Tubeimoside B liposome as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of vaccine adjuvants, and particularly relates to tubeimoside B liposome as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving phospholipid, cholesterol and PEGylated phospholipid in an organic solvent to form a mixed solution, and then evaporating the mixed solution to form a film; and hydrating the film by using an aqueous solution of tubeimoside B to finally prepare the tubeimoside B liposome. Compared with free tubeimoside B, the lipidosome has the advantages that the hemolysis percentage is obviously reduced, and antigen-specific humoral immune response and / or cellular immune response can be effectively enhanced when the lipidosome is used as a vaccine adjuvant. In an H1N1 influenza mouse model, the lipidosome combined antigen shows an excellent protection effect, which is shown in that the mouse weight fluctuation is relatively small and the lung virus load is remarkably reduced. The invention provides important theoretical basis and practical examples for developing low-toxicity and high-efficiency plant-derived vaccine adjuvants, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine adjuvants, and particularly relates to tubeimoside II liposomes, a preparation method thereof, and uses thereof. Background Art

[0002] As a non-specific immune enhancer, an immune adjuvant can stimulate the body to produce a stronger immune response through synergistic antigen, or regulate the type of immune response, and plays a key role in vaccine research and development. An ideal adjuvant needs to take into account multiple factors such as enhancing immunogenicity, optimizing the bias of immune response, and ensuring the safety of the preparation.

[0003] Due to their unique glycoside structure, saponin compounds exhibit the dual advantages of simultaneously activating humoral immunity and cellular immunity, and have become a research hotspot for new adjuvants. Among them, the purified component QS-21 of Quil A (saponin A) as a representative saponin adjuvant significantly increases antibody titers by promoting the activation of antigen-presenting cells and cytokine secretion, and has been commercially applied in multiple vaccines. However, QS-21 still has obvious defects: ① insufficient ability to activate Th2-type immune responses, limiting its protective effect against specific pathogens; ② the inherent hemolytic toxicity of saponins leads to a narrow clinical dose window; ③ the preparation process relying on natural plant extraction has problems such as unstable raw material supply. This has prompted researchers to turn to the development of new saponin adjuvants to break through the existing technical bottlenecks.

[0004] Bolbostemma paniculatum, as a traditional Chinese medicine, one of its active ingredients, tubeimoside II (TBMII or TBM2), has a cyclic bisaccharide chain triterpenoid saponin structure. Previous studies have shown that it can exert adjuvant activity by activating the TLR4 / NF-κB signaling pathway. However, there are two major application obstacles for natural TBMII: ① hemolytic toxicity that causes the rupture of erythrocyte membranes at high concentrations, making it difficult to balance the safety dose and the effective dose; ② unstable metabolism in vivo, resulting in low bioavailability.

[0005] Although attempts have been made in the prior art to improve its performance through chemical modification, it generally results in the loss of activity or the introduction of new toxic side effects. For example, existing studies have attempted to remove the aglycone of the sugar chain in tubeimoside II to improve solubility, but it will lose the ability to activate dendritic cells; for another example, existing studies have attempted to increase the hydrophobicity of tubeimoside II through esterification reaction to extend the in vivo half-life, but the modified molecule is more likely to insert into the erythrocyte membrane, resulting in a hemolysis rate more than twice that of the natural compound. Summary of the Invention

[0006] The present invention combines tubeimoside II with a liposome delivery system to solve formulation problems such as high hemolytic toxicity, thereby enhancing bioavailability.

[0007] To achieve the above object, the present invention may adopt the following technical solutions:

[0008] On the one hand, the present invention provides a method for preparing liposome of tuberoside B, which comprises: dissolving phospholipid, cholesterol and PEGylated phospholipid in an organic solvent to obtain a mixed solution, evaporating the mixed solution to obtain a film; hydrating the film to obtain liposome of tuberoside B; the hydration medium is an aqueous solution of tuberoside B ethyl ester.

[0009] Preferably, in the above preparation method, the mixed solution may further include monophosphoryl lipid A.

[0010] Preferably, the above preparation method satisfies one or more of the following conditions: (i) the phospholipid is selected from one or more combinations of DOPC, DSPC, DMPC or DMPG; (ii) the PEGylated phospholipid is selected from one or more combinations of DMG-PEG2000, DSPE-PEG5000 or DMG-PEG2000; (iii) the organic solvent is a mixed solvent of chloroform and methanol.

[0011] More preferably, in the above preparation method, the phospholipid is selected as DOPC and DSPC, and the mass ratio of DOPC, DSPC, cholesterol and PEGylated phospholipid is (0.8-1.2):(0.8-1.2):(0.8-1.2):0.5.

[0012] More preferably, in the above preparation method, the volume ratio of chloroform to methanol in the organic solvent may be 5:(1-5).

[0013] On the other hand, the present invention provides a liposome of tuberoside B prepared by the preparation method in the present invention.

[0014] On yet another aspect, the present invention provides an immunoadjuvant, which comprises the liposome of tuberoside B in the present invention.

[0015] On yet another aspect, the present invention provides an immunization vaccine, which comprises the immunoadjuvant and an antigen in the present invention.

[0016] Preferably, in the above immunization vaccine, the antigen is H1N1 influenza virus antigen, OVA antigen or melanoma antigen.

[0017] On yet another aspect, the present invention provides a use of the liposome of tuberoside B in the present invention, and the uses include: (i) the use of the liposome of tuberoside B in the preparation of an immunoadjuvant; (ii) the use of the liposome of tuberoside B in the preparation of an immunization vaccine.

