Liposome vaccine and its preparation method and application

Abstract

The application discloses a kind of liposome vaccine and preparation method thereof, belong to biological medicine technical field.The vaccine is composed of antigen, adjuvant and lipid component, wherein adjuvant includes iNKT cell agonist and STING receptor agonist.Preparation method includes: adjuvant and lipid component are dissolved in organic solvent to obtain adjuvant solution, and lipid film is obtained by removing solvent under reduced pressure;Add antigen solution and ultrasonic treatment to obtain antigen-loaded adjuvant encapsulated liposome crude particles.Crude particles are homogenized by liposome extruder, then loaded into dialysis bag to remove free antigen by dialysis, and after purification, liposome vaccine nanosuspension is obtained.The vaccine can induce mice to produce specific IgG and IgM antibodies against OVA antigen.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a liposomal vaccine, its preparation method, and its application. Background Technology

[0002] Tumor immunotherapy, which activates the body's own immune system to specifically kill tumor cells, has become a research hotspot in the prevention and treatment of malignant tumors. As an active immunotherapy method, tumor vaccines can deliver tumor antigens to induce the body to produce specific T cell responses. However, the immunogenicity of a single antigen is relatively weak, making it difficult to stimulate a strong and long-lasting anti-tumor immune response. Therefore, they usually need to be combined with immune adjuvants to synergistically enhance the immune response.

[0003] Existing single-target adjuvants can only activate a single immune pathway, resulting in limited immune activation effects. Among them, STING agonists can activate dendritic cells to secrete pro-inflammatory factors and initiate innate immunity; iNKT cell agonists can rapidly activate iNKT cells and simultaneously regulate innate and adaptive immunity. The two types of adjuvants have complementary pathways of action, but free adjuvants are easily degraded by enzymes in vivo and their systemic distribution can easily cause systemic toxicity, limiting their clinical application.

[0004] Liposome nanocarriers possess advantages such as good biocompatibility, the ability to co-encapsulate multiple active substances, and targeted delivery, making them excellent delivery carriers for antigens and adjuvants. However, most existing liposomal tumor vaccines only load a single adjuvant, lacking dual-adjuvant co-delivery systems that simultaneously load both STING and iNKT agonists. Furthermore, conventional liposome preparation processes result in uneven particle size and high levels of residual free antigen, which can easily lead to decreased immunogenicity and increased side effects.

[0005] Currently, there is still a lack of dual-adjuvant co-loaded liposome tumor vaccines that can simultaneously activate dual immune pathways, have controllable particle size, and high purity. Therefore, developing a homogeneous liposome vaccine that co-encapsulates iNKT cell agonist, STING receptor agonist, and tumor antigen, as well as its standardized preparation process, has important clinical value for improving the immunogenicity of tumor vaccines and expanding the treatment options for malignant tumors. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing tumor vaccines, such as insufficient immunogenicity, single adjuvant-based immune activation pathways, poor in vivo stability of free immune agonists, uneven particle size of ordinary liposomes, and numerous impurities in free antigens. This invention provides a liposomal vaccine co-loaded with an iNKT cell agonist and a STING receptor agonist, along with a standardized preparation method and its anti-tumor pharmaceutical applications. This invention co-encapsulates two complementary immune pathways with antigens in a liposomal carrier, simultaneously activating dual immune pathways and enhancing the body's anti-tumor immune response. Through a membrane hydration-gradient extrusion combined with dialysis purification process, precise controllable liposome particle size and high product purity are achieved, reducing systemic toxicity and making it suitable for the prevention and treatment of various solid tumors and hematological malignancies.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a liposomal vaccine comprising an antigen, an adjuvant, and a lipid component; wherein the adjuvant comprises an iNKT cell agonist and a STING receptor agonist.

[0008] This invention encapsulates adjuvants and antigens that complement two immune pathways in a liposome carrier, thereby significantly enhancing the body's anti-tumor immune response by simultaneously activating both innate and adaptive immune pathways.

[0009] Preferably, the iNKT cell agonist is at least one of KRN7000 and 7DW8-5, or at least one of the stereoisomers of KRN7000 and 7DW8-5, pharmaceutical salts, hydrates, and solvates.

[0010] The general formulas for KRN7000 and 7DW8-5 are shown in (Ⅰ): (I).

[0011] Preferably, the STING receptor agonist is at least one of DMXAA, c-di-GMP, c-di-AMP, and diABZI, or at least one of the stereoisomers, pharmaceutical salts, hydrates, and solvates of DMXAA, c-di-GMP, c-di-AMP, and diABZI.

[0012] The general formulas for DMXAA, c-di-GMP, c-di-AMP, and diABZI are shown in (II): (II).

[0013] More preferably, the iNKT cell agonist is KRN7000 and the STING receptor agonist is DMXAA.

