Ionizable cationic lipid as well as preparation method and application thereof

Abstract

The invention provides an ionizable cationic lipid as well as a preparation method and application thereof. The ionizable cationic lipid, auxiliary phospholipid, cholesterol and PEG lipid can be self-assembled to form lipid nanoparticles. Compared with a commercially available drug ionizable cationic lipid, the formed lipid nanoparticles can more efficiently deliver mRNA into animal bodies, protein can be more efficiently expressed in the bodies, and the safety index meets the commercially available medicinal requirements; the mRNA vaccine prepared based on the nanoparticles can induce high-level virus specific antibody, specific T cell immunity and memory B cell response reaction, and is very suitable for mRNA vaccine and drug development.

Description

[0001] This application claims the priority benefit of a prior application with the application number 202410563216.2 and the title "An Ionizable Cationic Lipid and Its Preparation Method and Application", which was filed with the China National Intellectual Property Administration on May 8, 2024. The full text of the prior application is incorporated herein by reference. Technical Field

[0002] The present invention relates to the technical field of cationic lipids, and particularly to an ionizable cationic lipid with high efficiency, low toxicity, and high specific immunogenicity, as well as its preparation method and application. Background Art

[0003] Ionizable cationic lipids are key components in lipid nanoparticle (LNP) technology and play a crucial role in the delivery of mRNA vaccines and therapeutic drugs.

[0004] Ionizable cationic lipids have the following characteristics:

[0005] (1) pH sensitivity, capable of changing their charge state at different pH values. At physiological pH (about 7.4), they are neutral, reducing toxicity to cell membranes. In an acidic environment, such as the endosome (pH about 5 - 6), they become positively charged, facilitating interaction with negatively charged mRNA.

[0006] (2) Cell penetration ability: Since mRNA molecules are negatively charged and difficult to cross the cell membrane, which is also negatively charged. The positively charged nature of ionizable cationic lipids in an acidic environment enables them to form a stable complex with mRNA, protecting mRNA from degradation and promoting its entry into cells through the cell membrane.

[0007] (3) Reduced immunogenicity: Traditional positively charged liposomes may cause a strong immune response, while ionizable cationic lipids, due to their pH sensitivity, reduce immunogenicity under physiological conditions, improving the safety of LNPs.

[0008] (4) Stability of LNPs: Ionizable cationic lipids contribute to the stability of LNPs and play an important role in the LNP structure, ensuring the stability and effectiveness of mRNA in vivo.

[0009] Currently, ionizable cationic lipids have been used in the clinical applications of various LNP platforms, including the treatment of genetic diseases and the development of COVID-19 mRNA vaccines, such as the marketed Dlin-MC3-DMA, SM-102, and ALC-0315. Although ionizable cationic lipids have shown great potential in mRNA delivery, there is still a need to continuously explore and optimize such lipids to improve their performance, reduce potential side effects, and develop new delivery systems. Summary of the Invention

[0010] In view of the deficiencies in the prior art, the present invention provides an ionizable cationic lipid, a preparation method thereof, and an application thereof.

[0011] On the one hand, the present invention provides an ionizable cationic lipid having the following structure of Formula I:

[0012]

[0013] According to an embodiment of the present invention, in Formula I,

[0014] R1 is selected from C 1-6 alkyl;

[0015] R2 is selected from C 1-12 alkyl;

[0016] R3 and R4 are the same or different and are each independently selected from unsubstituted or optionally halogen-substituted C 1-20 alkyl;

[0017] R is selected from H or halogen; m = 0, 1, 2, 3, 4;

[0018] According to an embodiment of the present invention, R1 is selected from C 1-3 alkyl; for example, R1 is selected from methyl, ethyl, n-propyl, and isopropyl.

[0019] According to an embodiment of the present invention, R2 is selected from C 1-6 alkyl; preferably, R2 is selected from C 3-6 alkyl; for example, R2 is selected from methyl, ethyl, n-propyl, isopropyl, n-pentyl, n-butyl, and n-hexyl.

[0020] According to an embodiment of the present invention, R3 and R4 are the same or different and are each independently selected from unsubstituted or optionally halogen-substituted C 1-12 alkyl; preferably, R3 and R4 are the same or different and are each independently selected from unsubstituted or optionally halogen-substituted C 6-10 alkyl; for example, R3 and R4 are each independently selected from C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C 10 alkyl, C 11 alkyl, C12 Alkyl. More preferably, R3 and R4 are each independently selected from n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0021] According to an embodiment of the present invention, the halogen is selected from F, Cl, Br, and I.

[0022] According to an embodiment of the present invention, the formula I is selected from the following structures:

[0023]

[0024] In a second aspect of the present invention, there is provided a method for preparing the ionizable cationic lipid described above, the method comprising one or both of the following synthesis steps:

[0025] (i) Reacting M-1 with R1-NH2 to form M-2

[0026]

[0027] (ii) Reacting M-2 with M-3 to obtain formula I

[0028]

[0029] Wherein, R1, R2, R3, R4, R, and m are as defined in formula I above.

[0030] According to an embodiment of the present invention, in step (i), the reaction is carried out in the presence of an organic solvent A; the organic solvent A is selected from alcohols, preferably ethanol; the reaction temperature is 20 - 30 °C, and the reaction time is 1 - 5 hours, preferably 2 - 4 hours, for example 3 hours.

[0031] According to an embodiment of the present invention, in step (ii), the reaction is carried out in the presence of an organic solvent B and a basic reagent; the organic solvent B is selected from tetrahydrofuran; the basic reagent is selected from sodium bicarbonate, sodium carbonate, potassium bicarbonate, and potassium carbonate; the reaction temperature is 20 - 35 °C, and the reaction time is 10 - 24 hours, preferably 14 - 18 hours, for example 16 hours.

[0032] According to an embodiment of the present invention, the method for preparing the ionizable cationic lipid comprises one or both of the following steps:

[0033] Step (1a): Reacting compound 11-1 with CH3-NH2 to form compound 11;

[0034]

[0035] Step (2a): Reacting compound 10 with compound 11 to form FS01

[0036]

[0037] In the third aspect of the present invention, there is provided a lipid nanoparticle comprising the above-mentioned ionizable cationic lipid.

[0038] According to an embodiment of the present invention, the lipid nanoparticle further comprises: one or several of co-lipids, sterol lipids, and PEG lipids.

[0039] According to an embodiment of the present invention, the co-lipid comprises phospholipid. Preferably, the phospholipid includes but is not limited to dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyl oleoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoyl phosphatidylcholine distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. In certain embodiments, the neutral phospholipids are selected from distearoyl phosphatidylcholine (DSPC) and dimyristoyl phosphatidylethanolamine (DMPE), preferably distearoyl phosphatidylcholine (DSPC).

[0040] According to an embodiment of the present invention, the sterol lipid is selected from one or more of cholesterol, cholesterol esters, steroid hormones, steroid vitamins, and phytosterols, preferably from one or more of cholesterol, cholesterol esters, and phytosterols, more preferably cholesterol.

