Anionic lipid nanoadjuvant and its preparation method and application

Abstract

The present invention provides an anionic lipid nanoadjuvant, its preparation method, and application. The anionic lipid nanoadjuvant of the present invention is anionic lipid nanoparticles containing anionic lipids, PEG-lipids, cholesterol compounds, and auxiliary lipids; wherein the surface potential of the anionic lipid nanoadjuvant is between 5mV and 45mV. The anionic lipid nanoadjuvant of the present invention has a specific surface potential distribution range and exhibits an immune adjuvant effect, capable of increasing the body's immune response to antigens, thereby enhancing the immune activation effect of co-administered antigens, and has good biosafety.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an anionic lipid nanoadjuvant and a preparation method and application thereof. Background Art

[0002] Malignant tumors and various infectious diseases seriously threaten human life and health, and there is an urgent need to develop safe and effective treatments to improve the prevention and treatment of malignant tumors and infectious diseases. Effective prevention and treatment of various malignant tumors and infectious diseases can be achieved by stimulating the patient's own immune response. By enhancing the processing and presentation of disease-related antigens or specific antigens and activating antigen-specific immune responses, the body's immune system can effectively clear and kill tumor cells or pathogens. The effectiveness of vaccines depends on efficient disease antigens and immune adjuvants. Furthermore, nanoformulations are expected to achieve precise delivery of antigens and adjuvants, thereby improving the immune efficiency of antigen presenting cells (APCs). Antigens with high immunogenicity are recognized by APCs such as dendritic cells (DC), bound to major histocompatibility complex (MHC) molecules, presented on the surface of APCs, and bound to CD4 + or CD8 + It binds to receptors on the surface of T cells and activates the immune response. At present, the development of vaccines still faces many challenges. First, the processing and presentation of antigens by APCs is limited by the efficiency of antigen delivery in the body, and APCs cannot effectively activate and complete antigen presentation; second, there is an urgent need to develop highly effective immune adjuvants to further enhance the immune effect of antigens. Adjuvants can reduce the number of vaccinations, improve the stability of antigens, and stimulate long-lasting and high-intensity immune responses. However, the number of immune adjuvants currently approved for marketing is limited, and cannot meet the needs of immune prevention and treatment of multiple diseases. Therefore, there is an urgent need to develop an immune adjuvant that meets the needs of immune prevention and treatment of multiple diseases. Summary of the Invention

[0003] To address the above issues, the applicant has provided a specifically formulated anionic lipid nanoadjuvant, which has the effect of activating the immune system and enhancing the immune response as an immune adjuvant, and can be used for combined administration of vaccines.

[0004] In a first aspect, the present invention provides an anionic lipid nanoadjuvant, which is an anionic lipid nanoparticle, and the anionic lipid nanoparticle contains anionic lipids, PEG-lipids, cholesterol compounds and auxiliary lipids; wherein the surface potential of the anionic lipid nanoadjuvant is -5mV to -45mV.

[0005] In another preferred embodiment, the surface potential of the anionic lipid nanoadjuvant is -10 mV to -45 mV; preferably -20 mV to -40 mV; more preferably -30 mV to -35 mV.

[0006] In another preferred embodiment, the anionic lipid, PEG-lipid, cholesterol compound and auxiliary lipid are complexed to form membrane particles.

[0007] In another preferred embodiment, the film-like particles have a hollow structure.

[0008] In a preferred embodiment, the anionic lipid is selected from the group consisting of DSPA, DPPA, DMPA, DLPA, DOPA, or a combination thereof;

[0009] The cholesterol compound is selected from the group consisting of CHO-HP, vitamin D2, vitamin D3, β-sitosterol, brassicasterol, ergosterol, or a combination thereof;

[0010] The auxiliary lipid is selected from the group consisting of DOPE, DSPE, DPPE, DMPE, DLPE, or a combination thereof; and / or

[0011] The PEG-lipid is selected from the group consisting of DMG-PEG, DSG-PEG, DSPE-PEG, DSPE-MPEG, DPPE-MPEG, or a combination thereof.

[0012] In another preferred embodiment, the molecular weight of PEG is 400-20,000, preferably 500-10,000, more preferably 1,000-5,000, and most preferably 2,000-3,000.

[0013] In another preferred embodiment, the molar ratio of the anionic lipid to the PEG-lipid is (0.1-80):1; preferably (0.2-70):1; more preferably (0.5-60):1; most preferably (0.5-50):1.

[0014] In a preferred embodiment, the molar ratio of the cholesterol compound to the PEG-lipid is (35-45):1; preferably (37-43):1; more preferably (38-42):1; most preferably (39-41):1.

[0015] In another preferred embodiment, the molar ratio of the auxiliary lipid to the PEG-lipid is (1-100):1; preferably (2-80):1; more preferably (3-70):1; most preferably (5-60):1.

[0016] In another preferred embodiment, the molar ratio of the anionic lipid, cholesterol compound, auxiliary lipid and PEG-lipid is (0.1-80):(35-45):(1-100):1; preferably (0.2-70):(37-43):(2-80):1; more preferably (0.5-60):(38-42):(3-70):1; most preferably (0.5-50):(39-41):(5-60):1.

[0017] In another preferred embodiment, the molar ratio of the anionic lipid to the cholesterol compound is (0.01-1.5):(0.9-1.1), preferably (0.02-1.5):(0.9-1.1).

[0018] In another preferred embodiment, the molar ratio of the auxiliary lipid and the cholesterol compound is (0.1-1.8):(0.9-1.1), preferably (0.2-1.5):(0.9-1.1).

