Liposome nano vaccine taking glycyrrhiza polysaccharide as adjuvant as well as preparation method and application of liposome nano vaccine

Abstract

The invention belongs to the technical field of biological medicines, and provides a lipidosome nano vaccine taking glycyrrhiza polysaccharide as an adjuvant, which comprises a lipidosome shell consisting of phospholipid and cholesterol, and glycyrrhiza polysaccharide and mode antigen OVA257-264 entrapped in a lipidosome core. The invention also provides a preparation method of the liposome nano vaccine taking glycyrrhiza polysaccharide as the adjuvant. The invention also provides an application of the liposome nano vaccine in preparation of antitumor drugs. The lipidosome nano vaccine taking glycyrrhiza polysaccharide as the adjuvant can target lymph nodes, induce an organism to generate cellular immune response and form immune memory, so that the immunotherapy effect on tumors is remarkably enhanced. As a vaccine adjuvant, glycyrrhiza polysaccharide not only has the advantages of being good in biocompatibility, safe and non-toxic, but also can effectively make up for the limitation that a current tumor vaccine adjuvant is low in efficiency, high in toxicity and insufficient in Th1 response.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a liposome nano-vaccine with glycyrrhizic polysaccharide (NGUP) as an adjuvant, and a preparation method and application thereof. Background Art

[0002] As an important tool for tumor immunotherapy, the efficacy of tumor vaccines depends primarily on the efficiency of antigen delivery and the ability of adjuvants to activate the immune system. Currently, tumor vaccine development faces the following key challenges: 1) Low antigen delivery efficiency: Free antigens are easily degraded and difficult to target in lymphoid tissues, resulting in insufficient uptake by antigen-presenting cells (APCs); 2) Adjuvant limitations: Commonly used adjuvants in clinical practice, such as aluminum adjuvants, primarily induce Th2 immune responses and insufficiently activate Th1 cellular immunity, particularly cytotoxic T lymphocytes (CTLs), which are crucial for tumor clearance; 3) Safety concerns: Potent adjuvants such as MF59 are often associated with significant inflammatory responses, limiting their clinical application.

[0003] Licorice polysaccharide is a natural active ingredient extracted from the traditional Chinese medicine licorice. It has immunomodulatory and anti-tumor effects: 1) It activates dendritic cells (DCs) through the TLR4 / NF-κB pathway, promoting IL-12 secretion and Th1 polarization; 2) It has excellent biocompatibility and no significant toxic side effects; 3) It can regulate macrophage polarization to M1 type and enhance the immune response of the tumor microenvironment.

[0004] Liposomes can serve as delivery vehicles for encapsulating antigens and promoting antigen presentation. However, the immune-enhancing effects of liposome vaccines alone remain suboptimal, requiring the use of highly effective, low-toxic adjuvants. Reverse evaporation can efficiently encapsulate hydrophilic components, while repeated freeze-thaw cycles can further enhance encapsulation efficiency. Currently, there are no reports of co-encapsulating glycyrrhizic polysaccharides and antigens in liposomes for use as tumor vaccines.

[0005] Therefore, the development of a liposome nanovaccine with glycyrrhizic polysaccharide as an adjuvant can not only improve the efficiency of antigen delivery but also enhance the immune response, and has important clinical application value. Summary of the Invention

[0006] In response to the above-mentioned technical problems in the prior art, the present invention provides a liposome nanovaccine with glycyrrhizic acid polysaccharide as an adjuvant, a preparation method and application thereof. The liposome nanovaccine with glycyrrhizic acid polysaccharide as an adjuvant, a preparation method and application thereof are intended to solve the technical problem that the immune enhancement effect of the simple liposome vaccine in the prior art is not ideal.

[0007] The present invention provides a liposome nano-vaccine with glycyrrhizic acid polysaccharide as an adjuvant, comprising:

[0008] 1) Liposome shell composed of phospholipids and cholesterol;

[0009] 2) Licorice polysaccharide and model antigen OVA encapsulated in the liposome core 257-264 .