[0018] Preferably, the above uses include: (a) the use of tuberoside B liposome in enhancing the body's humoral immunity and / or cellular immune response; (b) the use of tuberoside B liposome in enhancing the body's specific Th17 cell response; (c) the use of tuberoside B liposome in promoting the generation of germinal center B cells and / or follicular helper T cells in the body.

[0019] The beneficial effects of the present invention at least include:

[0020] (1) The hemolysis percentage of the tuberoside B liposome provided by the present invention can reach below 5%, which is much lower than the hemolysis percentage (100%) of tuberoside B.

[0021] (2) The tuberoside B liposome provided by the present invention can effectively enhance the body's humoral immune response and / or cellular immune response, drive Th1-biased immune responses, and can promote the generation of germinal center B cells and / or follicular helper T cells in the body.

[0022] (3) In the H1N1 influenza model, the tuberoside B liposome provided by the present invention shows excellent protective effects in combination with antigens, manifested as smaller body weight fluctuations and significantly reduced viral loads in the lungs, confirming its significant adjuvant effect. Description of the Drawings

[0023] Figure 1a is the particle size distribution diagram of liposome TBMⅡ-LP;

[0024] Figure 1b is the particle size distribution diagram of liposome (TBMⅡ+MPLA)-LP;

[0025] Figure 1c is the particle size distribution diagram of liposome MPLA-LP;

[0026] Figure 1d is the transmission electron microscopy image of the liposome;

[0027] Figure 2 is the hemolytic toxicity evaluation of different substances;

[0028] Figure 3a is the serum specific IgG antibody level of different groups;

[0029] Figure 3b is the serum specific IgG1 antibody level of different groups;

[0030] Figure 3c is the serum specific IgG 2a antibody level;

[0031] Figure 3d is the serum antibody IgG of different groups2a / IgG1 ratio;

[0032] Figure 4a The situation of IFN-γ-secreting cells after stimulation with whole antigen in different groups;

[0033] Figure 4b Bar chart of the number of IFN-γ-secreting cells after stimulation with whole antigen in different groups;

[0034] Figure 5a For different groups of OVA 323-339 The situation of IFN-γ-secreting cells after stimulation with peptide;

[0035] Figure 5b For different groups of OVA 323-339 Bar chart of the number of IFN-γ-secreting cells after stimulation with peptide;

[0036] Figure 6a For different groups of OVA 257-264 The situation of IFN-γ-secreting cells after stimulation with peptide;

[0037] Figure 6b For different groups of OVA 257-264 Bar chart of the number of IFN-γ-secreting cells after stimulation with peptide;

[0038] Figure 7a The situation of cells inducing Th17 response by different groups of helper antigens;

[0039] Figure 7b Bar chart of the number of cells inducing Th17 response by different groups of helper antigens;

[0040] Figure 8a For different groups of helper-induced CD4 + IL-17 + Flow cytometry distribution map of the number;

[0041] Figure 8b For different groups of helper-induced CD4 + IL-17 + Bar chart of the number;

[0042] Figure 9a For different groups of helper-induced CD4 + IL-4 + Flow cytometry distribution map of the number;

[0043] Figure 9b For different groups of helper-induced CD4 + IL-4 + Bar chart of the number;

[0044] Figure 10a Flow cytometry plots of the number of CD4 + IFN-γ + induced by different groups of adjuvants;

[0045] Figure 10b Flow cytometry plots of the number of CD4 + IFN-γ + induced by different groups of adjuvants;

[0046] Figure 11a Flow cytometry plots of the number of CD8 + IFN-γ + induced by different groups of adjuvants;

[0047] Figure 11b Flow cytometry plots of the number of CD8 + IFN-γ + induced by different groups of adjuvants;

[0048] Figure 12a Flow cytometry plots of the number of GcB cells induced by different groups;

[0049] Figure 12b Bar charts of the number of GcB cells induced by different groups;

[0050] Figure 13a Flow cytometry plots of the number of Tfh cells induced by different groups;

[0051] Figure 13b Bar charts of the number of Tfh cells induced by different groups;

[0052] Figure 14 Survival of mice in different groups after inoculation with B16-OVA cells;

[0053] Figure 15a HA-specific IgG levels in mice in different groups after inoculation with H1N1 influenza virus;

[0054] Figure 15b HA-specific IgG 2a levels in mice in different groups after inoculation with H1N1 influenza virus;

[0055] Figure 15c HA-specific IgG1 levels in mice in different groups after inoculation with H1N1 influenza virus;

[0056] Figure 16a Body weights of mice in different groups after sub-lethal challenge with H1N1 influenza virus;

[0057] Figure 16b Body weights of mice in different groups after lethal challenge with H1N1 influenza virus;

[0058] Figure 17 Survival of mice in different groups after lethal challenge with H1N1 influenza virus;

[0059] Figure 18 Viral load in the lungs of mice in different groups. Detailed implementation mode

[0060] The examples given are for better illustration of the present invention, but the content of the present invention is not limited to the examples given only. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation modes based on the above-mentioned inventive content still fall within the protection scope of the present invention.

[0061] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. Unless having a significantly different meaning in the context, the expressions in the singular form include the plural form. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and it is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations may exist or can be added. As used herein, depending on the situation, " / " can be interpreted as "and" or "or".

[0062] An embodiment of the present invention provides a preparation method of tuberoside B liposome, which includes: dissolving phospholipid, cholesterol and PEGylated phospholipid in an organic solvent to obtain a mixed solution, evaporating the mixed solution to obtain a film; hydrating the film to obtain tuberoside B liposome; the hydration medium is an aqueous solution of tuberoside B.