[0019] In another preferred embodiment, the molar ratio of the PEG-lipid to the cholesterol compound is (0.005-0.05):(0.9-1.1), preferably (0.01-0.03):(0.9-1.1).

[0020] In another preferred embodiment, the molar number of the anionic lipid is 0.01 to 1.5, preferably 0.02 to 1.5, calculated based on the molar number of the cholesterol compound being 1.

[0021] In another preferred embodiment, the molar number of the auxiliary lipid is 0.1 to 1.8, preferably 0.2 to 1.5, calculated based on the molar number of the cholesterol compound being 1.

[0022] In another preferred embodiment, the molar number of the PEG-lipid is 0.005-0.05, preferably 0.01-0.03, calculated based on the molar number of the cholesterol compound being 1.

[0023] In another preferred embodiment, the average hydrated particle size of the anionic lipid nanoadjuvant is 100-500 nm; preferably 150-400 nm; more preferably 200-300 nm.

[0024] In a preferred embodiment, the polydispersity coefficient of the anionic lipid nanoadjuvant is 0.2-0.8; preferably 0.2-0.6; more preferably 0.2-0.5 or 0.2-0.4.

[0025] In another preferred embodiment, the anionic lipid nanoadjuvant contains substantially no or contains amphoteric lipids, and substantially no or contains cationic lipids.

[0026] In another preferred embodiment, in the anionic lipid nanoadjuvant, the content of the amphoteric lipid is ≤0.01, calculated based on the molar number of cholesterol (CHO-HP) compounds being 1.

[0027] In another preferred embodiment, in the anionic lipid nanoadjuvant, the content of cationic lipid is ≤0.01, calculated based on the molar number of cholesterol (CHO-HP) compounds being 1.

[0028] According to a second aspect of the present invention, there is provided a method for preparing the anionic lipid nanoadjuvant according to the first aspect of the present invention, comprising the following steps:

[0029] S1. The anionic lipid, PEG-lipid, cholesterol compound and auxiliary lipid are dissolved in an organic solvent and dried by rotary evaporation under reduced pressure to form a film;

[0030] S2. The thin film obtained in step S1 is hydrated and homogenized in an aqueous phase to obtain the anionic lipid nanoadjuvant as described in the first aspect of the present invention.

[0031] In another preferred embodiment, in step S2, a buffer solution or pure water is added to the thin film obtained in step S1, and the film is hydrated and homogenized to obtain the anionic lipid nanoadjuvant.

[0032] In another preferred embodiment, the ratio of the film to the aqueous phase is 5-10 mg (dry weight): 1-10 mL.

[0033] In another preferred embodiment, step S1 further includes the step of placing the mixture at a temperature above 56° C. for 5 to 15 seconds; preferably, placing the mixture in a water bath at a temperature above 56° C. for 5 to 15 seconds.

[0034] In another preferred embodiment, in step S2, the hydration comprises the steps of: stirring at a temperature above 56°C for 0.5 to 3 hours; preferably, stirring in a water bath in hot water above 56°C for 0.5 to 3 hours.

[0035] In another preferred embodiment, in step S2, the homogenization includes the steps of: ultrasonic homogenization at room temperature for more than 5 times, wherein the ultrasonic time is 1 to 3 seconds, the interval time is 2 to 4 seconds, and the ultrasonic power is 100 to 300w.

[0036] In a preferred embodiment, in step S1, the organic solvent is selected from the group consisting of chloroform, dichloromethane, methanol, or a combination thereof.

[0037] In a preferred embodiment, in step S1, the temperature of the reduced pressure rotary evaporation drying is 20-80°C; preferably 25-75°C; more preferably 30-60°C.

[0038] In another preferred embodiment, in step S1, the time for the reduced pressure rotary evaporation drying is 1 to 15 minutes, preferably 1 to 12 minutes, and more preferably 2 to 7 minutes.

[0039] The third aspect of the present invention provides use of the anionic lipid nanoadjuvant according to the first aspect of the present invention in preparing a vaccine composition.

[0040] In another preferred embodiment, the vaccine composition is used to prevent and treat diseases selected from the following groups: tumors and infectious diseases.

[0041] The fourth aspect of the present invention provides a vaccine composition, which contains (a) an immunogen or a nucleic acid molecule encoding the immunogen, (b) the anionic lipid nanoadjuvant, and (c) a pharmaceutically acceptable carrier.

[0042] In another preferred embodiment, the vaccine composition contains substantially no or contains amphoteric lipids, and substantially no or contains cationic lipids.

[0043] In another preferred embodiment, in the vaccine composition, the content of amphoteric lipids in the anionic lipid nanoadjuvant is ≤0.01% (preferably ≤0.001%, or 0), based on the total weight (dry weight) of the vaccine composition.

[0044] In another preferred embodiment, in the vaccine composition, the content of cationic lipid in the anionic lipid nanoadjuvant is ≤0.01% (preferably ≤0.001%, or 0), based on the total weight (dry weight) of the vaccine composition.

[0045] In another preferred embodiment, the vaccine composition is used to prevent and treat diseases selected from the following groups: tumors and infectious diseases.

[0046] In another preferred embodiment, the immunogen includes proteins, polypeptides, epitope peptides, virus particles, and VLPs.

[0047] In another preferred embodiment, the nucleic acid molecule encoding the immunogen includes mRNA, plasmid, viral vector, or a combination thereof.

[0048] The fifth aspect of the present invention provides use of the anionic lipid nanoadjuvant according to the first aspect of the present invention in preparing a preparation for enhancing the ratio of dendritic cell maturation.

[0049] The sixth aspect of the present invention provides use of the anionic lipid nanoadjuvant according to the first aspect of the present invention in the preparation of a preparation for promoting the uptake and processing of antigens by dendritic cells.