[0010] Furthermore, the vaccine is loaded with licorice polysaccharide by reverse evaporation method and OVA is co-loaded by repeated freeze-thaw method. 257-264。

[0011] Furthermore, the molar ratio of the phospholipid to cholesterol is (10-30):1; and the phospholipid is soybean lecithin.

[0012] Furthermore, the licorice polysaccharide is extracted from the Chinese medicinal licorice slices, and the polysaccharide purity is ≥ 90%, the molecular weight of the licorice polysaccharide is 14.68 kDa, and the monosaccharides in the licorice polysaccharide are composed of glucose, arabinose and galactose. In the licorice polysaccharide, the mass percentage concentration of the glucose is 80.88%, the mass percentage concentration of the arabinose is 14.30% and the mass percentage concentration of the galactose is 4.82%; the model antigen OVA 257-264 The sequence is SIINFEKL.

[0013] Furthermore, the glycyrrhizic acid polysaccharide accounts for 5.03% of the total mass of the liposome, and the OVA 257-264 The mass ratio of the vaccine to licorice polysaccharide is 1:10; the particle size of the vaccine is 104.53 ± 2.81 nm, the polydispersity index is less than 0.2, and the encapsulation efficiency is ≥ 80%.

[0014] The present invention also provides a method for preparing the above-mentioned liposome nanovaccine using glycyrrhizic acid as an adjuvant, comprising the following steps:

[0015] (1) Weigh each substance according to the material ratio;

[0016] (2) Dissolving phospholipids and cholesterol in an organic solvent to form an organic phase;

[0017] (3) dissolving licorice polysaccharide in an aqueous solution;

[0018] (4) Adding the aqueous phase solution to the organic phase and ultrasonically emulsifying to form a W / O emulsion;

[0019] (5) Remove the organic solvent by rotary evaporation to form a liposome suspension;

[0020] (6) Extrusion homogenization to obtain liposomes containing glycyrrhizic polysaccharide;

[0021] (7) Place OVA 257-264 The liposomes were added to the liposomes obtained in the previous step and subjected to repeated freeze-thaw treatment for 2 to 6 cycles to obtain the liposome nanovaccine with glycyrrhizic acid polysaccharide as an adjuvant.

[0022] Furthermore, in step (3), the ultrasonic power is 50-150 W and the time is 1-5 minutes; in step (5), the freeze-thaw temperature is -20°C to 37°C; and in step (6), the product is extruded through a 0.45 μm microporous filter membrane and a 0.22 μm microporous filter membrane.

[0023] The present invention also provides the use of the liposome nano-vaccine in the preparation of anti-tumor drugs.

[0024] Furthermore, the anti-tumor drug is used to prevent or treat melanoma, breast cancer or lung cancer.

[0025] The present invention also provides a pharmaceutical composition comprising the above-mentioned liposome nanovaccine and a pharmaceutically acceptable carrier.

[0026] The nano-vaccine of the present invention consists of two parts: a liposome shell and an inner core. The liposome shell is composed of phospholipids and cholesterol, forming a stable structure with a particle size of about 100 nm. The inner core is composed of glycyrrhizic acid polysaccharide (NGUP) and the model antigen OVA co-encapsulated by the reverse evaporation-freeze-thaw method. 257-264 Among them, NGUP as an adjuvant accounts for 5.03% of the total mass of liposomes; OVA 257-264 As a model antigen, the encapsulation efficiency was ≥ 80%.

[0027] The liposome nanovaccine, adjuvanted with glycyrrhizic acid (Glycyrrhizic acid) polysaccharide, can target lymph nodes, induce a cellular immune response, and form immune memory, significantly enhancing the immunotherapy effect against tumors. Glycyrrhizic acid polysaccharide, as a vaccine adjuvant, not only offers excellent biocompatibility and safety but also effectively overcomes the limitations of current tumor vaccine adjuvants, which include low efficiency, high toxicity, and insufficient Th1 response.