[0041] According to an embodiment of the present invention, the PEG lipids include PEG-dilauroyl glycerol, PEG-dimyristoyl glycerol (PEG-DMG), PEG-dipalmitoyl glycerol, PEG-distearoyl glycerol (PEG-DSPE), PEG-dilauroyl glycerol amide, PEG-dimyristoyl glycerol amide, PEG-dipalmitoyl glycerol amide and PEG-distearoyl glycerol amide, PEG-cholesterol (1-[8′-(cholest-5-en-3[β]-yloxy)carboxamido-3′,6′-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-di-tetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMPE) or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA) and 1,2-distearoyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In some embodiments, the PEG lipid can be PEG2k-DMG. In some embodiments, the PEG lipid can be PEG2k-DSG. In some embodiments, the PEG lipid can be PEG2k-DSPE. In some embodiments, the PEG lipid can be PEG2k-DMA. In some embodiments, the PEG lipid can be PEG2k-C-DMA. In some embodiments, the PEG lipid can be PEG2k-DSA. In other embodiments, the PEG lipid can be PEG2k-C11. In some embodiments, the PEG lipid can be PEG2k-C14. In some embodiments, the PEG lipid can be PEG2k-C16. In some embodiments, the PEG lipid can be PEG2k-C18.

[0042] According to an embodiment of the present invention, the molar ratio of the ionizable cationic lipid, co-lipid, sterol lipid, and PEG lipid is (20-100):(5-20):(20-50):(0.5-5); preferably, the molar ratio is (30-60):(5-15):(30-40):(0.5-3). For example, in one embodiment of the present invention, the molar ratio of the ionizable cationic lipid, co-lipid, sterol lipid, and PEG lipid is 50:10:38.5:1.5.

[0043] According to an embodiment of the present invention, compared with lipid nanoparticles prepared from ionizable lipids Dlin-MC3-DMA and ALC-0315, the above lipid nanoparticles (empty LNPs) induce weaker innate immune responses and non-specific inflammatory responses, thus ensuring that the immune response is mainly driven by the bioactive agent rather than the carrier itself, thereby improving vaccine specificity and predictability. At the same time, for the treatment of expressing foreign genes, the above lipid nanoparticles do not cause persistent inflammation, which helps to stably express genes.

[0044] In a fourth aspect of the present invention, there is provided a composition comprising the above ionizable cationic liposome, lipid nanoparticle, and bioactive agent.

[0045] According to an embodiment of the present invention, the bioactive agent is one or more of nucleic acids, peptides, proteins, and small molecules.

[0046] According to an embodiment of the present invention, the peptide or protein includes an antigen or a fragment thereof, an antibody, or an antigen-binding fragment.

[0047] According to an embodiment of the present invention, the nucleic acid is RNA or DNA. In some embodiments, the nucleic acid includes antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer, etc. Preferably, the nucleic acid is mRNA.

[0048] According to an embodiment of the present invention, the encoded protein of the mRNA includes a reporter gene, a target protein, a gene editing protein, an antigenic protein, such as including eGFP, mCHERRY, Luciferase, IL-2, Cas9, OVA, SARS-CoV-2 S protein, varicella-zoster gE protein. Preferably, the encoded protein of the mRNA is varicella-zoster gE protein and hepatitis B virus HBsAg protein.

[0049] According to an embodiment of the present invention, the lipid nanoparticle encapsulates the bioactive agent.

[0050] In a fifth aspect of the present invention, there is provided a vaccine comprising the above cationic liposome, lipid nanoparticle, and composition.

[0051] In one embodiment of the present invention, the vaccine is an mRNA vaccine.

[0052] In one embodiment of the present invention, the vaccine can be an influenza vaccine, an AIDS (HIV) vaccine, a viral pneumonia (SARS, SARS-CoV-2, etc.) vaccine, a tuberculosis vaccine, a respiratory syncytial virus (RSV) vaccine, an enterovirus (such as EV71) vaccine, a lung cancer vaccine, a varicella-zoster virus (VZV) vaccine, a chronic hepatitis B virus vaccine, etc. In a preferred embodiment of the present invention, the vaccine is a varicella-zoster virus (VZV) vaccine and a chronic hepatitis B (HBV) vaccine.

[0053] In one embodiment of the present invention, compared with the vaccines prepared from ionizable lipids SM-102, Dlin-MC3-DMA, and ALC-0315, the vaccine has higher immunogenicity and / or activity.

[0054] In one embodiment of the present invention, compared with the vaccines prepared from ionizable lipids SM-102, Dlin-MC3-DMA, and ALC-0315, the vaccine induces higher antigen-specific antibody titers after the first immunization.

[0055] In one embodiment of the present invention, compared with the vaccines prepared from ionizable lipids SM-102, Dlin-MC3-DMA, and ALC-0315, the vaccine promotes more significant activation of specific memory B cells.

[0056] In one embodiment of the present invention, compared with the vaccines prepared from ionizable lipids SM-102, Dlin-MC3-DMA, and ALC-0315, the vaccine induces stronger antigen-specific T cells to secrete IFN-γ, TNF, and IL-2.

[0057] In one embodiment of the present invention, compared with the vaccines prepared from ionizable lipids SM-102, Dlin-MC3-DMA, and ALC-0315, the varicella-zoster virus vaccine activates a stronger Th1-type cell response.

[0058] In one embodiment of the present invention, the vaccine further comprises one or more pharmaceutically acceptable excipients. The excipients include any components other than the above-mentioned compounds and lipid components. The excipients can impart functional (e.g., controlling the drug release rate) and / or non-functional (e.g., adjuvants or diluents) characteristics to the drug or biologic. The choice of excipients will largely depend on factors such as the specific administration mode, the impact of the excipients on solubility and stability, and the dosage form. For example, when the drug or biologic is aqueous, the excipients include sugars (including but not limited to glucose, mannitol, sorbitol, etc.), salts, carbohydrates, and buffers (preferably at a pH of 3 to 9), etc.

[0059] In one embodiment of the present invention, the administration route of the vaccine can be intravenous administration, subcutaneous administration, intramuscular administration, inhalation administration, intranasal administration, etc. In a preferred embodiment of the present invention, the administration route of the vaccine is subcutaneous administration.

[0060] In the sixth aspect of the present invention, there is provided the use of the above ionizable cationic lipid, lipid nanoparticle, and composition for delivering a drug, wherein the drug is a bioactive agent.

[0061] In one embodiment of the present invention, the bioactive agent is one or more of nucleic acid, peptide, protein, and small molecule.

[0062] In the seventh aspect of the present invention, there is provided the use of the above ionizable cationic lipid, lipid nanoparticle, composition, and vaccine in the preparation of drugs for treating, diagnosing, or preventing diseases.