[0050] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The surface potential distribution of anionic lipid nanoadjuvants is shown.

[0052] Figure 2 The hydrated particle size distribution of anionic lipid nanoadjuvants is shown.

[0053] Figure 3 Transmission electron microscopy images of anionic lipid nanoadjuvants are shown.

[0054] Figure 4 The in vitro stability of anionic lipid nanoadjuvants was demonstrated.

[0055] Figure 5 The effects of anionic lipid nanoadjuvants on DC maturation are shown.

[0056] Figure 6 The effects of anionic lipid nanoadjuvants on DC antigen uptake function are shown.

[0057] Figure 7 Shows the distribution of anionic lipid nanoadjuvants in mice.

[0058] Figure 8 Shown are the effects of anionic lipid nanoadjuvants on biochemical parameters in mice.

[0059] Figure 9 The in vivo immune response evaluation of anionic lipid nanoadjuvants is shown, wherein Figure A shows CD8 + Figure 3 shows the proportion of T cells, and panel B shows the proportion of mature DCs.

[0060] Figure 10 Pharmacodynamic evaluation of anionic lipid nanoadjuvants is shown.

[0061] In the figure, LNP represents anionic lipid nanoadjuvant. DETAILED DESCRIPTION

[0062] After extensive and lengthy research and extensive screening, the inventors developed an anionic lipid nanoadjuvant for the first time. This anionic lipid nanoadjuvant can significantly activate DCs, promote their maturation, induce a stronger immune response, and enhance antigen cross-presentation. To further illustrate its efficacy in treating and preventing diseases, using therapeutic tumor vaccines as an example, it can enhance the efficacy of tumor vaccines without the need for additional adjuvant molecules, reducing the cost of tumor vaccines and streamlining their preparation, thus facilitating their development and use. This is the basis for the present invention.

[0063] As used herein, the terms “comprises,” “comprising,” “including,” “containing,” and variations thereof will be understood to include stated steps or components but not to exclude other steps or components, unless otherwise expressly stated.

[0064] As used herein, the term "room temperature" generally refers to 4 to 30°C, preferably 15 to 25°C.

[0065] As used herein, the term "anionic lipid nanoparticles" and "anionic lipid nanoadjuvants" refer to the same.

[0066] Abbreviations

[0067] DSPA: Distearoylphosphatidic acid

[0068] DPPA: Dipalmitoylphosphatidic acid

[0069] DMPA: 1,2-dimyristoylphosphatidic acid

[0070] DLPA: Dilauroylphosphatidic acid

[0071] DOPA: dioleoylphosphatidic acid

[0072] CHO-HP: High Purity Cholesterol

[0073] DOPE: dioleoylphosphatidylethanolamine

[0074] DSPE: Distearoylphosphatidylethanolamine

[0075] DPPE: Dipalmitoylphosphatidylethanolamine

[0076] DMPE: 1,2-dimyristoylphosphatidylethanolamine

[0077] DLPE: Dilauroylphosphatidylethanolamine

[0078] DMG-PEG: 1,2-dimyristoyl-rac-glycerol-3-polyethylene glycol, also known as PEGylated dimyristoylglycerol

[0079] DSG-PEG: Distearoyl-rac-glycerol-polyethylene glycol

[0080] DSPE-PEG: Distearoylphosphatidylethanolamine-polyethylene glycol

[0081] DSPE-MPEG: methoxy-polyethylene glycol-distearoylphosphatidylethanolamine

[0082] DPPE-MPEG: methoxy-polyethylene glycol-dipalmitoylphosphatidylethanolamine

[0083] DSPC: Distearoylphosphatidylcholine

[0084] DOTAP: 1,2-dioleoyl-3-coacetylcholine

[0085] BMDC: mouse bone marrow-derived dendritic cells

[0086] GM-CSF: Granulocyte-macrophage colony-stimulating factor

[0087] IL-4: interleukin-4

[0088] OVA: ovalbumin

[0089] PE: Phycoerythrin

[0090] LNP: lipid nanoparticles

[0091] PEG: polyethylene glycol

[0092] cholesterol

[0093] Cholesterol plays a crucial role in self-assembled membrane structures. Cholesterol has been shown to be an effective substance for increasing the fluidity of lipid membranes while maintaining their stability. Cholesterol analogs include vitamin D2, vitamin D3, β-sitosterol, brassicasterol, and ergosterol.

[0094] Cholesterol is known to be (3β)-cholest-5-en-3-ol, cholester-5-en-3β-ol, and is an essential component of higher animal cell membranes. As used herein, "cholesterol compounds" include cholesterol, cholesterol derivatives, and cholesterol analogs. Cholesterol derivatives refer to natural or synthetic cholesterol derivatives, and examples include acyl cholesterol, which is an ester with a fatty acid bonded to the hydroxyl group. Cholesterol analogs refer to natural or synthetic cholesterol analogs, and examples include plant sterols such as sitosterol, stigmasterol, fuccasterol, spinasterol, campesterol, and brassicasterol, and fungal ergosterol.

[0095] Cholesterol, cholesterol derivatives, and cholesterol analogs are all classified as steroids, belonging to the subgroup known as sterols (sterols).