[0028] The technical solution adopted in the present invention is:

[0029] 1) Reverse evaporation method: Dissolve phospholipids and cholesterol in an organic phase (e.g., chloroform:ethanol = 2:1), add the aqueous phase solution containing NGUP, perform ultrasonic emulsification to form a W / O emulsion, and remove the organic solvent by rotary evaporation to form a liposome suspension. Homogenize by extrusion to obtain liposomes containing glycyrrhizic polysaccharide.

[0030] 2) Repeated freeze-thaw encapsulation of OVA 257-264 : Freeze the initial suspension at -20℃ and then thaw rapidly at 37℃ for 2 to 6 cycles to increase the concentration of NGUP and OVA. 257-264 The encapsulation efficiency was achieved by extruder and homogenized by passing through 0.45 μm and 0.22 μm microporous membranes in sequence.

[0031] 3) Lymph node targeting: Nanosized particles (< 200 nm) allow the vaccine to be directly concentrated in lymph nodes via lymphatic vessels;

[0032] 4) DC maturation: Nanovaccines can promote DC cell maturation and enhance their ability to take up and cross-present antigens;

[0033] 5) Antigen-specific T cell immune response: Nanovaccines can increase the activation and proliferation of T cells in the spleen and enhance the killing effect of T cells on tumor cells.

[0034] 6) As a preventive or therapeutic vaccine for tumor immunotherapy, such as melanoma, breast cancer and other solid tumors or hematological tumors.

[0035] Compared with the existing technology, the technical effects of the present invention are positive and obvious:

[0036] 1. Process advantage: Reverse evaporation-repeated freeze-thaw method makes NGUP and OVA 257-264 The co-encapsulation efficiency was increased to 85%, far exceeding the conventional hydration method (≤ 50%).

[0037] 2. Immune enhancement: Licorice polysaccharide can promote DC maturation and Th1 immune response, significantly enhance antigen cross-presentation, and enhance cytotoxic T cell activity.

[0038] 3. Stable delivery: Liposomes protect antigens from degradation and target lymphoid tissues, improving antigen utilization.

[0039] 4. Safe and low toxicity: Natural licorice polysaccharide has good biocompatibility and reduces the side effects of traditional adjuvants.

[0040] 5. The vaccine has the ability to target lymph nodes, enhance cellular immune responses, and is suitable for preventing or treating tumors.

[0041] 6. This invention provides research ideas and preliminary data for the development of new vaccine adjuvants with high efficiency and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The process of vaccine preparation.

[0043] Figure 2 NDUPL@OVA 257-264 representation.

[0044] Figure 3 It is involved in DCs cell maturation, antigen uptake and presentation.

[0045] Figure 4 For T cell activation, proliferation and killing.

[0046] Figure 5 NDUPL@OVA 257-264 distribution in the body.

[0047] Figure 6 NDUPL@OVA 257-264 Immune activation effect in vivo.

[0048] Figure 7 NDUPL@OVA 257-264 The preventive and therapeutic effects of B16-OVA on melanoma. DETAILED DESCRIPTION

[0049] Example 1:

[0050] like Figure 1 As shown, the present invention provides a method for preparing a liposome nanovaccine with glycyrrhizic polysaccharide (NGUP) as an adjuvant, comprising the following steps:

[0051] (1) Soy lecithin (24 mg), cholesterol (2.18 mg), and Tween-80 (2 μl) were mixed in chloroform (4 mL) and ether (8 mL) to achieve the desired ratio, and then glycyrrhizic acid polysaccharide (NGUP 1 mg) was dissolved in phosphate-buffered saline (PBS, 4 mL, pH 7.3). The organic and aqueous phases were thoroughly mixed using ultrasound in an ice bath to form a stable water-in-oil (W / O) emulsion.

