulation rate × 100%

[0062] The specific test results are shown in Table 1 below.

[0063] Table 1

[0064]

[0065] As shown in Table 1, the encapsulation efficiency of Examples 1-5 was significantly higher than that of Comparative Examples 1-3, with Example 1 achieving the highest encapsulation efficiency of 97.2%. This may be because phytosphingosine can tightly embed itself within the lipid bilayer composed of hydrogenated soybean phospholipids and cholesterol. Its amino and hydroxyl groups strongly interact with the lipid membrane, significantly enhancing membrane density and forming a more complete drug encapsulation barrier. The synergistic use of sulfobutyl-β-cyclodextrin and phytosphingosine allows sulfobutyl-β-cyclodextrin to encapsulate drugs or lipids using its hydrophobic cavity. Under acidic hydration conditions, sulfobutyl-β-cyclodextrin electrostatically attracts the positively charged phytosphingosine, jointly stabilizing the nascent liposome structure and further reinforcing it, thus significantly improving the encapsulation efficiency. Compared to phytosphingosine, sphingomyelin lacks a primary amino group, resulting in weaker electrostatic anchoring with sulfobutyl-β-cyclodextrin, thus leading to a slightly lower encapsulation efficiency. Furthermore, DSPE-PEG is too hydrophilic, failing to effectively embed itself within the membrane for stabilization and even interfering with the orderly arrangement of lipid molecules, leading to decreased membrane integrity and significantly lower encapsulation efficiency. Compared to sulfobutyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin lacks a sulfonate negative charge, failing to generate strong electrostatic attraction with phytosphingosine and exhibiting poor binding strength to liposomes, resulting in a slightly lower encapsulation efficiency. Compared to Example 1, Comparative Examples 1-2 lack the inclusion and anchoring capabilities of cyclodextrin, failing to stabilize the structure in the early stages of formation, leading to significant drug loss. Comparative Example 3, lacking phytosphingosine, resulted in a significant decrease in lipid membrane density.

[0066] Table 1 shows that the accelerated stability test data indicates that the encapsulation efficiency of Examples 1-5 decreased significantly less than that of Comparative Examples 1-3, with Example 1 showing the smallest decrease in encapsulation efficiency and exhibiting better stability. This may be because phytosphingosine imparts higher rigidity and phase transition temperature to the lipid membrane. During accelerated stability testing, the membrane fluidity of this formulation of liposomes increased relatively little, better maintaining structural integrity and effectively inhibiting drug leakage due to increased molecular thermal motion. Simultaneously, the sulfobutyl-β-cyclodextrin anchored to the liposome surface via electrostatic interaction provides sustained steric stabilization and electrostatic stability during storage, effectively preventing liposome aggregation and fusion. In Example 3, dextran sulfate was introduced; its linear molecular structure lacks the inclusion-anchoring ability of cyclodextrin, making it prone to failure during long-term storage, thus its stability is inferior to Example 1. The liposomes in the comparative examples have a loose structure and rapidly deteriorate under thermal stress.

[0067] Test Example 2

[0068] Hemolytic test

[0069] Solution preparation:

[0070] Fresh rabbit blood was collected, washed and centrifuged three times with PBS buffer at pH 7.4, and a 2% (v / v) red blood cell suspension was prepared. 5 mL of the liposomal vaccine adjuvants prepared in Examples 1-5 and Comparative Examples 1-3 were accurately measured and then diluted with PBS buffer at pH 7.4 to an equivalent QS-21 concentration of 50 μg / mL as liposomal vaccine adjuvant samples.

[0071] Test tube setup:

[0072] Add 0.5 mL of liposomal vaccine adjuvant sample and 0.5 mL of 2% red blood cell suspension to a 5 mL test tube and record it as the test sample tube;

[0073] Add 0.5 mL of pH 7.4 PBS buffer and 0.5 mL of 2% red blood cell suspension to a 5 mL test tube and record it as a negative control tube;

[0074] Add 0.5 mL of 1% Triton X-100 aqueous solution and 0.5 mL of 2% erythrocyte suspension to a 5 mL test tube and record it as a positive control tube;

[0075] Add 0.5 mL of liposome vaccine adjuvant sample and 0.5 mL of pH 7.4 PBS buffer to a 5 mL test tube and record it as a sample blank tube;

[0076] After gently mixing the above test tubes, incubate them in a 37℃ water bath for 1 hour. After incubation, centrifuge all test tubes at 3000×g for 5 minutes. Take the supernatant and measure the absorbance at a wavelength of 540nm. Then calculate the hemolysis rate (%) using the following formula:

[0077] Hemolysis rate (%) = [(OD) 供试品 -OD 样品空白 )-OD 阴性对照 ] / (OD 阳性对照 -OD 阴性对照 )×100%

[0078] The specific test results are shown in Table 2 below.