[0096] Anionic lipid nanoadjuvants

[0097] The anionic lipid nanoadjuvant described herein is a lipid nanoparticle, i.e., a liposome. Liposomes are commonly used drug carriers with good biocompatibility. The present invention proposes an anionic lipid nanoadjuvant, comprising an anionic lipid nanoparticle formed by the hydrophilic end of the PEG-lipid outwardly forming a lipid nanoparticle shell, the hydrophobic end of the PEG-lipid is combined with the hydrophobic end of the anionic lipid, and cholesterol compounds and auxiliary lipids help stabilize the skeleton structure. In the present invention, the anionic lipid nanoadjuvant can be used as a carrier to construct a vaccine by physically mixing with a polypeptide or protein antigen. The carrier of this vaccine itself (i.e., the anionic lipid nanoadjuvant of the present application) can promote the maturation of DCs and the presentation function of antigens, enhance the anti-tumor immune response, exert the effect of the adjuvant, and achieve the goal of improving the therapeutic effect of the vaccine.

[0098] In certain embodiments, the particle size and polydispersity index (PDI) of the anionic lipid nanoadjuvants of the present application were analyzed using a particle size analyzer, revealing an average particle size of 200-300 nm and a PDI of 0.2-0.4. Particles of this size can accumulate in lymphoid organs such as lymph nodes and spleen. Furthermore, since their size is similar to that of pathogens, they are easily taken up by antigen-presenting cells (APCs), activating anti-tumor immune responses.

[0099] In certain embodiments, the anionic lipid nanoadjuvant of the present application can be observed to be a round sphere by transmission electron microscopy. Figure 2 As shown, the particle size is 200 to 300 nm.

[0100] In certain embodiments, the anionic lipid is 1,2-Distearoyl-sn-glycero-3-phosphate (DSPA);

[0101] The cholesterol compound is high-purity cholesterol (Cholesterol (for injection), CHO-HP);

[0102] The auxiliary lipid is dioleoylphosphatidylethanolamine (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE);

[0103] The PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG), wherein the molecular weight of PEG is 2000.

[0104] In certain embodiments, the molar ratio of DSPA, CHO-HP, DOPE, and DMG-PEG is 50:40:9:1 to 1:40:58:1 (the molar ratio of CHO-HP and DMG-PEG is fixed at 40 and 1); the addition of cholesterol compounds can increase the rigidity and stability of lipid nanoparticles, and PEG lipids can provide a spatial barrier and stabilize the structure.

[0105] Preparation of anionic lipid nanoadjuvants

[0106] The present invention also relates to a method for preparing the above-mentioned anionic lipid nanoadjuvant, which comprises at least the following steps:

[0107] S1. The anionic lipid, PEG-lipid, cholesterol compound and auxiliary lipid are dissolved in an organic solvent and dried by rotary evaporation under reduced pressure to form a film;

[0108] S2. The thin film obtained in step S1 is hydrated and homogenized in an aqueous phase to obtain the anionic lipid nanoadjuvant described in the present application.

[0109] In another preferred embodiment, in step S2, a buffer solution or pure water is added to the thin film obtained in step S1, and the film is hydrated and homogenized to obtain the anionic lipid nanoadjuvant.

[0110] In another preferred embodiment, the ratio of the film to the aqueous phase is 5-10 mg (dry weight): 1-10 mL.

[0111] In another preferred embodiment, step S1 further includes the step of placing the mixture at a temperature above 56° C. for 5 to 15 seconds; preferably, placing the mixture in a water bath at a temperature above 56° C. for 5 to 15 seconds.

[0112] In another preferred embodiment, in step S2, the hydration comprises the steps of: stirring at a temperature above 56°C for 0.5 to 3 hours; preferably, stirring in a water bath in hot water above 56°C for 0.5 to 3 hours.

[0113] In another preferred embodiment, in step S2, the homogenization includes the steps of: ultrasonic homogenization at room temperature for more than 5 times, wherein the ultrasonic time is 1 to 3 seconds, the interval time is 2 to 4 seconds, and the ultrasonic power is 100 to 300w.

[0114] In another preferred embodiment, in step S1, the temperature of the reduced pressure rotary evaporation drying is 30-60°C.

[0115] In another preferred embodiment, in step S1, the time of the reduced pressure rotary evaporation drying is 2 to 7 minutes.

[0116] In certain embodiments, the preparation method comprises the following steps:

[0117] S1. Dissolve DSPA, CHO-HP, DOPE, and DMG-PEG in an organic solvent according to the appropriate proportions. Place in a water bath at 56°C or higher for a few seconds. Then, dry by rotary evaporation at 30-60°C. After approximately 2-7 minutes, a thin film of lipid material can be observed adhering to the wall of the round-bottom flask.

[0118] S2. Add 2 mL of PBS to the round-bottom flask, then stir in a water bath at 56° C. or above for 0.5 to 3 h to fully hydrate, and ultrasonically homogenize at room temperature for more than 5 times (ultrasonic time 2 s, interval time 3 s, ultrasonic power 100 to 300 w) to obtain the anionic lipid nanoadjuvant.

[0119] In certain embodiments, in S1, DSPA, CHO-HP, DOPE and DMG-PEG are added to an organic solvent in proportion and dissolved. The organic solvent is selected from an organic solvent that can dissolve the above substances, preferably one or more of chloroform, dichloromethane and methanol.

[0120] In certain embodiments, in S2, homogenization is performed using an ultrasonic cell disruptor, and the ultrasonic conditions are as follows: ultrasonication for 2 seconds, interval for 3 seconds, and power of 100-300W.

[0121] Application of anionic lipid nanoadjuvants

[0122] The present invention also relates to the use of the anionic lipid nanoadjuvant in preparing vaccines for malignant tumors and various infectious diseases.

[0123] To further illustrate its disease prevention and treatment effects, using therapeutic tumor vaccines as an example, the present invention proposes a method for preparing tumor vaccines. The delivery vector exhibits an adjuvant effect, activating DCs, increasing antigen cross-presentation, and promoting anti-tumor immune responses. Therefore, the anionic lipid nanoadjuvant of the present invention is of great significance, providing a platform for preparing tumor vaccines without the need for additional adjuvant components.