[0063] It should be noted that tuberoside B, also known as tuberoside II, has the following structural formula: With the CAS number of 115810-12-3, preparing tuberoside B into liposome in the present invention can significantly reduce the hemolysis percentage of tuberoside B, thereby enhancing the bioavailability.

[0064] In some specific examples, in the above preparation method, the mixed solution further includes monophosphoryl lipid A (MPLA).

[0065] It should be noted that the mixed solution in the present invention may further include monophosphoryl lipid A (MPLA), which can be used as an immunomodulatory molecule and co-delivered with tuberoside B.

[0066] In some specific examples, the above preparation method satisfies one or more of the following conditions:

[0067] (i) The phospholipid is selected from one or a combination of DOPC, DSPC, DMPC, or DMPG; specifically, the phospholipids in the present invention can be well-known in the art, such as DOPC, DSPC, DMPC, or DMPG, and the two can be used alone or in combination. When used in combination, the mass ratio can be 1:1;

[0068] (ii) The PEGylated phospholipid is selected from one or a combination of DMG-PEG2000, DSPE-PEG5000, or DMG-PEG2000; specifically, the PEGylated phospholipids in the present invention are well-known in the art, such as DMG-PEG2000, DSPE-PEG5000, or DMG-PEG2000;

[0069] (iii) The organic solvent is a mixed solvent of chloroform and methanol.

[0070] It should be noted that for the above conditions (i) to (iii), the preparation method of the present invention can meet one of the conditions or can meet all of the above conditions simultaneously.

[0071] In some specific examples, in the above preparation method, the phospholipid is selected as DOPC and DSPC, and the mass ratio of DOPC, DSPC, cholesterol, and PEGylated phospholipid can be (0.8 - 1.2):(0.8 - 1.2):(0.8 - 1.2):0.5, more preferably 1:1:1:0.5.

[0072] In some specific examples, the volume ratio of chloroform to methanol in the organic solvent in the above preparation method can be 5:(1 - 5), such as 5:2, 5:3, or 5:4, etc.

[0073] The embodiment of the present invention also provides a liposome of zhebeinine ethyl prepared by the preparation method in the present invention.

[0074] It should be noted that the liposome of zhebeinine ethyl prepared by the preparation method of the present invention has a lower hemolysis percentage and higher water solubility.

[0075] The embodiment of the present invention also provides an immunoadjuvant, which includes the liposome of zhebeinine ethyl in the present invention.

[0076] It should be noted that the liposome of zhebeinine ethyl in the present invention has the function of enhancing the immune response of the body and can be prepared into an immunoadjuvant; of course, it should be understood that the immunoadjuvant can also include commonly used reagents for immunoadjuvants, such as stabilizers or preservatives, etc.

[0077] The embodiment of the present invention also provides an immunization vaccine, which includes the immunoadjuvant and an antigen in the present invention.

[0078] It should be noted that, as described above, the liposome of tuberoside B in the present invention can be used as an immune adjuvant, and then the immune adjuvant and an antigen for immune stimulation can be combined to prepare an immune vaccine.

[0079] In some specific examples, in the above immune vaccine, the antigen is an H1N1 influenza virus antigen, an OVA antigen or a melanoma antigen.

[0080] It should be noted that the vaccine adjuvant in the present invention and the H1N1 influenza virus antigen, the OVA antigen or the melanoma antigen can be prepared into different vaccines. When the antigen is the H1N1 influenza virus antigen, the vaccine is a viral vaccine; when the antigen is the melanoma antigen, the vaccine is a tumor vaccine, which can prevent and treat tumor vaccines.

[0081] The embodiment of the present invention also provides a use of the liposome of tuberoside B in the present invention, and the uses include:

[0082] (i) The use of the liposome of tuberoside B in preparing an immune adjuvant;

[0083] (ii) The use of the liposome of tuberoside B in preparing an immune vaccine.

[0084] In some specific examples, the above uses include:

[0085] (a) The use of the liposome of tuberoside B in enhancing the humoral immunity and / or cellular immune response of the body;

[0086] (b) The use of the liposome of tuberoside B in enhancing the specific Th17 cell response of the body;

[0087] (c) The use of the liposome of tuberoside B in promoting the generation of germinal center B cells and / or follicular helper T cells in the body.

[0088] It should be noted that the liposome of tuberoside B in the present invention can enhance the humoral immune response and / or cellular immune response of the body, enhance the specific Th17 response of the body and promote the generation of germinal center B cells and / or follicular helper T cells in the body, thereby enhancing the immune response of the body.

[0089] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with specific examples, but the content of the present invention is not limited to the following examples only.

[0090] In the following examples, the preparation method of the injected vaccine is as follows: OVA and vaccine adjuvants (note: MPLA-LP, TBMⅡ, TBMⅡ-LP, (TBMⅡ + MPLA)-LP, or (QS-21 + MPLA)-LP are all in liquid state, so the dosage is expressed in μL / mice, Alum is a commercial preparation, so the dosage is expressed in μg / mice) are dispersed in sterile PBS (NaCl 8g, KCl 0.2g, Na2HPO4 1.15g, KH2PO4 0.2g, CaCl2 0.1g, MgCl2·6H2O 0.1g, pH = 7.4, the same below) to obtain a dispersion, which is the injected vaccine.

