A liposome vaccine adjuvant and a preparation method thereof

Abstract

The application discloses a liposome vaccine adjuvant and a preparation method thereof, and belongs to the technical field of vaccine adjuvants. The adjuvant comprises hydrogenated soybean phospholipid, cholesterol, saponin adjuvant QS-21, membrane functional lipid and a stabilizer. The preparation method comprises the following steps: dissolving lipid components in an organic solvent to form a film, hydrating the film with a buffer solution containing the stabilizer, forming blank liposomes through high-pressure homogenization, incubating the blank liposomes with a QS-21 solution after purification to load drugs, and finally purifying and sterilizing to obtain the liposome vaccine adjuvant. Through the synergistic cooperation of the above components and the process, the technical problems of low encapsulation efficiency, poor storage stability and high hemolyticity existing in the traditional liposome encapsulation of QS-21 are successfully solved. Moreover, the prepared liposome vaccine adjuvant passes strict acute toxicity and local irritation tests, has good biological safety, and has important clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of vaccine adjuvants, in particular to a liposome vaccine adjuvant and a preparation method thereof. BACKGROUND

[0002] Vaccine adjuvants, as an important auxiliary material for enhancing the immunogenicity of antigens, have attracted widespread attention in the development of new vaccines in recent years. QS-21 is a highly effective saponin vaccine adjuvant that can strongly stimulate cellular and humoral immunity and is widely used in the development of new vaccines. However, it has the defect of poor chemical stability, and is easily hydrolyzed or aggregated in aqueous solution, and QS-21 has strong hemolytic toxicity, which seriously limits its clinical safety.

[0003] Currently, liposome technology is one of the ideal strategies for encapsulating QS-21, but the existing technology still has deficiencies: first, the encapsulation efficiency is low, resulting in a large amount of drug waste and potential toxicity; second, the stability is poor, and the drug is easily leaked from the liposome during storage, and the burst release phenomenon is obvious; finally, the conventional lipid membrane is difficult to completely shield the hemolytic activity of QS-21, and the introduction of polyethylene glycol and other components to improve stability may bring new immunogenicity risks. Therefore, it is urgent to develop a QS-21 liposome that can simultaneously achieve high encapsulation, high stability and high safety, in order to improve its adaptability in complex vaccine environments and industrial conversion prospects.

[0004] WO2025124295A1 discloses an immune adjuvant composition, a preparation method and use thereof, which comprises an immunologically active agent and a liposome for loading the immunologically active agent, and the immunologically active agent comprises saponin and CpG oligodeoxynucleotide. By using liposomes to load QS-21 and CpG oligodeoxynucleotide, the two have good synergistic effect in promoting cellular immunity, and can induce stronger cellular immune response level compared with commercial vaccines. However, the QS-21 loaded by the liposome has poor storage stability, and is prone to drug leakage or liposome aggregation, affecting the product potency and shelf life.

[0005] CN119971022A discloses a QS-21 composite adjuvant and its preparation method and application, the QS-21 composite adjuvant comprises the following components in parts by weight: QS-21: 0.1-0.5 parts; liposome: 4-6 parts; CpG oligonucleotide: 0.05-0.1 parts; freeze-drying protective agent: 0.1-0.3 parts; stabilizer: 0.2-0.5 parts; propylene glycol: 3-5 parts; ethanol: 1-2 parts; organic solvent chloroform: 5-15 parts; deionized water: 100-200 parts, and the liposome comprises: distearoyl phospholipid: 3-4 parts; amino lipid: 1-2 parts. The application improves the loading efficiency, immune effect stability and recovery after freeze-drying of the immune adjuvant, and enhances the durability and efficacy of the immune response by compounding the liposome carrier with QS-21, adding CpG oligonucleotide and optimizing the freeze-drying protective agent. However, the composite adjuvant cannot effectively shield the hemolytic toxicity of QS-21, and the clinical safety needs to be evaluated. SUMMARY

[0006] The purpose of the present application is to overcome the defects of the prior art, solve the problems of low encapsulation efficiency, drug waste and inaccurate dosage of QS-21 liposome adjuvant prepared by traditional methods; poor storage stability, easy drug leakage or liposome aggregation, affecting product efficacy and shelf life, and the risk of insufficient biological safety caused by the release of free adjuvant. The present application provides a liposome vaccine adjuvant with high encapsulation efficiency, good stability and effective shielding of QS-21 hemolytic toxicity by optimizing the lipid composition and preparation process.