[0124] Main advantages of the present invention

[0125] 1. The immune adjuvant effect of the anionic lipid nanoadjuvant of the present invention is mainly affected by the negative surface potential of the liposome itself. Experiments have found that the anionic lipid nanoadjuvant of the present invention can exert a highly effective immune adjuvant effect when its own surface potential is between -5mV and -45mV.

[0126] 2. The immune adjuvant effect of the anionic lipid nanoadjuvant of the present invention is independent of pre-loaded antigens or adjuvants. Previously reported liposome vaccines enhance immunity by improving the intracellular delivery efficiency of carrier-loaded antigens or adjuvants. The application form, function, and mechanism of action of the anionic lipid nanoadjuvant of the present invention differ significantly from these reported cases.

[0127] 3. The anionic lipid nanoadjuvants of the present invention exhibit excellent biosafety. Although liposomes with positive surface potential can promote dendritic cell maturation (see Example 4 for details), these liposomes exhibit significant cytotoxicity (see Example 3 for details), making them unsuitable as safe immunoadjuvants. The anionic lipid nanoadjuvants of the present invention exhibit excellent safety both in vitro and in vivo in animals.

[0128] 4. The anionic lipid nanoadjuvant of the present invention has broad application prospects. It can be administered sequentially, in combination, or physically mixed with tumor or infectious disease antigens to enhance antigen-induced immune responses and improve immunotherapy efficacy. This application is independent and scalable, independent of antigen loading or antigen-adjuvant formulation development, and will develop a new, broadly applicable, safe, and effective immune adjuvant.

[0129] 5. The component composition, ratio and key properties of the anionic lipid nanoadjuvant of the present invention are clear and quantifiable, with excellent production and quality control standards, and have the potential to achieve large-scale production and application.

[0130] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are percentages by weight and parts by weight.

[0131] Experimental reagents

[0132] The anionic lipids used in the examples, 1,2-distearoyl-sn-glycero-3-phosphate (DSPA), high-purity cholesterol (CHO-HP), auxiliary lipids dioleoylphosphatidylethanolamine (DOPE), and PEG-coated dimyristoylglycerol (DMG-PEG) were all purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd. OVA ovalbumin was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. OVA 257-264SIINFEKL peptide was purchased from Hefei Guopi Biotechnology Co., Ltd. C57BL / 6 mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. MC38-OVA cells (MC38 mouse colon cancer cell line modified with the chicken OVA gene) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Fluorescent dyes DiR and Cy5 and erythrocyte lysate were purchased from Dalian Meilun Biotechnology Co., Ltd. GM-CSF and IL-4 were purchased from Sino-Biotech Co., Ltd. T-Select H-2Kb OVA Tetramer-SIINFEKL-PE was purchased from Beijing Boerma Biotechnology Co., Ltd. Zombie Aqua™ Fixable Viability Kit, anti-CD45-FITC, anti-CD11c-PerCP-cy5.5, anti-MHCII-BV421, anti-CD80-PE, anti-CD86 antibody-APC, anti-CD3-FITC, and anti-CD8a-PE / Cyanine7 flow cytometry antibodies were purchased from BioLegend, USA. In this application, unless otherwise specified, all other reagents and solvents used are commercially available.

[0133] Experimental equipment

[0134] In the examples, an ultrasonic cleaner (SB-120DT, Ningbo Xinzhi) was used to ultrasonically promote the dissolution of the material. A rotary evaporator (R-100, Buchi, Switzerland) was used to reduce pressure and remove the organic solvent. A magnetic heating stirrer (LC-MSA-D, Shanghai Lichen) was used to hydrate the phospholipid membrane. An ultrasonic cell crusher (JYD-650L, Shanghai Zhixin) was used to homogenize the anionic lipid nanoparticles. A particle size analyzer (ZS90, Malvern) was used to detect the particle size, PDI, and zeta potential of the liposomes. Transmission electron microscopy images of the liposomes were taken using a transmission electron microscope (Talos L120C, FEI). Biochemical indicators were detected using a fully automatic biochemical analyzer (Chemray 240, Shenzhen Leidu). A flow cytometer (BD Fortessa, USA) was used to examine DC uptake and maturation as well as the immune response of mice. A small animal in vivo imaging instrument (PE IVIS SPECTRUM, PERKIN ELMER) was used to examine the distribution of liposomes in mice.

[0135] Example 1: Preparation of anionic lipid nanoadjuvant

[0136] Table A Prescription (unit: mg):

[0137] prescription 1 2 3 4 5 6 7 8 9 10 DSPA 3.63 3.27 2.91 2.44 2.1 1.75 0.7 0.35 0.14 0.07 CHO-HP 1.55 1.55 1.55 1.55 1.55 1.55 1.55 1.55 1.55 1.55 DOPE 0.67 1.04 1.41 1.79 2.16 2.53 3.65 4.02 4.24 4.32 DMG-PEG 0.2509 0.2509 0.2509 0.2509 0.2509 0.2509 0.2509 0.2509 0.2509 0.2509

[0138] Table B Prescription (unit: mol):

[0139] prescription 1 2 3 4 5 6 7 8 9 10 DSPA 4.993 4.498 4.003 3.356 2.889 2.407 0.963 0.481 0.193 0.096 CHO-HP 4.008 4.008 4.008 4.008 4.008 4.008 4.008 4.008 4.008 4.008 DOPE 0.901 1.398 1.895 2.406 2.903 3.400 4.906 5.403 5.699 5.806 DMG-PEG 0.100 0.100 0.100 0.100 0.100 0.100 0.100 0.100 0.100 0.100