[0063] In one embodiment of the present invention, the diseases include one or more of cancer, genetic diseases, infectious diseases, genetic metabolic diseases, central nervous system diseases, digestive system diseases, cardiovascular diseases, immunodeficiency diseases, and skin diseases. In some embodiments, the infectious diseases include viral infectious diseases. Preferably varicella-zoster virus infectious disease and chronic hepatitis B virus infectious disease. In some embodiments, the cancer includes one or more of lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, or prostate cancer. In some embodiments, the genetic diseases include one or more of hemophilia, thalassemia, and Gaucher's disease. In some embodiments, the diseases also include phenylketonuria, atherosclerosis, multiple sclerosis, hypertension, etc.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] Compared with the ionizable cationic lipids of marketed drugs, the lipid nanoparticles formed by the ionizable cationic lipid FS01 of the present invention can deliver mRNA to animals more efficiently, express proteins more efficiently in vivo, and have higher in vivo safety, and its liver toxicity safety meets the requirements for marketing drugs. The mRNA vaccine prepared based on FS01 nanoparticles can induce high levels of virus-specific antibodies, specific T cell immunity, and memory B cell response, and is very suitable for mRNA vaccine and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 A shows the particle size detection results of FS01 and three marketed ionizable lipids, Figure 1 B shows the polydispersity index (PDI), Figure 1 C shows the Zeta potential, Figure 1D is the encapsulation efficiency;

[0067] Figure 2 A is the flow cytometry detection result of in vitro expression after incubating HEK-293T cells with EGFP mRNA encapsulated by FS01 and three commercially available ionizable lipids for 12 h respectively; Figure 2 B is the flow cytometry detection result of in vitro expression after incubating for 24 h;

[0068] Figure 3A is the delivery effect of intramuscular injection of FS01 and three commercially available ionizable lipids; Figure 3B is the delivery effect of intravenous injection; Figure 3C is the delivery effect of subcutaneous injection;

[0069] Figure 4A is the total luminescence value of the main organs after intramuscular injection of FS01 and three commercially available ionizable lipids; Figure 4B is the total luminescence value of the main organs after intravenous injection; Figure 4C is the luminescence value of the muscle at the injection site after intramuscular injection;

[0070] Figure 5 A is the antibody titer 7 days after the first immunization of the varicella-zoster virus (VZV) mRNA vaccine prepared with FS01 and three commercially available ionizable lipids; Figure 5 B is the antibody titer 7 days after the second immunization;

[0071] Figure 6 is the detection result of VZV gE + specific memory B cells activated by the VZV mRNA vaccine prepared with FS01 and three commercially available ionizable lipids;

[0072] Figure 7 A is the result of the VZV mRNA vaccine prepared with FS01 and three commercially available ionizable lipids inducing antigen-specific T cells to secrete IFN-γ; Figure 7 B is the result of the vaccine inducing antigen-specific T cells to secrete IL-2;

[0073] Figure 8A is the result of the VZV mRNA vaccine prepared with FS01 and three commercially available ionizable lipids inducing antigen-specific CD4 + memory T cells to secrete IFN-γ; Figure 8B is the result of the vaccine inducing antigen-specific CD4 + memory T cells to secrete TNF; Figure 8C is the result of the vaccine inducing antigen-specific CD4 + memory T cells to secrete IL-2.

[0074] Figure 9A is the antibody titer 7 days after the first immunization of the chronic hepatitis B (HBV) mRNA vaccine prepared with FS01 and three commercially available ionizable lipids; Figure 9 B is the antibody titer 7 days after the second immunization; Figure 9 C is the antibody titer 14 days after the second immunization;

[0075] Figure 10 A is the result of the HBV mRNA vaccine prepared with FS01 and three commercially available ionizable lipids inducing antigen-specific T cells to secrete IFN-γ; Figure 10 B is the result of the vaccine inducing antigen-specific T cells to secrete IL-2;

[0076] Figure 11A is the result of the HBV mRNA vaccine prepared with FS01 and three commercially available ionizable lipids inducing antigen-specific CD4 + memory T cells to secrete IFN-γ; Figure 11B is the result of the vaccine inducing antigen-specific CD4 + memory T cells to secrete TNF; Figure 11C is the result of the vaccine inducing antigen-specific CD4 + memory T cells to secrete IL-2;

[0077] Figure 12A is the median fluorescence intensity (MFI) of the expression of CD40, CD80, CD86, and MHC-II in the dendritic cell (DC) subset cDC1; Figure 12B is the MFI of the expression of CD40, CD80, CD86, and MHC-II in cDC2;

[0078] Figure 13 A is the survival curve of the mice in Example 8; Figure 13 B is the result of the curve of the body weight change of the mice after administration;

[0079] Figure 14A is the serum ALT level of the mice in Example 8 after three administrations and 14 days after administration; Figure 14B is the AST level in the serum;

[0080] Figure 15A is the level of the inflammatory factor IL-6 in the serum of the mice in Example 8 after three administrations and 14 days after administration; Figure 15B is the MCP-1 level in the serum; Figure 15C is the TNF-α level in the serum; Detailed implementation manners

[0081] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0082] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

[0083] Preparation of Example 1 FS01

[0084] 1. Preparation of Compound 2

[0085]

[0086] To a solution of Compound 1 (30.0 g, 117 mmol, 1.00 equivalent) and 8-bromooctanoic acid (28.7 g, 129 mmol, 1.10 equivalents) in dichloromethane (150 mL) was added DMAP (1.43 g, 11.7 mmol, 0.10 equivalent) and EDCI (24.7 g, 129 mmol, 1.10 equivalents), and the mixture was stirred at 25 - 35 °C for 16 hours. TLC showed the formation of a new spot. The solution was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to obtain Compound 2 (43.7 g, crude product) as a colorless oil.

[0087] 1 H NMR: (400 MHz, CDCl3) δ 4.87 (t, 1H), 3.40 (t, 2H), 2.29 (t, 2H), 1.86 (quin, 2H), 1.69 - 1.60 (m, 2H), 1.56 - 1.16 (m, 34H), 0.88 (t, 6H).

[0088] 2. Preparation of Compound 3

[0089]

[0090] At 25 - 35 °C, to a solution of Compound 2 (20.0 g, 43.3 mmol, 1.00 equivalent) in i-PrOH (100 mL) was added N-(3-aminopropyl)carbamic acid tert-butyl ester (37.8 g, 217 mmol, 37.8 mL, 5.00 equivalents), and then the solution was stirred at 55 - 65 °C for 16 hours. TLC showed complete consumption of Compound 2 to obtain the desired compound. The reaction was cooled to 20 - 35 °C and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to obtain Compound 3 (13.0 g, crude product) as a yellow oil.

[0091] 1 1H NMR: (400 MHz, CDCl3) δ 5.19 (br s, 1H), 4.86 (t, 1H), 3.19 (br d, 2H), 2.66 (t, 2H), 2.57 (t, 2H), 2.27 (t, 2H), 1.73 - 1.57 (m, 4H), 1.55 - 1.39 (m, 17H), 1.38 - 1.14 (m, 31H), 0.87 (t, 6H)

[0092] 3. Preparation of Compound 11

[0093]

[0094] Methylamine (2 M, 21.11 mL, 1.20 equiv) was added to a solution of Compound 11-1 (5.00 g, 35.2 mmol, 1.00 equiv) in EtOH (500 mL). The mixture was stirred at 20 - 30 °C for 3 h. TLC indicated the complete consumption of Compound 11-1. The reaction mixture was filtered and concentrated under reduced pressure to afford Compound 11 (5.00 g, crude) as a white solid.