[0007] The present application provides a preparation method of a liposome vaccine adjuvant, comprising the following steps:

[0008] Step 1, accurately weigh 550-600 mg of hydrogenated soybean phospholipid, 180-220 mg of membrane functional lipid and 250-350 mg of cholesterol, then add a mixed solvent of 16-24 mL of anhydrous ethanol and 8-12 mL of chloroform to mix and dissolve, and heat in a 45-55°C water bath for 1-2 min to obtain a mixed solution; transfer the mixed solution to a round-bottom flask, and use a rotary evaporator to evaporate the organic solvent under reduced pressure in a 40-50°C water bath until a uniform lipid film forms on the bottle wall; place the lipid film in a vacuum drying oven overnight;

[0009] Step 2, hydrate the lipid film of step 1 with preheated 18-22 mL of citric acid buffer solution containing 0.5-2 wt% stabilizer and 4-6 wt% sucrose at a concentration of 280-320 mmol / L, and oscillate at 150-300 rpm in a 50-60°C water bath for 50-80 min to obtain a primary milk; homogenize the obtained primary milk to obtain a blank liposome suspension;

[0010] Step 3, the blank liposome suspension is replaced with pre-cooled PBS buffer by ultrafiltration centrifugation to replace the outer water phase, and the replacement is repeated for 5-8 times, and then the volume is fixed to obtain the liposome suspension; 28-33 mg QS-21 is added into 1-2 mL PBS buffer to be mixed and dissolved to obtain a QS-21 solution, and then the QS-21 solution is added into the liposome suspension under stirring at 80-150 rpm, and then incubated in the dark to obtain the drug-loaded liposome;

[0011] Step 4, the drug-loaded liposome is centrifuged at 12000-15000 x g for 25-35 min at 2-5 ℃, the filtrate is discarded, and the PBS buffer pre-cooled to 2-5 ℃ is added to the concentrated liposome to the original volume; then the above operation is repeated for 1-3 times, and then the volume is fixed; then filtered through a 0.22-0.45 μm sterile filter membrane, and then stored in the dark at 1-8 ℃ to obtain the liposome 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, the step 2 uses the pre-heated citric acid buffer solution at 50-60 ℃ for hydration.

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

[0016] Preferably, the step 3 replaces the outer water phase by ultrafiltration centrifugation, that is, the blank liposome suspension is transferred to the pre-cooled ultrafiltration centrifuge tube at 2-5 ℃, centrifuged at 12000-15000 x g for 25-35 min, the filtrate is discarded, and the PBS buffer pre-cooled to 2-5 ℃ is added to the concentrated liposome to the original volume.

[0017] Preferably, the step 3 incubates in the dark for 50-70 min in a 45-55 ℃ water bath.

[0018] The application also provides a liposome vaccine adjuvant prepared by the above method.

[0019] The application has the following beneficial effects:

[0020] Compared with the prior art, the present application successfully realizes the synergistic promotion of multiple beneficial effects by optimizing the lipid component and the preparation process. In the preparation method of the liposome vaccine adjuvant, key components such as membrane functional lipids and stabilizers are introduced to synergistically act, wherein the membrane functional lipids are selected from one of plant sphingosine, sphingomyelin and DSPE-PEG, and the stabilizer is selected from one of sulfobutyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin and dextran sulfate. Not only is the encapsulation rate of QS-21 significantly improved to more than 90%, effectively reducing drug loss, but also by enhancing the rigidity of the lipid membrane and providing a surface stable barrier, the product exhibits excellent stability under accelerated storage conditions, while the hemolysis rate is reduced to a low level. In addition, the liposome vaccine adjuvant provided by the present application, on the basis of high encapsulation efficiency, excellent stability and low hemolysis, passes the strict acute toxicity and local irritation tests, and proves its reliable biological safety, providing strong technical support for the development of safe and efficient vaccine products. DETAILED DESCRIPTION

[0021] Parameters for using specific chemicals, sources.