[0052] (2) The emulsion was transferred to a round-bottom flask and subjected to rotary evaporation to form a gel. PBS was then added to rehydrate the gel for 15 minutes and homogenized using an ultrasonic cell disruptor for 20 minutes. Finally, the liposome suspension was filtered through 0.45 μm and 0.22 μm filter membranes to ensure sterility to obtain glycyrrhizic polysaccharide liposomes (NGUPL), which were then stored at 4°C for subsequent experiments.

[0053] (3) Mix NUGPL and antigen solution at a volume ratio of 10:1: add 0.1 ml of OVA to 1 ml of NUGPL 257-264 (Purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number S7951)) The mixture was then frozen at -20°C for 12 hours; then transferred to a thermostatic shaker and shaken at 37°C and 100 rpm for 30 minutes. This operation was repeated three times to obtain NDUPL@OVA. 257-264 Blank liposomes are BL@OVA without the addition of licorice polysaccharide. 257-264 .

[0054] Example 2: NDUPL@OVA 257-264 Characterization

[0055] NGUP entrapment efficiency (EE) was analyzed using a Sephadex G-50 microcolumn centrifugation method: the liposome suspension was loaded onto the column and centrifuged (1,500 rpm, 5 minutes) to separate free NGUPs. After disrupting the liposomes with 10% Triton X-100, total NGUPs and encapsulated NGUPs were quantified using the phenol-sulfuric acid method. EE% was calculated as: EE% = (encapsulated NGUPs / total NGUPs) × 100%.

[0056] For OVA 257-264 , Determine the entrapment efficiency by ultracentrifugation: The liposome solution was centrifuged (12,000 rpm, 1 h), and the supernatant was collected to measure the free protein content (Cs) using a BCA protein assay kit.

[0057] After liposome disruption, the total protein content (Ct) was measured and EE% was calculated as follows: EE% = (1 - Cs / Ct) × 100%. The drug loading (W%) was calculated using the following formula: W% = [(encapsulated NGUP and OVA) / ... 257-264 (total mass of membrane components, including soybean lecithin and cholesterol)] × 100%.

[0058] Dynamic light scattering (DLS) was used to detect the particle size and PDI of the nanovaccine, and laser Doppler electrophoresis (Zetasizer Nano ZS, Malvern) was used to detect the zeta potential.

[0059] like Figure 2 As shown, the results showed that nanoparticles 257-264 It showed good encapsulation efficiency (83.83 ± 8.81%) and drug loading (5.03 ± 0.38%). DLS analysis showed that NDUPL@OVA 257-264 The average hydrodynamic diameter of the NDUPL@OVA nanoparticles was 104.53 ± 2.81 nm (A), the PDI was 0.155 ± 0.04 (B), and the zeta potential was -40 ± 0.47 mV (C). Transmission electron microscopy images showed that the NDUPL@OVA nanoparticles were spherical in shape with uniform size and clear round shape without obvious aggregation (C). 257-264 The nanovaccine exhibited excellent stability in a physiological environment, maintaining stable particle size, PDI (D, E), and zeta potential (F) over 48 hours.

[0060] Example 3: DCs cell maturation, antigen uptake and presentation

[0061] DCs cell maturation, antigen uptake and presentation were detected by flow cytometry. DCs were plated at 1×106 The cells were seeded at a density of 100 cells / mL in a 12-well plate in a culture medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng mL -1 ). Treated for 24 hours according to the different groups. Then, cells were harvested and centrifuged at 400 × g for 5 minutes at 4°C. Nonspecific binding sites on the cells were blocked with 3% BSA for 15 minutes on ice. The cells were then rinsed with PBS and incubated in staining buffer containing anti-mouse CD11c-PE-Cy7, anti-mouse CD80-APC, anti-mouse CD86-PE, and anti-mouse SIINFEKL-PE, on ice for 30 minutes. Finally, after rinsing with PBS, the cells were resuspended in staining buffer and analyzed by FCM.