[0095] 1 1H NMR: (400 MHz, CDCl3) δ 8.86 - 8.10 (m, 1H), 4.39 - 4.10 (m, 3H), 3.16 - 2.86 (m, 3H)

[0096] 4. Preparation of Compound 6

[0097]

[0098] To a mixed solution of Compound 4 (10.0 g, 43.65 mmol, 1.00 equiv) in dichloromethane (100 mL) and water (10.0 mL) were added cyclopentyl(diphenyl)phosphine dichloropalladium catalyst (3.19 g, 4.37 mmol, 0.10 equiv), butylboronic acid (6.68 g, 65.5 mmol, 1.50 equiv) and potassium carbonate (18.1 g, 131 mmol, 3.00 equiv). The mixture was stirred at 90 - 110 °C for 14 h. TLC indicated the complete consumption of Compound 4. The reaction mixture was quenched by adding water (50 mL) and extracted with ethyl acetate. The combined organic layers were washed with saturated NaCl solution (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue, affording Compound 6 (20.0 g, crude) as a black solid. Compound 6 was purified by column chromatography to give Compound 6 (7.30 g) as a yellow oil.

[0099] 11H NMR: (400 MHz, CDCl3) δ 7.23 (br d, 4H), 3.79 - 3.61 (m, 5H), 2.77 - 2.54 (m, 2H), 1.65 - 1.53 (m, 2H), 1.50 - 1.37 (m, 2H), 0.98 (t, 3H).

[0100] 5. Preparation of Compound 7

[0101]

[0102] To a solution of compound 6 (7.30 g, 35.4 mmol, 1.00 equiv) in THF (73.0 mL) at -5 - 5 °C was added LAH (2.5 M, 14.2 mL, 1.00 equiv). The mixture was stirred at 20 - 30 °C for 3 h. TLC indicated complete consumption of compound 6. The reaction mixture was quenched by adding 30.0 mL of saturated sodium potassium tartrate solution and extracted with ethyl acetate. The combined organic layers were washed with 50.0 mL of saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 7 as a yellow oil (5.88 g, 33.0 mmol, 93.2%).

[0103] 1 1H NMR: (400 MHz, CDCl3) δ 7.23 - 7.10 (m, 4H), 3.85 (br d, 2H), 2.93 (t, 2H), 2.75 - 2.59 (m, 2H), 1.64 - 1.53 (m, 3H), 1.49 - 1.36 (m, 2H), 0.95 - 0.93 (m, 1H), 0.97 (t, 2H)

[0104] 6. Preparation of Compound 8

[0105]

[0106] To a solution of compound 7 (5.88 g, 33.0 mmol, 1.00 equiv) and compound 7A (8.09 g, 36.3 mmol, 1.10 equiv) in DCM (30.0 mL) was added DMAP (403 mg, 3.30 mmol, 0.10 equiv) and EDCI (6.96 g, 36.3 mmol, 1.10 equiv), and then the solution was stirred at 25 - 35 °C for 7 h. TLC showed complete consumption of the starting materials. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to afford compound 8 as a yellow oil (9.52 g).

[0107] 11H NMR: EW43795-269-P1A (400 MHz, CDCl3) δ 7.24 - 7.11 (m, 4H), 4.27 (t, 2H), 3.41 (t, 2H), 2.98 (t, 2H), 2.71 - 2.58 (m, 2H), 2.31 (t, 2H), 1.94 - 0.79 (m, 2H), 1.68 - 1.55 (m, 4H), 1.49 - 1.38 (m, 4H), 1.33 (td, 4H), 0.97 (t, 3H)

[0108] 7. Preparation of Compound 9

[0109]

[0110] To a solution of Compound 8 (4.89 g, 12.8 mmol, 1.20 eq.) and Compound 3 (6.28 g, 10.6 mmol, 1.00 eq.) in i-PrOH (30.0 mL) was added sodium carbonate (3.38 g, 31.9 mmol, 3.00 eq.), and then the solution was stirred at 80 - 90 °C for 20 h. LCMS showed complete consumption of Compound 8 and formation of the desired compound. The solution was cooled to 25 - 35 °C, water (10.0 mL) was added thereto, and then the mixture was extracted with ethyl acetate. The combined organic layers were concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to afford Compound 9 (5.14 g) as a pale yellow oil.

[0111] 1 1H NMR: (400 MHz, CDCl3) δ 7.23 - 7.10 (m, 4H), 5.66 (br s, 1H), 4.87 (quin, 1H), 4.26 (t, 2H), 3.18 (br d, 2H), 2.96 (t, 2H), 2.72 - 2.59 (m, 2H), 2.45 (br t, 2H), 2.38 - 2.24 (m, 8H), 1.67 - 1.55 (m, 8H), 1.54 - 1.48 (m, 4H), 1.47 - 1.37 (m, 15H), 1.36 - 1.21 (m, 37H), 0.95 (t, 3H), 0.88 (t, 6H).

[0112] 8. Preparation of Compound 10

[0113]

[0114] At 20 - 30 °C, HCl / EtOAc (2 M, 30.0 mL, 10.0 equiv) was added to a solution of compound 9 (5.14 g, 6.00 mmol, 1.00 equiv) in DCM (15.0 mL), and then the solution was stirred for 1 hour. LCMS showed that compound 9 was completely consumed and the desired compound was obtained. The reaction mixture was diluted with water (50.0 mL) and adjusted to pH 7 - 8 with saturated sodium bicarbonate. The mixture was extracted with DCM, and the combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give compound 10 as a yellow oil (4.30 g).

[0115] 1 H NMR: (400 MHz, CDCl3) δ 7.20 - 7.07 (m, 4H), 4.86 (t, 1H), 4.41 (br s, 2H), 4.25 (t, 2H), 2.95 (t, 2H), 2.89 (t, 2H), 2.69 - 2.60 (m, 2H), 2.54 (t, 2H), 2.47 - 2.37 (m, 4H), 2.28 (q, 4H), 1.72 - 1.65 (m, 2H), 1.64 - 1.54 (m, 6H), 1.53 - 1.46 (m, 4H), 1.46 - 1.36 (m, 6H), 1.35 - 1.13 (m, 37H), 0.94 (t, 3H), 0.87 (t, 6H)

[0116] 9. Synthesis of Compound FS01

[0117]

[0118] To a solution of compound 10 (4.00 g, 5.25 mmol, 1.00 equiv) in THF (27.0 mL) was added compound 11 (964 mg, 6.83 mmol, 1.30 equiv) and sodium bicarbonate (34.8 g, 41.4 mmol, 16.1 mL, 10%, 7.89 equiv), and then the solution was stirred at 40 - 50 °C for 16 hours. LCMS showed that compound 10 was completely consumed and the desired compound was obtained. The mixture was extracted with ethyl acetate, and the combined organic layers were concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to obtain compound FS01 as a gummy white solid (2.18 g).