[0140] The molecular weights of the above components are as follows: (unit: Da)

[0141] DSPA: 726.98

[0142] CHO-HP: 386.70

[0143] DOPE: 744.03

[0144] DMG-PEG: 2509.20

[0145] Preparation process:

[0146] S1. Using the formula in Table A, DSPA was dissolved in a mixed solvent of chloroform (325 μL) and methanol (175 μL). Ultrasonic cleaning was performed for 10 seconds to promote the complete dissolution of DSPA. CHO-HP, DOPE, and DMG-PEG were then dissolved in chloroform. All the above materials were transferred to a round-bottom flask, incubated in a 60°C water bath for several seconds, and then dried by vacuum rotary evaporation at 37°C in a water bath. After about 4-5 minutes, the lipid material was observed to form a thin film attached to the wall of the round-bottom flask.

[0147] S2. Add 2 mL of PBS to the round-bottom flask, then stir in a 60°C water bath for 2 h to fully hydrate, and then ultrasonically homogenize 10 times (ultrasound 2 s, interval 3 s, power 200 w) to obtain 10 anionic lipid nanoparticles with different formulations, recorded as anionic lipid nanoparticles (also called anionic lipid nanoadjuvants) No. 1 to No. 10.

[0148] The particle size and zeta potential of the lipid nanoadjuvants were measured using a particle size analyzer (ZS90, Malvern), and the lipid nanoadjuvants were observed using transmission electron microscopy. The anionic lipid nanoadjuvant from Example 1 was stored at 4°C, and its in vitro stability was investigated by characterizing changes in particle size. Sampling was performed at 1, 2, 3, 5, and 7 days.

[0149] Experimental results

[0150] The potentials of the prepared anionic lipid nanoadjuvants No.1 to No.10 with different formulations ranged from -5mV to -45mV ( Figure 1 ), hydrated particle size is between 200 and 300 nm ( Figure 2 ), PDI is between about 0.2 and 0.5 ( Figure 2 ).

[0151] The transmission electron microscope photo of anionic lipid nanoadjuvant No.2 is as follows: Figure 3The results showed that the particles were spherical in shape and had a particle size of about 200 to 300 nm.

[0152] The stability test results showed that the particle size of the anionic lipid nanoadjuvant did not change significantly within 7 days, indicating that the anionic lipid nanoadjuvant of Example 1 had good stability at 4°C. The test results of anionic lipid nanoadjuvant No. 2 were as follows: Figure 4 shown.

[0153] Example 2: Preparation of different lipid nanoparticles

[0154] According to the preparation method of the anionic lipid nanoadjuvant of Formulation 2 of Example 1, a variety of lipid nanoparticles with different electrical properties were obtained. The specific formulations are shown in Table 1 below, where Formulation 2 of Example 1 is Formulation A, and the other lipid nanoparticle formulations are respectively designated as Control Formulation B, Control Formulation C, and Control Formulation D.

[0155] The experimental results show that different formulations have different hydrated particle sizes and surface potentials, as shown in Table 2 below.

[0156] Table 1. Different prescriptions

[0157] lipids CHO-HP DOPE DMG-PEG Prescription A DSPA 3.27mg 1.55mg 1.04mg 250.9 μg Control prescription B DSPC 2.38mg 1.55mg 2.16mg 250.9 μg Control prescription C DOTAP 3.14mg 1.55mg 1.04mg 250.9 μg Control prescription D DOTAP 1.75mg 1.55mg 2.53mg 250.9 μg

[0158] Table 2. Particle size and potential distribution of lipid nanoadjuvants with different formulations

[0159]

[0160]

[0161] The lipid contained in the control formulation B is an amphoteric lipid, which, although exhibiting a certain negative potential, has essentially no adjuvant effect (see Example 4).

[0162] The control formulations C and D, which contain a high concentration of cationic lipids and a low concentration of cationic lipids, respectively, exhibited a potential of approximately 0 mV and a positive potential of approximately 18 mV, respectively.

[0163] Example 3: Effects of lipid nanoparticles with different formulations on BMDC cell viability

[0164] Anionic lipid nanoadjuvants and four other control lipid nanoparticles were prepared according to Examples 1 and 2, and bone marrow cells of C57BL / 6 mice were obtained and induced to differentiate into BMDCs using GM-CSF (20 ng / mL) and IL-4 (10 ng / mL). BMDCs were cultured at a rate of 5×10 4Cells were seeded at a density of 100 μL / mL in a 96-well cell culture plate, and GM-CSF and IL-4 were supplemented. After overnight culture, lipid nanoadjuvants (500 μg / mL) with different formulations were added. After further culture for 48 h, the cells were collected and the cell viability was determined.

[0165] The results are shown in Table 3 below.

[0166] Table 3. Effects of lipid nanoadjuvants with different formulations on BMDC cell survival.

[0167]

[0168] The experimental results showed that the anionic lipid nanoadjuvant had the highest cell survival rate. In contrast, the control formulation C (positive potential of about 18 mV) and the control formulation D (potential of about 0 mV) had significant cytotoxicity.