[0062] like Figure 3 As shown, flow cytometry (FCM) results showed that NDUPL@OVA 257-264 The expression of DCs maturation markers CD80 and CD86 was significantly upregulated (A). In addition, FCM detected more FITC-OVA 257-264 DCs and SIINFEKL + DCs (B) with free OVA 257-264 and BL@OVA 257-264 In comparison, NDUPL@OVA 257-264 Can significantly enhance DCs' ability to resist OVA 257-264 intake (C).

[0063] Example 4: T cell activation, proliferation and killing

[0064] Splenocytes were isolated from the spleens of 6-week-old C57BL / 6 mice by lymphocyte density gradient centrifugation. DCs treated with the above drugs were harvested and cocultured with splenic lymphocytes at a 1:5 ratio for 24 hours. The cells were harvested and centrifuged at 400 × g for 5 minutes at 4°C. Flow cytometry (FCM) was performed using anti-mouse CD3-PE, anti-mouse CD8a-APC, anti-mouse CD69-PE-Cy7, and anti-mouse CFSE-FITC staining according to the flow cytometry (FCM) manufacturer's instructions. The treated T cells were then added to B16-OVA tumor cells and cultured for a further 24 hours. The T cell cytotoxicity was assessed using a crystal violet assay.

[0065] like Figure 4 As shown, flow cytometry (FCM) results showed that NDUPL@OVA 257-264 Increase CD69 + T / CFSE +T cell expression level (A), significantly promoted T cell activation and proliferation (B); In addition, the crystal violet experiment statistical results showed that NDUPL@OVA 257-264 Enhanced the killing ability of T cells against B16-OVA tumor cells (C).

[0066] Example 5: NDUPL@OVA 257-264 Distribution in the body

[0067] Observation of NDUPL@OVA using in vivo imaging 257-264 Distribution in mice. Experimental methods: 4-6 week old female C57BL / 6 mice were fed for one week and randomly divided into 3 groups. 257-264 The vaccine Cy7-NGUPL@OVA was prepared by the above method with Cy7 labeled NGUP. 257-264 -Cy5.5 was injected at a dose of 0.1 mL per mouse, and the drug distribution in vivo was observed under a small animal living imaging device at different times.

[0068] like Figure 5 As shown, the results showed that OVA in live imaging mice 257-264 -Cy5.5 fluorescence intensity gradually increased over time and reached a peak at 8 h (A). 257-264 Compared with the group, NDUPL@OVA 257-264 The fluorescence intensity of NGUP-Cy7 group was significantly higher (B). 257-264 -Cy5.5 fluorescence intensity trends are very similar (C). These findings indicate that NDUPL@OVA 257-264 Efficiently targets and accumulates in mouse lymph nodes with prolonged retention.

[0069] Example 6: NDUPL@OVA 257-264 In vivo immune activation effect

[0070] Twenty-five 4-6 week old C57BL / 6 female mice were purchased from Shanghai Model Organisms Center Co., Ltd. The mice were divided into five groups and immunized after one week of adaptive feeding.

[0071] The grouping and immunization procedures were as follows: the first group was a blank control group, and each mouse was subcutaneously injected with 0.1 mL of normal saline solution; the second group was BL@OVA 257-264 In the group, each mouse was subcutaneously injected with 0.1 mL BL@OVA 257-264 (20 μg OVA 257-264 ) solution; the third group was NDUPL (low)@OVA 257-264 (50 μg NGUP + 20 μg OVA257-264 ); The fourth group is NDUPL (high) @ OVA 257-264 (200 μg NGUP + 20 μg OVA 257-264 The fifth group was the positive control AlumO (25 μg Alum + 20 μg OVA 257-264 ). Booster immunization injections are given once a week for a total of three immunization injections, with the dose of each injection remaining the same.