[0091] I. Liposome Preparation and Characterization

[0092] (I) Liposome Preparation

[0093] Example 1

[0094] (1) DOPC (1,2-dioleoyl phosphatidylcholine), DSPC (1,2-distearoyl phosphatidylcholine), cholesterol, and DMG-PEG2000 (dimyristoyl glycerol polyethylene glycol 2000) are weighed separately according to a mass ratio of 1:1:1:0.5. Among them, 1.6 mg of DOPC, DSPC, and cholesterol are weighed respectively, and 0.8 mg of DMG-PEG2000 is weighed; then they are all dissolved in a mixed solvent of chloroform and methanol (the volume ratio of chloroform to methanol is 5:3, 5 mL of chloroform, 3 mL of methanol), and vortexed until fully dissolved to obtain a mixed solution;

[0095] (2) The above mixed solution is placed in a 25 mL round-bottom flask, the water bath temperature is set at 50 °C, the solvent is removed by rotary evaporation, and a film is formed in the round-bottom flask;

[0096] (3) The round-bottom flask is removed and dried overnight in a constant temperature vacuum drying oven at 37 °C to completely remove the residual organic solvents;

[0097] (4) TBMⅡ (also known as TBM2) is dissolved in 1 mL of physiological saline as a hydration medium (the concentration of TBMⅡ is 1 mg / mL), then added to the round-bottom flask, and sonicated in a water bath at 50 °C (ultrasonic power is 110 W) until the film peels off to obtain a hydration medium solution;

[0098] (5) The hydration medium solution is transferred to a 1.5 mL EP tube for probe sonication (ultrasonic power is 110 W, sonication for 4 s, pause for 6 s, total working time (including stop time) is 5 min) to obtain liposome TBMⅡ-LP.

[0099] Example 2

[0100] (1) DOPC (1,2-dioleoyl phosphatidylcholine), DSPC (1,2-distearoyl phosphatidylcholine), cholesterol, and DMG-PEG2000 (dimyristoyl glycerol polyethylene glycol 2000) were weighed separately according to a mass ratio of 1:1:1:0.5. Among them, 1.6 mg of DOPC, DSPC, and cholesterol were weighed respectively, and 0.8 mg of DMG-PEG2000 was weighed. Then they were all dissolved in a mixed solvent of chloroform and methanol (the volume ratio of chloroform to methanol was 5:3, 5 mL of chloroform and 3 mL of methanol). Then MPLA (monophosphoryl lipid A) was added to make the final concentration of MPLA 25 μg / mL, and vortexed until fully dissolved to obtain a mixed solution;

[0101] (2) The above mixed solution was placed in a 25 mL round-bottom flask, the water bath temperature was set at 50 °C, and the solvent was removed by rotary evaporation to form a film in the round-bottom flask;

[0102] (3) The round-bottom flask was removed and dried overnight in a constant temperature vacuum drying oven at 37 °C to completely remove the residual organic solvents;

[0103] (4) TBMⅡ was dissolved in 1 mL of physiological saline as a hydration medium (the concentration of TBMⅡ was 1 mg / mL), and then added to the round-bottom flask. It was sonicated in a water bath at 50 °C (ultrasonic power was 110 W) until the film peeled off to obtain a hydration medium solution;

[0104] (5) The hydration medium solution was transferred to a 1.5 mL EP tube and subjected to probe sonication (ultrasonic power was 110 W, sonicated for 4 s, stopped for 6 s, and the total working time (including the stop time) was 5 min) to obtain liposomes (TBMⅡ + MPLA)-LP.

[0105] Control Example 1

[0106] (1) DOPC (1,2-dioleoyl phosphatidylcholine), DSPC (1,2-distearoyl phosphatidylcholine), cholesterol, and DMG-PEG2000 (dimyristoyl glycerol polyethylene glycol 2000) were weighed separately according to a mass ratio of 1:1:1:0.5. Among them, 1.6 mg of DOPC, DSPC, and cholesterol were weighed respectively, and 0.8 mg of DMG-PEG2000 was weighed. Then they were all dissolved in a mixed solvent of chloroform and methanol (the volume ratio of chloroform to methanol was 5:3, 5 mL of chloroform and 3 mL of methanol). Then MPLA (monophosphoryl lipid A) was added to make the final concentration of MPLA 25 μg / mL, and vortexed until fully dissolved to obtain a mixed solution;

[0107] (2) The above mixed solution was placed in a 25 mL round-bottom flask, the water bath temperature was set at 50 °C, and the solvent was removed by rotary evaporation to form a film in the round-bottom flask;

[0108] (3) Remove the round-bottom flask and dry it overnight in a constant temperature vacuum drying oven at 37 °C to thoroughly remove the residual organic solvents;

[0109] (4) Add 1 mL of normal saline as the hydration medium to the round-bottom flask and ultrasonicate it in a water bath at 50 °C (ultrasonic power: 110 W) until the film peels off to obtain a hydration medium solution;

[0110] (5) Transfer the hydration medium solution to a 1.5 mL EP tube and perform probe sonication (ultrasonic power: 110 W, sonication for 4 s, pause for 6 s, total working time (including pause time): 5 min) to obtain liposomes MPLA-LP.

[0111] Control Example 2

[0112] The difference between Control Example 2 and Example 2 is that in Control Example 2, TBMⅡ in Example 2 was replaced with QS-21 (a vaccine adjuvant, purchased from DesertKing) and the final concentration of QS-21 was 25 μg / mL. Other steps were the same as in Example 2, and liposomes (QS-21 + MPLA)-LP were prepared.

[0113] (II) Liposome Characterization

[0114] Dilute the above liposomes 1:100 with pure water respectively, and then measure the liposome particle size using DLS and observe the liposome morphology using a transmission electron microscope.

[0115] The particle size distributions of liposomes TBMⅡ-LP, (TBMⅡ + MPLA)-LP, and MPLA-LP are shown respectively in Figure 1a 、 Figure 1b and Figure 1c The results show that the average particle size of TBMⅡ-LP is 99.41, and the PDI (the smaller the PDI value, the more uniform the particle size distribution; conversely, the larger the PDI value, the more uneven the particle size distribution) is 0.222; the average particle size of (TBMⅡ + MPLA)-LP is 110.1 nm, and the PDI is 0.305; the average particle size of MPLA-LP is 136.1 nm, and the PDI is 0.142.