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

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

[0024] DSPE-PEG, wherein PEG is PEG2000, item number: R-1028-2K, from Xi'an Ruishi Biological Technology Co., Ltd.;

[0025] Hydrogenated soybean phospholipid, model number: HSPC-95A, from Aevitop (Shanghai) Pharmaceutical Technology Co., Ltd.;

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

[0027] Example 1

[0028] A preparation method of a liposome vaccine adjuvant, comprising the following steps:

[0029] Step 1, accurately weigh 600 mg of hydrogenated soybean phospholipid, 200 mg of plant sphingosine and 300 mg of cholesterol, then add a mixed solvent of 20 mL of anhydrous ethanol and 10 mL of chloroform to mix and dissolve, and heat in a 50°C water bath for 2 min to obtain a mixed solution; transfer the mixed solution to a round-bottom flask, and use a rotary evaporator to evaporate the organic solvent under reduced pressure in a 45°C water bath until a uniform lipid film is formed on the bottle wall; place the lipid film in a vacuum drying oven overnight;

[0030] Step 2, the lipid film of step 1 above is hydrated with 20 mL of a citric acid buffer solution containing 1.5 wt% sulfobutyl-β-cyclodextrin and 5 wt% sucrose at a concentration of 300 mmol / L and pH 4.0 preheated to 55°C, oscillated at 200 rpm for 60 min in a 55°C water bath to obtain a milk; the obtained milk is homogenized 6 times at a pressure of 800 bar, followed by 6 times at a pressure of 1000 bar to obtain a blank liposome suspension;

[0031] Step 3, the blank liposome suspension is transferred to an ultrafiltration centrifuge tube pre-cooled to 4°C, centrifuged at 14000 x g for 30 min at 4°C, the filtrate is discarded, and the concentrated liposomes are mixed with PBS buffer pre-cooled to 4°C and pH 7.4 to the original volume, and the replacement is repeated 5 times, and the volume is adjusted to 100 mL with PBS buffer at pH 7.4 to obtain a liposome suspension; 30 mg of QS-21 is dissolved in 1.5 mL of PBS buffer at pH 7.4 to obtain a QS-21 solution, which is added to the liposome suspension under stirring at 100 rpm, and then incubated at 50°C in a water bath for 60 min in the dark to obtain drug-loaded liposomes;

[0032] Step 4, the drug-loaded liposomes are centrifuged at 14000 x g for 30 min at 4°C, the filtrate is discarded, and the concentrated liposomes are mixed with PBS buffer pre-cooled to 4°C and pH 7.4 to the original volume; then the above operation is repeated twice, and the volume is adjusted to 100 mL with PBS buffer at pH 7.4; then filtered through a 0.22 μm sterile filter membrane, and stored at 4°C in the dark to obtain a liposome vaccine adjuvant.

[0033] Example 2

[0034] A method for preparing a liposome vaccine adjuvant differs from example 1 only in that step 2 is:

[0035] The lipid film of step 1 above is hydrated with 20 mL of a citric acid buffer solution containing 1.5 wt% sulfobutyl-β-cyclodextrin and 5 wt% sucrose at a concentration of 300 mmol / L and pH 4.0 preheated to 55°C, oscillated at 200 rpm for 60 min in a 55°C water bath to obtain a milk; the obtained milk is homogenized 6 times at a pressure of 800 bar, followed by 6 times at a pressure of 1000 bar to obtain a blank liposome suspension.

[0036] Example 3

[0037] A method for preparing a liposome vaccine adjuvant differs from example 1 only in that step 2 is:

[0038] Hydrate the lipid film of Step 1 above with 20 mL of a citric acid buffer containing 1.5 wt% dextran sulfate and 5 wt% sucrose at a concentration of 300 mmol / L and pH 4.0 preheated to 55°C, and shake at 200 rpm in a 55°C water bath for 60 min to obtain a preliminary emulsion; homogenize the obtained preliminary emulsion 6 times at a pressure of 800 bar, and then 6 times at a pressure of 1000 bar to obtain a blank liposome suspension.

[0039] Example 4

[0040] The preparation method of a liposome vaccine adjuvant differs from Example 1 only in that Step 1 is:

[0041] Accurately weigh 600 mg of hydrogenated soybean phospholipid, 200 mg of DSPE-PEG2000, and 300 mg of cholesterol, then add a mixed solvent of 20 mL of anhydrous ethanol and 10 mL of chloroform to dissolve and mix, and heat 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 bottle wall; place the lipid film in a vacuum drying box overnight.

[0042] Example 5

[0043] The preparation method of a liposome vaccine adjuvant differs from Example 1 only in that Step 1 is:

[0044] Accurately weigh 600 mg of hydrogenated soybean phospholipid, 200 mg of DSPE-PEG2000, and 300 mg of cholesterol, then add a mixed solvent of 20 mL of anhydrous ethanol and 10 mL of chloroform to dissolve and mix, and heat 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 bottle wall; place the lipid film in a vacuum drying box overnight.