[0014] Preferably, the mass ratio of the iNKT cell agonist to the STING receptor agonist is (0.5~1.5):1.

[0015] Preferably, the lipid components include SM-102, DSPC (1,2-distearyl-sn-propanetriyl-3-phosphocholine), cholesterol, and DSPE-PEG2000.

[0016] The four lipid components mentioned above together constitute the liposome carrier framework. Among them, SM-102, as an ionizable cationic lipid, helps to encapsulate and deliver active substances intracellularly, DSPC provides membrane structural stability, cholesterol regulates membrane fluidity, and DSPE-PEG2000 endows liposomes with long-term cycling performance and reduces non-specific protein adsorption.

[0017] Preferably, the molar ratio of SM-102, DSPC, cholesterol and DSPE-PEG2000 is (40~60):(5~20):(30~50):(1~3).

[0018] More preferably, the molar ratio of SM-102, DSPC, cholesterol and DSPE-PEG2000 is 50:10:38.5:1.5.

[0019] Secondly, the present invention provides a method for preparing the liposome vaccine, comprising the following steps: (1) iNKT cell agonist, STING receptor agonist and lipid components were dissolved together in an organic solvent to prepare an adjuvant mixed solution; (2) The adjuvant mixture obtained in step (1) is concentrated under reduced pressure to remove the organic solvent and form a lipid film on the inner wall of the container; (3) Add the antigen solution to the container with the attached lipid film, and sonicate to fully hydrate and peel off the lipid film to prepare coarse liposome particles with encapsulated adjuvant and loaded antigen. (4) The coarse liposome particles are graded and extruded using a liposome extruder to achieve uniform particle size, thereby obtaining liposomes with a particle size of 80~200nm. (5) The liposomes obtained in step (4) are subjected to dialysis to remove unencapsulated free antigens. After purification, liposome nanosuspension is obtained, which is the liposome vaccine.

[0020] Preferably, the specific process of vacuum concentration in step (2) is as follows: first, a rotary evaporator is used to remove most of the organic solvent at 40~60℃, and then the oil pump is used to dry under reduced pressure for 6~24h at room temperature to completely remove the residual solvent and obtain a dry lipid film.

[0021] Preferably, the specific process of the graded extrusion particle size homogenization treatment in step (4) is as follows: first, use a 400nm polycarbonate membrane to repeatedly extrude coarse particles until there is no resistance to extrusion, then replace the 200nm polycarbonate membrane and continue to extrude repeatedly until there is no resistance to obtain homogeneous liposomes with a particle size of 80~200nm.

[0022] Preferably, the organic solvent is selected from one or more of dichloromethane, methanol, and chloroform.

[0023] Thirdly, the present invention provides the use of the liposomal vaccine in the preparation of medicaments for the prevention and / or treatment of tumors.

[0024] Preferably, the tumor is selected from one or more of the following: breast cancer, ovarian cancer, lung cancer, uterine cancer, liver cancer, prostate cancer, melanoma, colorectal cancer, renal cell carcinoma, cellular lymphoma, pancreatic cancer, thyroid cancer, brain cancer, stomach cancer, and leukemia.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) Synergistic effect of dual adjuvants: The present invention adopts a dual adjuvant co-load design of iNKT cell agonist and STING receptor agonist. The immune pathways of the two types of adjuvants are complementary, which can simultaneously activate the body's innate immunity and adaptive immunity. Compared with the single adjuvant system, the present invention can significantly enhance the antigen-specific anti-tumor immune response and overcome the deficiency of the limited immune activation effect of single adjuvant in the prior art.

[0026] (2) Liposome carriers enhance safety and bioavailability: In this invention, liposome carriers can encapsulate adjuvants and antigens inside, avoiding rapid degradation and systemic diffusion of active substances after they are released, effectively reducing systemic inflammatory adverse reactions and improving bioavailability.

[0027] (3) Precise and controllable particle size: This invention uses a combined thin-film hydration and graded extrusion process to precisely control the liposome particle size within the range of 80-200 nm. This particle size range is beneficial for the accumulation of the vaccine in lymph nodes, thereby more effectively activating the immune response.

[0028] (4) High product purity: This invention removes free antigens through dialysis, reduces non-specific immune interference, and greatly improves product purity and vaccine stability.