[0119] 11H NMR: (400 MHz, CDCl3) δ 7.22 - 7.10 (m, 4H), 4.86 (t, 1H), 4.25 (t, 2H), 3.66 (br s, 2H), 3.28 (br d, 3H), 2.96 (t, 2H), 2.70 - 2.59 (m, 2H), 2.53 (br t, 2H), 2.46 - 2.35 (m, 4H), 2.34 - 2.24 (m, 4H), 1.81 - 1.70 (m, 2H), 1.66 - 1.47 (m, 10H), 1.41 (qd, 7H), 1.34 - 1.18 (m, 36H), 0.95 (t, 3H), 0.88 (t, 6H)

[0120] Example 2 Preparation of Liposome Nanoparticles Encapsulating mRNA and Detection of Its Particle Size and Zeta Potential

[0121] Preparation of Lipid Solution: Dissolve the ionizable cationic lipid (SM - 102 / Dlin - MC3 - DMA / ALC - 0315 / FS01):DSPC:cholesterol:mPEG2000 - DMG at a molar ratio of 50:10:38.5:1.5 in an ethanol solution;

[0122] Preparation of mRNA Solution: Dissolve a certain mass of Luciferase mRNA in 10 mM citric acid buffer solution with pH = 4.0;

[0123] Preparation of Lipid Nanoparticles: Use a syringe to separately draw 1 mL of Luciferase mRNA and 3 mL of lipid solution, insert them into a microfluidic chip, and set the parameters as: Volume: 4.0 mL; Flowrate ratio: 3:1, Total flow rate: 18 mL / min, and mix to obtain a lipid nanoparticle solution;

[0124] Solution Replacement: Add the lipid nanoparticle solution to an ultrafiltration tube for centrifugal ultrafiltration, and replace it multiple times with phosphate buffer solution to obtain the finished product.

[0125] Particle Size and Potential Detection: Dilute the above - prepared finished product, and use a Brookhaven particle size analyzer to detect the particle size, Zeta potential, and polydispersity index (PDI) of the LNP prepared with four ionizable lipids, and calculate the encapsulation efficiency of LNP. The experimental results are as Figure 1 shown.

[0126] The results show that compared with the three commercially available ionizable lipids, the LNP - mRNA prepared by encapsulating Luciferase mRNA with FS01 has a smaller particle size and PDI ( Figure 1 A, 1B), and the Zeta potential is within the normal value range (Figure 1 C), the encapsulation efficiency is comparable to that of SM-102 and Dlin-MC3-DMA.

[0127] Example 3 In vitro delivery effect of FS01-LNP

[0128] EGFP mRNA was introduced as a reporter gene to observe the mRNA transfection effect of the prepared LNPs at the cellular level. After transfection of EGFP mRNA into cells, enhanced green fluorescent protein can be expressed intracellularly. The emission wavelength of this protein is 509 nm, and the excited green fluorescence can be detected by a FACScelesta flow cytometer.

[0129] Experimental method: Resuscitate HEK-293T cells. When the cell density reaches 80-100%, seed the cells in a 12-well plate; after 12 hours when the cells grow to 70% density, add four LNP solutions encapsulating 1 μg of EGFP mRNA to HEK-293T cells respectively. Cells added with an equal volume of PBS solution are used as negative controls. Place them in an incubator at 37 °C. Start harvesting cells after incubating for 12 / 24 hours. Aspirate the culture medium, add 1 ml of PBS and gently pipette the cells. Collect the cell suspension into a 1.5 ml centrifuge tube. Centrifuge at 1500 rpm for 5 minutes. After centrifugation, discard the supernatant, add 400 μl of PBS to resuspend the cells, and detect the average fluorescence intensity of the cells by flow cytometry. The experimental results are as Figure 2 shown in Figures A and 2B.

[0130] The results showed that, compared with the three commercially available ionizable lipids, at the dosage of 1 μg of LNP-mRNA, after incubating for 12 h ( Figure 2 Figure A) and 24 h ( Figure 2 Figure B), the in vitro delivery effect of the LNP-mRNA prepared by encapsulating EGFP mRNA with the ionizable lipid FS01 can reach the same level as that of the commercially available ionizable lipid SM-102, and is higher than that of Dlin-MC3-DMA and ALC-0315.

[0131] Example 4 In vivo delivery effect of FS01-LNP

[0132] LNP preparations prepared by encapsulating Luciferase-mRNA with four ionizable lipids (SM-102 / Dlin-MC3-DMA / ALC-0315 / FS01) were injected into mice by three injection methods: intramuscular, intravenous, and subcutaneous, for bioluminescence imaging analysis. The specific operations are as follows:

[0133] 1) Inject the four LNP preparations into mice by intramuscular injection, intravenous injection, and subcutaneous injection respectively at a dose of 5 μg per mouse;

[0134] 2) After 6 h, 12 h, 24 h, 48 h, and 72 h of administration, 200 μL of 15 mg / mL potassium D - luciferin solution was intraperitoneally injected into each mouse, and the mouse was anesthetized with isoflurane.

[0135] 3) After 10 min, a small animal in - vivo imager was used to observe the expression of Luciferase - mRNA delivered by lipid nanoparticles in the mouse body through bioluminescence, so as to compare the in - vivo delivery effects of the four LNP formulations. The results are as Figure 3A 、 3B 、shown in Figure 3C.

[0136] The results showed that in intramuscular administration ( Figure 3A ), the delivery effect of FS01 - LNP was slightly lower than that of SM - 102, but both were higher than those of Dlin - MC3 - DMA - LNP and ALC - 0315 - LNP; in subcutaneous administration ( Figure 3B ), the delivery effect of FS01 - LNP was higher than that of the other three commercially available LNPs; in intravenous injection ( Figure 3C ), the delivery effect of FS01 - LNP was comparable to that of SM - 102 - LNP, and both were higher than those of Dlin - MC3 - DMA - LNP and ALC - 0315 - LNP.

[0137] Example 5 In - vivo distribution of FS01 - LNP

[0138] The LNP formulations prepared by encapsulating Luciferase - mRNA with four ionizable lipids (SM - 102 / Dlin - MC3 - DMA / ALC - 0315 / FS01) were injected into mice by two injection methods, intramuscular and intravenous, for bioluminescence imaging analysis. The specific operations are as follows:

[0139] 1) Four LNP formulations were injected into mice by intramuscular injection and intravenous injection at a dose of 5 μg per mouse.

[0140] 2) After 3 h, 6 h, 12 h, and 24 h of administration, 200 μL of 15 mg / mL potassium D - luciferin solution was intraperitoneally injected into each mouse.

[0141] 3) After 8 min, the mice were dissected, and the heart, liver, spleen, lung, kidney, and the muscle at the injection site of the mice were taken. A small animal in - vivo imager was used to observe the expression and distribution of Luciferase - mRNA delivered by lipid nanoparticles in the main organs of the mouse body through bioluminescence. The experimental results are as Figure 4A 、 4B 、shown in Figure 4C.

[0142] The results showed that from the total luminescence value of the main organs ( Figure 4A)It can be seen that in intramuscular administration, the delivery effect of FS01-LNP is slightly lower than that of SM-102-LNP, but both are higher than those of Dlin-MC3-DMA-LNP and ALC-0315-LNP, which is consistent with the in vivo imaging results; the results of intravenous administration( Figure 4B )also show a trend similar to that of in vivo imaging; analyzing the luminescence signal of the muscle at the administration site in intramuscular administration( Figure 4C ), it is found that the FS01-LNP group and the SM-102-LNP group can reach the same level, and both are higher than the Dlin-MC3-DMA-LNP group and the ALC-0315-LNP group.