[0045] Comparative Example 1

[0046] The preparation method of a liposome vaccine adjuvant differs from Example 1 only in that Step 2 is:

[0047] Hydrate the lipid film of Step 1 above with 20 mL of a citric acid buffer containing 1.5 wt% L-glutamine and 5 wt% sucrose at a concentration of 300 mmol / L and pH 4.0 preheated to 55°C, and shake at 200 rpm in a 55°C water bath for 60 min to obtain a preliminary emulsion; homogenize the obtained preliminary emulsion 6 times at a pressure of 800 bar, and then 6 times at a pressure of 1000 bar to obtain a blank liposome suspension.

[0048] Comparative Example 2

[0049] A preparation method of a liposome vaccine adjuvant is only different from that of Example 1 in that the step 2 is:

[0050] The lipid film of step 1 is hydrated with 20 mL of a citric acid buffer solution containing 5 wt% sucrose, having a concentration of 300 mmol / L and a pH of 4.0, preheated to 55°C, and oscillated at 200 rpm in a 55°C water bath for 60 min to obtain a preliminary emulsion; the obtained preliminary emulsion is homogenized 6 times under a pressure of 800 bar, and then homogenized 6 times under a pressure of 1000 bar to obtain a blank liposome suspension.

[0051] Comparative Example 3

[0052] A preparation method of a liposome vaccine adjuvant is only different from that of Example 1 in that the step 1 is:

[0053] Accurately weigh 800 mg of hydrogenated soybean phospholipid and 300 mg of cholesterol, then add and dissolve in a mixed solvent of 20 mL of anhydrous ethanol and 10 mL of chloroform, and heat 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 bottle wall; place the lipid film in a vacuum drying box overnight.

[0054] Test Example 1

[0055] Encapsulation efficiency test

[0056] Accurately take 1.0 mL of the liposome vaccine adjuvant prepared in Examples 1-5 and Comparative Examples 1-3 as samples, respectively, and set up 5 parallels each; place each sample in an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa, and centrifuge at 4°C and 10000 rpm for 15 min; free QS-21 will pass through the filter membrane, and encapsulated QS-21 will be trapped in the liposome; accurately take the filtrate, and determine the concentration of free QS-21 using high performance liquid chromatography, and record it as C 游离 ; in addition, accurately take 1.0 mL of the liposome vaccine adjuvant stock solution, and demulsify it with 10% Triton X-100 aqueous solution to completely dissolve the liposome vaccine adjuvant and release all QS-21; after being diluted to 5 mL with methanol, the total QS-21 concentration is also determined using high performance liquid chromatography, and recorded as C 总 ; then calculate the encapsulation efficiency (EE%) of QS-21, and the specific calculation formula is as follows:

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

[0058] The specific test results were averaged, and the specific results are shown in Table 1 below.

[0059] Acceleration stability test

[0060] 1 mL of the liposome vaccine adjuvant prepared in Examples 1-5 and Comparative Examples 1-3 was respectively taken as a sample and divided into a Schlenk bottle, which was sealed and then placed in a constant temperature box at 4°C, 25°C and 60°C respectively to store in the dark, and recorded as Examples 1-5 and Comparative Examples 1-3 groups, and each group was set up in five parallel; samples were taken on the 0th day (initial) and the 28th day after storage; then, the encapsulation rate at each time point was determined according to the test method in the above encapsulation rate test; the decrease (%) of the encapsulation rate on the 28th day compared with the initial encapsulation rate was calculated, and the specific calculation formula was as follows:

[0061] Encapsulation rate decrease (%) = (initial encapsulation rate - encapsulation rate on the 28th day) / initial encapsulation rate x 100%

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

[0063] Table 1

[0064]