[0116] In addition, the transmission electron microscope image of liposomes (TBMⅡ + MPLA)-LP is shown in Figure 1d The results show that it is a regular spherical vesicle structure with good morphological characteristics.

[0117] II. Hemolytic Toxicity Evaluation of Liposomes

[0118] The above liposomes were diluted 1:100 with pure water to obtain liposome dilutions. The liposome dilutions were mixed with 2% mouse red blood cells to obtain suspensions. The resulting suspensions were added to flat-bottom 96-well plates (200 μL per well). A PBS solution (including 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.2–7.4, the same below, also abbreviated as PBS hereinafter) was used as the negative control, and 2% TritonX-100 (TritonX-100 was dissolved in the PBS solution, and the volume fraction of TritonX-100 was 2%) was used as the positive control. After incubation at 37 °C for 1 h, the mixtures were centrifuged at 1500 rpm for 8 min to obtain supernatants. The hemolytic activity was determined by measuring the absorbance of the supernatants at 570 nm (tested using a microplate reader, the model of the microplate reader was ThermoFisher VarioskanLUX2, the same below). Among them, the hemolysis percentage was calculated as: H(%) = (OD570nm sample - OD570nm PBS solution) / (OD570nm water - OD570nm PBS solution) * 100.

[0119] The test results are as Figure 2 shown. The results showed that the hemolysis percentage of the TBMⅡ aqueous solution was comparable to that of 2% TritonX-100, up to 100%, while the hemolytic toxicity of TBMⅡ-LP decreased significantly, and the hemolysis percentage was less than 5%. Compared with TBMⅡ-LP, the hemolytic toxicity of (TBMⅡ + MPLA)-LP increased, but the hemolysis percentage was less than 10%, much lower than that of the TBMⅡ aqueous solution. The above results suggest that delivering TBMⅡ using liposomes can significantly reduce the hemolytic toxicity of TBMⅡ and has good in vitro safety.

[0120] III. Liposome Immune Response Test

[0121] In the following tests, Alum was Aluminum Hydroxide Gel from Sigma-Aldrich.

[0122] (I) Animal Immunization

[0123] Female SPF-grade Balb / c mice (6 - 8 weeks old) were randomly divided into 8 groups of 5 mice each. The groups were the PBS group, OVA group, OVA + MPLA-LP group, OVA + TBMⅡ group, OVA + TBMⅡ-LP group, OVA + (TBMⅡ + MPLA)-LP group, OVA + Alum group, and OVA + (QS-21 + MPLA)-LP group. Each group was intramuscularly injected with the vaccine on days 0, 14, and 28. The specific details are shown in Table 1 below (in Table 1, X / mice refers to the dosage per mouse. For example, 5μg / mice means the dosage per mouse is 5μg, and the same applies hereinafter).

[0124] Table 1 Vaccines and injection conditions for different groups

[0125]

[0126]

[0127] Two weeks after the last immunization, the mice were sacrificed and ocular blood and splenic lymphocytes were collected for further analysis.

[0128] (II) Enzyme-linked immunosorbent assay (ELISA)

[0129] The OVA-specific IgG level was determined by standard indirect ELISA, which specifically included: coating a 96-well flat-bottom plate with OVA and incubating overnight at 4°C; after blocking with PBST + 5% BSA (blocking was incubated at 37°C for 2 h), the diluted samples (ocular serum, diluted with PBS solution, serially diluted, starting dilution factor was 20,000-fold dilution) were added to the plate and incubated at 37°C for 1 h; HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a were used as secondary antibodies; 50 μL of TMB substrate solution was added to each well and incubated for about 10 min; then, the reaction was terminated by adding the termination solution (2M H2SO4 solution). Finally, the absorbance value at 450 nm was detected on an enzyme-linked immunosorbent assay reader (ThermoFisher Varioskan LUX2).

[0130] The ELISA test results are as Figure 3a 、 Figure 3b 、 Figure 3c and 3d shown. The results showed that TBMⅡ, TBMⅡ-LP, and (TBMⅡ + MPLA)-LP all significantly increased the level of serum-specific IgG antibodies, and TBMⅡ-LP induced a stronger specific IgG response than the aqueous solution of TBMⅡ, while the serum-specific IgG level induced by (TBMⅡ + MPLA)-LP was 4.8 times that of the aluminum adjuvant; IgG 2aand IgG1 can be used as markers of Th1 and Th2 cell responses respectively. Therefore, the levels of OVA-specific IgG1 and IgG were measured. 2a The levels of serum specific IgG1 were significantly enhanced by both TBMⅡ-LP and (TBMⅡ+MPLA)-LP, and more significant specific IgG1 responses were also induced compared with the aqueous solution of TBMⅡ. (TBMⅡ+MPLA)-LP could induce higher levels of IgG. 2a When observing the IgG / IgG1 ratio of liposomal formulations, it was found that liposomal formulations could drive a more effective Th1-biased immune response. 2a

[0131] (III) Detection of cellular immune responses of liposomes

[0132] (1) ELISPOT assay

[0133] To further investigate whether liposomal formulations could enhance specific cellular immune responses, splenocytes from immunized mice were collected and incubated in ELISpot plates for 48 hours in the absence or presence of 20 μg / mL OVA whole antigen, OVA 257-264 peptide or OVA 323-339 peptide (the dosages of OVA whole antigen, OVA 257-264 peptide or OVA 323-339 peptide were the same as those in Table 1 above, and the immunization method was the same as that in Table 1 above). The specific numbers of cells secreting IFN-γ or IL-17a were detected using an ELISPOT detection kit (from Mebtech) (the detection method was carried out according to the instructions of the ELISPOT detection kit). Among them, the number of spots generated after treatment with whole antigen or polypeptide minus the number of spots generated without treatment was used to represent the specific numbers of cells secreting IFN-γ or IL-17a in splenocytes.