[0072] like Figure 6 As shown in the figure, flow cytometry analysis of the changes in DCs and T cells in the lymph nodes and spleen of immunized mice showed that after the injection of NDUPL@OVA 257-264 After 3 days, we observed that 257-264 and AL+OVA 257-264 Compared with the group, NDUPL@OVA 257-264 Significantly increased the proportion of mature DCs in inguinal ILN (A). 257-264 On the 5th day after treatment, FCM results showed that SIINFEKL-H2K b The expression level on DCs in ILN was significantly increased (B). These findings indicate that NDUPL@OVA 257-264 It can promote DC maturation, enabling mature DC to effectively capture and process OVA 257-264 , and then presents peptide-MHC complexes on its surface for T cell recognition. This phenomenon is similar to NDUPL@OVA 257-264 The results of in vitro experiments on immune cell immunomodulation were consistent. On the 7th day after vaccination, OVA 257-264 The results of restimulation experiments showed that NDUPL@OVA 257-264 Significantly upregulated IFNγ + CD8 + The proportion of T cells (C). Triggered a strong specific T cell immune response. In addition, NDUPL@OVA was detected 21 days after vaccination. 257-264 Promote CD4 + / CD8 + The generation of memory T cells (D, E) indicated that NDUPL@OVA 257-264 Induced effective immune memory.

[0073] Example 7: NDUPL@OVA 257-264 The preventive and therapeutic effects of B16-OVA on melanoma

[0074] Immunization protocol for tumor prevention models: 30 4-6 week-old C57BL / 6 female mice were purchased from Shanghai Model Organisms Center Co., Ltd. The mice were divided into 6 groups and immunized after one week of adaptive feeding.

[0075] The grouping and immunization procedures were as follows: the first group was a blank control group, and each mouse was subcutaneously injected with 0.1 mL of normal saline solution; the second group was BL@OVA 257-264 In the group, each mouse was subcutaneously injected with 0.1 mL BL@OVA 257-264 (20 μg OVA 257-264 ) solution; the third group was NDUPL (low)@OVA 257-264 (50 μg NGUP + 20 μg OVA 257-264 ); The fourth group was NGUP (high) + OVA 257-264 (200 μg NGUP + 20 μg OVA 257-264 ); The fifth group is NDUPL (high) @ OVA 257-264 (200 μg NGUP + 20 μg OVA 257-264 ); The sixth group was the positive control AlumO (25 μg Alum + 20 μg OVA 257-264 ). Booster immunization injections are given once a week for a total of three immunization injections, with the dose of each injection remaining the same.

[0076] Immunization protocol for tumor model treatment: 30 4-6 week old female C57BL / 6 mice were purchased from Shanghai Model Organisms Center Co., Ltd. The mice were divided into 6 groups and immunized after one week of adaptive feeding.

[0077] The grouping and immunization procedures were as follows: the first group was a blank control group, and each mouse was subcutaneously injected with 0.1 mL of normal saline solution; the second group was BL@OVA 257-264 In the group, each mouse was subcutaneously injected with 0.1 mL BL@OVA 257-264 (20 μg OVA 257-264 ) solution; the third group was NDUPL (low)@OVA 257-264 (50 μg NGUP + 20 μg OVA 257-264 ); The fourth group was NGUP (high) + OVA 257-264 (200 μg NGUP + 20 μg OVA 257-264 ); The fifth group is NDUPL (high) @ OVA 257-264 (200 μg NGUP + 20 μg OVA 257-264); The sixth group was the positive control AlumO (25 μg Alum + 20 μg OVA 257-264 The first immunization injection was performed on the third day after tumor loading, and a booster injection was performed every four days thereafter, for a total of three immunization injections, with the immunization dose for each injection remaining the same.