[0029] (5) Mature technology and wide applicability: The complete film hydration and extrusion purification process in this invention is mature, stable, and highly repeatable, making it easy to scale up production. The selected lipid components have good biocompatibility and high clinical translational value. At the same time, this liposomal vaccine can achieve preventive and therapeutic effects against a variety of solid tumors and hematological malignancies, and has a wide range of clinical applications. Attached Figure Description

[0030] Figure 1 This is a particle size and distribution diagram of the liposome vaccine prepared in Example 1 of the present invention; Figure 2 This is a graph showing the IgG antibody titer evaluation of the liposome vaccine prepared in Example 1 of the present invention; Figure 3 This is a graph showing the IgM antibody titer evaluation of the liposome vaccine prepared in Example 1 of the present invention. Detailed Implementation

[0031] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0032] Example 1 This embodiment provides a method for preparing a liposomal vaccine adjuvant, including the following steps: 1) Lipid phase formulation The total lipid concentration was fixed at 10 mg / mL, and the molar ratio of lipid components SM-102:DSPC:cholesterol:DSPE-PEG2000 was 50:10:38.5:1.5. KRN7000 (iNKT agonist) single adjuvant group, DMXAA (STING agonist) single adjuvant group, and KRN7000+DMXAA dual adjuvant group were set up, with an adjuvant concentration of 0.12 mg / mL and a dual adjuvant mass ratio of 1:1. Each lipid raw material and the corresponding dose of the two adjuvants were accurately weighed according to the formula. Using a dichloromethane-methanol mixture (3:1 volume ratio), all lipids and adjuvant components were thoroughly dissolved in a round-bottom flask to obtain a homogeneous lipid-adjuvant mixture.

[0033] 2) Preparation of lipid films The round-bottom flask containing the mixture was connected to a rotary evaporator. The water bath temperature was set to 45°C and the process was kept completely dark. The mixture was then rotated at low speed for 2 hours to completely remove the organic solvent mixture and form a dry and uniform lipid film on the inner wall of the flask.

[0034] 3) Preparation of crude liposome suspension by membrane hydration Preheat PBS buffer to 45°C, prepare an antigen solution with a final OVA concentration of 2 mg / mL, and add it to a flask with an attached lipid film. Place the flask in an ultrasonic device and sonicate for 20 min to promote the full hydration, detachment, and dispersion of the lipid film, thus obtaining a coarse liposome suspension co-encapsulated with dual adjuvants and loaded with OVA antigen.

[0035] 4) Extrusion homogenization regulates liposome particle size The crude liposome suspension was transferred to a syringe, and a liposome extruder was assembled. The suspension was repeatedly squeezed with 400 nm and 200 nm polycarbonate membranes until the squeezing resistance disappeared, thus completing the particle size homogenization process and obtaining a monolayer lipid vesicle suspension with a particle size of 80–200 nm.

[0036] 5) Dialysis purification and sample preservation Collect the liposome suspension after extrusion, put it into a dialysis bag with a molecular weight cutoff of 50 kDa, and dialyze it in sufficient distilled water at 4℃ for purification. Replace the dialysis fluid every 4 to 6 hours and continue dialysis for 24 hours to fully remove the unencapsulated free OVA antigen in the system. After dialysis, collect the purified liposome nanosuspension.

[0037] 6) Determination of particle size, distribution, and zeta potential The liposome sample was diluted 100-fold with PBS. 1 ml of the diluted solution was transferred to a multi-laser nanoparticle tracking analysis system (NS300) to determine the particle size distribution and concentration. The liposome sample was also diluted 100-fold with Coulter nanoparticle size analyzer-specific diluent. 200 μL of the diluted solution was transferred to the particle size potential chip of the Coulter nanoparticle size analyzer, and the chip was placed in the analyzer to determine the sample particle size and distribution. Results are as follows: Figure 1 As shown, all liposomes have a particle size of less than 200 nm, mainly distributed between 70-110 nm. Liposomes of this size are more easily recognized and phagocytosed by macrophages and dendritic cells, which helps the vaccine to be taken up and presented to the immune system, thereby improving the immune activity of the vaccine.

[0038] Experimental Example 1 In this experiment, Example 1, mice were immunized with the liposome vaccine prepared in Example 1, and its immune effect was preliminarily evaluated by ELSA experiment.

[0039] 1. ELISA Immunoassay 1) Mouse immunization: C57BL / 6 mice aged 6-8 weeks were randomly divided into 5 groups of 6 mice each. The prepared liposome vaccine was administered subcutaneously to the mice on days 0, 14, 21, and 28, with each mouse receiving 0.1 mL of the vaccine. The control group received an equal volume of PBS solution. On day 38, blood was collected from the mice, incubated at 0°C for 1 hour, and then centrifuged. The clear serum supernatant was used for ELISA analysis.