[0143] Example 6 Pharmacokinetics and Immunogenicity of FS01-LNP

[0144] 1. Immunogenicity of Herpes Zoster mRNA Vaccine

[0145] The immunogenicity of mRNA vaccines encoding varicella-zoster virus gE protein (the preparation method of the mRNA vaccine can refer to Patent CN116474086A) respectively prepared from four ionizable lipid molecules and FS01 that have been marketed was systematically evaluated in mice. The specific steps are as follows:

[0146] 1.1 Animal Immunization

[0147] C57BL / 6 mice (female, initial immunization age 6 weeks, purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) were used for vaccine immunogenicity detection. An immunization dose of 1 μg was used, and double-dose immunization (immunization on days 0 and 14) was carried out. There were a total of 5 groups, 4 mice in the PBS control group, and 6 mice in each of the other groups. After the vaccine was diluted with PBS to reach the specified concentration, it was intramuscularly injected through the thigh, with an injection volume of 50 μl and unilateral injection.

[0148] On days 7 and 21 after immunization, blood was collected, left standing at room temperature for more than 1 hour, centrifuged at 3000 rpm for 10 minutes, the serum was separated, and the samples were stored at -80 °C for subsequent immunological analysis.

[0149] On day 21 after immunization, all animals were dissected, the spleens were placed in a 40 μm cell strainer and ground, erythrocyte lysate (purchased from Solarbio) was added and placed at room temperature for 5 minutes, then 10 ml of PBS was added, centrifuged at 1600 rpm for 5 min, and resuspended with PBS to prepare a single-cell suspension.

[0150] 1.2 Antibody Titer Detection

[0151] The gE antigen (purchased from Acro Biosystem) was diluted with PBS at a concentration of 0.5 μg / ml for coating. 100 μl was added to each well of a 96-well plate and incubated in an air bath at 4 °C for 12 hours. 200 μl of washing solution was added to each well for washing, and the washing was repeated three times. The plate was blocked with a blocking solution. 200 μl was added to each well and incubated in an air bath at 27 °C for 2 hours. After blocking, the blocking solution was discarded. The samples were diluted with a sample diluent according to the serum dilution gradient (100 / 200 / 400 / 800 / 1600 / 3200 / 6400 / 12800 / 25600 / 51200 / 102400 / 204800 / 409600). 100 μl of the sample was added to each well and incubated in an air bath at 27 °C for 2 hours. The plate was washed 3 times with the washing solution, 200 μl each time. The secondary antibody Goat anti-mouse IgG:HRP (purchased from Abcam) was diluted 1:50000 with the sample diluent. 100 μl of the diluted secondary antibody was added to each well and incubated in an air bath at 27 °C for 1 hour. The ELISA plate was washed 4 times, 200 μl per well, and the liquid in the plate was completely removed during the last wash. For the washed plate, 100 μl of TMB chromogenic solution was added to each well for color development, and the color development status was observed in real time. 100 μl of stop solution was added to each well for termination, and then the plate was detected on the machine. The detection indicators of the microplate reader at double wavelengths: The OD value was read at a wavelength of 450 nm, and the dilution ratio corresponding to the OD value greater than 4.1 times that of the negative control (Cut off) was recorded as the antibody titer. The results are as Figure 5 shown in Figures A and 5B.

[0152] The results showed that 7 days after the first immunization, compared with the vaccine groups prepared with three commercially available ionizable lipids, the vaccine group prepared with the ionizable lipid FS01 induced the highest antigen-specific antibody titer. 7 days after the second immunization, the antigen-specific antibody titer induced by the vaccine group prepared with the ionizable lipid FS01 was close to that of the vaccine group prepared with SM-102, and both were higher than those of the other two experimental groups.

[0153] 1.3 Detection of specific memory B cells

[0154] The gE protein was labeled with biotin using a Super Biotin Quick Labeling Kit (purchased from Frdbio), and then conjugated with gE-Biotin and fluorophores (APC, BV421). The spleen lymphocytes isolated from the dissected mice mentioned above were counted, and 2.5×10 6Spleen lymphocytes were transferred into a sterile flow tube, and the cells were washed once with PBS at 1500 rpm for 5 min. Then, 5 μl of gE-biotin-APC and gE-biotin-BV421, along with 40 μl of FACS buffer, were added, and the mixture was incubated at 4°C for 20 min. Without washing, surface staining was directly performed. 50 μl of a live / dead dye diluted with PBS (purchased from thermo Fisher) was added, and the cells were stained at room temperature for 5 min. Subsequently, 50 μl of an anti-mouse antibody mixture diluted with PBS was added, including APC / Cyanine7 anti-mouse IgM Antibody, BrilliantViolet 605TM anti-mouse IgD Antibody, PerCP-Cy5.5 anti-mouse / human CD45R / B220 Antibody, PE anti-mouse CD19 Antibody, AF700 CD38 (all purchased from Biolegend), and the cells were stained at 4°C in the dark for 20 min. The cells were washed once with 1×PBS and then subjected to detection on the machine. The experimental results are as Figure 6 shown.

[0155] It can be seen from the results that compared with the vaccine groups prepared with three commercially available ionizable lipids, the vaccine group prepared with the ionizable lipid FS01 can activate more significant specific memory B cells.

[0156] 1.4 Detection of specific T cells

[0157] The IL-2 and IFN-γ detection kits (both purchased from MABTECH) were used and the operations were carried out according to the instructions. The specific steps are as follows: Dilute the coating antibody with PBS to a concentration of 15 μg / ml, and calculate and prepare the volume according to the requirement of 100 μl per well. Take out the PVDF plate, pre-wet it with 35% ethanol, 15 μl / well, for at most 1 min. Wash the plate 5 times with sterile water, 200 μl / well. Add 100 μl / well of the coating antibody, incubate overnight at 4 - 8 °C. Remove the coating solution, wash 4 times with sterile PBS, 200 μL / well; block with 1640 complete medium containing 10% FBS, 200 μL / well, place it in the cell culture incubator, and let it stand for more than 30 min. Dilute the protein concentration to 2 μg / mL with 1640 complete medium. Take out the ELISpot plate, discard the medium, add 100 μL / well of gE protein (final concentration 1 μg / mL), and add 100 μL of medium to the negative control. Add 100 μL / well of the above-separated spleen cell suspension (cell number 2×105 / well), place it in the cell culture incubator for 18 - 24 hours. Take out the ELISpot plate, discard the liquid, wash 5 times with PBS, 200 μL / well, and pat dry. Dilute the primary antibody (detection antibody conjugated with biotin) with the antibody diluent at a ratio of 1:1000 (final concentration: 1 μg / mL), add 100 μL to each well, and incubate at room temperature for 2 hours. Discard the primary antibody, wash 5 times with PBS, 200 μL / well, and pat dry. Dilute the secondary antibody (Streptavidin-ALP) with the antibody diluent at a ratio of 1:1000, add 100 μL to each well, and incubate at room temperature for 1 hour. Discard the secondary antibody, wash 5 times with PBS, 200 μL / well, and pat dry. Add 100 μL of the filtered chromogenic solution BCIP / NBT-plus to each well, avoid light, and observe the chromogenic state in real time. Rinse the plate thoroughly with running water, dry it, store it in the dark at room temperature, and read the plate using an ELISpot reader. The experimental results are as Figure 7 shown in A and 7B.