[0079] Table 2

[0080]

[0081] As shown in Table 2, the hemolysis rates of Examples 1-5 were significantly lower than those of Comparative Examples 1-3, with Example 1 exhibiting the lowest hemolysis rate. This is likely because the synergistic use of phytosphingosine and sulfobutyl-β-cyclodextrin provided high encapsulation efficiency and excellent stability, ensuring that the vast majority of hemolytically active QS-21 molecules were safely encapsulated within the liposomes. Therefore, during the incubation process for the hemolysis test, the release of free QS-21 was minimized, reducing damage to the erythrocyte membrane at its source. Furthermore, the sulfobutyl-β-cyclodextrin anchored on the liposome surface formed a hydration protective layer on its hydrophilic outer surface, shielding the direct interaction between the hydrophobic surface of the liposome and the erythrocyte membrane, thus avoiding membrane fusion or disturbance that might be caused by physical contact, resulting in a significantly lower hemolysis rate. Compared to Example 1, the membrane structure integrity of the other examples was slightly weaker and the protective layer effect was slightly inferior, therefore their hemolysis rates were all slightly higher than those of Example 1. In contrast, the liposomes in Comparative Examples 1-3 had serious structural defects and insufficient stability. During the incubation process of the hemolysis test, they were more likely to release free QS-21 and membrane components, resulting in high hemolysis.

[0082] Test Example 3

[0083] Biosafety testing

[0084] To assess the in vivo safety of the liposomal vaccine adjuvant of this invention, the following experiments were conducted in accordance with the Good Laboratory Practice (GLP) guidelines for non-clinical drug studies.

[0085] SPF-grade BALB / c mice, 6-8 weeks old and weighing 18-22 grams, were selected, with an equal number of males and females. The experimental animals were acclimatized for 7 days in a barrier environment with a temperature of 22±2℃, humidity of 50±10%, and a 12-hour light cycle.

[0086] Preparation of test sample: Take the liposome vaccine adjuvant prepared in Examples 1-5, reconstitute it with 0.9% sodium chloride injection 20 minutes before use, and store it at 4°C for later use.

[0087] Preparation of reference standard: physiological saline (0.9% sodium chloride injection)

[0088] I. Acute toxicity test

[0089] Experimental Design: Thirty healthy mice were randomly divided into three groups of 10 each, with half males and half females. A high-dose group (1000 mg / kg, equivalent to 8 times the clinical dose), a low-dose group (250 mg / kg, equivalent to 2 times the clinical dose), and a blank control group (equal volume of physiological saline) were established. Administered via subcutaneous injection at a volume of 20 mL / kg.

[0090] Observation indicators: Animal behavior was recorded every 15 minutes for 2 hours after drug administration, with a focus on acute toxicity symptoms such as tremors, convulsions, respiratory rate, piloerection, and diarrhea. For 14 consecutive days, daily records were kept of changes in animal weight, food intake, activity level, coat condition, and ocular and nasal secretions. At the end of the experiment, animals were euthanized, and dissections were performed to observe the macroscopic morphology of major organs (heart, liver, spleen, lungs, and kidneys), and organ coefficients were calculated.

[0091] Experimental Results: No mice in any of the treatment groups died during the observation period, and no typical acute toxicity symptoms were observed. The weight gain curves were basically consistent with the control group, and no pathological changes such as congestion, edema, or necrosis were observed in any organs; there were no statistically significant differences in organ coefficients. The results indicate that the liposomal vaccine adjuvant of this invention did not exhibit acute toxicity characteristics at the experimental dose.

[0092] II. Local Irritation Test

[0093] Experimental design: Twelve healthy mice were randomly divided into an experimental group and a control group, with six mice in each group (half male and half female). The experimental group received a subcutaneous injection of 1000 mg / kg of liposomal vaccine adjuvant, while the control group received an equal volume of physiological saline.

[0094] Evaluation method: Erythema and induration were scored at the injection site at four time points: 1, 24, 48, and 72 hours after drug administration. The scoring criteria are shown in Table 3 below:

[0095] Table 3

[0096]

[0097] Histological examination: Skin and subcutaneous tissue were taken from the injection site 72 hours later. After formalin fixation, paraffin embedding, and HE staining, the integrity of the epidermis, dermal inflammatory cell infiltration, and subcutaneous tissue pathological changes were observed.