[0078] Figure 7 NDUPL@OVA 257-264 The preventive and therapeutic effects of B16-OVA on melanoma. The tumor growth curve and tumor weight in the preventive model showed that the PBS control group exhibited rapid tumor progression and had no preventive effect. 257-264 NGUP+OVA 257-264 and AL+OVA 257-264 The prevention effect of the groups on melanoma in mice was poor (A, B, C). It is worth noting that NDUPL (high)@OVA 257-264 The group maintained significantly smaller mean tumor volume at all measured time points, achieving excellent preventive effects with a tumor inhibition rate of 97.10%. The tumor growth curves and resected tumor weights in the treatment model showed that NDUPL (high)@OVA 257-264 The group showed the best anti-tumor activity (tumor inhibition rate: 83.69%), compared with BL@OVA 257-264 and AL+OVA 257-264 Compared with the other groups, the therapeutic effect was significantly superior (D, E, F).

Claims

1. A liposome nano vaccine with glycyrrhizic acid polysaccharide as adjuvant, characterized in that include: 1) Liposome shell composed of phospholipids and cholesterol; 2) Licorice polysaccharide and model antigen OVA encapsulated in the liposome core 257-264 .

2. The liposome nanovaccine with glycyrrhizic acid polysaccharide as adjuvant according to claim 1, characterized in that: The vaccine is loaded with licorice polysaccharide by reverse evaporation method and OVA is co-loaded by repeated freeze-thaw method. 257-264 .

3. The liposome nanovaccine with glycyrrhizic acid polysaccharide as adjuvant according to claim 1, characterized in that: The molar ratio of the phospholipid to cholesterol is (10-30):1; the phospholipid is soybean lecithin.

4. The liposome nanovaccine with glycyrrhizic acid polysaccharide as adjuvant according to claim 1, characterized in that: The licorice polysaccharide is extracted from the Chinese medicinal licorice slices, and the polysaccharide purity is ≥ 90%. The molecular weight of the licorice polysaccharide is 14.68 kDa. The monosaccharides in the licorice polysaccharide are composed of glucose, arabinose and galactose. In the licorice polysaccharide, the mass percentage concentration of the glucose is 80.88%, the mass percentage concentration of the arabinose is 14.30% and the mass percentage concentration of the galactose is 4.82%. The model antigen OVA 257-264 The sequence is SIINFEKL.

5. The liposome nanovaccine with glycyrrhizic acid polysaccharide as adjuvant according to claim 1, characterized in that: The licorice polysaccharide accounts for 5.03% of the total mass of the liposome, and the OVA 257-264 The mass ratio of the vaccine to licorice polysaccharide is 1:10; the particle size of the vaccine is 104.53 ± 2.81 nm, the polydispersity index is less than 0.2, and the encapsulation efficiency is ≥ 80%.

6. The method for preparing a liposome nano-vaccine with glycyrrhizic acid polysaccharide as an adjuvant according to any one of claims 1 to 5, characterized in that The following steps are involved: 1) Weigh each substance according to the material ratio; 2) dissolving phospholipids and cholesterol in an organic solvent to form an organic phase; 3) dissolving the glycyrrhiza polysaccharide in the aqueous solution; 4) adding the aqueous phase solution to the organic phase and ultrasonically emulsifying to form a W / O emulsion; 5) removing the organic solvent by rotary evaporation to form a liposome primary suspension; 6) extrusion and homogenization to obtain liposomes containing glycyrrhizic polysaccharide; 7) Place OVA 257-264 The liposomes were added to the liposomes obtained in the previous step and subjected to repeated freeze-thaw treatment for 2 to 6 cycles to obtain the liposome nanovaccine with glycyrrhizic acid polysaccharide as an adjuvant.

7. The method for preparing a liposome nano-vaccine with glycyrrhizic acid polysaccharide as an adjuvant according to claim 6, characterized in that: In step (3), the ultrasonic power is 50-150 W and the time is 1-5 minutes; in step (5), the freeze-thaw temperature is -20°C to 37°C; in step (6), the product is extruded through a 0.45 μm microporous filter membrane and a 0.22 μm microporous filter membrane.

8. Use of the liposome nanovaccine according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that The anti-tumor drug is used for preventing or treating melanoma, breast cancer or lung cancer.

10. A pharmaceutical composition comprising the liposome nanovaccine according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

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