[0169] Example 4: Effect of anionic lipid nanoadjuvants on DCs maturation

[0170] Anionic lipid nanoparticles were prepared according to Example 1. Bone marrow cells of C57BL / 6 mice were obtained and induced to differentiate into BMDCs using GM-CSF (20 ng / mL) and IL-4 (10 ng / mL). 6 Cells were seeded at a density of 100 μg / mL in 24-well cell culture plates, with 1 mL per well, supplemented with GM-CSF and IL-4, and cultured overnight. Anionic lipid nanoadjuvants were added at a concentration gradient (500 μg / mL). After 48 h of culture, cells were collected and centrifuged at 300 g for 5 min. Dead cells were labeled using the Zombie Aqua™ Fixable Viability Kit and then incubated with antibody diluents (anti-CD45-FITC, anti-CD11c-PerCP-cy5.5, anti-MHCII-BV421, anti-CD80-PE, anti-CD86 antibody-APC) at 4°C for 30 min. The proportion of mature DCs (mDCs) in different groups was determined by flow cytometry (Zombie - anti-CD45-FITC + anti-CD11c-PerCP-cy5.5 + anti-MHCII-BV421 + anti-CD80-PE + anti-CD86-APC + ), preliminarily evaluated the adjuvant effect of anionic lipid nanoadjuvants.

[0171] The experimental results are as follows Figure 5As shown in the results, compared with the blank control group, the anionic lipid nanoadjuvant can promote the maturation of DCs and increase the proportion of mDCs, indicating that the anionic lipid nanoadjuvant can exert an adjuvant effect.

[0172] At the same time, the effects of lipid nanoparticles prepared with other formulations in Example 2 on DCs maturation were also investigated. The results are shown in Table 4 below.

[0173] Table 4. Effects of different lipid nanoparticle formulations on DC maturation

[0174] Potential / mV mDCs (%) Prescription A -32.50±2.40 65.1 Control prescription B -15.57±0.67 45.7 Control prescription C 17.97±0.23 72.8 Control prescription D -0.69±0.35 59.7

[0175] The experimental results showed that prescription A has excellent effect in promoting DCs maturation and improving the adjuvant effect on mDCs.

[0176] Control formulations C and D can also promote DCs maturation and enhance the adjuvant effect on mDCs. However, as shown in Example 3, control formulations C and D have significant cytotoxicity and low cell survival rates.

[0177] Example 5: Effect of anionic lipid nanoadjuvants on DC antigen uptake

[0178] Anionic lipid nanoadjuvants, namely LNPs, were prepared according to the method in Example 1. Before the experiment, SIINFEKL / LNPs were prepared by physical mixing with SIINFEKL, wherein the mass ratio of SIINFEKL to LNP was approximately 1:3.

[0179] Bone marrow cells were obtained from C57BL / 6 mice and GM-CSF (20 ng / mL) and IL-4 (10 ng / mL) were used to induce BMDC differentiation. 6 The cells were seeded at a density of 1 μg / mL in a 24-well cell culture plate, 1 mL per well, and supplemented with GM-CSF and IL-4. After overnight culture, SIINFEKL (1 μg / mL) and OVA / LNP were added respectively. After further culture for 48 hours, the cells were collected, washed once with PBS, centrifuged at 300 g for 5 minutes, and incubated with T-Select H-2Kb OVA Tetramer-SIINFEKL-PE at 4°C for 2 hours. The proportion of MHC-I class complexes bound to SIINFEKL was determined by flow cytometry, and the effect of the anionic lipid nanoparticles of the present application on the antigen uptake function of DC was preliminarily evaluated.

[0180] Depend on Figure 6 As shown in Table 5 below, the PE-positive ratio of cells in the SIINFEKL / LNP group was higher than that in the PBS and SIINFEKL groups.

[0181] Table 5. Effects of anionic lipid nanoadjuvants on DC antigen presentation function (relative PE positive ratio).

[0182]

[0183] Unexpectedly, the PE intensity of the immunogen alone group increased by only 0.087 compared with PBS, while when the anionic lipid nanoadjuvant of the present invention was added, the PE intensity increased by 5.080 (increased by about 58 times, 5.080 / 0.087≈58.6)

[0184] This indicates that the anionic lipid nanoparticles (LNPs) of the present invention have very excellent immune adjuvant function.

[0185] Example 6: Distribution of anionic lipid nanoadjuvants in mice

[0186] In cellular uptake experiments, anionic lipid nanoparticles (LNPs) were labeled with the cell membrane near-infrared fluorescent probe DiR. The preparation process was similar to that in Example 1, except that 0.4 mg of DiR was added to the chloroform in S1 to produce DiR LNPs. The LNPs were injected subcutaneously into mice at the tail base, and the fluorescence distribution in the mice was imaged using a small animal in vivo imaging system. The excitation wavelength of DiR was 740 nm, and the emission wavelength was 780 nm.

[0187] The results are as follows Figure 7 As shown in the figure, the DiR fluorescence encapsulated by the anionic lipid nanoadjuvant is not only concentrated in the lymph nodes (indicated by the red arrow), but also distributed in more parts of the body, indicating that the anionic lipid nanoadjuvant can increase the multi-organ distribution of exogenous antigens throughout the body, especially to achieve targeted enrichment of lymph nodes.

[0188] Example 7: Effects of anionic lipid nanoadjuvants on biochemical indicators in mice

[0189] Eighteen 4-week-old female Balb / c mice were randomly divided into three groups. PBS, OVA, and OVA / LNP were injected subcutaneously at the tail base of the Balb / c mice on days 0, 7, and 14, respectively. Three days after the last injection, blood was collected from each group of mice, and serum was separated. Liver function (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and renal function (urea (Urea), creatinine (Crea)) were measured using an automatic biochemical analyzer. The results are shown in Figure 3. Figure 8 shown.

[0190] Depend on Figure 8As can be seen, there were no significant differences in ALT, AST, Urea, and Crea between the OVA and OVA / LNP groups compared to the PBS group. This demonstrates that the anionic lipid nanoparticles of the present invention have no effect on the liver and kidney function of mice, confirming their biosafety and contributing to their widespread application as immune adjuvants.