[0065] As can be seen from Table 1, the encapsulation rate of Examples 1-5 is significantly higher than that of Comparative Examples 1-3, and Example 1 reaches the highest encapsulation rate of 97.2%, and the analysis reason may be that the phytosphingosine can be closely embedded in the lipid bilayer composed of hydrogenated soybean phospholipid and cholesterol, and can interact with the lipid membrane through its amino and hydroxyl groups, significantly enhancing the membrane compactness, and forming a more complete drug wrapping barrier. The introduction of sulfobutyl-β-cyclodextrin and phytosphingosine for synergistic use, the sulfobutyl-β-cyclodextrin uses its hydrophobic cavity to include drugs or lipids, and in the acidic aqueous environment, it produces electrostatic attraction with the positively charged phytosphingosine, which together stabilizes the structure of the primary liposome, further strengthens the structure of the liposome, and thus significantly improves the encapsulation rate. Compared with phytosphingosine, sphingomyelin lacks primary amino groups, and the electrostatic anchoring effect with sulfobutyl-β-cyclodextrin is weaker than that of phytosphingosine, so the encapsulation rate is slightly lower; and DSPE-PEG is too hydrophilic to better embed in the membrane to play a stabilizing role, and even can interfere with the ordered arrangement of lipid molecules, leading to a decrease in membrane integrity, so the encapsulation efficiency is significantly lower. Compared with sulfobutyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin lacks sulfonate negative charge and cannot produce strong electrostatic attraction with phytosphingosine, so the combination with the liposome is poor, and thus the encapsulation rate is slightly lower. Compared with Example 1, the lack of cyclodextrin inclusion and anchoring ability in Comparative Example 1-2 cannot stabilize the structure at the initial stage, leading to a large amount of drug loss, and the lack of phytosphingosine in Comparative Example 3 leads to a significant decrease in the compactness of the lipid membrane.

[0066] From Table 1, the accelerated stability test data shows that the encapsulation efficiency of Examples 1-5 decreases significantly lower than Comparative Examples 1-3, in which the encapsulation efficiency of Example 1 decreases the least, having better stability. The reason for analysis may be that phytosphingosine gives the lipid membrane higher rigidity and phase transition temperature, and during the accelerated stability test, the membrane fluidity of the formula liposome increases relatively less, which can better maintain the structural integrity and effectively inhibit the leakage of the drug due to the intensification of molecular thermal motion. At the same time, the sulfobutyl-β-cyclodextrin anchored on the surface of the liposome provides a persistent steric hindrance and electrostatic stabilization effect during storage, effectively preventing the aggregation and fusion of the liposome during storage. The linear molecular structure of dextran sulfate in Example 3 lacks the inclusion anchoring ability of cyclodextrin, which is easy to fail in long-term storage, so the stability is not as good as Example 1. The liposome structure of the comparative example is loose, which is quickly destroyed under thermal stress.

[0067] Test Example 2

[0068] Hemolytic test

[0069] Test solution configuration:

[0070] Freshly collected rabbit blood was washed with pH 7.4 PBS buffer and centrifuged 3 times to prepare a 2% (v / v) red blood cell suspension; 5 mL of liposome vaccine adjuvant prepared from Examples 1-5 and Comparative Examples 1-3 was accurately measured, respectively, and then diluted with pH 7.4 PBS buffer to an equivalent QS-21 concentration of 50 μg / mL as a liposome vaccine adjuvant sample;

[0071] Test tube setup:

[0072] 0.5 mL of liposome vaccine adjuvant sample and 0.5 mL of 2% red blood cell suspension were added to a 5 mL test tube, which was recorded as the test sample tube;

[0073] 0.5 mL of pH 7.4 PBS buffer and 0.5 mL of 2% red blood cell suspension were added to a 5 mL test tube, which was recorded as the negative control tube;

[0074] 0.5 mL of 1% Triton X-100 aqueous solution and 0.5 mL of 2% red blood cell suspension were added to a 5 mL test tube, which was recorded as the positive control tube;

[0075] 0.5 mL of liposome vaccine adjuvant sample and 0.5 mL of pH 7.4 PBS buffer were added to a 5 mL test tube, which was recorded as the sample blank tube;

[0076] After mixing the above test tubes gently, incubate at 37℃ water bath for 1h; after incubation, centrifuge all test tubes at 3000xg for 5min; take the supernatant, measure the absorbance value at 540nm wavelength; then calculate the hemolysis rate (%), the calculation formula is as follows:

[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 ) x 100%

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

[0079] Table 2

[0080]