[0134] The results are shown as follows:

[0135] After stimulation with whole antigen, the specific numbers of cells secreting IFN-γ in the OVA+TBMⅡ-LP group and the OVA+(TBMⅡ+MPLA)-LP group were both significantly increased, and were significantly higher than those in the TBMⅡ aqueous solution group and the aluminum adjuvant group, indicating that TBMⅡ-LP and (TBMⅡ+MPLA)-LP could assist antigens in eliciting stronger T cell responses (see Figure 4a and Figure 4b );

[0136] OVA 323-339 After peptide stimulation, the specific numbers of cells secreting IFN-γ in each group were comparable, suggesting that the abilities of each adjuvant to elicit specific CD4+ T cell responses were comparable (see Figure 5a and Figure 5b );

[0137] while OVA 257-264 After peptide stimulation, the number of cells specifically secreting IFN-γ in the (TBMⅡ + MPLA)-LP group was significantly higher than that in other groups, suggesting that (TBMⅡ + MPLA)-LP induced CD8 + The ability of T cell response was significantly better than that of other adjuvant groups (see Figure 6a and Figure 6b );

[0138] In addition, compared with the aqueous solution of TBMⅡ, TBMⅡ-LP could assist antigens to induce a more significant Th17 response, and among all adjuvants, (TBMⅡ + MPLA)-LP had the strongest ability to enhance antigen-specific Th17 response (see Figure 7a and Figure 7b ).

[0139] (2) FCM (Flow cytometry)

[0140] After the spleen lymphocytes of immunized mice were stimulated with the whole antigen for 18 hours, then blocked with a Golgi blocker (protein transport inhibitor mixture (500X) from invivogen's eBioscience TM , diluted to 1x with PBS, and then 100 μL per well) for 6 hours, the cells were first stained with L / D (FVS700) at 4°C for 15 min, and then stained with CD3-PE, CD4-FITC, CD8-PerCp for 30 min. Finally, after fixation and permeabilization with BDCytofix / Cytoperm TM Fixation / PermeabilizationKit, they were stained with IL-17A-BV510, IL-4-APC, IFN-γ-BV421 and IL-2-PE / Cy7 simultaneously for 30 min; after resuspension with PBS, the expression of intracellular cytokines was detected by a flow cytometer (FACS CantoII flow cytometer from BD, 332116, the same below).

[0141] The detection results are as follows:

[0142] CD4 + IL-17 + The production situation is as Figure 8a and Figure 8b shown. The results showed that the proportion of CD4 + IL-17 + induced by (TBMⅡ + MPLA)-LP to assist OVA was higher than that of TBMⅡ-LP, TBMⅡ-LP was higher than MPLA-LP, and MPLA-LP was higher than the aluminum adjuvant (Alum);

[0143] CD4+ IL-4 + production is as shown in Figure 9a and Figure 9b The results show that (TBMⅡ+MPLA)-LP, TBMⅡ-LP and MPLA-LP can all assist in inducing the production of CD4 + IL-17 + ;

[0144] CD4 + IFN-γ + production is as shown in Figure 10a and Figure 10b The results show that (TBMⅡ+MPLA)-LP assisted OVA in inducing the production of CD4 + IL-17 + The proportion was higher than that of MPLA-LP, MPLA-LP was higher than that of TBMⅡ-LP, and TBMⅡ-LP was higher than that of aluminum adjuvant (Alum);

[0145] CD8 + IFN-γ + production is as shown in Figure 11a and Figure 11b The results show that (TBMⅡ+MPLA)-LP assisted OVA in inducing the production of CD4 + IL-17 + The proportion was higher than that of MPLA-LP, MPLA-LP was higher than that of TBMⅡ-LP, and TBMⅡ-LP was higher than that of aluminum adjuvant (Alum).

[0146] From the above data, it can be known that (TBMⅡ+MPLA)-LP, TBMⅡ-LP and MPLA-LP can all assist in inducing the production of CD4 + IL-17 + , especially (TBMⅡ+MPLA)-LP, indicating that it can significantly enhance Th1, Th17 and cytotoxic T lymphocyte (CTL) responses and has the potential to enhance the killing effect on target cells.

[0147] (III) Detection of Liposome Germinal Center B Cells and Follicular Helper T Cells

[0148] Since long-lived plasma cells and memory B cells that secrete antibodies are differentiated from germinal center B cells (GcB), the GC reaction (germinal center reaction) is the basis for inducing high-quality and long-lasting B cell responses. In addition, follicular helper T cells (Tfh) that assist B cells can promote the proliferation, survival and differentiation of GcB cells by transmitting co-stimulatory molecules and cytokines, thereby regulating the GC reaction. Therefore, it is necessary to explore whether the liposomes in the present invention can regulate the immune response by increasing the levels of germinal center B cells and follicular helper T cells.