[0098] Experimental Results: No obvious erythema or induration was observed at the injection sites in any of the experimental group animals at any time point, and the stimulation score was 0. Histological examination showed that the epidermal structure was intact, with no keratinocyte necrosis or shedding, no significant inflammatory cell infiltration in the dermis, and no edema or fibrin exudation in the subcutaneous tissue. The results indicate that the formulation of this invention is non-irritating to the injection site.

[0099] Conclusion: Through systematic review of acute toxicity and local irritation tests, the liposomal vaccine adjuvant prepared in this invention has been confirmed to have good biocompatibility under the test conditions and meets the safety requirements for clinical application of vaccine adjuvants.

Claims

1. A method for preparing a liposomal vaccine adjuvant, characterized in that, Includes the following steps: Step 1: Accurately weigh 550-600 mg of hydrogenated soybean lecithin, 180-220 mg of membrane-functional lipids, and 250-350 mg of cholesterol. Then, add them to a mixed solvent of 16-24 mL of anhydrous ethanol and 8-12 mL of chloroform to dissolve them. Heat the mixture in a water bath at 45-55°C for 1-2 minutes to obtain a mixed solution. Transfer the mixed solution to a round-bottom flask and evaporate the organic solvent under reduced pressure using a rotary evaporator in a water bath at 40-50°C until a uniform lipid film forms on the flask wall. Place the lipid film in a vacuum drying oven overnight. Step 2: Hydrate the lipid film from Step 1 with 18-22 mL of preheated citrate buffer containing 0.5-2 wt% stabilizer and 4-6 wt% sucrose at a concentration of 280-320 mmol / L. Shake at 150-300 rpm for 50-80 min in a water bath at 50-60°C to obtain the colostrum. Homogenize the obtained colostrum to obtain the blank liposome suspension. Step 3: Replace the external aqueous phase of the blank liposome suspension with pre-cooled PBS buffer via ultrafiltration centrifugation, repeating the replacement 5-8 times, and bring the volume to a final level to obtain the liposome suspension; add 28-33 mg of QS-21 to 1-2 mL of PBS buffer to dissolve and obtain the QS-21 solution, add the QS-21 solution to the liposome suspension while stirring at 80-150 rpm, mix, and then incubate in the dark to obtain the drug-loaded liposomes; Step 4: Centrifuge the drug-loaded liposomes at 12000-15000×g for 25-35 min at 2-5℃, discard the filtrate, and add PBS buffer pre-cooled to 2-5℃ to the concentrated liposomes to the original volume and mix. Repeat the above operation 1-3 times and make up the volume. Then filter through a 0.22-0.45μm sterile filter membrane and store at 1-8℃ in the dark to obtain the liposomal vaccine adjuvant.

2. The method for preparing the liposome vaccine adjuvant according to claim 1, characterized in that, The membrane functional lipids are selected from one of phytosphingosine, sphingomyelin, and DSPE-PEG.

3. The method for preparing the liposome vaccine adjuvant according to claim 1, characterized in that, The stabilizer is selected from one of sulfobutyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and dextran sulfate.

4. The method for preparing the liposome vaccine adjuvant according to claim 1, characterized in that, Step 2 involves hydration using a citrate buffer solution preheated to 50-60°C.

5. The method for preparing the liposome vaccine adjuvant according to claim 1, characterized in that, In step 2, the obtained colostrum is first homogenized 5-8 times at a pressure of 800-1000 bar, and then homogenized again 5-8 times at a pressure of 1000-1200 bar to obtain a blank liposome suspension.

6. The method for preparing the liposome vaccine adjuvant according to claim 1, characterized in that, Step 3 involves replacing the external aqueous phase using ultrafiltration centrifugation. The blank liposome suspension is transferred to an ultrafiltration centrifuge tube pre-cooled to 2-5°C. The tube is centrifuged at 12000-15000×g for 25-35 minutes at 2-5°C. The filtrate is discarded, and PBS buffer pre-cooled to 2-5°C is added to the concentrated liposomes to bring the volume to the original volume.

7. The method for preparing the liposome vaccine adjuvant according to claim 1, characterized in that, Step 3, the light-protected incubation, involves placing the container in a water bath at 45-55℃ and incubating it in the dark for 50-70 minutes.

8. A liposomal vaccine adjuvant, characterized in that: Prepared by the method described in any one of claims 1-7.

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