[0040] 2) ELISA Immunoassay: Prepare a 2.0 µg / mL solution of OVA antigen using carbonate buffer and add 100 µL to each well of a 96-well ELISA plate. Incubate at 4°C for 12 hours. Remove the plate and incubate for 1 hour. Wash the plate three times with PBST. After washing, add 2% skim milk (250 µL) to each well and incubate at room temperature for 1 hour. Wash the plate three times. Dilute mouse serum samples by half-maximal concentration and add 100.0 µL to each well of a sealed 96-well plate. Incubate for 1.5 hours and wash the plate three times. Add secondary antibody (HRP-labeled IgG or HRP-labeled IgM) (100.0 µL) to each well and incubate for 1 hour. Wash the plate three times. Add TMB solution and incubate at room temperature in the dark for 15 minutes. Finally, add 0.5 M H2SO4 solution to stop the color development and immediately measure the absorbance using a microplate reader at a wavelength of 450 nm and a background wavelength of 570 nm.

[0041] The absorbance (OD) value at 450 nm wavelength minus the OD value at 570 nm background wavelength yields the OD value of the antibody produced in vaccine-induced mice. The results are shown in Table 1. Figure 2-3 As shown.

[0042] Table 1. Average absorbance (OD) of OVA antibodies produced in vaccine-induced mice From Table 1 and Figure 2-3 As can be seen, the liposomal vaccine prepared in Example 1 of this invention, compared with the blank group, can induce high titers of IgG and IgM antibodies in different adjuvant groups, indicating that the liposomal vaccine can effectively improve the immunogenicity of OVA antigen and induce mice to produce an immune response against OVA antigen; in particular, the OD values ​​of IgG and IgM antibodies induced by the liposomal vaccine in the DMXAA+KRN7000 double adjuvant group are significantly higher than those in the DMXAA and KRN7000 single adjuvant groups, indicating that the DMXAA+KRN7000 double adjuvant can coordinate and enhance the immunogenicity of the vaccine, thereby improving the activity of the vaccine.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A liposomal vaccine, characterized in that, The liposomal vaccine comprises an antigen, an adjuvant, and a lipid component; the adjuvant includes an iNKT cell agonist and a STING receptor agonist.

2. The liposome vaccine as described in claim 1, characterized in that, The iNKT cell agonist is at least one of KRN7000 and 7DW8-5, or at least one of the stereoisomers of KRN7000 and 7DW8-5, pharmaceutical salts, hydrates, and solvates.

3. The liposome vaccine as described in claim 1, characterized in that, The STING receptor agonist is at least one of DMXAA, c-di-GMP, c-di-AMP, and diABZI, or at least one of the stereoisomers, pharmaceutical salts, hydrates, and solvates of DMXAA, c-di-GMP, c-di-AMP, and diABZI.

4. The liposome vaccine as described in claim 1, characterized in that, The mass ratio of the iNKT cell agonist to the STING receptor agonist is (0.5~1.5):

1.

5. The liposome vaccine as described in claim 1, characterized in that, The lipid components include SM-102, DSPC, cholesterol, and DSPE-PEG2000.

6. The liposome vaccine as described in claim 5, characterized in that, The molar ratio of SM-102, DSPC, cholesterol and DSPE-PEG2000 is (40~60):(5~20):(30~50):(1~3).

7. The method for preparing a liposomal vaccine according to any one of claims 1-6, characterized in that, Includes the following steps: (1) iNKT cell agonist, STING receptor agonist and lipid components were dissolved together in an organic solvent to prepare an adjuvant mixed solution; (2) The adjuvant mixture obtained in step (1) is concentrated under reduced pressure to remove the organic solvent and form a lipid film on the inner wall of the container; (3) Add the antigen solution to the container with the attached lipid film, and sonicate to fully hydrate and peel off the lipid film to prepare coarse liposome particles with encapsulated adjuvant and loaded antigen. (4) The coarse liposome particles are graded and extruded using a liposome extruder to achieve uniform particle size, thereby obtaining liposomes with a particle size of 80~200nm. (5) The liposomes obtained in step (4) are subjected to dialysis to remove unencapsulated free antigens. After purification, liposome nanosuspension is obtained, which is the liposome vaccine.

8. The preparation method according to claim 7, characterized in that, The specific process of the graded extrusion particle size homogenization treatment in step (4) is as follows: First, use a 400nm polycarbonate membrane to repeatedly extrude coarse particles until there is no resistance to extrusion, then replace it with a 200nm polycarbonate membrane and continue to extrude repeatedly until there is no resistance, so as to obtain homogeneous liposomes with a particle size of 80~200nm.

9. The use of the liposomal vaccine as described in any one of claims 1-6 in the preparation of a medicament for the prevention and / or treatment of tumors.

10. The application as described in claim 9, characterized in that, The tumor is selected from one or more of the following: breast cancer, ovarian cancer, lung cancer, uterine cancer, liver cancer, prostate cancer, melanoma, colorectal cancer, renal cell carcinoma, cellular lymphoma, pancreatic cancer, thyroid cancer, brain cancer, stomach cancer, and leukemia.

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