[0149] (1) Flow cytometry detection of GcB

[0150] Fourteen days after the second immunization, the inguinal lymph nodes of mice were collected and single-cell suspensions were prepared (the mice were sacrificed by cervical dislocation, the skin on the outer side of the thigh was disinfected with 75% alcohol, the subcutaneous connective tissue was gently dissected, in the lower abdominal inguinal region, three blood vessels could be seen near the junction of the genital and the thigh, and the confluence of the three blood vessels was the inguinal lymph node. The adipose tissue was bluntly dissected with curved forceps, and the lymph node was carefully dissected out. It could be verified in PBS first whether the dissection was correct. The lymph node should sink in the PBS solution. If it floated, it was adipose tissue. After confirmation, the lymph node was ground into a cell suspension in a 70μm cell sieve with a grinding pestle, the same below); the single-cell suspension was first stained with L / D-BV510 at 4°C for 15 min, and then stained with Fas-CD95, GL-7-PerCp / Cy5.5 and CD19-PE / Cy7 for 30 min respectively; after washing with staining Buffer (PBS + 1% BSA by volume fraction) and resuspending with PBS, the expression of GcB markers was detected by flow cytometry.

[0151] The detection results are as Figure 12a and Figure 12b shown. The results showed that TBMⅡ-LP could induce a high level of GcB cells in the lymph nodes, and (TBMⅡ + MPLA)-LP could further increase the number of GcB cells in both the lymph nodes and the spleen, suggesting that inducing a high level of GCB cells might be one of the mechanisms by which TBMⅡ-LP and (TBMⅡ + MPLA)-LP exerted their immune adjuvant effects.

[0152] (2) Flow cytometry detection of Tfh

[0153] Fourteen days after the second immunization, the inguinal lymph nodes of mice were collected and single-cell suspensions were prepared; the single-cell suspension was first stained with L / D-FVS700 at 4°C for 15 min, and then stained with CD3-PE, CD4-PerCP / Cy5.5, CD44-PE / Cy7, ICOS-PB, CXCR5-APC and APCStreptavidin for 30 min respectively; after washing with staining Buffer and resuspending with PBS, the expression of Tfh (Tfh is a key regulatory cell in the GC reaction, CD4 + CD44 + expressing CXCR5 and PD-1 or CXCR5 and ICOS is Tfh) markers was detected by flow cytometry.

[0154] The detection results are as Figure 13a and Figure 13bAs shown, the results showed that, consistent with the detection results of GcB cell responses, TBMⅡ-LP and (TBMⅡ+MPLA)-LP could significantly increase the Tfh level in lymph nodes, suggesting that TBMⅡ-LP and (TBMⅡ+MPLA)-LP could regulate the GC response through the Tfh level.

[0155] IV. Evaluation of the therapeutic effect of liposomes on B16-OVA

[0156] Mice (C57BL / J6, SPF grade) were randomly divided into 7 groups with 10 mice in each group. The groups were PBS, OVA, OVA+MPLA-LP, OVA+TBMⅡ-LP, OVA+(TBMⅡ+MPLA)-LP, OVA+(QS-21+MPLA)-LP, and OVA+Alum. On day 0, B16-OVA cells were inoculated at a dose of 5×10 5 / mice, and the corresponding vaccines were administered on days 4, 8, and 12 respectively; the survival rate was recorded. Among them, the drug use situation of vaccine treatment is shown in Table 2 below.

[0157] Table 2 Vaccines and injection situations for different groups

[0158]

[0159] (I) Survival curve

[0160] Starting from the day when cell inoculation was completed as day 0, the growth changes of tumors were recorded once a day. When the tumor volume exceeded 2000 mm 3 the mice were sacrificed and considered dead by default.

[0161] The survival of the mice was as Figure 14 shown. The results showed that within the 32-day observation period, all mice in the PBS group and the OVA group died; the survival rates of the mice in the OVA+MPLA-LP group, the OVA+TBMⅡ-LP group, and the OVA+aluminum adjuvant group were comparable, all being 40%; the survival rate of the mice in the OVA+(TBMⅡ+MPLA)-LP group was 50%, and the survival rate of the positive control OVA+(QS-21+MPLA)-LP group was 60%; the above results indicated that both TBMⅡ-LP and (TBMⅡ+MPLA)-LP adjuvants could improve the survival rate of mice and prolong the survival time.

[0162] V. Evaluation of the anti-influenza virus effect of liposome preparations

[0163] H1N1 is a common seasonal influenza virus, and its large-scale prevalence has posed a great threat to human health. To more comprehensively evaluate the preventive adjuvant effect of liposomal preparations, mice were randomly divided into 7 groups: PBS, HA (provided by Changchun Veterinary Research Institute), HA+MPLA-LP, HA+TBMⅡ-LP, HA+(TBMⅡ+MPLA)-LP, HA+(QS-21+MPLA)-LP, and HA+Alum. An immunization schedule of day 0 and day 14 was adopted; among them, the immunization schedule is shown in Table 3 below.

[0164] Table 3 Vaccines and injection conditions for different groups

[0165]

[0166] On day 28, lethal and sub-lethal challenges with H1N1 influenza virus were carried out (the lethal dose was 10 LD 50 , and the sub-lethal dose was 1 LD 50 ).

[0167] (1) ELISA test

[0168] Coat a 96-well flat-bottom plate with influenza antigen hemagglutinin HA and incubate overnight at 4°C; after blocking with PBST + 5% BSA (incubate at 37°C for 2 h), add the diluted samples (gradient dilution, starting dilution factor of 20,000-fold dilution, and the diluent is PBST + 0.5% BSA) to the plate and incubate at 37°C for 1 hour; HRP-conjugated goat anti-mouse IgG, IgG1, IgG 2a was used as the secondary antibody (the final concentration of the secondary antibody was 0.1 μg / mL); add 50 μL of TMB substrate solution to each well and incubate for about 10 min; then, terminate the reaction by adding the termination solution (2 M H2SO4). Finally, measure the absorbance value at 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader.