[0158] As can be seen from the results, the vaccine group prepared with the ionizable lipid FS01 induced stronger IFN-γ- and IL-2-secreting antigen-specific T cells compared to the vaccine groups prepared with the three commercially available ionizable lipids.

[0159] 1.5 Intracellular cytokine detection

[0160] Take the above-separated spleen lymphocytes, count them, and transfer 3×10 6Cells were plated in 96-well U-bottom plates, 100 μL of the system, gE protein (5 μg / ml) and 1×BFA (purchased from Biolegend) were added, 100 μL of the system. Stimulation was performed for 10-12 hours. Washed once with 1×PBS. 50 μL of dead-alive dye (purchased from Thermo Fisher) diluted in PBS was added, and staining was performed at room temperature for 5 minutes. Then 50 μL of anti-mouse antibody mixture diluted in PBS, including FITC anti-mouse CD3, BV785 anti-mouse CD4, PEanti-mouse CD44, APC anti-mouse CD62L (all purchased from Biolegend), and FcR blocking (purchased from Miltenyi Biotech) were added, and staining was performed at room temperature for 20 minutes. Washed cells with 1×PBS, discarded the supernatant, and tried to ensure that there was no PBS residue. 200 μL of fixation buffer (BD) was added to resuspend the cells, and gently pipetted to mix. Place at room temperature away from light for 20 minutes; centrifuge and discard the supernatant; add 1 mL 1×perm / wash buffer (purchased from BD) for intracellular cytokine staining, add 50 μl 1×perm / wash buffer diluted BV421 anti-mouse IFN-γ, BV605 anti-mouse TNF-α, PE-Cy7 anti-mouseIL-2 (all purchased from Biolegend), place at room temperature away from light for 20 minutes; wash once with 1×PBS and detect on the machine.

[0161] The experimental results are shown in Figure 8. The vaccine group prepared with ionizable lipid FS01 induced stronger secretion of IFN-γ than the vaccine groups prepared with ionizable lipids Dlin-MC3-DMA and ALC-0315 ( Figure 8A ), TNF( Figure 8B ) and IL-2( Figure 8C ) Antigen-specific CD4 + Memory T cells, thus the vaccine group prepared with ionizable lipid FS01 can activate a stronger Th1 cell response.

[0162] 2. Immunogenicity of chronic hepatitis B mRNA vaccine

[0163] The immunogenicity of mRNA vaccines encoding hepatitis B virus HBsAg made from four types of ionizable lipid molecules and FS01 were systematically evaluated in mice. The specific steps are as follows:

[0164] 2.1 Animal immunization

[0165] C57BL / 6 mice (female, initial immunization age 6 weeks, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.) were used for vaccine immunogenicity detection. An immunization dose of 1 μg was adopted, with double-dose immunization (immunization on days 0 and 14), a total of 5 groups, with 6 mice in each group. After the vaccine was diluted with PBS to reach the specified concentration, intramuscular injection was performed through the thigh area, with an injection volume of 50 μl and unilateral injection.

[0166] On days 7, 21, and 28 after immunization, blood was collected, left to stand at room temperature for more than 1 hour, centrifuged at 3000 rpm for 10 minutes, the serum was separated, and the samples were stored at -80 °C for subsequent immunological analysis.

[0167] On day 28 after immunization, all animals were dissected, the spleens were placed in a 40-μm cell strainer and ground, erythrocyte lysate (purchased from Solarbio) was added and left at room temperature for 5 minutes, then 10 ml of PBS was added, centrifuged at 1600 rpm for 5 min, and resuspended with PBS to prepare a single-cell suspension.

[0168] 2.2 Antibody titer detection

[0169] A kit for detecting hepatitis B virus surface antibody (enzyme-linked immunosorbent assay, Beijing Wantai Biological Pharmacy Co., Ltd.) was used to detect the content of antigen-specific antibodies in mice at different time points after immunization.

[0170] The results showed that 7 days after the first immunization, the antigen-specific antibody titer induced by the FS01 HBV vaccine group was comparable to that of the SM-102 HBV vaccine group, and higher than that of the Dlin-MC3-DMA and ALC-0315 vaccine groups ( Figure 9 A); 7 days and 14 days after the second immunization, the antibody titers were significantly higher than those of the Dlin-MC3-DMA HBV vaccine group and were comparable to those of the other two experimental groups ( Figure 9 B, 9C).

[0171] 2.3 Specific T cell detection

[0172] The experimental method was the same as 1.4. The experimental results were as Figure 10 shown. The FS01 HBV vaccine group induced more antigen-specific T cells secreting IFN-γ and IL-2 than the Dlin-MC3-DMA HBV vaccine group, and was slightly lower than the SM-102 HBV vaccine group and the ALC-0315 HBV vaccine group.

[0173] 2.4 Intracellular cytokine detection

[0174] The experimental method was the same as 1.5. The experimental results are shown in Figure 11. The FS01 HBV vaccine induced antigen-specific CD4 secreting IFN-γ, TNF-α, and IL-2 +The number of memory T cells was slightly lower than that in the SM-102 HBV vaccine and ALC-0315 HBV vaccine groups, and was similar to the number induced by the Dlin-MC3-DMA HBV vaccine. These results indicate that the FS01 HBV vaccine has strong immunogenicity and is superior to the Dlin-MC3-DMA HBV vaccine.

[0175] Example 7 Innate Immune Response Induced by Empty LNP

[0176] The ionizable lipid molecules SM-102, Dlin-MC3-DMA, ALC-0315, and FS01 were prepared into empty LNPs (eLNPs) according to the aforementioned method and injected intramuscularly into C57BL / 6J mice at a dose of 1000 μg. PBS was used as a placebo, and the TLR7 / 8 agonist (25 μg / mouse) was used as a positive control. The mice were sacrificed 12 hours after injection, and the draining lymph nodes (dLNs) were collected for flow cytometry analysis.

[0177] The experimental results are shown in Figure 12. Under the stimulation of all eLNPs and the TLR7 / 8 agonist, dendritic cell (DC) subsets, especially cDC1 and cDC2, had a strong activation effect. The median fluorescence intensity (MFI) of CD40, CD80, CD86, and MHC-II expression in these subsets was measured. The results showed that all eLNPs induced high levels of CD40 and MHC-II expression in cDC1 cells, consistent with the TLR7 / 8 agonist. The expression levels of CD80 and CD86 induced by FS01 eLNP and SM-102 eLNP were slightly lower than those induced by MC3 eLNP and ALC-0315 eLNP ( Figure 12A ). A similar trend was also observed in cDC2 cells ( Figure 12B ). In summary, compared with Dlin-MC3-DMA and ALC-0315, FS01 and SM-102 induced a weaker innate immune response.