[0081] As can be seen from Table 2, the hemolysis rates of Examples 1-5 are significantly lower than those of Comparative Examples 1-3, and the hemolysis rate of Example 1 is the lowest. The possible reason for this is that the synergistic use of phytosphingosine and sulfobutyl-β-cyclodextrin provides high encapsulation efficiency and excellent stability, ensuring that most of the QS-21 molecules with hemolytic activity are safely encapsulated inside the liposomes. Therefore, during the incubation process of the hemolysis test, the amount of free QS-21 released is minimized, which fundamentally reduces the damage to the red blood cell membrane, and the sulfobutyl-β-cyclodextrin anchored on the surface of the liposome forms a layer of hydration protection layer on its hydrophilic outer surface, which can shield the direct interaction between the hydrophobic surface of the liposome and the red blood cell membrane, thereby avoiding the membrane fusion or disturbance that may be caused by physical contact, so the hemolysis rate is significantly reduced. Compared with Example 1, the membrane structural integrity of other examples is slightly weaker and the protective layer effect is slightly worse, so the hemolysis rate is slightly higher than that of Example 1. The liposome structure of Comparative Examples 1-3 has serious defects, and the stability of the liposome is insufficient, which is more likely to release free QS-21 and membrane components during the incubation process of the hemolysis test, resulting in high hemolysis.

[0082] Test Example 3

[0083] Biological safety test

[0084] To evaluate the in vivo safety of the liposome vaccine adjuvant of the present application, the following experiments were carried out in accordance with the Good Laboratory Practice for Nonclinical Studies of Drugs.

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

[0086] Preparation of test sample: The liposome vaccine adjuvant prepared in Example 1-5 was reconstituted with 0.9% sodium chloride injection 20 minutes before use, and stored at 4°C.

[0087] Preparation of control sample: physiological saline (0.9% sodium chloride injection)

[0088] I. Acute toxicity test

[0089] Test design: 30 healthy mice were randomly divided into three groups, 10 in each group, half male and half female. The high-dose group (1000 mg / kg, equivalent to 8 times the clinical dose), the low-dose group (250 mg / kg, equivalent to 2 times the clinical dose) and the blank control group (equal volume of physiological saline) were set up. The subcutaneous injection route was used for administration, and the administration volume was 20 mL / kg.

[0090] Observation index: The behavior of the animals was recorded every 15 minutes within 2 hours after administration, and the acute toxicity symptoms such as tremor, convulsion, respiratory rate, piloerection, diarrhea, etc. were observed. The body weight change, food intake, activity state, fur condition and eye and nose secretions of the animals were recorded daily for 14 consecutive days. The animals were euthanized at the end of the experiment, the macroscopic morphology of the main organs (heart, liver, spleen, lung, kidney) was observed, and the organ coefficients were calculated.

[0091] Test results: All mice in the administration groups had no death during the observation period, and no typical acute toxicity symptoms were observed. The body weight growth curve was basically consistent with that of the control group, and no pathological changes such as congestion, edema and necrosis were observed in the organs, and there was no statistical difference in the organ coefficients. The results showed that the liposome vaccine adjuvant of the present application did not show acute toxicity characteristics at the test dose.

[0092] II. Local irritation test

[0093] Test design: 12 healthy mice were randomly divided into experimental and control groups, 6 in each group, half male and half female. The experimental group was subcutaneously injected with 1000 mg / kg dose of liposome vaccine adjuvant, and the control group was injected with equal volume of physiological saline.

[0094] Evaluation method: At four time points of 1, 24, 48 and 72 hours after administration, the injection site was scored for erythema and induration. The scoring criteria are as follows in Table 3:

[0095] Table 3

[0096]

[0097] Histological examination: The skin and subcutaneous tissue at the injection site were taken after 72 hours, fixed with formalin, embedded in paraffin, and stained with HE, and the epidermal integrity, dermal inflammatory cell infiltration and subcutaneous tissue pathological changes were observed.

[0098] Test results: All time point experimental group animals injection site were not obvious erythema or induration formation, the stimulation score was 0. Histological examination showed that the epidermis structure was complete, there was no keratinocyte necrosis and shedding, there was no significant inflammatory cell infiltration in the dermis layer, and there was no edema and fibrin exudation in the subcutaneous tissue. The results show that the preparation has no irritation to the injection site.

[0099] Conclusion: Through the systematic evaluation of acute toxicity test and local irritation test, it is confirmed that the liposome vaccine adjuvant prepared by the application has good biological safety under the test conditions, which meets the safety requirements of clinical application of vaccine adjuvant.

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. The membrane functional lipid is phytosphingosine; The stabilizer is sulfobutyl-β-cyclodextrin.

2. 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.

3. 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.

4. 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.

5. 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.

6. A liposomal vaccine adjuvant, characterized in that: Prepared by the method described in any one of claims 1-5.

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