[0169] On day 14 after the last immunization, ELISA was used to detect the levels of HA-specific IgG, IgG1, IgG 2a in the sera of mice, as shown in Figure 15a , Figure 15b and Figure 15c . The results showed that TBMⅡ-LP and (TBMⅡ+MPLA)-LP could significantly increase the titers of influenza antigen HA-specific IgG, IgG1, and IgG 2a antibodies, and the levels of IgG 2a in TBMⅡ-LP and (TBMⅡ+MPLA)-LP were significantly higher than those in the aluminum adjuvant group, suggesting an induction of Th1-biased cellular immune responses.

[0170] (2) Monitoring of body weight and survival curve after challenge

[0171] Fourteen days after the final immunization, five mice in each group were respectively subjected to lethal and sub-lethal challenge (the lethal dose was 10LD 50 , and the sub-lethal dose was 1LD 50 ). The body weights of the mice were monitored daily, and the results are as shown in Figure 16a and Figure 16b : For sub-lethal challenge, the body weights of the mice in each group decreased to a certain extent within 6 days after the challenge. However, the body weight decrease in the TBMⅡ-LP and (TBMⅡ+MPLA)-LP adjuvant groups was the lowest, and there were extremely significant differences compared with the simple antigen group (p<0.0001) (see Figure 16a ); for lethal challenge, the body weight decrease in the TBMⅡ-LP and (TBMⅡ+MPLA)-LP groups was also the lowest, and there were highly significant (p<0.01) and extremely significant differences respectively compared with the simple antigen group (p<0.0001) (see Figure 16b ).

[0172] In addition, the survival rates of the mice in the T-LP and (M+T)-LP adjuvant groups were significantly increased after lethal challenge, both reaching 100% (see Figure 17 ).

[0173] (3) PCR detection of viral load in the lungs of mice after challenge

[0174] After the mice were sacrificed, the lung tissues were aseptically removed, quickly minced in pre-cooled PBS, and 1 mL was added and homogenized on ice until the tissues were completely lysed. Centrifugation was performed at 12000 rpm for 5 min at 4℃ to remove debris, and the supernatant was successively subjected to chloroform layering (ice for 5 min), isopropanol precipitation (pre-cooled, ice for 10 min), and two washes with 75% ethanol (prepared with DEPC water) (centrifugation at 17500 rpm for 5 min at 4℃). Finally, it was air-dried and dissolved in nuclease-free water to measure the RNA concentration (30 - 50 μL); a detection system was constructed with HiScript II U+One Step qRT-PCR Probe Kit, and probe primers were designed for influenza virus (provided by Changchun Veterinary Research Institute), and sensitive detection of viral RNA was achieved by real-time fluorescence quantitative PCR. The whole process was carried out on ice and nuclease-free consumables were used to ensure RNA integrity. As can be seen from Figure 18 , both TBMⅡ-LP and (TBMⅡ+MPLA)-LP can reduce the viral load in the lung tissues of mice. Among them, the viral load in the lungs of the (TBMⅡ+MPLA)-LP group was the least, and there was a significant difference compared with the aluminum adjuvant group (P<0.001), suggesting that (TBMⅡ+MPLA)-LP has a protective effect on acute lung injury induced by influenza virus.

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Preparation method of terrestrinone B liposome, characterized in that, Comprising: Dissolve phospholipids, cholesterol, and PEGylated phospholipids in an organic solvent to form a mixed solution, and evaporate the mixed solution to obtain a thin film; the thin film is hydrated to obtain liposome of zhebeinine ethyl saponin; the hydration medium is an aqueous solution of zhebeinine ethyl saponin.

2. The preparation method according to claim 1, characterized in that, The mixed solution further comprises monophosphoryl lipid A.

3. The preparation method according to claim 1 or 2, characterized in that, The preparation method satisfies one or more of the following conditions: (i) The phospholipids are selected from one or more combinations of DOPC, DSPC, DMPC, or DMPG; (ii) The PEGylated phospholipids are selected from one or more combinations of DMG-PEG2000, DSPE-PEG5000, or DMG-PEG2000; (iii) The organic solvent is a mixed solvent of chloroform and methanol.

4. The preparation method according to claim 3, characterized in that, The phospholipids are selected as DOPC and DSPC, and the mass ratio of DOPC, DSPC, cholesterol, and PEGylated phospholipids is (0.8 - 1.2):(0.8 - 1.2):(0.8 - 1.2):0.5; and / or in the organic solvent, the volume ratio of chloroform to methanol is 5:(1 - 5).

5. Liposome of zhebeinine ethyl saponin prepared by the preparation method according to any one of claims 1 to 4.

6. An immunoadjuvant, characterized in that, Comprising the liposome of zhebeinine ethyl saponin according to claim 5.

7. An immune vaccine, characterized in that, Comprising the immunoadjuvant and antigen according to claim 6.

8. The immunogenic vaccine according to claim 7, wherein, The antigen is H1N1 influenza virus antigen, OVA antigen, or melanoma antigen.

9. Use of liposome of zhebeinine ethyl saponin, the use comprising: (i) Use of liposome of zhebeinine ethyl saponin in the preparation of an immunoadjuvant; (ii) Use of liposome of zhebeinine ethyl saponin in the preparation of an immunization vaccine.

10. The use according to claim 9, characterized in that the use Comprising: (a) Use of liposome of zhebeinine ethyl saponin in enhancing the humoral immune response and / or cellular immune response of the body; (b) Use of liposome of zhebeinine ethyl saponin in enhancing the specific Th17 cell response of the body; (c) Use of liposome of zhebeinine ethyl saponin in promoting the generation of germinal center B cells and / or follicular helper T cells in the body.

Images