[0191] Example 8: In vivo immune response evaluation of anionic lipid nanoadjuvants

[0192] The spleen of the mice in Example 7 was collected to prepare a single cell suspension, and CD8 + T(CD3 + CD8 + )、mDC(CD45 + CD11c + CD80 + CD86 + ) in terms of quantity and proportion.

[0193] Depend on Figure 9 It can be seen that compared with the PBS group and the OVA group, the CD8 + T cell( Figure 9 A), mDC( Figure 9 B) were significantly increased, further illustrating that the anionic lipid nanoadjuvant of the present invention can increase the immunogenicity of various exogenous antigens, promote the immune response brought about by the corresponding vaccine, and have the effect of an immune adjuvant.

[0194] Example 9: In vivo pharmacodynamic evaluation of anionic lipid nanoadjuvants

[0195] Eighteen 4-week-old female C57BL / 6 mice were randomly divided into three groups. PBS, OVA, and OVA / LNP were injected subcutaneously at the tail base of the C57BL / 6 mice on days 0, 5, and 10, respectively. Three days after the last injection, MC38-OVA cells, a mouse colon cancer cell line modified with the chicken OVA gene, were inoculated subcutaneously on the right side of their back. Blood was collected 30 days later, and red blood cells were lysed and washed once with PBS. After centrifugation at 300 g for 5 min, the cells were incubated with T-Select H-2Kb OVATetramer-SIINFEKL-PE antibody purchased from Beijing Borma Biotechnology Co., Ltd. at 4°C for 2 h. The proportion of MHC class I complexes bound to SIINFEKL was determined by flow cytometry to evaluate the OVA antigen-specific CD8 + The proportion of T cells.

[0196] Depend on Figure 10 It can be seen that compared with the PBS group and the OVA group, the OVA antigen-specific CD8 +The proportion of T cells was significantly increased, indicating that the anionic lipid nanoadjuvant LNP can effectively process and present antigens and activate the body's immune response.

[0197] In summary, the present invention provides an anionic lipid nanoadjuvant with a specific formulation, component ratio, and negatively charged surface potential. It can promote the maturation of dendritic cells, an important antigen-presenting cell, improve the presentation efficiency of combined antigens, activate adaptive immune responses, and treat diseases, which has significant clinical significance.

[0198] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anionic lipid nanoadjuvant, characterized in that The anionic lipid nanoadjuvant is anionic lipid nanoparticles, which are composed of anionic lipids, PEG-lipids, cholesterol compounds and auxiliary lipids; Wherein, the anionic lipid is DSPA; The PEG-lipid is DMG-PEG; The cholesterol compound is CHO-HP; The auxiliary lipid is DOPE; The average hydrated particle size of the anionic lipid nanoadjuvant is 200 to 300 nm; The surface potential of the anionic lipid nanoadjuvant is -30mV to -35mV.

2. The anionic lipid nanoadjuvant according to claim 1, characterized in that The molar ratio of the cholesterol compound to the PEG-lipid is (35-45):

1.

3. The anionic lipid nanoadjuvant according to claim 1, characterized in that The molar ratio of the cholesterol compound to the PEG-lipid is (37-43):

1.

4. The anionic lipid nanoadjuvant according to claim 1, characterized in that The molar ratio of the cholesterol compound to the PEG-lipid is (38-42):

1.

5. The anionic lipid nanoadjuvant according to claim 1, characterized in that The molar ratio of the cholesterol compound to the PEG-lipid is (39-41):

1.

6. The anionic lipid nanoadjuvant according to claim 1, characterized in that The polydispersity coefficient of the anionic lipid nanoadjuvant is 0.2-0.

8.

7. The anionic lipid nanoadjuvant according to claim 1, characterized in that The polydispersity coefficient of the anionic lipid nanoadjuvant is 0.2-0.

6.

8. The anionic lipid nanoadjuvant according to claim 1, characterized in that The polydispersity coefficient of the anionic lipid nanoadjuvant is 0.2 to 0.

5.

9. The anionic lipid nanoadjuvant according to claim 1, characterized in that The polydispersity coefficient of the anionic lipid nanoadjuvant is 0.2-0.

4.

10. A method for preparing the anionic lipid nanoadjuvant according to claim 1, characterized in that: The following steps are involved: S1. The anionic lipid, PEG-lipid, cholesterol compound and auxiliary lipid are dissolved in an organic solvent and dried by rotary evaporation under reduced pressure to form a film; S2. The thin film obtained in step S1 is hydrated and homogenized in an aqueous phase to obtain the anionic lipid nanoadjuvant according to claim 1.

11. The preparation method according to claim 10, characterized in that: In step S1, the organic solvent is selected from the group consisting of chloroform, dichloromethane, methanol, or a combination thereof.

12. The preparation method according to claim 10, characterized in that In step S1, the temperature of the reduced pressure rotary evaporation drying is 20-80°C.

13. The preparation method according to claim 10, characterized in that In step S1, the temperature of the reduced pressure rotary evaporation drying is 25-75°C.

14. The preparation method according to claim 10, characterized in that In step S1, the temperature of the reduced pressure rotary evaporation drying is 30-60°C.

15. Use of the anionic lipid nanoadjuvant according to claim 1 in preparing a vaccine composition.

16. A vaccine composition, characterized in that The vaccine composition contains (a) an immunogen or a nucleic acid molecule encoding the immunogen, (b) the anionic lipid nanoadjuvant according to claim 1, and (c) a pharmaceutically acceptable carrier.

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