[0178] Example 8 In Vivo Safety Evaluation of eLNP

[0179] To compare the safety of eLNPs prepared with FS01 with those prepared with commercially available ionizable lipids (SM-102, Dlin-MC3-DMA, and ALC-0315), they were injected into male C57BL / 6J mice at a high dose of 2000 μg per mouse. The immunization was divided into three times, administered once every 3 days, and the mice were sacrificed 7 days after the third administration. Biochemical and physiological parameters were monitored, including survival, body weight, biochemical indices, and cytokine levels.

[0180] The results are as Figure 13, as shown in Figures 14 and 15, all mice in the Dlin-MC3-DMA eLNP group died during the experimental period; 1 mouse in the SM-102 eLNP group died after the first administration, while no mouse death was observed in the FS01 and ALC-0315 eLNP groups( Figure 13 A). Compared with the PBS group, all eLNP groups showed weight loss. The weight loss of the FS01 eLNP was slightly greater than that of the SM-102 and ALC-0315 eLNP, but lower than that of the Dlin-MC3-DMA eLNP( Figure 13 B). After three administrations, the serum ALT levels in the FS01 and SM-102 eLNP groups were within the normal range, lower than those in the Dlin-MC3-DMA and ALC-0315 eLNP groups( Figure 14A ); The serum AST test results after three administrations showed that except for a single mouse in the Dlin-MC3-DMA eLNP group with a slightly higher AST level than the normal value after the first administration, the serum AST levels in other groups were within the normal range( Figure 14B ); The detection results of inflammatory factors IL-6, MCP-1, and TNF-α showed that the secretion levels of inflammatory factors in the FS01 eLNP group were lower than those in the Dlin-MC3-DMA eLNP group. Similar to the ALT and AST levels, all inflammatory cytokine levels returned to the baseline level 7 days after the third administration( Figure 15A -C). These results indicate that FS01 eLNP has high safety and is superior to Dlin-MC3-DMA eLNP.

[0181] 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 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 purpose 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. An ionizable cationic lipid, characterized in that It has the following structure: Wherein, R1 is selected from C 1-6 alkyl; R2 is selected from C 1-12 alkyl; R3 and R4 are the same or different and are independently selected from C 1-20 alkyl; R is selected from H or halogen; m=0, 1, 2, 3, 4.

2. An ionizable cationic lipid as claimed in claim 1, characterized in that R1 is selected from C 1-3 Alkyl; preferably, R1 is selected from methyl, ethyl, n-propyl, isopropyl; R2 is selected from C 1-6 Alkyl; preferably, R2 is selected from C 3-6 Alkyl; for example, R2 is selected from methyl, ethyl, n-propyl, isopropyl, n-pentyl, n-butyl, n-hexyl; R3, R4 are the same or different, each independently selected from unsubstituted or optionally substituted by halogen C 1-12 Preferably, R3, R4 are the same or different, each independently selected from unsubstituted or optionally substituted by halogen C 6-10 Alkyl; more preferably, R3 and R4 are each independently selected from n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl; the halogen is selected from F, Cl, Br, and I; Preferably, the formula I is selected from the following structures:

3. The method for preparing the ionizable cationic lipid according to claim 1 or 2, characterized in that: The preparation method comprises one or two of the following synthesis steps: (i) Reaction of M-1 with R1-NH2 to generate M-2 (ii) reacting M-2 with M-3 to obtain formula I Wherein, R1, R2, R3, R4, R, and m are as defined in Formula I; Preferably, in step (i), the reaction is carried out in the presence of an organic solvent A; the organic solvent A is selected from alcohols, preferably ethanol; the reaction temperature is 20-30° C., the reaction time is 1-5 hours, preferably 2-4 hours, more preferably 3 hours; Preferably, in the step (ii), the reaction is carried out in the presence of an organic solvent B and an alkaline agent; the organic solvent B is selected from tetrahydrofuran; the alkaline agent is selected from sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate; the reaction temperature is 20-35°C, and the reaction time is 10-24 hours, preferably 14-18 hours, and more preferably 16 hours. Preferably, the method for preparing the ionizable cationic lipid comprises one or two of the following steps: Step (1a): reacting compound 11-1 with CH3-NH2 to generate compound 11; Step (2a): Compound 10 is reacted with compound 11 to generate FS01 4. A lipid nanoparticle, characterized in that: The invention comprises the ionizable cationic lipid according to claim 1 or 2.

5. A lipid nanoparticle according to claim 4, characterized in that, The lipid nanoparticles further include: one or more of auxiliary lipids, sterol lipids, and PEG lipids; Preferably, the molar ratio of the ionizable cationic lipid, the auxiliary lipid, the sterol lipid, and the PEG lipid is (20-100):(5-20):(20-50):(0.5-5).

6. A composition, characterized in that Comprising the ionizable cationic lipid according to claim 1 or 2, the lipid nanoparticle according to claim 4 or 5, and a bioactive agent; Preferably, the bioactive agent is one or more of nucleic acids, peptides, proteins and small molecules; more preferably, the nucleic acid is RNA or DNA; further preferably, the nucleic acid is mRNA; Preferably, the protein encoded by the mRNA includes a reporter gene, a target protein, a gene editing protein, and an antigen protein; more preferably, the protein encoded by the mRNA is varicella-zoster gE protein and chronic hepatitis B virus HBsAg protein.

7. A vaccine, characterized in that Comprising the ionizable cationic lipid according to claim 1 or 2, the lipid nanoparticle according to claim 4 or 5, and the composition according to claim 6; Preferably, the vaccine is an mRNA vaccine; more preferably, the vaccine is a varicella-zoster virus (VZV) vaccine or a chronic hepatitis B (HBV) vaccine.

8. The vaccine according to claim 7, characterized in that Compared to vaccines prepared with ionizable lipids SM-102, Dlin-MC3-DMA and ALC-0315, the vaccine has higher immunogenicity and / or activity; Preferably, the vaccine induces higher antigen-specific antibody titers after one immunization compared to vaccines prepared with ionizable lipids SM-102, Dlin-MC3-DMA and ALC-0315; Preferably, the vaccine promotes more significant specific memory B cell activation compared to vaccines prepared with ionizable lipids SM-102, Dlin-MC3-DMA and ALC-0315; Preferably, the vaccine induces stronger secretion of IFN-γ, TNF and IL-2 by antigen-specific T cells compared to vaccines prepared with ionizable lipids SM-102, Dlin-MC3-DMA and ALC-0315; Preferably, the varicella-zoster virus vaccine activates a stronger Th1 cell response than vaccines prepared with ionizable lipids SM-102, Dlin-MC3-DMA and ALC-0315; Preferably, the vaccine further comprises one or more pharmaceutically acceptable excipients.

9. The ionizable cationic lipid according to claim 1 or 2, the lipid nanoparticle according to claim 4 or 5, or the composition according to claim 6 is used for delivering a drug, wherein the drug is a bioactive agent.

10. Use of the ionizable cationic lipid according to claim 1 or 2, the lipid nanoparticle according to claim 4 or 5, the composition according to claim 6, or the vaccine according to claim 7 or 8 in the preparation of a drug for treating, diagnosing or preventing a disease; Preferably, the disease is varicella-zoster virus infectious disease or chronic hepatitis B virus infectious disease.

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